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Brealey y PN veut Re Tr aa ener) ant vind Mesut ¥ Lata am me “ZOOLOGICAL SCIENCE An International Journal ZOOLOGICAL SCIENCE The Official Journal of the Zoological Society of Japan Bers CMe The Zoological Society of Japan: POON LEVIS 8) Tosi) Toshin-building, Hongo 2—27-2, Bunkyo-ku, MidestnicobayastiaaTok yo) Tokyo 113, Japan. Phone (03) 3814-5675 Managing Editor: Ose USERS Narmayen (Oleie me) President: Hideo Mohri (Chiba) exssistanl Editors: : Secretary: Hideo Namiki (Tokyo) Vosm ase Lamsinima (Qlsayara) Treasurer: Makoto Okuno (Tokyo) Masaki Saket Csyane) Librarian: Masatsune Takeda (Tokyo) Akiyoshi Nida (Okayama) a Editorial Board: Howard A. Bern (Berkeley) Walter Bock (New York) Aubrey Gorbman (Seattle) Horst Grunz (Essen) Robert B. Hill (Kingston) Yukio Hiramoto (Chiba) Susumu Ishii (Tokyo) Yukiaki Kuroda (Tokyo) John M. Lawrence (Tampa) Koscak Maruyama (Chiba) Roger Milkman (Iowa) Kazuo Moriwaki (Mishima) Richard S. Nishioka (Berkeley) Chitaru Oguro (Toyama) Tokindo S. 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ZOOLOGICAL SCIENCE 9: 679-695 (1992) © 1992 Zoological Society of Japan REVIEW Hormonally Induced Synaptic Plasticity in the Adult Neuroendocrine Brain AKIRA MATSUMOTO Department of Anatomy, Juntendo University School of Medicine, Hongo, Tokyo 113, Japan CONTENTS MNGKOG WC HO Wee see eerie as nse nese eesaeisek tastes tetcctasiclee sires selaranistishaer sists jos aljceleisieininenintisiesinaynainslseubiepeainsleineten 679 ESHOsenicnesulatlonion synaptic plasticibyssseessesrsse-eseesare este sen dec ceceses-eeeeeercee-esecnerecesesesseceere 680 (i) Synaptic plasticity in the hypothalamus with brain lesion ...................0cceeeeceeeeecneceee eee ees 680 (ii) Synaptic plasticity in the hypothalamus without brain lesion ...................cecceeeeeeeeeeeeen ees 682 Gea) Synaptic plasticity mite VIMIN aii. fis. le osaetecGehdnceorl sane otsecnebensssentuautnssbtneeescce stencils 682 Gib) Synapticiplasticity mithe th OAG ea creeeec ee ceatein sec sensoh odes cetoecdadserdecurescdcceeceesecsecsss 683 (li-c) Synaptic plasticity during the estrous cycle ................. cee ceee cece eee eec eee eeeeeeeeeeeeseeeseeens 683 Androgenic regulation of synaptic plasticity ............. 0c. cece eee ee cece c eee ec eec eee eee eeu ereeneeeeeeeeereees 684 @ eS ynapticrplasticityaimithe:SNBccnas.cc-e heir ee pee scm once bariepct torhjans sesesepemauademeaeeceaesenciiee see 684 (ii) Plasticity of gap junctions in the SNB ............ceceeceececeeeeee eer eeneeeeteceee eee eeneeeeeeenereeeeees 685 Sex steroidal regulation of mRNA expression of structural proteins ..................cseceeeeeeeeeee eer eees 686 Gee xpressiomioige-actimvmRNA saicorecscsen crepe ceca sen eri oes sccenerinesiceessasnasherceccsnumececsranes 687 (i) aE xpressionlofocaphjunctionmmlRINAgeeescerre eee aterteeeeseesaecadeceeeseeteeeeeee cea seeee cae ceeeeae eae 688 (iii) Possible mechanisms of sex steroids for the expression of f-actin and gap junction IRIRINIANS sc nconesaboaaaedeb soba anSaeHeodndes seGaagtsepaP ental cScosadccnedr san sense Coreanadd Seas doce nama e er eee en 689 (COMGINGION '5.cooadadbaanoasbaceran Sap ROAUHBAB ENE CHGbn AGP BASAe nace Raa tc SPeUnACHE MICA SLEn tn ioe oan ana SR ener seers aren 690 IRNGRETEN CES Reeeee ereee reset Cierny are aamrae ayers rasa tail nian’ set clase aafo sili astiais aaiisieleeiisie Muir awe tinelnadnrediata sluineina 691 neuronal number [15-17], in dendritic morphology INTRODUCTION It is well established that sex steroids play a crucial role in reproductive neuroendocrine func- tions such as gonadotropin secretion and copula- tory behavior. The effects of gonadal steroid hormones on the sex steroid-accumulating neuro- nal structures have been classically subdivided into organizational and activational ones [1-3]. Estrogen or aromatizable androgen is responsible for modulating neuronal development and neuro- nal circuit formation during perinatal period [4- 10]. In certain brain regions these organizational actions of sex steroids can induce permanent sex- ual dimorphism in nuclear volume [11-14], in Received April 30, 1992 [18, 19], in neuronal membrane organization [20], in the distribution patterns of serotoninergic [21] and vasopressinergic fibers [22], in synaptic forma- tion [23-28] and in neuronal connectivity [29, 30]. In contrast, activation effects of sex steroids on adult brain tissues are considered to be imperma- nent and reversible. Although the effects have been thought to involve changes in neurophysiologi- cal and/or neurochemical events in the neuro- endocrine brain, evidence is now accumulating that even impermanent effects of sex steroids can in- volve major structural alterations in neuronal ele- ments and circuits, reflecting several kinds of re- productive neuroendocrine functions. Sex steroids have been found to regulate nuclear volumen [31, 32], neuronal number [33], neuronal size [34-36], 680 A. MAtTsumMoTo dendritic morphology [35, 37-43], vesopres- sinergic innervation patterns [44], and synaptic remodeling [45-51] in several areas of adult rodent neuroendocrine brain. It has also been reported that androgen regulates nuclear volume [52], dendritic length [53] and synaptic reformation [54, 55] in a portion of adult canary striatum. In the present paper, to clarify the activation effects of sex steroids on neuronal circuit forma- tion, the focus is placed on synaptogenic action of sex steroids on adult neuronal structures of the neuroendocrine brain such as hypothalamus and spinal cord which participate in reproductive func- tions [56-60]. ESTROGENIC REGULATION OF SYNAPTIC PLASTICITY The hypothalamic arcuate nucleus (ARCN), hypothalamic ventromedial nucleus (VMN) and preoptic area (POA), which are sex steroid- accumulating neuronal groups [61-64] are consid- ered to play a significant role in regulating gonado- tropin secretion and sexual behavior [56, 57]. Most of these functions are sex steroid-dependent and sexually dimorphic. Recently, the volume of the POA [11-14] and VMN [12], and the synaptic organization in the POA [23, 24], ARCN [25, 26] and VMN [27, 28] have been reported to be sexually dimorphic and are modified by neonatal sex steroid environment. Estrogen during neo- natal or prepubertal periods has a facilitatory effect on neuritic growth of mouse hypothalamic explants [65] or rat cultured hypothalamic cells [66, 67], dendritic spine formation in the VMN [68] and synaptogenesis in the ARCN [69-74], VMN [27, 28] and POA [75]. Since these hypothalamic neural tissues in neonatal animals are in an unde- veloped state [28, 69, 75-80], estrogen may act as growth-promoting factors on the developing hypothalamic neural components and play a criti- cal role in modulating postnatal neuronal matura- tion and neural circuit formation in the hypo- thalamus. (i) Synaptic plasticity in the hypothalamus with brain lesion Synaptogenic action of estrogen on several neu- ral tissues is not limited only at neonatal or pre- pubertal period. Estrogen has been found to have a facilitatory effect on synapse formation in the ARCN of adult [45-49] and aged female rate [47]. Matsumoto and Arai [45-47] have pointed out that estrogen has a facilitatory effect on synap- togenesis in the ARCN of adult hypothalamus. When the medial basal hypothalamus (MBH), including the ARCN, is isolated completely by means of Halasz knife (MBH-island), a remark- able reduction in the number of axodendritic shaft and spine synapses (Fig. la and b) in the ARCN of ovariectomized females with MBH-islands is caused by transection of the afferent fibers con- verging to the ARCN [81]. As shown in Table 1, the number of shaft and spine synapses decrease to 45% and 73% of the intact level, respectively. Administration of estrogen for three weeks follow- ing brain surgery can bring about a dramatic increase in the number of shaft and spine synapses in the ARCN of ovariectomized MBH-island females. Shaft synapses restore to almost 75% of the intact level, and the incidence of spine synapses is significantly greater than that in control animals. These suggest the possibility that estrogen can enhance the plasticity of intact arcu- ate neurons in adult brain, presumably stimulating not only axonal sprouting but also dendritic spine formation in intact arcuate neurons. There are a number of vacated synaptic sites in the ARCN after brain surgery. Estrogen fails to increase in the number of shaft and spine synapses in the ARCN of ovariectomized females without brain lesion (Table 1). This evidence indicates that synaptogenic potential of estrogen can only be expressed in the adult arcuate neurons when synaptic sites are vacated due to lesion-induced degeneration of axon terminals. In the ARCN, most axon terminals are associated with only one dendritic element, a shaft or a spine (Fig. la and b). In addition to these synapses, axon terminals contacting two postsynaptic elements in the plane (double synapse) are seen in the ARCN (Fig. Ic). Although the incidence of double synapses is usually very low, three types of double synapse, that is, spine-spine, spine-shaft and shaft-shaft double synapses, are recognized. Treatment of results Synaptic Plasticity in Response to Sex Steroids 681 nbd Poe One a Hf Fic. 1. (a) Axodendritic shaft synapse in the ARCN of a female rat at two months of age. « 33,000. (b) Axoden- dritic spine synapse in the ARCN of a female rat at two months of age. S= spine. 28,000. (c) Spine-spine dou- ble synapse in the ARCN of an ovar- iectomized MBH-island female rat treated with estrogen for three weeks. S=spine. 24,000. (Reproduced with permission from Ref. [46]) TABLE 1. Effect of estrogen on numbers of axodendritic shaft and spine synapses in the arcuate nucleus (ARCN) with or without complete deafferentation of the medial basal hypothalamus (Reproduced with permission from Ref. [46]) Axodendritic synapse Group ae ; Shaft synapse Spine synapse Intact controls 8 1544+91* 215+23 OVX 6 1539 + 87 207 +23 OVX+EB 6 1686 + 153 241 +37 MBH-island+ OVX 9 696+ 46 156+ 20 MBH-island+OVX-+ EB 8 1159+70 380+ 24 * Mean+S.E.M. MBH-island=complete deafferentation of the medial basal hypothalamus; OVX=ovariec- tomy; EB=treatment of estradiol benzoate. 682 A. Matsumoto TABLE 2. Effect of estrogen on numbers of double synapses in the arcuate nucleus (ARCN) with or without complete deafferentation of the medial basal hypothalamus (Reproduced with permission from Ref. [46]) Number Double synapse Guo of rats . Spine-spine Spine-shaft Shaft-shaft Intact controls 8 1.87+0.58* 3.75 +1.01 4.75+1.47 OVX 6 1.66+0.67 2.83+0.79 3.00 + 1.00 OVX+EB 6 2.83 £0.94 7-33 ste1e45 5.00+0.51 MBH-island+OVX 9 1.44+0.50 6.77 + 1.43 4.11+1.12 MBH-island+OVX+EB 8 15.00 +3.45 ES ae silk 6.00 + 1.03 * Mean+S.E.M. MBH-island=complete deafferentation of the medial basal hypothalamus, OVX=ovariectomy, EB= treatment with estradiol benzoate. estrogen causes a dramatic increase in the numbers of spine-spine and spine-shaft double synapses in the ARCN of ovariectomized MBH-island rats (Table 2). Although the total number of double synapses is very low as compared to regular types of synapses, an increase in the number of spine- spine and spine-shaft double synapses, but not of shaft-shaft double synapses, is remarkable in the deafferented ARCN of estrogen-treated females (Talbe 2). The findings provide further support for the ability of estrogen to stimulate synaptogenesis in the adlut ARCN. Collateral sproutings from the axon terminal, which have already synapsed on a shaft or a spine may not only reoccupy a vacated shaft or spine, but may also synapse with a spine newly formed by estrogen stimulation. Much remains to be clarified concerning the functional significance of this plastic response of adult neural tissues to estrogen. It seems unlikely that synaptic connections established at early developmental period persist throughout the life. In this context, it is worthy to note the coexistence of growth cones and degenerating axon terminals in the adult VMN [82], indicating that spontaneous synaptic degen- eration and synaptic remodeling can occur in adult brain. Estrogen might be responsible for such a process of synaptic reorganization. (ii) out brain lesion Synaptic plasticity in the hypothalamus with- Estrogen has been reported to exert a stimula- tory influence on synaptogenesis in the VMN [51, 83], in the lateral septum [84] and in the midbrain . central gray [85] of adult female rats even if the neural tissues are never lesioned. (ii-a) Synaptic plasticity in the VMN The VMN plays a crucial role in female sexual bahavior or lordosis, which is activated by estrogen and progesterone during the estrous cycle [57, 58]. Since the VMN contains a number of estrogen- accumulating neurons [61-64], and implants of estrogen into the VMN facilitate lordosis [58], the VMN is thought to be a direct site for estrogenic effect on lordosis. Administration of estrogen into ovariectomized female hamsters for two days in- duces a 48% increase in the dendritic length of VMN neurons compared with VMN neurons of control females [42]. The formation of dendritic spines on VMN neurons of ovariectomized female rats is also stimulated by estrogen treatment for two days [39, 40]. This estrogenic regulation of dendritic membrane area implies a concomitant change in synaptic inputs to dendrites. Ultra- structural study reveals estrogenic influence on synaptic inputs to VMN neurons [51, 83]. Accord- ing to Frankfurt and McEwen [51], axodendritic synapses cover 81% of the dendritic membrane in ovariectomized rats treated with estrogen for two days, and 53% in ovariectomized ones. The num- ber of axodendritic synapses per unit area of the VMN is 2.93+0.13 in ovariectomized rats treated with estrogen, and 1.79+0.11 in ovariectomized ones. Changes in the dendritic length, dendritic spine density and axodendritic synaptic population in the VMN have been detected as early as two Synaptic Plasticity in Response to Sex Steroids 683 days after estrogen treatment. These morpholo- gical findings suggest that the VNM might be responsive to even daily changes in estrogen level. (ii-b) Synaptic plasticity in the POA The luteinizing hormone-releasing hormone (LHRH) neurons in the POA form the final com- mon pathway regulating the gonadotropin secre- tion from the anterior pituitary [86]. The activity of LHRH neurons is considered to be regulated by intra- and extrahypothalamic neural elements as well as steroidal environment [56, 57]. Since LHRH neurons do not accumulate estrogen [87], it seems likely that other estrogen-sensitive inter- neurons in the POA transduce steroidal signals and in turn, alter the activity of LHRH neurons. In fact, LHRH-immunoreactive terminals have been reported to make synaptic contact with estrogen receptor-immunoreactive neurons in the POA [88]. Immunohistochemical ultrastructural investigations have demonstrated that LHRH neurons themselves in the POA are contacted with axon terminals containing amino acid, mono- amines and neuropeptides such as y-aminobutyric acid [89, 90], dopamine and norepinephrine [91, 92], serotonin [94], @-endorphin [92, 93], subst- ance P [95], pro-opiomelanocortin [96], adreno- corticotropic hormone [97] corticotropin-releasing factor [98], and LHRH [99-102]. According to Chen et al. [93], there is sexual dimorphism in the synaptic input to LHRH neurons in the rat POA. LHRH neurons in females receives approximately twice the number of synapses as do those of males. f-endorphin-immunoreactive terminals contribute to this dimorphism. Physiological differences in regulation of gonadotropin secretion may be reflected in a sexual dimorphic connectivity of the LHRH system. Witkin et al. [102] have demonstrated that synaptic input and glial apposition to LHRH neurons in the POA of adult rhesus monkeys is sensitive to circulating estrogen. Removal of estrogen by ovariectomy results in a significant increase in the apposition of glial processes to LHRH-immunoreacive neurons and a significant decrease in their innervation in the POA, and these changes are prevented by estrogen replace- ment. This anatomical study indicates the possibil- ity that LHRH system responds to alterations in the gonadal steroid environemtn with reversible changes in synaptic input and glial ensheathing. In relation to this finding, it is noteworthy that so- mata of LHRH-immunoreactive neurons in the POA of anestrous sheep are less innervated than non-immunoreactive cells around LHRH-im- munoreactive neurons, and that somata and de- ndrites of LHRH-immunoreactive neurons are almost entirely surrounded by glial processes [103]. Thus, natural seasonal changes in estrogen level probably influence the synaptic organization of LHRH system. (ii-c) Synaptic changes during the estrous cycle As mentioned above, estrogenic effects on changes in neuronal elements in the VMN occur two days after estrogen treatment. It seems quite possible that daily fluctuations in the level of estrogen during the estrous cycle cause significant alterations in the amount of membrane available for synaptic input and in the number of synapses. The dendritic spine density of VMN neurons of female rats at proestrus has been reported to be significant higher than that at estrus and diestrus [39]. It has not yet determined whether these changes in dendritic spines reflect changes in synaptic population in the VMN during the estrous cycle. On the other hand, Olmos et al. [104] found that the number of axosomatic synapses in the ARCN decreases at estrus when compared to other days of the estrous cycle. There is a reduc- tion of 31% in axosomatic synapses between proestrus and estrus. Because estrogen has a facilitatory effect on synaptic formation in the adult ARCN, it is reasonable to assume that synaptic remodeling of the ARCN occur during the estrous cycle. It seems likely, therefore, that the neuronal plasticity which has been demon- strated to occur during the estrous cycle may be a natural consequence of sex steroid feedback, and that the adult pattern of synaptic connections can be altered in the hypothalamus in relation to reproductive neuroendocrine and behavioral func- tions. Although the hippocampus is not considered to be directly involved in regulatory mechanisms of neuroendocrine functions, estrogen exerts a stim- 684 A. Matsumoto ulatory influence on dendritic spine formation of pyramidal neurons in the hippocampus (CAI re- gion) of ovariectomized adult rats [105]. More- over, spine density of the CA1 pyramidal neurons [106] and synaptic population in the neuropil of CA1 region alter during the estrous cycle (McEwen, B. S., personal communication). These evidence further supports the possibility that neuronal elements and synaptic connections reorganize during the estrous cycle. ANDROGENIC REGULATION OF SYNAPTIC PLASTICITY The spinal nucleus of the bulbocavernosus (SNB) of the rat lumbar spinal cord is a sexually dimorphic motoneuron pool [107-111]. The SNB motoneurons innervate the perineal striated mus- cles bulbocavernosus and levator ani that attach to the penis. They have an important role in copula- tory behavior, which is sensitive to alterations in circulating levels of androgen in adulthood [59, 60]. Both neuronal and muscular elements contain androgen receptors [107-114]. Castration of adult male rats rsults in a significant decrease in both somatic size [34, 36] and dendritic length [37, 38, 41] of these motoneurons, and androgen treatment of castrates reverses this effect. According to Kurz et al. [43], androgenic influences on somatic size and dendritic length are also detected in another androgen-sensitive motoneuron pool in the lumbar spinal cord, the dorsolateral nucleus (DLN), which innervates the ischiocavernosus and urethral sphincter muscles [113, 115]. These evidence suggests that the adult SNB and DLN motoneurons retain a great deal of plasticity to androgen, which is expressed when androgen levels fluctuate. This androgenic regulation of somatic and dendritic membrane area implies a Fic. 2. Percentage of SNB membrane covered by synaptic contacts (a), size of synaptic contacts (b) and frequency of synaptic contacts per 1 ~m of SNB neuronal membrane (c) of control (Controls), cas- trated (Castrates) and castrated male rats treated with testosterone for four weeks (Castrates + Testos- terone). Vertical lines indicate S.E.M. Numbers in the bottom of columns refer to the number of rats examined. (Reproduced with permission from Ref. [2]) concomitant change in synaptic inputs to these membranes. (i) Synaptic plasticity in the SNB Quantitative electron microscopic study reveals a | Soma | | Proximal dendrite 30+ 155 5 5 5 Controls Castrates Castrates Percent Covering of Synaptic Contacts + Testosterone b EI Soma HEE Proximal dendrite (um) 2.05 io iS) 2 r= cS) ra 9] a ° c bal a r) ao pe 7) 5 5 5 Controls Castrates Castrates + Testosterone Cc =] Soma HEE Proxima! denarite 9 ty a (o} r 0.100- Frequency of Synaptic Contacts (per jum of Neuronal Membrane) 5 5 5 Costrates Controls Castrates + Testosterone Synaptic Plasticity in Response to Sex Steroids 685 androgenic influences on synaptic inputs to SNB motoneurons [48, 50]. In intact adult males, synaptic structures cover about 40% and 47% of somatic and proximal dendritic membranes of SNB motoneurons, respectively (Fig. 2a) [48]. Four weeks after castration, these values are re- duced to about 14% and 19% of somatic and proximal dendritic membranes, respectively. However, treatment of testosterone for four weeks after castration prevents this decline. Castration and testosterone treatment also influence the synaptic size (Fig. 2b) and number of synaptic contacts per unit length of somatic and proximal dendritic membranes (Fig. 2c). These results indi- cate that androgen is critical for maintaining the reformation of synaptic inputs to SNB motoneurons in adult male rats. Together, the earlier light microscopic studies [34, 36-38, 41] and ultrastructural ones suggest that the SNB system can retain a great deal of plasticity in adulthood. Because androgen levels in rodents fluctuate with seasons [38], stress and aging [116, 117], the synaptic changes induced by castration may reflect an ongoing process of synaptic or- ganization and reorganization regulated by androgen throughout the adult life. Previous studies have suggested which kinds of neurotransmitters or neuromodulators may be contained in affectes to SNB motoneurons. Im- munohistochemical studies have suggested that the presence of norepinephrine, serotonin, substance P, leu- and met-enkephalin, thyrotropin-releasing hormone (TRH) and calcitonin gene-related pep- tide (CGRP) in fibers and terminals in close prox- imity to somata and dendrites of motoneurons in the rat SNB [118, 119], and in the cat or baboon Onuf’s nucleus which is homologous to the SNB [120-124]. These data indicate that the terminals of these fibers may make synaptic contact with SNB neurons. In fact, serotonin- and tyrosine hydroxylase-immunoreactive terminals make synaptic connection with SNB motoneurons [125]. Motoneurons in the Onuf’s nucleus are contacted with axon terminals containing norepinephrine, serotonin, substance P, leu- and met-enkephalin, TRH and CGRP [120, 124]. It has been clearly established that synaptic inputs to the SNB motoneurons can be regulated by androgenic in- fluence. Further studies are needed to determine the chemical identity of neuronal inputs to the SNB motoneurons which are affected by androgen. (ii) Plasticity of gap junctions in the SNB The SNB motoneurons exist in a cluster, and their somata come into contact with each other [107-111]. Numerous number of longitudinally oriented bundles of dendrites are packed in the neuropil of the SNB [126, 127]. Gap junctional plaques are found between these motoneurons, Fic. 3. A gap junction (arrow and inset) can be seen along the apposed membranes of a proximal den- drite (PD) of an SNB motoneuron and another SNB dendrite (D) in a testosterone-treated male rat four weeks after castration. Cholera toxin conjugated to HRP (CT-HRP) was injected into the bulbo- cavernosus muscles two day before autopsy, and the spinal cords containing SNB were dissected, proces- sed with a modified tetramethylbenzidine method for visualization of retrogradely transported CT- HRP. CT-HRP crystals (arrowheads) are found in both dendrites. x 16,400 (inset: x 175,500). (Re- produced with permission from Ref. [128]) 686 A. Matsumoto TABLE 3. Effect of androgen on the incidence and size of gap junctions between SNB motoneurons in control and experimental animals (Reproduced with permission from Ref. [128]) Number of neurons Number of gap Number - : : Length of gap containing ga t : ; Group of rats Finclone P ai junctions (nm) Controls 5 2.6+0.4* 3.4+0.4 168.8+25.2 Castrates 0.8+0.2' On On 64.74 8.1 Castrates + testosterone 2.8+0.5 3.6+0.2 154.4+19.1 * Mean+S.E.M. * P<0.001, * P<0.0005 (vs. Controls or Castrates+ testosterone). using thin-section and freeze-fracure technique [128]. The presence of gap junctions between SNB motoneurons is also ientified with electrophysiolo- gical and Lucifer yellow dye coupling techniques [129]. Translucent electron microscopic study re- veals that gap junctions show a characteristic fea- ture of pentalaminar structure (Fig. 3). Freeze- fracture replicas demonstrate plaques of in- tramembranous particles of the protoplasmic and complementary pits on the extracellular fracture faces. These particles are considered to be chan- nels of gap junctions, permitting electrical and/or metabolic coupling between motoneurons. Of the gap junctions observed in the SNB, 45% were somatodendritic, 35% dendro-dendritic and 20% somatosomatic. As shown in Table 3, the removal of testosterone by castration dramatically reduces the number and size of the junctional plaques, and these changes are prevented by testosterone re- placement. Because the size of the junctional plaques is related to the number of gap junctional channels between the cells [130], the findings that androgen regulates the total number of gap junc- tional channels would indicate that the degree of electrical and/or metabolic coupling is regulated by androgen. The enhanced electrical and/or metabolic coupling between SNB motoneurons may promote synchronous neuronal activity in this nucleus. Gap junctional plaques are also found between motoneurons in the DLN [131]. In addition to SNB motoneurons, gonadal ster- oids have been reported to regulate the size or frequency of gap junctional plaques in various kinds of cells. For example, estrogen increases gap junctions between granulosa cells of the rat ovary [132, 133], between smooth muscle cells of rodent uterus [134-139] and between neurons in the rat ARCN [140]. In contrast, removal of estrogen by ovariectomy induces an increase in the incidence of Lucifer yellow dye coupling between mag- nocellular neurons in the supraptic nucleus of the rat hypothalamus [141]. On the other hand, androgen stimulates the incidence of Lucifer yel- low dye coupling between magnocellular neurons in the paraventricular nucleus of the rat hypothala- mus [142]. Female Xenopus frogs possess dye- coupled laryngeal muscle fibers, whereas adult males do not [143]. Androgen treatment of females reduces the coupling in females. These evidence indicates that gonadal steroids regulates the number of gap junction channels in a wide variety of reproductive tissues, including the gonads, nervous system, and smooth and striated muscles. These tissues have in common the pre- sence of gonadal hormone receptors. Indeed, it become reasonable to ask whether steroid control of gap junctions and electrical coupling is a com- mon feature of all such steroid-sensitive cells. SEX STEROIDAL REGULATION OF mRNA EXPRESSION OF STRUCTURAL PROTEINS Although the molecular mechanisms underlying the neuronal plasticity in response to sex steroids are poorly understood, evidence is now accumulat- ing that sex-steroids play a significant role in regulating gene expression of structural proteins in sex steroid-sesitive neuronal tissues [144-149]. Synaptic Plasticity in Response to Sex Steroids 687 Riri ae of Bee bea) & eget teeta ra ; pate soir ea eH eRe ar ” + Ea a Aghines ones Eng re Boies se iat, ian z ea iets Fic. 4. Autoradiographic signals of £-actin mRNA on SNB motoneurons in a castrated male rat treated with testosterone for four weeks (a), and in a castrated male rat (b). X390. (Reproduced with permission from Ref. [149]) (i) Expression of B-actin mRNA Actin is one of the important cytoskeletal pro- teins that has been identified in neurons of the central nervous system [150-152]. Actin filaments are found to associate particularly with the neuro- nal membrane and postsynaptic density. As men- tioned before, androgen regulates the somatic and dendritic membrane area of SNB motoneurons and the number of synapses onto these motoneurons. It seems likely, therefore, that actin might be involved in the hormonally induced neuronal plasticity in the SNB motoneurons. In situ hybridization histochemical study using complementary DNA encoding chick /-actin re- veals androgenic influences on the expression of f-actin mRNA in SNB motoneurons of adult male rats [149]. Hybridizable G-actin mRNA are found to localized in the somata and proximal dendrites of SNB motoneurons (Fig. 4a and b). As shown in Figs. Sa, 5b and 5c, removal of androgen by castra- tion significantly reduces the neuronal area of SNB motoneurons and the expression level of -actin mRNA in SNB motoneurons, whereas these changes are prevented by testosterone treatment for four weeks. On the contrary, castration or testosterone treatment does not induce any change in the expression level of ~-actin mRNA in the motoneurons of the retrodorsolateral nucleus (RDLN) (Fig. 5a-c). The RDLN motoneurons innervating hindlimb musculature are examined, because the RDLN motoneurons are known to be androgen-insesitive [148, 149, 153]. These findings suggest that androgen exerts a regulatory influence on the f-actin mRNA expression in the SNB motoneurons. {-tubulin is known to be main component of microtubules which are another major cytoskeletal element in neuronal cells. The expression of #-tubulin mRNA in the SNB moto- nourons is also regulated by androgen (Matsumo- to, A. et al., unpublished data). Recent studies provide evidence for an increase in expression of cytoskeletal proteins such as actin, tubulin, and their mRNAs in neural plasticity processes like neuronal growth and synapse forma- tion [154-158]. In the SNB, androgen has been shown to regulate the somatic and dendritic size of motoneurons [34, 36-38, 41] and the number and size of synapses onto these motoneurons [48, 50], suggesting that f-actin participates in the under- lying mechanisms of plastic response of SNB motoneurons. Estrogen has been reported to exert a stimulatory influence on the expression of actin [159] and f£-actin mRNA in immature rat uterus [160]. These findings indicate the possibility that sex steroids generally play a significant role in the expression of G-actin genes in sex steroid-sensitive cells. 688 A. Matsumoto ‘E fs it 2 Ss sy} o = i) © Cc [e) i. > oO Zz SNB RDLN (b) IL ~ 400 oO o L oO ~ a Yc £9 S25 i= ‘op © Lez fe} i= o a iS =) z oO o SS Yn U0 is) De wo ee fo) = co) we} E 2) z RDLN Fic. 5. Neuronal area (a) and numbers of grains of B-actin mRNA per neuronal area (b) and per unit area (c) of motoneurons in the SNB and RDLN. Solid bars, control males; hatched bars, castrated males; open bars, castrated males treated with tes- tosterone. Six animals were examined in each group. Values given are the mean+S.E.M. (Repro- duced with permission from Ref. [149]) (11) Expression of gap junction mRNA Recent biochemical and molecular biological studies make possible to raise polyclonal and monoclonal antisera against several kinds of gap junction proteins (connexins) and to clone cDNAs coding for them [161-163]. It has been reported that neuronal tissues react immunohistochemically with antibody against gap junction protein of the rat liver (connexin 32) [164-169], and that the expression of gap junction mRNA is demonstrated in the neural tissues by a Northern blot analysis [170, 171] and by in situ hybridization histochemis- try using complementary DNA (cDNA) encoding connexin 32 [148, 149, 169, 172]. Androgenic influences on the expression of gap junction mRNA in the SNB motoneurons have been clarified by recent in sity hybridization his- tochemical studies using cDNA encoding connexin 32 [148, 149]. Hybridizable gap junction mRNA are localized on the somata and proximal dendrites of SNB motoneurons (Fig. 6a and b) [148]. Re- moval of androgen by castration significantly re- duces the expression level of gap junction mRNA in the SNB motoneurons, whereas this decline is prevented by testosterone treatment for two days (Fig. 7a-c). The recovered expression level is almost the same as that treated with testosterone for four weeks [149]. This evidence indicates that androgen can enhance and maintain the expression level of gap junction mRNA in the SNB motoneurons. On the contrary, castration or tes- tosterone replacement does not induce any change in the expression level of gap junction mRNA in the motoneurons of the RDLN (Fig. 7a-c). These results indicate that androgenic regulation of gap junction gene expression is rather specific to the SNB. The recovery of expression level of the gap junction mRNA is detected in the SNB motoneurons of castrates treated with testoster- one. This evevation of gap junction transcript is thought to be associated with the synthesis of gap junction protein, reflecting the formation of gap junction channels and its plaques. Indeed, we have recently pointed out that androgen increases the number and size of junctional plaques between SNB motoneurons [128]. In the uterine myomet- rial cells, an increase in the number and size of gap junctional plaques has been observed at parturi- tion [173]. Plasma levels of estrogen [174] and number of estrogen receptors in these cells [175] increase markedly, preceding the increment of gap junctions observed at parturition. Formation of gap junction plaques between these cells is en- hanced by treatment with estrogen [134-139]. In addition to these obervations, as increase in the expression of gap junction mRNA had been de- tected in these cells at parturition [176]. These Synaptic Plasticity in Response to Sex Steroids 689 ees Baas ce ees RAYS et at Ro Se ewhaces S — eh". Fic. 6. Autoradiographic signals of gap junction mRNA on SNB motoneurons in a castrated male rat treated with testosterone for two days (a), and in a castrated male rat (b). X390. (Reproduced with permission from Ref. [148]) Neuronal area( x10 °mm’) Number of grains/Neuronal area 500 250 ” Z 00) D S) [— Z SNB RDLN Number of grains/Unit area Lia Ya SNB RDLN Fic. 7. Neuronal area (a) and numbers of grains of gap junction mRNA per neuronal area (b) and per unit area (c) of motoneurons in the SNB and RDLN. Solid bars, control males; hatched bars, castrated males; open bars, castrated males treated with tes- tosterone for two dyas. Six animals were examined in each group. Values given are the mean+S.E.M. (Reproduced with permission from Ref. [148]) findings indicate that sex steroids generally exert influences on regulating the expression of gap junction genes in sex steroid-sensitive cells. (iii) Possible mechanisms of sex steroids for the expression of B-actin and gap junction mRNAs As mentioned above, removal of androgen by 690 A. MATSUMOTO castration reduces the expression of f-actin and gap junction mRNAs in SNB motoneurons and this decline is prevented by testosterone treat- ment. In contrast to the SNB motoneurons, androgen does not induce any change in the ex- pression of @-actin and gap junction rRNAs in the RDLN motoneurons. These evidence indicate there is regional difference in regulatory mecha- nisms of androgen on the ejxpression of /-actin and gap junction mRNAs in these lumber moto- neurons. According to Breedlove and Arnold [107, 113] and Jordan et al. [114], there is a regional differ- ence in the pattern of androgen accumulation by lumbar motoneurons, that is, the motoneurons in the SNB accumulate androgen more frequently and densely than those in the RDLN. This evi- dence may be correlated with the difference in androgenic influence on the expression of /-actin and gap junction mRNAs between these two motoneuron pools. It seems likely, therefore, that androgenic regulation of the expression of these genes is specific to the SNB, and that the express- ion of #-actin and gap junction genes in androgen- accumulating neurons is sensitive to androgen. The mechanism by which sex steroids influence on the expression of f-actin and gap junction genes is not yet known. The observed modulation of their mRNA expression may be attributed to al- teration in transcription rate of genes or in stability of mRNAs [177]. Steroid hormones have been shown to regulate the gene expression first by binding to their specific receptors and then by interaction of the hormone-receptor complex with specific steroid hormone responsive elements on the DNA which act as transrcriptional enhancers [178-181]. To clarify the regulatory mechanisms of the #-actin and gap junction gene expression, it will be important to identify the steroid hormone responsive elements in these genes. However, the steroid hormone responsive elements have not yet been detected in the rat Z-actin gene [182]. It is possible that there are no steroid hormone respon- sive elements in these genes. The conjecture may be associated with the idea that expression of these genes is mediated by some gene products which are under the direct influence of the androgen- receptor complex. There is another possible explanation for the regional difference in androgenic regulation of the expression of #-actin and gap junction mRNAs between SNB and RDLN motoneurons. It has been suggested that the androgenic regulation of the size of SNB motoneurons is mediated through their target muscles [183]. According to Araki et al. [184], androgen does not induce any change in neuronal size of the SNB motoneurons which have innervated androgen-insensitive soleus muscle. In contrast, the size of SNB motoneurons which have reinnervated to their androgen-sensitive target muscles is reduced by castration and enlarged by testosterone treatment. Recently, it is reported that androgen is involved in modulating the ex- pression of calcitonine gene-related peptide mRNA in SNB motoneurons through the effects on their target muscles [185]. On the other hand, in the RDLN system, Leslie et al. [153] pointed out that castration has no effect on the size of RDLN motoneurons and their target muscle, flexor digi- torum brevis. These findings indicate the possibil- ity that, in the SNB system, androgen plays a significant role in the production of muscle-derived trophic factors which can regulate the size of SNB motoneurons. It seems likely, therefore, that androgen regulates not only the neuronal size of SNB motoneurons but also the expression of /- actin and gap junction genes in them through its action on their target muscles. CONCLUSION The present paper provides ultrastructural evi- dence suggesting that sex steroids play a significant role in modulating neural circuit formation in sex steroid-sensitive areas in adult neuroendocrine brain. Electrical coupling and/or cell-to-cell com- munication via gap junctions may also be modified by sex steroid environment. Moreover, we now undertake to clarity the molecular mechanisms underlying the neurnal plasticity in response to sex steroids. Androgen exerts potent influences on modulating gene expression of structural proteins such as #-actin, 8-tubulin and gap junction in the SNB motoneurons. Expression of growth- associated protein 43 kDa (GAP-43) mRNA in the VMN is reported to be regulated by estrogen Synaptic Plasticity in Response to Sex Steroids 691 [147]. In relevant to these evidence, it is of interest to note that the expression of actin and tubulin mRNAs is facilitated by neonatal treatment with androgen in the preptic area-hypothalamus [145] and limbic system of female rats [144]. The expression pattern of @-tubulin mRNA in the developing rat hypothalamus is sexually dimorphic [146]. Exposure to androgen dring neonatal period has a permanent organizing effect on neuronal morphology and synaptic pattern in these regions [1-10] which contain abundant receptors for sex steroids [186, 187]. Thus, sex steroids may be involved in neuronal plasticity, that is, organi- zation and reorganizatin of neuronal elements and synaptic connections, through regulation of gene expression for neuronal structural proteins such as actin, tubulin, GAP-43 and gap junction protein. ACKNOWLEDGMENTS The present author’s studies were carried out in col- laboration with Prof. Y. Arai and Mrs. S. Murakami, Department of Anatomy, Juntendo University School of Medicine, Tokyo, Prof. A. Urano, Ocean Research Institute, University of Tokyo, Tokyo, Dr. S. Hyodo, Department of Biology, College of Arts and Sciences, University of Tokyo, Tokyo, and Profs. A. P. Arnold and P. E. Micevych, Laboratory of Neuroendocrinology, Brain Research Institute, University of California at Los Angeles, Los Angeles. These studies were supported by the grants from Ministry of Education, Culture and Science of Japan, Narishige Neuroscience Foundation, Yamada Science Foundation and NIH (NS23468 and HD15021). REFERENCES 1 Arnold, A. P. and Breedlove, S. M. (1985) Horm. Behav., 19: 469-498. 2 Matsumoto, A. (1991) Psychoneuroendocrinolo- gy, 16: 25-40. 3 Matsumoto, A. (1992) Rev. Neurosci., in press. 4 Goy, R. W. and McEwen, B. S. (1980) “Sexual Differentiation of the Brain”. 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Anat., 140: 589-593. mae foe of oman —— 1 180 min following subcutaneous injection of the 100 ug/g dose of apomorphine (Table 1). In C. pyrrhogaster, one of six animals vomited in 40.5 min at the 300 ug/g dosage, though it was uncertain if this was caused by apomorphine proper or by the high dose of saline. In this regard, one out of five control newts vomited after injection of 0.06 ml/g of 0.65% NaCl alone, but only after 180 min. At all other dosages of apomorphine, including the case of intraperitoneal injection, ejection of gastric con- tents did not occur. Nevertheless many specimens of both species, in particular C. pyrrhogaster, showed an atypically wide gaping of their mouth for a few seconds, plus strong lateral bending, ventral flexion, or complex twisting of the body following apomorphine injections at dosages higher than 5 ug/g. Furthermore, no subcutane- ous administration of 0.65% NaCl alone in a volume of 0.01 or 0.02 ml/g induced any trace of these same extreme postures in control specimens. Administration of copper sulfate, in contrast to apomorphine, dramatically induced vomiting in both species (see Table 1). An 0.65% NaCl admi- nistration was not effective. Cynops pyrrhogaster reacted slightly quicker than H. nebulosus to orally administered copper sulfate. At comparable dosages, C. pyrrhogaster tre- sponded much quicker to oral than subcutaneous administered copper sulfate. Hynobius nebulosus, however, did not show a similar difference in its response time to copper sulfate that could be associated with the route of administration. Although less extensively tested, antimony] potas- sium tartrate also proved to be an effective emetic (Table 1). Oral administration of this agent in- duced vomiting in all animals tested in both spe- cies. When emesis occurred, the behaviors associated with ejection of gastric contents were the same in both species and did not vary with the drug used or its pathway for administration. Ejection of gastric contents always occurred in a surge associated with one to several episodes of bending or twisting of the torso. In the extreme, the salamanders coiled into a circular, sigmoidal or reversed-sigmoidal posture (Figs. 1, 2 and 3). During the contortions associated with emesis the torso of C. pyrrhogaster shortened by an average of 12.8% (N=5, range 7.3-15.8%). In contrast, the maximum width of the body increased on average by only 3.6% (N= 5, range 0-8%). Thus the animals were becoming shorter without a proportionate expansion of cross-sectional area to maintain constant volume. Prolapse of the esophagus or stomach outside the mouth was never detected. The actual vomiting act took from about one to eighty sec depending Emesis in Urodeles 715 TABLE 1. The responses of salamanders to various emetic drug treatments Cynops pyrrhogaster Hynobius nebulosus No. vomited Reaction No. vomited Reaction No. tested times (min)* No. tested times (min)* Apomorphine HCI** 0.5 ug/g S.c. 0/6 2 0/6 5 0/7 0/5 20 0/8 50 0/6 0/5 100 0/6 2/5 101.7, >180 200 0/6 300 1/6 40.5 Copper Sulfate 0.2 mg/g s.c. 1/3 >180 3/3 38.9+11.8 1.0 mg/g s.c. 2/3 62.9, >180 2.0 mg/g s.c. 3/3 42.0 (n=2), >180 0.2 mg/g p.o. 3/3 26.5+3.2 4/4 31.4+10.6 0.4 mg/g p.o. 2/2 42.1, 6.6 2.0 mg/g p.o. 3/3 4.4+1.9 3/3 13.1+5.0 Antimony! potassium tartrate 0.4 mg/g p.o. 4/4 1.6+0.3 (n=3), TG 138.0+19.5 (n=6), >180 (n=1) >180 (n=1) * Mean+SE given for all animals that responded in less than 180 minutes. ** Intraperitoneal injections of 50, 100, or 200 ug/g did not induce vomiting in any of the 2-3 C. pyrrhogaster which received them. Note: s.c.=subcutaneous, p.o.=per oral. *** Includes 4 animals previously used in apomorphine tests. 3cm Fic. 1. Bending posture during vomiting in salamanders. Left: C. pyrrhogaster; right: H. nebulosus. Arrow indicates vomitus just appearing. Left and right correspond to Fig. 2, D and Fig. 3, C, respectively. (Images taken directly from videofilm using Sony Multiscan Video Printer UP-930). 716 T. NaAITOH AND R. J. WASSERSUG 444 O 3.5 2cm Fic. 2. Vomiting behavior in C. pyrrhogaster induced by orally administered copper sulfate at a dose of 2.0 mg/g body mass. A . Resting state, 50.0 sec before the start of the vomiting act. B through F=1.2, 3.5, 4.41, 4.44, 4.8 sec, respectively, after the start of vomiting. Vomitus (stippled) outside the mouth appears first in D to the left of the head. It is completely ejected by F. Note the characteristic bending of the body in D through E. Drawn from a videofilm. -—4.0 sec on the specimen and the concentration of the emetic administered. Often episodes of weak bending or twisting were repeated before culmi- nating in vomiting and gastric content ejection. DISCUSSION Apomorphine and copper sulfate are well estab- lished emetics in mammals [9]. The former is known to act centrally, while the latter, centrally and peripherally depending on how it is adminis- tered [10]. Antimony! potassium tartrate is also an effective emetic in mammals [9, 11]. In our previous study surveying the vomiting response in ten species of anurans [8], Xenopus laevis was typical for frogs in its sensitivity to apomorphine hydrochloride. Twenty ug/g and 50 ug/g were effective in Xenopus, whereas Rana Japonica and Rana ornativentris were less respon- sive to apomorphine even at dosages of 50 and 100 ug/g body mass. In contrast only two urodele studied here ejected its gastric contents after apo- morphine administration at these dosages, though gaping of the mouth and bending of the body commonly occurred in both species. Thus, these amphibians are an order of magnitude or more less sensitive to this agent than are carnivorous mam- mals on a zg per g body mass basis. However, the important point is that urodeles unequivocally vomit in response to relatively low doses of copper sulfate and antimonyl potassium tartrate. Mellin- ger [5], who seems to be the only author to previously explore emesis in a urodele, concluded that newts (in his case Triturus) could not vomit. Our present results with another salamandrid genus, as well as a hynobiid, do not support this conclusion. The response of urodeles to both copper sulfate Fic. 3. Vomiting behavior in H. nebulosus induced by copper sulfate administered orally at a dose of 0.4 mg/g body mass. A. Resting state, 4.0 sec before the start of the vomiting act. B through H=0.8, 31.3, 32.8, 35.0, 63.1, 69.4 and 72.3 sec, respective- ly, after the start of vomiting. Vomitus (stippled) first appears in C and is completely ejected from the mouth by H. Sigmoidal posture (C) and twisting of the body (C to E) are shown. In D, abdominal contraction is marked. Drawn from a videofilm. Emesis in Urodeles WAG and antimony! potassium tartrate leads us to tenta- tively suggest that most, if not all, amphibians possess an emetic capability. This would be consis- tent with the fact that salamanders and frogs both feed on relatively large and potentially toxic prey, with little or no oral processing. In mammals, carnivores that swallow large boluses of unmasti- cated food have a strong emetic response, whereas rodents, which extensively process and reduce food within their oral cavity exhibit a taste aver- sion response (see Fox, 1990 and other references cited therein [12]), but not the ability to vomit. It remains to be seen whether caecilians, which feed on quite different prey and in a quite different manner from frogs and salamanders [13], can vomit. Both subcutaneous injection and oral adminis- tration of copper sulfate were potently effective in inducing vomiting. The neurobiology of emesis in amphibians remains unstudied, but on pharmaco- logical grounds it seems similar to that of mammals described by Wang and Borison [10]. One of six newts vomited after subcutaneous injection of apomorphine at a very high (300 g/g) dose. However, this may not be the effect of apomorphine, since vomiting was also observed in one instance after a simple injection of the same volume of NaCl solution. It should be noted that relatively low doses of apomorphine, but not saline, induced marked behaviors such as twisting of the body and gaping of the mouth, without ejection of gastric contents. If such behaviors are indicative of retching, the neural mechanisms of apomorphine-induced vomiting may differ slightly from that of vomiting induced by systemically administered copper sulfate. The most conspicuous feature of vomiting in salamanders is the preparatory bending and twist- ing of the torso. These axial movements, in conjunction with tonic contraction of the muscles of the abdominal wall, elevate intra-abdominal pressure. Our observations suggest that salaman- ders raise intra-abdominal pressure by bending and twisting movements of the torso which com- press the abdominal cavity. Previous studies have shown that elevation of intra-abdominal pressure is necessary to eject stomach contents in frogs [2— 4]. Similar elevation of abdominal pressure seems necessary for emesis in urodeles as well. In this regard, emesis in amphibians is similar to that of mammals and is not a visceral response as simplis- tic as (reverse) antiperistalsis. Prolapse of stomach, however, which is common during emesis in anurans, was not observed in urodeles. This is likely due to the gross structural differences, such as the proportionately longer and narrower esophagus of salamanders and their longer bodies overall. This work was conducted following the “Guid- ing Principles for the Care and Use of Animals in the Field of Physiological Science” set by the Physiological Society of Japan. ACKNOWLEDGEMENTS We thank Mr. Masahiro Kirihara and Miss Motoko Matuura for maintaining the animals in the laboratory and their help in conducting the experiments. This work is part of a joint program on “Visceral Functions in Amphibians” supported by grants from the International Scientic Research Program of the Ministry of Education, Science and Culture (Japan) and the Natural Science and Engineering Research Council (Canada). REFERENCES 1 Tokita, K., Iwasaki, S. and Yui, F. (1953) On the Opening motion of mouth and vomiting of frogs after the digitalis preparations and on their mechanisms. Folia Pharmacol. Japon., 49: 138-142. 2 Hukuhara, T., Naitoh, T. and Kameyama, H. (1973) A peculiar phenomenon, the prolapsus of the esophagus-stomach, observed in the vomiting of the frog. Jpn. J. Smooth Muscle Res., 9: 1-8. 3 Naitoh, T., Nakai-Kameyama, H. and Hukuhara, T. (1981) The effect of denervation of abdominal musculature on the prolapse of esophagus and sto- mach in the vomiting of frogs. Mem. Fac. Sci. Shimane Univ., 15: 57-63. 4 Naitoh, T., Wassersug, R. J. and Leslie, R. A. (1989) The physiology, morphology, and ontogeny of emetic behavior in anuran amphibians. Physiol. Zool., 62: 819-843. 5 Mellinger, C. (1881) Beitrage zur Kenntniss des Erbrechens. Arch. Ges. Physiol. Pfltiger’s, 24: 232— 245. 6 Reeder, W. G. (1964) The Digestive System. In “Physiology of the Amphibia”. Ed. by J. A. Moore, Academic Press, New York, N.Y. pp. 99-149. 7 Nigam, H. C. (1977) Vomiting response in the Indian frogs Rana tigrina (Daud) and Rana cyanoph- 10 718 lyctis (Boulenger). Curr. Sci., 46: 791-792. Naitoh, T., Imamura, M. and Wassersug, R. J. (1991) Interspecific variation in the emetic response of anurans. Comp. Biochem. Physiol., 100C: 353- 359. Borison, H. L. and Wang, S. C. (1953) Physiology and pharmacology of vomiting. Pharmacol. Rev., 5: 193-230. Wang, S. C. and Borison, H. L. (1952) A new concept of organization of the central emetic mechanism: Recent studies on the sites of action of apomorphine, copper sulfate and cardiac glycosides. T. NAITOH AND R. J. 11 12 13 WASSERSUG Gastroenterology, 22: 1-12. Koppanyi, T. (1930) Studies on defecation, with special reference to a medullary defecation center. J. Lab. Clin. Med., 16: 225-238. Fox, R. A. (1990) Investigating motion sickness using the conditioned taste aversion paradigm. In “Motion and Space Sickness”. Ed. by G. H. Cramp- ton, CRC Press, Boca Raton, pp. 105-121. Bemis, W. E., Schwenk, K. and Wake, M. H. (1983) Morphology and function of the feeding apparatus in Dermophis mexicanus (Amphibia: Gymnophiona). Zool. J. Linn. Soc., 77: 75-96. ZOOLOGICAL SCIENCE 9: 719-724 (1992) Origin of Serotonergic Innervation of Olfactory Bulbs in the European Hamster, Cricetus cricetus: An Autoradiographic Study Using [°H]5-HT Retrograde Labelling JEAN Luc Miro!, SirviA ARANEDA~ and BERNARD CANGUILHEM Institute de Physiologie, Faculté de Médecine, Université Louis Pasteur, 67085 Strasbourg Cedex, France and URA CNRS 1332, and *Départment de Cytologie, Institut des Neurosciences, Université P. et M. Curie. 7, quai St-Bernard. 75252 Paris cedex 05, France ABSTRACT — Serotonergic afferents to the olfactory bulb of the European hamster, Cricetus cricetus were investigated by autoradiography following [*H]5-HT retrograde labelling techniques. Injections of [PH]5-HT into the main olfactory bulb resulted in radiolabelling of some nerve terminals in the granular and glomerular layers of the olfactory bulb, and in cell bodies of the median raphe and dorsal raphe. In the dorsal raphe, labelled cells were located in the dorso-ventral and lateral part of the nuclei. In the median raphe, labelled neurons were observed more caudally in the medial region. In this nucleus, the number of radiolabelled cells was a third of those in the raphe dorsalis. A few scattered radiolabelled cells were observed in the periaqueductal gray. The number of radiolabelled cells in both the raphe nuclei was not changed when animals were treated with a monoamine oxidase (MAO) inhibitor, Clorgyline. However, the intensity of labelling was increased in the treated animals. Treatment with 5,7-dihydroxytryptamine abolished the [°H]5-HT retrograde labelling in the olfactory bulb, as well as in the raphe cell bodies. It is concluded that in the Cricetus cricetus the granular and glomerular layers of © 1992 Zoological Society of Japan the ofactory bulbs receive a serotonergic innervation from the median and dorsal raphe. INTRODUCTION During the cold season hibernators display bouts of deep hibernation characterized by a low body temperature and a reduced basal metabolic rate. Hibernation is interrupted by spontaneous arousal and a return to an euthermic body temperature for a few hours or days. The time course of entrance into hibernation has only been studied in detail in few species by monitoring the body or hypotha- lamic temperature and recording electrical activ- ity. From these studies two main results have been obtained. Firstly, the decline in body or brain temperature is not a passive process but a control- led one [1]. Secondly, entrance into hibernation is thought to be an extension of normal slow wave Accepted April 21, 1992 Received November 12, 1991 1 Deceased on May 27th, 1992 sleep [2]. In certain species it seems that entrance into hibernation is achieved by way of.serotonergic system (see ref. in [3]). For example in the European hamster, Cricetus cricetus, serotonin (5- HT) concentrations in differents brain areas, espe- cially in the limbic system, were lower in hibernat- ing animals than in aroused animals in winter [4]. In addition, electrolytic or pharmacological lesion of the median but not of the dorsal raphe nucleus [3] as well as total ablation of the olfactory bulbs (OB) [5] has led to a suppression of hibernation. The evidence thus strongly suggests an anatomical connection between the raphe nuclei and the OB of the European hamster. Recently, retrograde axonal transport with triti- ated transmitters has been developed as a specific autoradiographic method for tracing pathways in the CNS [6]. Using this technique [*H]5-HT is specifically taken up at serotonergic nerve termi- nals and then transported by retrograde axonal 720 J. L. Miro, S. ARANEDA AND B. CANGUILHEM transport to their corresponding cell bodies [7-9]. The specificity of this methodology was dependent upon the concentration of injected [(PH]5-HT, and has been confirmed using combined studies of (SH]5-HT retrograde labelling and immuno- cytochemistry for serotonin [10]. Consequently, [(>H]5-HT retrograde radiolabelling is now re- garded as an important technique for studying the projections of serotonergic neurons of the mid- brain and medullary raphe in the rat [11]. In view of the involvement of the median raphe, olfactory bulb and serotonergic system in the process of entrance into hibernation, we have investigated the origin of the serotonergic innervation of the OB in the European hamster using [*H]5-HT retrograde labelling. It has been previously reported that the uptake [>H]amines by central catecholaminergic fibres is decreased in hibernating animals [12]. This result and those reported previously for the serotonergic system [3, 4, 14-16] suggest that the activity of the serotonergic neurons could be different between winter and summer hamsters. In order to test this hypothesis, we also designed experiments using retrograde axonal transport with [°H]S-HT in winter arising hamsters. MATERIALS AND METHODS Adult male European hamsters (Cricetus cri- cetus) were caught at the end of the hibernation period (April-May) in the field surrounding Stras- bourg (France). They were kept in individual cages with free access to food and water. For the summer experiments 19 hamsters were housed for one month (July) in a 12h light:12h dark cycle and constant temperature (20+1°C) before surgi- cal manipulation. The summer animals were di- vided into three groups. The first one was a control groups (n+5) which only received [?H]5- HT injections. The second group (n+5) was pretreated with clorgyline, a MAO inhibitor (10 mg/kg), 30 min before the [*H]5-HT injections in order to prevent the degradation of 5-HT into 5-hydroxyindole-3-acetic acid. The third group (n +9) was injected with 5,7-DHT (5,7-dihydro- xytryptamine creatinine sulfate, Regis Chemicals, 8 wg in 2 yl of saline with 0.1% ascorbic acid) which selectively destroys serotonergic nerve ter- minals. The 5,7-DHT solution was injected into the OB at the same stereotaxic cooredinates as the [>H]5-HT injections six days before. For the winter experiments, 4 animals were transferred into a cold room (7+1°C) with total darkness for one month (January) before the ex- periment. During this time hibernation was check- ed daily by visual inspection. Thirty min before the experiments, the hamsters were pinched in order to produce an arousal so that when the [H]S-HT injections began all the animals were normothermic. Hamsters from these experiments were not treated with any drugs before the [°H]5- HT injections. For the injections of [H]5-HT, animals were anaesthetised with Nembutal (50 mg/kg) and were placed in a David Kopf stereotaxic instrument. A mechanical obstruction of the olfactory diverticle was then carried out (AP=15, L=1, 2 and DV= 3, 4) in order to avoid difussion of the radioactive tracer as previously described in the rat by Ara- neday, ctqysalgauilail: [(H]-serotonin [°H]5- hydroxytryptamine creatinine sulfate, Amersham, 15 Ci/mol) was injected 20 wCi, 5x 10* M) in one or both OB (AP=17, L=1 and DV=3) over a Fic. 1. injections into the olfactory bulb. Autoradiograms of the olfactory bulb (A), dorsal raphe (B, C) and median raphe (D) following [*H]S-HT A) The PHI5-HT injection site in the olfactory bulb was located in the granular layer, where an intense autoradiographic reaction was observed which decreased in the plexiform and glomerular layers. Scale bar, 80 pm. B and C) Labelled cell bodies in the dorsal raphe, 24 hours after injections into the olfactory bulb. They were situated ipsilaterally when the [*H]5-HT was injected into one olfactory bulb. Some radiolabelled cells were located between both medial longitudinal bundles (mlb). Scale bar, 30 “m. D) In the median raphe, labelled cell bodies were observed in the mediolateral part of the nuclei and presented a dense autoradiographic labelling. Scale bar, 45 zm. Note that the silver grains were mainly distributed in the cytoplasm. The area indicated by arrows is enlarged in the inset. Scale bar, 20 um. Serotonin and Olfactory Bulbs in Hamster 722 J. L. Miro, S. ARANEDA AND B. CANGUILHEM period of 25 min. Twenty four hours later, animals were re-anaesthetised and the brains fixed by intra-aortic perfusion using glutaraldehyde (3.5%) in phosphate buffer (0.2 M, pH7.3). The whole brain was removed, dehydrated in ethanol and embedded in parafin-paraplast. Sections (7 ~m thick) were cut every 50 um. Then coated by dipping in Ilford KS emulsion (diluted 1:1), ex- posed for 10-30 days at 6°C, developed in phenisol (Ilford) and stained with cresyl violet. Light microscope observations were made using both bright and darkfield illumination. Radiolabelled cells were counted in the midbrain raphe and the Bartlett’s test for homogeneity of variance was used to determine statistical significance. RESULTS Injection site (H]5-HT injections into the OB of summer hamsters were characterised by a central zone of high density of silver grains confined to the granu- lar layer of the olfactory bulb. The intensity of this autoradiographic reaction gradually decreased towards the glomerular layer (Fig.1A), and caudally towards the anterior olfactory nucleus. A dense accumulation of silver grains was observed in the granular, plexiform and glomerular layers within 1 mm of the injection site. Accumulation of silver grains was restricted to the neuropile and not to the cellular components. The mitral tufted and granular cells were devoid of silver grains. Following 5,7-DHT treatment, a diffuse auto- radiographic reaction persisted but the accumula- tion of silver grains was abolished in all of the olfactory layers. Raphe cell bodies Dorsal and median raphe nuclei of the Euro- pean hamster are located in the mesencephalon ventral to the aqueduct. As has been described for Fic. 2. Schematic representation of dorsal (DR) and median raphe (MR) and the distribution of labelled cell bodies (dots) detected by autoradiography following retrograde axonal transport with [SH]5-HT. Brain sections were taken at 150 ~m intervals to be drawn using a camera lucida. Brain regions were identified with reference to the rat atlas of Paxinos and Watson (18). 3n: oculomotor nerve, mlb: medial longitudinal bundle, xscp: decussation of the superior cerebellar peduncle, CG: central gray, Aq: aqueduct, scp: superior cerebellar peduncle, TN: tegmental nucleus, ml: medial lemniscus, 5 Mo: motor trigeminal nucleus, LTN: lateral tegmental nucleus. Serotonin and Olfactory Bulbs in Hamster 723 the rat dorsal raphe [12], we also distinguished four principal regions in the dorsal raphe of the hamster: the ventromedian, the dorsomedian and the lateral groups, and the pars caudalis. The median raphe was situated ventrally to the supe- rior cerebellar peduncle in the mediolateral posi- tion. At the rostro-caudal levels, it extended from the interpenducular nucleus to the caudal levels of the tegmental nucleus (Fig. 2). Twenty four hours after the [>H]5-HT injections into the OB, a number (n=68) of dorsal raphe cell bodies showed specific autoradiographic labelling (Fig. 1B-D). Silver grains were distributed in the cytoplasm and sometimes extended to the de- ndrites but not to the nuclei. Radiolabelled cells were observed in the ventral position and scattered among many non-radiolabelled neurons. At the rostral level of the superior cerebellar peduncle, a moderate number of radiolabelled cells were observed in the medial region of the dorsal raphe. Caudally, there were more radiolabelled cells dis- tributed dorsal to and between the medial longitu- dinal bundle (Fig. 2). A few labelled cells were also observed in the adjacent central gray. In the median raphe, the largest number of retrogradely labelled cells (n=20) were observed in the ventral position, and situated more caudally to the labelled cells of the dorsal raphe (Fig. 2). In both raphe nuclei, radiolabelled cell bodies were situated mainly ipsilaterally when [*H]5-HT was injected into one OB (Fig. 1C). The locus coe- ruleus neurons were not radiolabelled. Results from the winter experiments indicate that the number of radiolabelled cells in the dorsal (n=45+20) and median raphe (n=17+9) were not statistically different from the summer hams- ters. The number of radiolabelled neurons in the dorsal raphe and median raphe of summer ham- sters was 50+18 and 17+9, respectively. DISCUSSION Injection of radiolabelled serotonin into the OB of European hamsters is followed by a selective accumulation of radioactivity in some cells of the dorsal and median raphe. This autoradiographic labelling depends upon uptake of the radioactive amine by the serotonergic nerve terminals since their selective destruction by 5,7-DHT did not result in a retrograde accumulation in the corres- ponding cell bodies. Moreover, the lack of label- ling of the noradrenergic neurons of the locus coeruleus indicated that this process was selective to 5-HT neurons. These results on the selectivity of labelling serotonergic projections are in good agreement with those obtained in the rat OB- raphe connections [10]. Using a concentration of 10* M [°H]5-HT, we have also previously demon- strated specific radiolabelling using a combination of autoradiography after [*H]5-HT retrograde transport and immunocytochemistry for serotonin in both the olfactory and spinal serotonergic pro- jections [11]. In the present study, inhibition of the enzyme monoamine oxidase by clorgyline did not affect the number of labelled neurons indicat- ing that the [*H]5-HT taken up by the terminals could be quickly bound or metabolized to another form in order to be transported retrogradely by axons as it has been described in the rat [9]. The present findings thus demonstrate a sero- tonergic innervation of the hamster OB by the dorsal and median raphe. Radiolabelling was located in the dorsomedian, ventromedian and lateral serotonergic cell groups in the dorsal raphe, and caudally in the median raphe. Moreover, radiolabelled neurons of both raphe innervate the ipsilateral OB. These results are in accordance with previous horseradish peroxidase anterograde- retrograde tracing [17] and [°H]5-HT retrograde autoradiographic studies in the rat [8, 11]. Although further studies will be required to deter- mine the precise anatomical site of serotonergic innervation of hamster OB structures, it appears likely from our results that many of the termina- tions are in the granular and glomerular layers. In the dorsal raphe, the distribution of sero- tonergic radiolabelled cells was mainly in the ven- tral and dorsal position which seems quite similar to that observed in the dorsal raphe of the rat [12]. In the median raphe, however, labelled neurons were located more caudally, and were not co- distributed in the same frontal section of the dorsal labelled cells as has been previously observed in the rat using the same methodology [11]. Our previous experiments have suggested that in the European hamster only specific groups of 724 serotonergic neurons in a small part of the median raphe are involved in the process of entrance into hibernation which may or may not be associated with the OB [3, 5]. Our present results lend some support to this assumption. If our hypothesis were true, however, we would expect to find a greater number of labelled cells in the summer than in the winter animals indicating increased accumulation of [PH|5-HT in serotonergic cell bodies before entrance into hibernation. This was not the case and further studies should thus be undertaken using true hibernating hamsters in order to address this question. In conclusion, we have demonstrated a sero- tonergic innervation of the hamster OB by a well defined population of neurons from the median and dorsal raphe. REFERENCES 1 Heller, H. C. (1979) Hibernation: neural aspect. Ann. Rev. Physiol., 41: 302-321. 2 Walker, J. M., Glotzbach, S. F., Berger, R. J. and Heller, H. C. (1977) Sleep and hibernation in ground squirrel (Citellus spp): electrophysiological observations. Am. J. Physiol., 233: R213—R221. 3 Canguilhem, B., Miro, J.-L., Kempf, E. and Schmitt, P. (1986) Does serotonin play a role in entrance into hibernation? Am. J. Physiol., 251: R755—R761. 4 Canguilhem, B., Kempf, E., Mack, G. and Schmitt, P. (1977) Regional studies of brain serotonin and norepinephine in the hibernating, awakening or active European Hamster, Cricetus cricetus, during winter. Comp. Biochem. Physiol., 57C: 175-179. 5 Miro, J.-L., Canguilhem, B. and Schmitt, P. (1980) Effects of bulbectomy on hibernation, food intake and body weight in the European hamster, Cricetus cricetus, Physiology and behaviour, 24: 859-862. 6 Cuenod, M., Bagnoli, P., Beaudet, A., Rustioni, A., Wiklund, L. and Streit, P. (1982) Transmitter specific retrograde labelling of neurons. In “Cytochemical Methods in Neuroanatomy”. Ed. by V. Chan-Palay and S. L. Palay, Alan R. Liss, New York, pp. 17-44. 7 Araneda, S., Bobillier, P., Buda, M. and Pujol, J. F. (1980) Retrograde axonal transport following injection of [*H]-serotonin in the olfactory bulb. I: 10 11 12 13 14 15 16 17 18 J. L. Miro, S. ARANEDA AND B. CANGUILHEM Biochemical study. Brain Res., 196: 405-415. Araneda, S., Font, C., Pujol, J. F. and Bobillier, P. (1983) Retrograde axonal transport after radioac- tive indole injections into the olfactory bulb. An autoradiographic study. Neurochem. Inter., 5: 741- 750. Araneda, S., Mermet, A., Buda, M., Bobillier, P., Pujol, J. F. (1984) Retrograde axonal transport after radioactive serotonin injections into the olfac- tory bulb. A biochemical analysis of radioactive material. Neurochem. Inter., 6: 27-39. Araneda, S., Magoul, R. and Calas, A. (1989) Tracing specific transmitter pathways in the rat CNS: combination of [*H]-serotonin retrograde labelling with immunocytochemical detection of en- dogenous transmitters. J. Neurosci. Methods, 30: 211-218. Magoul, R., Oblin, A., Calas, A. and Araneda, S. (1988) Serotonergic projections to the spinal cord but not those to the olfactory bulb also contain substance P. A combined immunocytochemical and autoradiographic study following retrograde axonal transport of [*H]-serotonin labeled products. Neurosci., 26: 959-969. Steinbucsh, H. W. M. and Niewenhuys, R. (1983) The raphe nuclei of the rat brainstem: a cytoar- chitectonic and immunocytochemical study. In “Chemical Neuroanatomy”. Ed. by P. C. Emson, Raven Press, New York, pp. 131-207. Faure, A. et Calas, A. (1976) Etude radioautog- raphique de l’incorporation in vitro de noradrena- line tritiée dans des fibres catécholaminergiques centrales chez I’hérisson actif et en hibernation C. R. Soc. Biol. 171: 136-140. Kempf, E., Mack, G., Canguilhen, B. and Mandel, P. (1978) Seasonal changes in the levels and the turnover of brain serotonin and noradrenaline in the European hamster kept under constant environ- ment, Experientia, 34: 1032. Miro, J.-L., Canguilhen, B. and Schmitt, P. (1979) Sérotonine cérébrale et sommeil hibernal, Brain Res., 170: 387-391. Miro, J.-L., Canguilhem, B. and Koch, A. (1986) Brain serotonin levels and metabolism in the non- hibernating bulbectomized European hamster. Life Sci., 39: 2551-2555. Mclean, H. J. and Shipley, M. (1987) Serotonergic afferents to the rat olfactory bulb: I. Origins and laminar specificity of serotonergic inputs in the adult rat. J. Neurosci., 7: 3016-3028. Paxinos, G. and Watson, C. (1982) The rat brain in stereotaxic coordinates. Academic Press, Sydney. ZOOLOGICAL SCIENCE 9: 725-731 (1992) Photoperiodic Control of the Determination of Two Different Seasonal Diphenisms of the Asian Comma Butterfly, Polygonia c-aureum L. KATSUHIKO ENDO, SATOSHI UENO, MiTSUNORI MATSUFUJI and YOSHIKI KAKUO Environmental Biology Laboratory, Biological Institute, Faculty of Science, Yamaguchi University, Yamaguchi 753, Japan ABSTRACT—The Asian comma butterfly, Polygonia c-aureum L. exhihits two different seasonal diphenisms in the morph and adult reproductive activity, both of which are governed by photoperiod and temperature existing during the post-embryonic development. In the complete and 8L-16D asymmetrical skeleton photoperiods at 20°C or 25°C, they formed similar response curves in the incidences of autumn morph and reproductive diapause in adult females. They required 4 and 5 16L-8D (long-day) days for achieving 50% occurrence of vitellogenic and summer morph adults, respectively. Critical stages of 16L-8D to 8L-16D (short-day) transfer obtained on 50% preventions of vitellogenic and summer morph female adults were located on day 0 and on day 2 of the 3rd instar. In contrast, the critical stages of adverse photoperiodic transfer greatly differed between these two kinds of seasonal diphenisms. The stages were day 3 of the Sth instar in the seasonal morphs and day 3 of pupae in the reproductive activity. Physiological mechanisms underlying photoperiodic control in P. c-aureum has been previously indicated to involve two neuroendrocrine systems secreting summer morph-producing hormone and/or a factor activating corpora allata in adults. Differential responsiveness of these two neuroendocrine systems to photoperiods may reflect on the difference in the incidences of the seasonal morphs and reproductive activity. In addition, the photoperiodic sensitive stage seems to be different with respect to © 1992 Zoological Society of Japan these two different seasonal diphenisms. INTRODUCTION In a number of insects, diapause and seasonal polyphenism are governed by environmental fac- tors, such as photoperiod and temperature they are subjected to after hatching [1-5]. Pupal diapause and spring morphs of the swal- lowtail, Papilio xuthus L., are induced by short days at 20°C. They become photoperiodically sensitive at around the first larval ecdysis [6]. The photoperiodic sensitiveness is lost at the middle of the 5th instar. In the photoperiod-sensitive stage, they require seven 8L-16D (SD: short-day) days for the 50% incidences (or require five 16L-8D (LD: long-day) days for the 50% preventions) of pupal diapause and spring morph development at 20°C [6]. The Asian comma butterfly, Polygonia c- aureum L., exhibits two different seasonal diphe- Accepted April 28, 1992 Received March 11, 1992 nisms, 1.€. seasonal morph and adult reproductive activity, both of which are determined by photo- period and temperature they are exposed to after hatching. Under LD at 20°C, P. c-awreum females developed into vitellognic/summer morph adults, whereas those held in SD at 20°C developed into reproductive diapause/autumn morph adults [7]. Physiological mechanism underlying photo- periodic control of these two different seasonal diphenisms involves cerebral factors secreted from corpus cardiacum and/or corpus allatum in the early pupal stages [7-9]. Critical stages at which summer-morph producers (long-day pupae) re- quire no more brain and/or nervi corporis cardiaci (I+1I) for summer morph and vitellogenic adult development lie at about 32 hr after larval-pupal ecdysis at 20°C in P. c-aureum [10, 11]. Our present study was initiated to determine the number of long-day required for the occurrence of summer morph and vitellogenic female adults of P. c-aureum. Then, the study was extended to deter- 726 K. Enpo, S. mine the beginning and the end of photoperiod- sensitive stages in the induction (or prevention) of these two different seasonal diphenism. MATERIALS AND METHODS Animals P. c-aureum collected in the Yama- guchi City was used. Eggs and larvae were held in transparent plastic containers of either 695 cm or 19x13xX5cm and were exposed to complete photoperiods, whose photophase was changed from 2hr (2L-22D) to 20hr (20L-4D) at 2-hr intervals. Rearing containers were placed in cabinets with constant temperature (20°C or 25°C) and were illuminated by two 20-W white fluores- cent tubes which were controlled by a 24-hour time-switch. In the light period, light intensity was at about 500 lux to the rearing containers. Larvae were fed on fresh leves of Humulus japonicus provided daily. Classification of seasonal morphs Summer- morph and autumn-morph females exhibit clear differences in background color of the wings. The wing background color of summer morph females is dark yellow on both sides, whereas that of autumn morph females is reddish-brown on the dorsal side and dark brown on the ventral side [12]. Seasonal morph classification was made only in females on the basis of the wing background color. Criterion for adult reproductive diapause When fed on 10% sucrose four days after emergence at 25°C, an vitellogenic (reproductive non-diapause) females accumulate green yolk in oocytes and/or produce eggs. In contrast, females entering repro- ductive diapause do not accumulate yolk at the same period. Female adults were caught on the day of emerg- ence, fed on 10% sucrose for 4 days at 25°C and dissected in 0.9% NaCl to see whether the oocytes in the ovaries accumulated yolk or not. Female adults accumulating green-colored yolk in oocytes were classified as vitellogenic, whereas others were classified as diapausing. UENO et al. RESULTS Fluctuations of the incidences of autumn morph and reproductive diapause female adults in the complete photoperiods at 20°C and 25°C To assess how the incidences of autumn morph and reproductive diapause adults fluctuate, groups of 150-200 larvae were subjected to complete photoperiods whose photophase was changed de- pending on larval groups from 2 hr (2L-22D) to 20 hr (20L-4D) at 2-hr intervals at 20°C or 25°C. Photoperiodic response curves were obtained on the basis of the percentages of autumn morph and reproductive diapause adult females at 20°C and 25°C (Fig. la and b). 100 Ape OE a ) rs) 7) a) £ ro) ~ 50 & v fo) o e a 0 @—@-—a—60 4 8 12 16 20 Photoperiod 100 b uw en U mS ee o os uv £ © 50 Soeike ~ fo) re $ wa o u a ot 8° @0—e0—00—00 4 8 12 16 20 Photoperiod Fic. 1. Photoperiodic response curves of P. c-aureum two different seasonal diphenisms in the complete photoperiods at 20°C (a) and 25°C (b). Open and solid circles show the incidences of autumn morph and reproductive diapause female adults, respec- tively. Seasonal Diphenisms of Polygonia 727 Under shorter than 12 hr photoperiodic regi- ments at 20°C, all females developed into autumn morph/reproductive diapause adults with an ex- ception of 2L-22D group in which 12 summer morph and 2 (out of 18) autumn morph adults were vitellogenic. In contrast, all females de- veloped into vitellogenic/summer morph adults under longer 14hr photoperiodic regimens at AIG: At 25°C, vetellogenic/summer morph adults appeared in all groups reared under complete photoperiods examined. Under short-day condi- tions at 25°C, percentages of autumn morphs varied in a range from 55% (2L-22D) to 95% (12L-12D) and 5% to 75% of the autumn morphs were judged as being vitellogenic. In contrast, all females developed into vitellogenic and summer morph adults under longer 14 hr photoperiodic regimens. Critical photoperiods for the determina- tion of these two different seasonal diphenisms were about 13 hr (13.1 hr and 12.7 hr) both at 20°C and at 25°C. The results indicate that P. c-aureum used the same critical daylength for determining both sea- sonal morphs and reproductive activity, in the complete photoperiods at 20°C and 25°C. How- ever, autumn morph development seems not al- ways to be accompanied by reproductive diapause at 25°C. Fluctuation of the incidences of autumn morph and reproductive diapause adult females in the asym- metrical skeleton photoperiods of 8L-16D To clarify how the incidences of autumn morph and reproductive diapause adults are fluctuated by a supplementary light pulse at 20°C, groups of 200-250 larvae were subjected to a photoperiodic regimen of 8L-16D, whose scottophase was inter- rupted by a supplementary light pulse of 0.5 hr. The giving time of light pulse was delayed from light-off of 8L-16D at 1-hr intervals. All females developed into autumn morph/re- productive diapause adults under 8L-16D at 20°C. All or a large part of females were prevented from exhibiting autumn morph and reproductive di- apause by a 0.5-hr supplementary light pulse on 3— 6hr after light-off of 8L-16D. However, a light pulse earlier or later in scotophase was ineffective where all or majority of females developed into autumn morph adults, 5% to 30% of which were vitellogenic (Fig. 2). WO eeeoes more -0-0~9-0-0-040-0 An 4 RO 2 © OS a awe @-0-0 vo 75) ro 2 / c= §50) H 5 é rod A ao (5) 2 a (oe P od osc Moen yaad 0) 4 8 12 16 20 2% Time of light pulse after dawn (hours) Fic. 2. Photoperiodic response curves of P. c-aureum two different seasonal diphenisms in the asymmet- rical skeleton photoperiods of 8L-16D at 20°C, where 0.5-hr light pulse interrupted the scotophase on the time given in the abscissa. Open and solid circels show the incidences of autumn morph and reproductive diapause female adults. Open trian- gles show the incidence of autumn morphs recorded under the corresponding complete photo-periods at 20°C, redrawn from Fig. 1. For the 50% prevention of autumn morph de- velopment, P. c-aureum has two clear critical photoperiods of 10.5-11.0 hr and 13.0—-13.5 hr in the asymmetrical skeleton photoperiods of 8L-16D at 20°C. The shorter critical daylength was almost the same value as, but the longer one was about 0.5-hr shorter than, the critical daylength of the 50% prevention of reproductive diapause (Fig. 2). Response curves to the number of 16L-8D (LD: long-day) days for inducing the development of summer morph and vitellogenic female adults To investigate how the effect of LD days is integrated and represented on the incidences of summer morph and vitelogenic adult females, 0- day-old 4th-instar larvae were selected from a stock culture raised from the egg stage under short days (SD-8L-16D) at 20°C. The LD-exposure was performed on 4th-instar or 4th- to 5th-instar larvae and the length of the exposure was changed from zero to 7 days depending on the groups. Any prevention in the incidence of autumn morph adults was not observed by a LD-exposure of shorter length than 3 days. But, a few percent of 728 K. Enpbo, S. UENo et al. the autumn morph adults became vitellogenic. Percentages of the summer morph adults rose gradually with the increase of the numbers of LD-exposure days. Fifty percent of the incidences of summer morph and vitellogenic adult females reached by a LD-exposure of five and four days, respectively. One hundred percents were achieved by the LD-exposure of seven days (Fig. 3). 100 @0 Percent of insects ol Ss) oO e on | ea oO OO Yo 1 3 5 7 Number of LD cycles Fic. 3. Response curves of P. c-aureum two different seasonal diphenisms to the number of long-day days, where 0-day-old 4th-instar larvae from SD condi- tions were exposed to LD cycles as given in the abscissa and then transferred to SD regime. Open and solid circles show the occurrence of summer morph and vitellogenic female adults at 20°C. Stage-dependent changes of the effects of LD-to-SD transfer on the incidences of summer morph and vitellogenic female adults To examine how the effects of LD-to-SD trans- fer on the incidences of summer morph and vitel- logenic adult females change with the developmen- tal stages, 0-day-old larvae of the 2nd, 3rd, 4th and Sth instar were selected from a stock culture raised from the egg stage under SD at 20°C. About two-hundred and fifty larvae of the same stages were grouped and subjected to a LD-to-SD trans- fer at 20°C. The LD-to-SD transfer was carried out at different/difinite developmental stages which were changed depending on groups at one- day intervals from day 2 of the first instar to day 2 of the Sth instar. When larvae were subjected to a LD-to-SD transfer before day 2 of the 2nd instar, all females responded to SD and developed into autumn morph adults, 13%-27% of which became vitel- logenic. Percentages of females showing a re- sponse to SD decreased gradually when the trans- ferred stages were delayed from day 2 of the 2nd instar. Fifty percent of the incidences of summer morph and vitellogenic adults reached by a group subjected to a LD-to-SD transfer on day 2 and day 0 of the 3rd instar, respectively. Critical stages of LD-to-SD transfer after which P. c-aureum did not show any response to SD were day 2 (for seasonal morphs) and day 0 of the 4th instar (for reproduc- tive activity) (Fig. 4). 100 76 Oe as vy) 3) cD) w) iS iz — ee — = 504 G =— 5days — © oa 0 J e“d-0-0 Lye Slimesl peleeant 0 10 i VO. a. keen Stage of Pee transfer (LD to SD) Fic. 4. Stage-dependent changes of the effects of LD- to-SD transfer on the determination of P. c-aureum two different seasonal diphenisms. Open and solid circles show the occurrence of summer morph and vitellogenic female adults at 20°C. “I0”, “II0”, “TII0”, “IVO” and “VO” show the first days of the first-, 2nd-, 3rd-, 4th- and Sth-instar larval stages, respectively. The results indicate that day 0 and day 2 of the 3rd instar seem to be the critical stages before which P. c-aureum can experience sufficient num- bers of LD days for achieving the 50% incidences of summer morph and vetellogenic adults, respec- tively. Stage-dependent changes of the effects of SD-to-LD transfer on the incidence of summer morph and vitellogenic adult females To assess how the effects of SD-to-LD transfer on the incidences of summer morph and vitel- Seasonal Diphenisms of Polygonia 729 logenic adults change with the developmental stages, 0-day-old 4th- and Sth-instar larvae and 0-day-old pupae were selected from a stock culture raised from the egg stage under SD at 20°C. Two-hundred larvae and two-hundred pupae of the same stages were grouped and were subjected to a SD-to-LD transfer at 20°C. In this experi- ment, the stage of the SD-to-LD transfer was changed depending on the groups at 1-day inter- vals from day 2 of the 4th instar to day 3 of pupa. All females introduced to LD on days preceding day 0 of the 4th instar developed into summer morph adults. Percentage of the summer morphs lowered quickly when the stage of the transfer was delayed from day 0 of the 4th instar. Fifty percent and one hundred percent of the incidences of the summer morphs were achieved by groups intro- duced to LD on day 3 and on day 5 of the Sth instar, which corresponded to 3 days and one day before larval-pupal ecdysis, respectively (Fig. 5). With respect to the vitellogenic activity deter- mination, P. c-aureum showed responses to a SD-to-LD transfer different from those in morphs. Seventy-four percent of females introduced to LD on day 2 of pupae became vitellogenic. Critical stage of the SD-to-LD transfer based on the 50%- incidence of vitellogenic adults was found to lie on day 2 of pupae, which is 5 days latter than that obtained on the 50%-incidence of the summer morphs (Fig. 5). The results indicate that the developmental stages at which P. c-aureum looses the photo- 100 5 un U o wo sg —4days Oo 504 = wo 1S) o - e ° si \oret sie, he TAP Tioger Vara 0 eae eee T ua T T T TT T T Teal: 1Y{0) vO PO Adult Stage of photoperiodic transfer (SD to LD) Fic. 5. Stage-dependent changes of the effects of SD- to-LD transfer on the determination of P. c-aureum two different seasonal diphenisms. Open and solid circles show the occurrence of summer morph and vitellogenic female adults at 20°C. “IV0”, “VO”, “PO” and “adult” show the first days of 4th-instar larval, Sth-instar larval, pupal and imagimal stages. periodic responsiveness seems to vary depending on the kinds of seasonal diphenisms of the insects. DISCUSSION Almost all adult females of autumn morphs in P. c-aureum developed under complete short-day photoperiods at 20°C entered reproductive di- apause. Critical daylengths for occurrence of autumn morph and reproductive diapause adult females were about 13 hr, as has been reported for morph determination in the same species [7]. The coincidental responses in the determination of these two kinds of seasonal diphenisms dis- appeared in the complete photoperiods at 25°C (Fig. 1b). Differential fluctuations of the inci- dences of these two kinds of seasonal diphenisms were also observed in the larval groups subjected to the asymmetrical skeleton photoperiods of 8L- 16D at 20°C (Fig. 2). Physiological mechanism underlyint the photo- periodic control of these two kinds of seasonal diphenisms may involve clock and neuroendocrine systems as had been demonstrated in many other insects [13-18]. For the regulation of two kinds of seasonal diphenisms, P. c-aureum may share clock components functioning time-measurement of photoperiod and accumulation of photoperiodic information. This suppositon is based on almost the same values of critical daylengths of these two kinds of seasonal diphenisms which P. c-aureum showed in the complete and asymmetrical skeleton photoperiods, respectively (Figs. 1 and 2). The differential responses of these two kinds of seasonal diphenisms mentioned as above may de- pend on differential responsiveness of neuroen- docrine systems secreting summer-morph-produ- cing hormone [12] and a factor activating corpora allata in adults, which seems to do similar function as allatotropin in Manduca sexta [19]. P. c-aureum required a LD-exposure of diffe- rent length (five and four days) for achieving 50% summer morph and vitellogenic adults (Fig. 3). Photoperiod-sensitive stage in which P. c-aureum shows the responsiveness to photoperiods seems to begin at around the first larval ecdysis (Fig. 6) as has been demonstrated in the determination of P. xuthus pupal diapause [6]. This supposition is 730 Peaks of hemolymph ecdysteroid titer LD-to-SD Critical stages transfer for seasonal morph determination Photoperiod-sensitive stage Critical stages for reproductive diapause determination LD-to-SD transfer Photoperiod-sensitive stage K. Enpbo, S. UENO et al. SD-to-LD Brain transfer NCCI+I for seasonal morph determination Brain SD-to-LD ag transfer for reproductive diapause determination 3rd instar First instar 4th instar 2nd instar Egg Fia. 6. 5th Pupa Adult instar Schematic representation of P. c-aureum photoperiod-sensitive stage with the critical stages of summer morph and vitellogenic adult development. “NCC(I+II)” shows the nervi corporis cardiaci. based on a hypothesis that groups of insects receiv- ing a LD-exposure of 5 and 4 days before LD-to- SD transfer should represent 50%-incidences of summer morph and vitellogenic adult females. On that day, P. c-aureum larvae undergo the first larval ecdysis at 20°C (Fig. 4). A similar explanation may be available for the end of the photoperiod-sensitive stage; the stage may come on day 1 of pupae, which is 5 days later than the critical day of SD-to-LD transfer of the incidences of summer morphs (Fig. 5). The stage is one-day earlier than the critical days at which summer-morph-producers no more require the brain and the nervi corporis cardiaci which are essential for summer morph and vitellogenic adult development (Fig. 6) [10]. However, P. c-aureum pupae are thought to have photoperiodic respon- siveness on days following larval-pupal ecdysis. This is based on the evidence that large numbers of pupae introduced to LD on 6 days following larval-pupal ecdysis showed a clear response and developed into vitellogenic (autumn morph) adults (Fig. 5). It may be that the a factor activating corpora allata in P. c-aureum adults may be se- creted in the late pupal stage according to the photoperiodic imformation accumulated from day 0 of the 2nd instar to two or three days before eclosion by the clock component (Fig. 6). Here, we could not provide any evidence about neuroendocrine system secreting a factor activat- ing corpora allata (allatotropin?) in P. c-aureum adults. But, it may become clear by further studies on physiological mechanism underlying the photo- periodic control of seasonal diphenisms of P. c- aureum. ACKNOWLEDGMENTS This work was supported in part by Grant-in-Aid from the Ministry of Education, Science and Culture, Japan No. 03640606 to K.E. REFERENCES 1 Tanaka, Y. (1950) Studies on hibernation with special reference to photoperiodicity and breeding of Chinese tussar silkworm. I. J. Seric. Jpn., 19: 358-371. 2 Miller, H. J. (1955) Die Saisonformenbildung von Araschnia levana, ein photoperiodisch gesteuerter Diapause-effekt. Naturwiss., 42: 134-135. 3 Lees, A. D. (1959) The role of photoperiod and temperature in the determination of parthenogene- tic and sexual forms in the aphid Megoura viciae Buckton—I. The influence of these factors on apter- ous virginoparae and their progeny. J. Insect Phys- iol., 3: 92-117. 4 Danilevskii, A. S. (1961) Fotoperiodism i sezonne razvitie nasekomykh. Izd. Leningradskogo Univer- sititeta, Leningrad, p. 238. 5 Beck, S. D., Clutier, E. J. and McLeod, D. G. R. 10 11 12 Seasonal Diphenisms of Polygonia (1962) Photoperiod and insect development. In “Proc. 23rd Biol. Colloq. Orgon State Univ.” pp. 43-64. Endo, K., Ito, T. and Chiba, Y. (1986) Photo- periodic control of pupal diapause in the swallow- tail, Papilio xuthus L. (Lepidoptera, Papilionidae) —Sensitive stage and number of required cycles. Zool. Sci., 3: 351-356. Hidaka, T. and Takahashi, H. (1967) Temperature condition and maternal effect as modifying factor in the photoperiodic control of seasonal forms in Poly- gonia c-aureum (Lepidoptera, Nymphalidae). Annot. Zool. Japon., 40: 200-204. Fukuda, S. and Endo, K. (1966) Hormonal control of the development of seasonal forms in the but- terfly, Polygonia c-aureum L. Proc. Japan Acad., 42: 1082-1087. Endo, K. (1970) Relation between ovarian matura- tion and activity of corpora allata in seasonal forms of the butterfly, Polygonia c-aureum L. Devel. Growth and Differ., 11: 279-304. Endo, K. (1972) Activation of the corpora allata in relation to ovarian maturation in the seasonal forms of the butterfly, Polygonia c-aureum L. Devel. Growth and Differ., 14: 263-274. Endo, K. (1984) Neuroendocrine regulation of the development of seasonal forms of the Asian comma butterfly, Polygonia c-aureum L. Devel. Growth and Differ., 26: 217-222. Endo, K., Masaki, T. and Kumagai, K. (1988) 13 14 15 16 17 18 19 731 Neuroendocrine regulation of the development of seasonal morphs in the Asian comma butterfly, Polygonia c-aureum L.: Difference in activity of summer-morph-producing hormone from _brain- extracts of the long-day and short-day pupae. Zool. Sci., 5: 145-152. Bunning, E. (1960) Circadian rhythms and time measurement in photoperiodism. Cold Spring Har- bor Symp. Quant. Biol., 25: 249-256. Tyshchenko, V. P., Gorshin, N. I. and Azaryan, A. G. (1972) The role of circadian processes in insect photoperiodism. Zhur. Obshch. Biol., 33: 21-31. Saunders, D. S. (1976) Insect Clock. Pergamon Press, New York, p. 279. Beck, S. D. (1980) Insect Photoperiodism. Academic Press, New York, 2nd ed., p. 387. Saunders, D. S. (1971) The temperature-compen- sated photoperiodic clock “programming” develop- ment and pupal diapause in the fleshfly, Sarcophaga argyrostoma. J. Insect Physiol., 17: 801-812. Gorshin, N. I. and Tyshchenko, V. P. (1970) Ther- mostability of the process of perception of photo- periodic information in the moth Acronucta rumicis (Lepidoptera, Noctuidae). Dokl Acad. Nauk. USSR, 193: 458-461. (in Russian) Kataoka, H., Toschi, A., Li, J. P., Carney, R. L., Schooly, D. A. and Kramer, S. J. (1989) Identifica- tion of an allatotropin from adult Manduca sexta. Science, 243: 1481-1483. hiiyt aditilieanlion 0 t ay We Adleprcty (st oe ‘ A orruatiatane & Buivess yu ‘ ile mepeleyhine va day thy telhins gic asl) Wh ah ‘a oc SRT Ree re b VGOMS) Vd rind (eet Title a by Maoist? AREA anit il Mia 3 lone wir ott faery ") hive fy See te natn PL) 4 Al ead we beh den ® a: sie | | ‘ had ae ha oe i Arete fl tr fl. t aie? FEA yepearte| LAE J PGE aT AYA bes Wi i t] eer it 1h aes inodirvobil peernraven staneena fi py bo ae 4 ed Punk ; State tn Ninbpn mererwsrsut eeatinl poumpy ite Elomi Ayu Agiopri a Pa {Ae a ot Op gabled wisn Stk ir Priuth b nema brshaaay von f tne 1. oulemt es "1 ea Mee ohhelas rH mialie Garris Sf] ke Gualt Step i ih bry PET UA ns ah UO Oi 1 so upobiers teat} 4 Wind ty ey weet 4 p i eel ot ek { ( erie: ¢ eiyay We ea es hy ue Ali la ae f iB r Apes dere Bol, bide (aeTy pine | j . a Tee ee be 1 “- > (Ne « r* Ly : a oe \ re ae Per ty Vind Big DETY « : 7 tut : red thal, ZOOLOGICAL SCIENCE 9: 733-739 (1992) Scanning Electron Microscopy of Differentiating Chick Ovaries during Embryonic Period ATSUMI UKESHIMA Kumamoto University, College of Medical Science, Kumamoto 862, Japan ABSTRACT— Asymmetrically developing right and left ovaries of the chick embryos from 2 to 19 days of incubation were observed by scanning electron microscope (SEM). Surface epithelium of undifferentiated gonad showed rough appearance with many vesicular protrusions and fillopodia, as compared with the other regions of coelomic surface. After the ovarian differentiation started, surface feature of the ovary was different between the right and left. In the left ovary, which made a normal development, the surface was still rugged, and possessed many vesicular protrusions and fillopodia like in undifferentiated period. In contrast, the right ovarian surface showed degenerating profile, possessing few numbers of cytoplasmic protrusions. The most significant feature of the right ovary was the fact that from 11 days onward, many holes were found on the surface. These holes exhibited a variety in size and continued to inner medullary lacunae. Sometimes, a cell or cell aggregate which, © 1992 Zoological Society of Japan supposedly, were abandoned germ cells, were found at the opening of the holes. INTRODUCTION In the female chick embryo, the ovary develops only on the left side, and the right degenerates to atrophic remnant during an embryonic period. This is due to the failure of the cortex formation in the right gonad. Although primary sex cords are equally formed on both sides during an early differentiating period of the ovary, the secondary cords, which is to form the cortex from 7 days of incubation onward, do not appear on the right. Eventurally the growth of the right ovary stops. Although some ultrastructural studies on the developing avian ovaries were present [1-4], they described on the inner structure by transmission electron microscopy (TEM). In the present study, changes of the surface morphology of the right and left ovaries which take the different pattern of formation with the advance of development, were compared by scanning electron microscopy (SEM). MATERIALS AND METHODS Chick ovaries from stage 16 (staged by Hambur- Accepted May 8, 1992 Received March 12, 1992 ger and Hamilton [5], about 2 days of incubation) to 19 days were employed for this study. As the growth of the right ovary stopped at the time of the occurrence of secondary cords, female gonads (Ovaries) were apparently different in size on the right and left sides. Therefore, identification of the gonadal sex was available. Ovaries removed from the embryos were fixed in 2.5% glutaraldehyde (phosphate buffer, pH 7.4) for 1.5 hr, and postfixed in 1% osmic acid (phosphate buffer pH 7.4) for 1 hr. Some ovaries were sectioned into thin pieces by free hand tech- nique to observe the cut face. After dehydration, materials were dried by critical point drying method, coated with platinum-paradium, and finally observed by JEOL scanning electron micro- scope (Model JSM-6400F). RESULTS In the present study, a period of the ovarian development is divided into two sections. The first is an undifferentiated period. At the initiation of this period (stage 16, about 2 days), gonadal anlages are composed of columnar (thickened) coelomic epithelium, or what is called the “germin- al ridges”. Germ cells (primordial germ cells) 734 A. UKESHIMA Fic. 1. Low magnification figure of the epithelial surface of undifferentiated gonad of a stage-16 embryo. Free surface of the cells is swollen out and possesses many fillopodia, vesicles and short microvilli. 2,400. Fic. 2. Mesonephric surface of a stage-16 embryo. As compared with the gonadal surface, few cytoplasmic processes are seen, although cells are swollen out like in the gonadal surface. 2,400. Fic. 3. Surface of the left ovary of a 8-day embryo. On the cell surface, numerous vesicular protrusions and some fillopodia are present. 3,600. Fic. 4. Right ovarian surface of a 8-day embryo. In contrast to the left ovary, the cell surface are flattened, and many short microvilli are lined at the border of each cell. 2,400. SEM of Embryonic Chick Ovaries 735 migrate into the germinal ridges via blood vessels _ epithelium is still thickned and pseudostratified, from the extraembryonic sites [6-8]. and contains many immigrated germ cells. In the The second is a differentiated period. At the right ovary, however, secondary cords are lacking. beginning (7 days) of this period, secondary cords _‘The epithelium exhibits a thin, squamous structure appear in the left ovary to form the cortex. The with no germ cells [4]. Fic. 5. Lower magnification figure of the left ovary of a 11-day embryo. The surface is very rugged, showing depression and jutting out. 540. Fic. 6. Left ovarian surface of a 16-day embryo. Cell surfaces are also covered with many vesicles, fillopodia as in younger embryo. The cells in center retain the trace of cell division (arrow). The mitotic features according to the ovarian growth were frequently observed, especially on the left ovary. 2,700. @ E mg Fic. 7. A cut surface of a 9-day left ovary. The thick surface epithelium (Ep) is composed of columnar cells. Note a germ cell (asterisk) enclosed by the epithelial cells. 2,400. Fic. 8. A sectioned view of a 9-day right ovary. Unlike the left ovary, the epithelium (Ep) is thin, and is composed of squamous cells. Under the epithelium, lacunae of various sizes (asterisk) are frequently seen. 2,400. 736 A. UKESHIMA Undifferentiated period Surface of the germinal ridges (gonadal anlages) as a whole showed a rough profile at low mag- nification (Fig. 1). This was based on the presence of vesicles of various sizes and fillopodia on the cell surface. Free surface of each epithelial cell, measuring 3.5 to 8 u~m in diameter, was swollen out into the coelomic cavity. These surface fea- tures of the undifferentiated gonads were apparently different from those on the other re- gions of the coelomic epithelium. For example, epithelial surface of mesonephros was smooth ow- ing to poor protrusions, although each cell was swollen out like in the gonadal surface (Fig. 2). Fic. 9. Whole view of the right ovary from a 16-day embryo. Many holes are found on the ovarian surface. Note a posterior end (arrows) of the ovary (Ov) incorporated to the mesonephros (Ms). X90. Fic. 10. oval shape. 250. Surface of a 19-day right ovary. Holes of various sizes are seen. The opening of the holes is usually round or SEM of Embryonic Chick Ovaries 737 Differentiated period From 7 days onward, which ocrresponded to the beginning of the secondary cord formation, surface of the developing left ovary still showed similar profile as in the undifferentiated gonads; each cell surface was usually swollen out, possessing vesicu- lar protrusions of various sizes (Fig. 3). Among those, long fiber-like structures were seen extend- ing from the edge of the cells. The free surface of the cell was 3 to 6 ~m in diameter, usually about 4 ym. On the other hand, the mght ovary, which ceased to make further development because of the failure of the secondary cord formation, exhi- bited a smooth and flattened surface (Fig. 4). The free surface of each cell was somewhat larger than that in the left ovary, measuring 4.6 to 14 wm in diameter. At the margin of each cell surface, short microvilli or protrusions were seen. The surface of the left ovary became more prominent with developmental stages, showing irregularly rugged features at lower magnification (Fig. 5). Some parts were protruded, the others hollowed. Small hollows were often observed. Higher magnification revealed the rough surface of the epithelial cells covered by many vesicular protrusions. Some cells were elongated and con- Fic. 11. nected with other cells by the intercellular bridge, showing the vestige of cell division (Fig. 6). The cut surface revealed difference in the epithelial composition of both ovaries. The epithe- lium of the left ovary was thickned like in earlier stages. Germ cells were frequently seen to be involved among these columniated epithelial cells (Fig. 7). In the right ovary, however, the epithe- lium was thin and composed of the squamous cells (Fig. 8), possessing no germ cells. Just under the epithelium, medullary lacunae were often obser- ved. The most prominent feature of the right ovary was the holes on the surface, which appeared from 11 days onward (Figs. 9 and 10). Although 11-day ovary contained several holes on the surface, they increased in number with the advance of develop- ment. In 17 days, almost whole surface of the right ovary were occupied with the holes. The holes varied in size, measuring 12 to 47 um in diameter. The right ovary in advanced stage of embryo was seen to be incorporated partly to the mesonephros, the border partly being obscured (Fig. 9). The epithelial surface was flat and scanty of microvilli. In the surroundings of the holes, however, the cytoplasmic protrusions such as fillopodia and mic- rovilli were seen increasing in number (Fig. 11). The cut surface of right ovary revealed the holes ie : j eee (12) A high magnification figure of a hole of the right ovary from a 13-day embryo. Around the hole many microvilli or long fillopodia are seen, although superficial epithelial cells have few microvilli. 1,800. Fic. 12. A sectioned view of the right ovary from a 19-day embryo. This figure shows that the hole on the ovarian surface continues with the medullary lacunae (La). Arrow: pathway of the hole to the lacunae. x700. 738 A. UKESHIMA Fic. 13. This figure shows a cell aggregate coming out of the opening of a hole on the right ovary from 19-day embryo. The aggregate is composed of several adhered cells. The arrows show the adhered portions of the cells. x3.600. continuing to the medullary lacunae (Fig. 12), while the hollows found on the left ovarian surface were not continuous to the inner structure. At the opening of the holes, free cells were occasionally found after 16 days of incubation. These cells were observed as a single cell, or a cell aggregate in which several cells adhered to each other (Fig. 13). The number of the cells were not alterable with increasing embryo age. DISCUSSION In the chick embryo, the ovaries were different in size between the right and left sides. This is due to the failure of development of the right ovary. The surface morphology of the asymmetrically developing ovaries were first revealed by SEM in the present study. On the surface of the right atrophic ovaries, many holes were present at later stage of develop- ment and continued to inner medullary lacunae. Although it is not clear what mechanism exists about this continuity in the medullary lacunae of the atrophic right ovary, it might be concerned with the degeneration of germ cells from under- standing in our previous study [4], which described on germ cells released in the medullary lacunae of degenerating right ovaries. In some cases, a cell or cell aggregate were found at the opening of the holes. Also judging from our previous study [4], the cells found at the opening may be exhibiting a terminal phase of those germ cells. More accurate identification of these cells, however, should be done by the histo- logical technique. As to birds, microscopic studies on the lacunar system of the normal left ovary have been per- formed by Callebaut er al. [9, 10]. They indicated that the lacunar system continued to the coelomic (peritoneal) cavity at the dorsal (proximal) side of the left ovary. In the present study, SEM observa- tion have been made only on the surface of both ovaries, and not on the dorsal dide, demonstrating that no holes were present on the left ovarian surface. The cell aggregates found at the opening of the holes in the right ovary were often adhered tightly to each other. This aggregate is, presumably, a mass made of germ cells and lacunar epithelial cells. Similar structures were found in the right medullary lacunae in our previous study [4]. At the beginning of ovarian differentiation, SEM of Embryonic Chick Ovaries germ cells were observed in the epithelium of the left ovary, but never in that of the right. Initially, migrating germ cells were equally existed in the columnar epithelium of both gonads. Thereafter, almost all germ cells in the right ovary have moved to the medulla with a part of the epithelium, while epithelial cells have changed to a squamous shape. Therefore, germ cells in the right ovary were localized in the medulla, but none in the epithe- hum. At the cut surface of the ovary, the germ cells within a epithelium were easily identified, because the germ cells in the epithelium were round in shape and larger than the mitotic epithelial cells [7, Sh, Lil], As secondary cords are formed into a cortex only on the left side, the left ovary developed larger in size than the right. At 7 days of incuba- tion, however, difference in ovarian size between the right and left sides was imperceptible, because it was just after initiation of the cortex formation. Therefore, materials from 7-day embryo were not employed. REFERENCES 1 Narbaitz, R. and Adler, A. (1966) Submicroscopic- al observations on the differentiation of chick gonads. J. Embryol. Exp. Morphol., 16: 41-47. 2 Yamada, K. and Amanuma, A. (1980) Fine struc- ture of Balbiani body in germ cells of the chick embryo. J. Predental Fac. Gifu Coll. Dentistry, No. 10 739 6: 87-103. (in japanese). Yamada, K. and Amanuma, A. (1985) Ultrastruc- ture of interstitial cells in the gonad of quail embryo. J. Liberal Arts Asahi Univ., No. 11: 113-125. Ukeshima, A. and Fujimoto, T. (1991) A fine morphological study of germ cells in asymmetrically developing right and left ovaries of the chick. Anat. Rec., 230: 378-386. Hamburger, V. and Hamilton, H. L. (1951) A series of normal stages in the development of the chick embryo. J. Morphol., 88: 49-92. Fujimoto, T., Ukeshima, A. and Kiyofuyi, R. (1976) The origin, migration and morphology of the pri- mordial germ cells in the chick embryo. Anat. Rec., 185: 139-154. Ukeshima, A. and Fyimoto, T. (1984) Ultrastruc- ture of primordial germ cells in the early chick embryo. In: Ultrastructure of Reproduction. (J. Van Blerkom and P. M. Motta eds.) Martinus Nijhoff Pub., Boston, 12-18 pp. Ukeshima, A., Yoshinaga, K. and Fujimoto, T. (1991) Scanning and transmission electron micros- copic observations of chick primordial germ cells with special references to the extravasation in their migration course. J. Electron Microsc., 40: 124-128. Callebaut, M. (1979) The avian ovary is an open organ.; A study of the lacunar system. Anat. Embryol., 158: 103-119. Callebaut, M., Meeussen, C. and Nassauw, L. V. (1988) The early development of the lacunar system in the avian ovary. Med. Sci. Res., 16: 1131-1133. Ukeshima, A. and Fujimoto, T. (1978) Scanning electron microscopy of the primordial germ cells in early chick embryos. J. Electron Microsc., 27: 19- 24. ahnet) ee om bia } 4 Teh his NGG OF Son x aI ih rh ab f RR TE A a sae Lie rere | han oo Si t a x p Meier Ferd airy nf oF 4 SW Heese ete mort 3 e be ok F vo 5 i i ; 1 j fad fF te al { { i hel of \ j 4 ah {rtaret & b a | Aeraes| . a \ 4 fs fi ~<. t- ? © = ¢ Yi ZOOLOGICAL SCIENCE 9: 741-747 (1992) © 1992 Zoological Society of Japan The Acid-Insoluble Organic Matrix of Spicules in the Sea Urchin Hemicentrotus pulcherrimus SHOGO NAKAMURA, RYOKO KAGOTANI, HIROE FUJISAKI and MANABU K. KoJIMA Department of Biology, Faculty of Science, Toyama University, Toyama 930, Japan ABSTRACT— The structure of the acid-insoluble organic matrix (ATOM) of the spicules of sea urchin larvae was examined by light microscopy and transmission electron microscopy. The spicules were isolated from pluteus larvae of Hemicentrotus pulcherrimus, and AJOM was obtained by demineraliza- tion of the spicules with 0.1 N acetic acid. We found that AIOM has many fine fibers (diameter: 16-27 nm) and two types of granules. INTRODUCTION A mineralized skeleton of spicules is one of the most conspicuous structures in sea urchin embryos. Previous investigations of the develop- ment of the skeleton in sea urchin larvae showed that the primary mesenchyme plays a major role in spicule formation [1-4]. However, the control of spicule formation has not yet been fully resolved. Calcite, CaCO3, and small amount of MgCO; are the inorgaic components of the spicules [5]. Oka- zaki [1] showed that spicules contain an organic matrix which was insoluble after acid-treatment. Benson ef al. [6] revealed that EDTA- or acid- insoluble organic matrix contains fibrous lamella. Although acid- or EDTA-soluble organic matrix had several glycoproteins and proteins [7, 8], the components of acid- or EDTA-insoluble matrix are not known. To obtain further information about biomineralization in the sea urchin embryo, we have observed the structure of the acid- insoluble organic matrix (AIOM) by light and electron microscopy. We found that AIOM con- tains many fibers and two types of granules. Accepted May 28, 1992 Received March 9, 1992 MATERIALS AND METHODS Gametes Hemicentrotus pulcherrimus were collected from the coast of Toyama Bay and were maintained at 14°C in aquaria with circulating sea water. Gametes were obtained by injecting 0.5 M KCl into the body cavity. Sperm were collected directly from the coelomic cavity and stored “dry” in the refrigerator. Eggs were collected and washed 2 times in filtered natural sea water (FNSW). Isolation of organic matrix Fertilized eggs were washed in FNSW and cultured to plutei at 18°C with constant stirring (~1lround/sec). Larvae were collected by centrifugation (at 3,000 g for 10 min) and washed once in cold (4°C) Ca*t- and Mg**-free sea water (CMFSW: NaCl 26.5 g, KCI 0.7 g, pH was adjusted to 8.2 by the addition of 0.5 M NaHCoO;) [9]. They were resuspended in CMFSW, shaken vigorously, then centrifuged at 1,000 g for 5 min. The pellet was resuspended in 10 vol of 10 mM Tris buffer (pH 7.5) which con- tained 0.5 mM phenylmethylsulfonyl fluoride, and pipetted vigorously. The suspension was centri- fuged at 1,000 g for 5 min. The pellet was resus- pended in 10 vol of 2% TritonX-100, 4% sodium deoxycholate, 20mM Tris buffer (pH7.4) and pipetted strongly, then centrifuged at 1,000 g for 5 min. This procedure was repeated 2-3 times to 742 S. Nakamura, R. KAGOTANI et al. obtain crude fraction of spicules as the pellet. The crude fraction was resuspended in 5vol of the same Tris buffer containing detergents and 5 vol of 5% sodium hypochlorite in 20 mM Tris buffer (pH 7.5). The suspension was pipetted and centrifuged at 25 g for 3 min. The pellet was treated with 5% sodium hypochlorite for 10min and washed 3 times with artificial sea water (ASW: NaCl 26.5 g, MgSO,:7H20 11.9g, KCl 0.7 g, CaCl 1.2 g, pH was adjusted to 8.2 by the addition of 0.5M NaHCOs) [9]. Micrographs of isolated spicules were taken by drak-field microscopy (Olypus BH2 microscope with a BH2-DCW dark-field condenser). Electron microscopy Isolated spicules were de- mineralized by resuspending them in equal vol of 0.1 N acetic acid and incubating for 30 min at 0- 4°C. The suspension was spun at 16,000 g for 10 min, and the acid-insoluble organic matrix (AIOM) was obtained as the pellet. The AIOM was fixed in 2% glutaraldehyde in 80% ASW for 1 hr at room temperature, and then fixed again in 1% glutaraldehyde in 0.1M sodium cacodylate (pH 7.0) for 1 hr. The samples were post-fixed in 1% OsOy, in 0.1 M sodium cacodylate (pH 7.2) for 1 hr. After rinsing 0.1 M sodium cacodylate buffer (pH 7.2), the samples were dehydrated through a ethanol series. They were infiltrated and embed- ded in epoxy resin (Epok 812, Oken Shoji Co., Ltd., Tokyo). Sections were cut with glass knives on a Sorvall MT1 Porter-Blum ultramicrotome, and stained with uranyl acetate and Reynold’s lead citrate. To survey the purity of isolaed spicule, whole mounts were prepared. Ultrathin sections and whole mounts were examined with a JEOL 100SX electron microscope operated at 80 kV. RESULTS We isolated many spicules in almost intact form from pluteus larvae of Hemicentrotus pulcherrimus (Fig. 1b). There was very little contamination in the fraction of isolated spicules (Fig. 1b) and the surface of the spicules was clean (Fig. 2). When the isolated spicules were treated with 0.1 N acetic acid for 30 min, demineralization occurred and thread-like structures remained (Fig. lc), as re- Fic. 1. sea urchin, Hemicentrotus pulcherrimus. Preparation of AIOM from pluteus larvae of the Pluteus larvae (a: 72 hr after the insemination) were used for isolation of spicules which appeared free of con- tamination by dark-field microscopy (b). After isolated spicules were demineralized with 0.1 N ace- tic acid, thread-like structures with granular varico- sities remained (c). Bar, 100 «~m. ported by Okazaki [1]. granules on the thread-like structures. There were many fine Those granules were never seen in the fraction of isolated spicules. We call this thread-like structure the acid-insoluble organic matrix (AIOM). AIOM was examined with transmission electron micros- copy. Figure 3 shows that AIOM has many fibers Organic Matrix of Spicules 743 which are twined loosely (diameter: ~27 nm) with a few filaments (Fig. 4a) or tightly (diameter: ~ 16 nm) (Fig. 4b). We could not see a periodic pattern on the fibers. The fibers ran parallel with each other in some regions and they made an eddy pattern in other regions. Moreover, AIOM contained two types of gran- ules (Fig. 3). Granules whose appearances re- minded us of a cell nucleus (N-granules) lacked limiting membranes (Figs. 3, 5a). Many fine fibrous structures were present in the electron Fic. 2. Whole mount EM of ioslated spicules. No translucent compartment of N-granules (Fig. 5a). contamination is evident around the spicule surface. | We often observed disintegrating N-granules with Bar, 5 wm. discharged fibrous structures (Fig. 5a) which looked like filaments in the loosely twined fibers (Fig. 4a). The other type of granules (V-granules) Fic. 3. A typical ultrathin section of AIOM. Many fine fibers and two types of granules were found in this specimen. The fibers run parallel in some regions, and in eddy patterns in other regions. N: N-granule. V: V-granule. Bar, 2 pm. Fic. 4. High magnification TEM views of the fibers in AIOM. The fibers are twined loosely with a few filaments (a) or more tightly (b). Bar, 0.2 ~m. Fic. 5. High magnification views of two types of granules in AIOM. The nucleus-like granule (N-granule) (a) has no clear membrane and many fine fibers in the electron translucent parts. This N-granule was disintegrating with discharged filaments (arrows). The other type of granules (V-granules) have vacuoles surrounded with electron dense deposite (b). Bar, 0.5 um. 744 S. Nakamura, R. KaGorani et al. Organic Matrix of Spicules 745 746 S. NAKAMURA, R. KAGOTANI et al. showed homogenous and low electron density, and had large vacuoles with peripheral deposits of electron dense materials in it (Figs.3, 5b). V- granules had no outer membrane and took various shapes. They were seen more frequently than N-granules. Preliminary observations show that N-granules and V-granules have various diameters (N-granules: 2.5 ~m-—3.2 um; V-granules: 1.4 ~m— 2.4 wm). DISCUSSION We have found that spicules of sea urchin embryos contain many fine fibers and two types of granules. Our preliminary observations show that the same types of fibers and granules that we observed in Hemicentrotus pulcherrimus embryos exist in spicules of Pseudocentrotus depressus embryos (data not shown). Benson ef al. [6] reported that the organic matrix of spicules had fibrous lamellae. However, they did not show the fine structure of the fibrous lamella and did not report the existence of granules. In the blastocoel of the sea urchin embryo, there is an abundunce of fibrillar matrix which may consist of collagen [10]. However, we believe that fibers and granules in our preparation are not contaminants of blastocoelic fibrillar matrices or substances, since (1) no contaminants were evident around the surface of isolated spicules by light and electron microsopy (Figs. 1b, 2), (2) we observed only one type of fiber and two types of granules in AIOM. Although the diameter of the fibers varied from 16 nm to 27 nm, we think they are made from the same material(s) and that the difference in diameter reflects their formation or assembly. It is well known that vertebrate bone predomi- nantly contains tvpe I collagen fiber. Pucci- Minafra et al. [11] have reported that the spicule of a sea urchin embryo contains collagen and which may play a role in the formation of the organic matrix of the spicules. Several investigators re- ported that collagen metabolism is required for spicule formation [12-14]. On the other hand, Benson et al. failed to detect the presence of hydroxyproline-rich collagen by structural [6] and biochemical studies [7], and they claimed that spicules isolated by the methods of Pucci-Minafra et al. were impure. We think the fibers seen in AIOM are probably not collagen since they did not show periodic banding, and the proteins did not have typical mobilities of collagen in SDS-PAGE (preliminary data not shown). We suppose the fibers give strength and flexibil- ity to the spicule and the granules are related to the fiber formation. Whole AIOM may have an important role in the formation of the spicule. Biochemical studies are under way to define their natures and functions. ACKNOWLEDGMENTS We thank Drs. S. L. Tamm and S. Pimple (BUMP, MBL, Woods Hole) for their critical readings of this manuscript. REFERENCES 1 Okazaki, K. (1960) Skeleton formation of sea urchin larvae. II. Organic matrix of the spicule. Embryologia, 5: 283-320. 2 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. 3 Okazaki, K. (1965) Skeleton formation of sea urchin larvae. V. Continuous observation of the process of matrix formation. Exp. Cell Res., 40: 585-596. 4 Okazaki, K. (1975) Spicule formation by isolated micromeres of the sea urchin embryo. Amer. Zool., 15: 567-581. 5 Okazaki, K. and Inoué, S. (1976) Crystal property of the larval sea urchin specicule. Develop., Growth and Differ., 18: 413-434. 6 Benson, S., Jones, E. M. E., Crise-Benson, N. and Wilt, F. (1983). Morphology of the organic matrix of the spicule of the sea urchin larva. Exp. Cell Res., 148: 249-253. 7 Benson, S. C., Benson, N. C. and Wilt, F. (1986) The organic matrix of the skeletal spicule of sea urchin embryos. J. Cell Biol., 102: 1878-1886. 8 Venkatesan, M. and Simpson, R. T. (1986) Isola- tion and characterization of spicule proteins from Strongylocentrotus purpuratus. Exp. Cell Res., 166: 259-264. 9 Okazaki, K. (1956) Skeleton formation of sea urchin larvae. I. Effect of Ca concentration of the medium. Biol. Bull., 110: 320-333. 10 Crise-Benson, N. and Benson, S. C. (1979) Ultra- structure of collagen in sea urchin embryos. Wilhelm 11 12 Organic Matrix of Spicules Roux’s Arch Dev. Biol., 186: 65-70. Pucci-Minafra, I., Casano, C. and La Rosa, C. (1972) Collagen synthesis and spicule formation in sea urchin embryos. Cell Differ., 1: 157-16513. Golob, R., Chetsanga, C. J. and Doty, P. (1974) The onset of collagen synthesis in sea urchin embryos. Biochim. Biophys. Acta, 349: 135-141. 13 14 747 Gould, D. and Benson, S. (1978) Selective inhibi- tion of collagen synthesis in sea urchin embryos by a low concentration of actinomycin D. Exp. Cell Res., 112: 73-78. Blankenship, J. and Benson, S. (1984) Collagen metabolism and spicule formation in sea urchin micromeres. Exp. Cell Res., 152: 98-104. ; | chee ; { eee wee . nia) bes 4 haa o i ‘Li vl a OG ROR pha so yeiy Hips aeirnaiedie Naveen WI oo “hy “ae Pal) wee aa, ett Vs ite i perenne pti & yeh destabal odes ub A wah nn, wadigdetian ‘ ih ert ea Lt 4 & ; : Ye ‘Bima tite: Shy): iit ine a die ‘inden G oe ne anil Wnfea Cah. (141 CONROY | Ea = at vm : ok, Gre So Tie ' : ; a : ee ee a — a fs 7, i Ss J . Pt - i T ay ) 4 i ZOOLOGICAL SCIENCE 9: 749-755 (1992) © 1992 Zoological Society of Japan Scleral Fibroblasts of the Chick Embryo Can Proliferate without Transferrin in Protein-Free Culture YosHie Ouya!, KAzUuo WATANABE!”*, NoBuo SHIMAMOTO2 and Minoru Amano! 'Cell and Developmental Biology Laboratory, Faculty of Integrated Arts and Sciences, Hiroshima University, Hiroshima 730, and 2DNA Research Center, National Institute of Genetics, Mishima 411, Japan ABSTRACT—Scleral fibroblasts of the chick embryo can proliferate in vitro without transferrin. We prepared a monoclonal antibody, TFY004, which was reactive against avian-transferrin, and checked the possibility of an autocrine secretion of transferrin in the cells. Dot-blot immunoassay of the conditioned medium by the use of TFY004 indicated that the conditioned medium did not contain detectable amount of transferrin. We tested muscle trophic activities of the conditioned medium to myogenic cells to detect transferrin activities in the conditioned medium. The results also indicated no presence of transferrin in the conditioned medium. Furthermore, the supplementation of transferrin had no effect for survival and proliferation of the scleral fibroblasts. All these results suggested that, in the scleral fibroblast culture, mechanisms different from the external transferrin-mediated one must be involved to uptake iron from medium into cells. INTRODUCTION Living cells inevitably requie iron. It is generally accepted that the cells utilize environmental iron by an efficient transport system with the use of transferrin, a soluble protein with high iron- binding capacity [1, 2]. One of the reason for adding serum in the cell cultures is to supply a sufficient amount of serum-transferrin. The trans- ferrin binds efficiently irons in the culture medium, and the iron-bound transferrin is internalized by endocytosis [3] after specific binding to the cellular receptor [4]. In consequence, the supplementation of high concentration of transferrin, 5-10 “g/ml, is a general format for serum-free cultures [5]. Interestingly, the scleral fibroblasts of chick embryo do not require supplementation of trans- ferrin to the protein-free culture medium for their proliferation [6]. Then, a question arises, how the Accepted May 28, 1992 Received May 6, 1992 3 To whom all correspondence should be addressed. scleral fibroblasts uptake environmental iron, which is necessary for cell survival. One possible mechanism in an autocrine secre- tion of transferrin by the cells themselves. In fact, we reported that the scleral fibroblasts proliferated by producing multiple autocrine growth factors into the conditioned medium [6, 7]. Dittmann et al. reported that HL-60 cells produced a transfer- rin-like protein as an autocrine factor, which reacted with anti-transferrin antibody [8], and also Kohgo et al., reported K562 cells could proliferate without transferrin by secreting immuno-reactive transferrin as an autocrine factor [9]. In this study, we tried to detect transferrin in the conditioned medium of the scleral fibroblast cul- ture. MATERIALS AND METHODS Chick embryos were obtained by incubating eggs of randomly bred Japanese fowl purchased from Yamagishism Association, Japan. 750 Y. Onya, K. WATANABE et al. 1. Cell culture Scleral fibroblasts Procedures for culturing scleral fibroblasts were described previously [6, 7]. In brief, the scleral fibroblast layers were isolated from the eyeballs of chick embryos (stage 38 or stage 39) [10], and incubated with 2 ml of 0.2% collagenase at 37°C for 40 min, and flushed gently in 2 ml of EF- medium (1:1 mixture of Dulbecco’s modified Eagle medium and Ham’s F-12 medium), using a Pasteur pipette to dissociate tissues into single cells. The dissociated cells were inoculated at an initial density of 5 x 10° cells in 100-mm plastic dish with 8ml of EF-medium and cultured in 5% CO,-95% moist air at 38°C. Myogenic cells Muscle tissues were isolated from the femoral region of stage 38 embryos. Procedures for dis- sociation were the same as in the case of scleral fibroblasts. The cells were inoculated at an initial density of 5X10? cells in 35-mm plastic dish (Fal- con 3001; Becton Dickinson Co., U.S.A.) with 2 ml of EF-medium supplemented with 10% horse serum (GIBCO, U.S.A.) and were incubated in humidified 5% CO,-incubator at 38°C. 2. Preparation of conditioned medium and SAF-I from the scleral fibroblast culture After the first medium change on the third day of culture, the conditioned media were harvested every second day, i.e., the fifth, seventh and nineth days of culture. Cell densities were 120 cells/mm? at the third day and 650 cells/mm” at the nineth day. The procedures for preparing the SAF-I (scleral autocrine factor-I) and its characterization were described previously [6]. In brief, pooled con- ditioned medium was concentrated by the use of ultrafiltration, removing molecules less than 10 kDa. The concentrated conditioned medium was applied to DEAE-Sepharose column chroma- tography at pH 8.0, and proteins were eluted with 0-0.35 M NaCl gradient. We called a fraction, eluted at about 0.2 M and showed a heat-stable growth-promoting activity, as SAF-I. 3. Preparation of ovotransferrin as an avian trans- ferrin Ovotransferrin was extracted from chick egg white after Kimura’s methods [11]. In brief, acetone powder of egg white was prepared and dissolved in FeCl; solution. The solution was subjected to preparative DEAE-Sepharose, and next to CM-Sepharose column chromatography. A fraction which showed a single peak (absorb- ance 280 nm), manifested also a clear single band with molecular weight of ca. 80kDa by SDS polyacrylamide-gel electrophoresis (SDS-PAGE) [12]. We call the fraction as purified ovotrans- ferrin. 4. Preparation of monoclonal antibody against transferrin As an antigen, 0.8 mg of the purified ovotrans- ferrin was dissolved in 0.25 ml of distilled water, mixed with the same volume of complete Freund’s adjuvant and injected intraperitoneally to a BALB/c mouse. Twelve days after the primary injection, booster injection was made with an adjuvant-free antigen. Three days later, spleen cells were harvested and fused with myeloma cells (Sp2/0-Ag-14) [13] by the use of polyethylenegly- co16000 (Koch-Light Co., Germany) as described by Galfre et al., [14]. HAT-selected hybridoma cells were cloned by limiting dilution and the supernatant media were screened by ELISA method using horse-radish peroxidase (HRP)- conjugated goat anti-mouse immunoglobulins (ZYMED LAB. Inc., U.S.A.). We obtained 15 hybridoma clones producing antibodies against transferrin. But unfortunately, most positive clones attenuated rapidly, i.e., they ceased to produce antibodies, or stopped to pro- liferate and died. Finally a hybridoma clone, TFY004 clone, was remained. We recloned the TFY004 cells, which left sufficient amount of hy- bridoma supernatant (TFY004), which specifically reacted with purified ovotransferrin on nitrocellu- lose filter (Fig. 1). 5. Dot-blot immunoassay Every one ml of the solutions containing various concentration of the test samples was blotted to Cell Proliferation without Transferrin 751 ovalbumin BSA va — Ing 300ng eS 100ng 30ng 10ng 3ng Fic. 1. Dot-blot immunoassay of purified ovotransfer- rin, ovalbumin and BSA (bovine serum albumin) with hybridoma supernatant, TFY004. Each 1 ml of serially-diluted ovalbumin (No. 300-00711; Wako, Japan), BSA (No. 017-12961; Wako) or purified ovotransferrin (Tf) were blotted to a nitrocellulose filter and reacted with TFY004. The concentrations were indicated at the right. nitrocellulose filter (No. 162-0115; BIO-RAD, U.S.A.) by the use of Milliblot (Millipore Ltd., U.S.A.). The filter was washed with Ca**- and Mg**-free phosphate buffered saline (PBS) and reacted with non-diluted hybridoma supernatant, TFY004, for 1hr. Then, 60-times dilution of HRP-labelled goat anti-mouse immunoglobulins (ZYMED LAB., Inc.) was added and visualized by diaminobenzidine. All procedures were done at room temperature and filters were washed with PBS containing 0.05% Tween-20. Non-specific binding of antibodies was blocked with PBS con- taining 3% of commercial skim milk. 6. Measurement of growth-promoting activity Growth-promoting activity was assayed as an enhancement in *H-thymidine incorporation into acid-insoluble fraction of the cells. Detailed condi- tions for culturing and labelling were given pre- viously [6, 7] and also in figure legends for Figure 5 and 6. After labelling, the cell monolayer was lysed with 0.1 N NaOH and lysate was poured onto a glass microfiber filter after Kawahara’s methods [15]. The filter was washed three times with 5% TCA and once with absolute ethanol. Radioactivity was counted with an Aloka LSC- 7000 liquid scintillation counter [15]. RESULTS Conditioned medium did not contain transferrin according to dot-blot immunoassay SDS-PAGE of the conditioned medium indi- cated the presence of a protein with molecular weight near 80 kDa, which was similar to transfer- Its concentration in the medium was esti- We purified rins. mated to be 150 ng/ml (Fig. 2). conditioned medium with DEAE-sepharose chro- CM SAF-| «— 80kDa—, aay hae sae 320° > Fic. 2. SDS-PAGE of the conditioned medium (CM) and the partially purified growth promoting factor (SAF-I) separated from the conditioned medium. Ten micro litter of the acid-insoluble materials of the conditioned medium was applied, corresponding to 0.1 ml of conditioned medium. In the case of SAF-I, 10 wl of the solution containing 4 ug of protein was applied. 752 Y. Onya, K. WATANABE et al. matography. The partially purified fraction named SAF-I [6], also showed a band of ca. 80kDa protein (Fig. 2). However, there found no substance to react with anti-transferrin antibody in the conditioned medium by dot-blot immunoassay. If transferrin existed, it should be less than 1 ng/ml in the conditioned medium (Figs. 1, 3). SDS-Tf tml 300, 30 pI 10 pl Sp! 1ng tpl 0.1ng Fic. 3. Dot-blot immunoassay of the conditioned medium of scleral fibroblasts with hybridoma super- natant, TFY004. Each 1 ml of serially-diluted con- ditioned medium were blotted to nitrocellulose filter. The volume of conditioned medium used was indicated at the left. As the controls, each | ml of ovotransferrin and SDS-treated one (SDS-Tf) were blotted to the same filter and reacted with TFY004. Their concentrations were indicated at the right. Conditioned medium had no effect on myogenic cells Addition of 3 “g/ml of purified ovotransferrin to myogenic cells brought about prominent muscle trophic effects (Fig. 4), as reported by Ozawa’s group [2, 16]. On the other hand, 50-fold concen- trated conditioned medium of the scleral fibro- blasts did not promote the proliferation and dif- ferentiation of myogenic cells (Fig. 4). Consistently, the conditioned medium and SAF- I, which showed DNA-synthesis promoting activ- ity to the scleral fibroblasts, did not promote any DNA synthesis of the myogenic cells, under the condition that the supplementation of ovotransfer- rin specifically promoted it (Fig. 5). The addition of ovotransferrin had no effect on the growth of scleral fibroblasts The addition of 6 4g/ml of ovotransferrin, a routine concentration in serum-free cultures, had no DNA-synthesis promoting effect to the scleral fibroblasts, although insulin had prominent effect to promote DNA synthesis (Fig. 6). Increasing the concentration of ovotransferrin from 6 ng/ml to 50 ug/ml, we obtained the same results (data not shown). DISCUSSION The scleral fibroblasts can rapidly proliferate in vitro under protein-free medium [6]. We tested a postulation that the scleral fibroblasts secreted transferrin into conditioned medium as an auto- crine factor. It is known that the biological activities of transferrins manifest class-specificity among verte- brate animals [2, 17], i.e., mammalian-type trans- ferrin does not manifest its biological activity to avian cells, and vice versa. So, we purified ovo- transferrin from chick egg white as an avian-type transferrin [11, 17] and used it for positive control experiments in the biological assays (Figs. 4, 5, 6). At the same time, we prepared an specific mono- clonal antibody, TFY004, against the ovotransfer- rin. Since the TFY004 could not recognize SDS- treated ovotransferrin (Fig. 3), the epitope of TFY004 seemed to be protein conformation, not to be carbohydrate moiety. The obtained results indicated; (1) the con- ditioned medium did not contain immunochemi- cally-detectable amount of transferrin. The dot- blot analysis indicated that it contained, if existed, less than 1 ng/ml (Figs. 1, 3), much less than the effective concentration, 800 ng/ml [18], of trans- ferrin on growth of myogenic cells. (2) The Cell Proliferation without Transferrin 753 conditioned medium did not mainfest any muscle trophic effects (Fig. 4) or growth-promoting effect (Fig. 5) upon myogenic cells even with the use of concentrated one, which estimated to contain ca. 7-10 ug/ml of the 80 kDa protein. These results indicated that there are no significant amount of transferrin in the conditioned medium, and lead us to discard the possibility of autocrine secretion of transferrin in the scleral fibroblasts. The protein with molecular weight of ca. 80 kDa in the con- ditioned medium was considered not to be trans- ferrin. 100 hm Fic. 4. Myogenic cell cultures treated with ovotransferrin (3 ~g/ml) or conditioned medium (final concentration was 50-fold concentrated from original conditioned medium), showing prominent differences in growth and differentiation on the 4th day after inoculation. For culture conditions, see Materials and Methods. Then, how do the scleral fibroblasts satisfy iron requirement for their survival and proliferation? Interestingly, the supplementation of 6 ng—S0 ug/ ml of ovotransferrin showed no effects on their proliferation. This fact seems to suggest the scleral fibroblasts lack transferrin receptor, since it is generally accepted that the transferrin receptors increase in the rapidly proliferating cells [2]. In another word, the fact implicates the uptake of iron without participation of soluble transferrin in the scleral fibroblasts. One possible mechanism is the scleral fibroblasts =) Nn ms 3H-Thymidine incorporation (cpm x 10-3) 3H-Thymidine incorporation (cpm x 10-2) = (o) , ] Y. Onya, K. WATANABE et al. BSA = oO Tf Fic. 5. SAF-| Control Insulin Fic. 6. BSA Tf Control 24 hr 48 hr 72 hr — Lt Fic. 5. Effects of ovotransferrin, BSA, conditioned medium and SAF-I, on *H-thymidine incorporation of myogenic cells. 5X10*cells/6-mm microwell (Falcon 3072) were inoculated with 2001 of DMEM (Dulbecco’s modified Eagle medium) con- taining 10% horse serum. After 12 hr cultivation, ovotransferrin (final concentration; 5 ug/ml), BSA (final concentration; 5 ug/ml), conditioned medium (30 wl/well) or SAF-I (5 pl/well), was added to become final volume of 200 wl. After further 12 hr cultivation, cells were labelled for 12 hr with 0.3 nCi of ?H-thymidine (Amersham Co., U.K., 40-60 Ci/ mmol) in 60 “«l of DMEM, and radioactivities were measured. Control means no addition of test sam- ple. The data were averages of three different cultures with SD. Fic. 6. Effects of insulin, BSA and ovotransferrin on 3H-thymidine incorporation of the scleral fibro- blasts. 510° cells/6-mm microwell (Falcon 3072) were inoculated with 200 «l of DMEM containing 6 yg/ml of insulin (from bovine pancreas; No. I-1882; Sigma Co., U.S.A.), BSA or ovotransferrin. After cultivation for 24, 48 and 72 hr, cells were labelled for 12 hr with 0.3 Ci of *H-thymidine and radioacti- vities were measured. Control means no addition of test sample. The data were averages of three different cultures with SD. uptake iron through membrane-bound transferrin. In fact, melano-transferrin (p 97), a membrane- bound transferrin like molecule, was found in human melanoma [19]. Unfortunately, we could not test the mechanism now, because hybridoma (TFY004) had already attenuated. On the other hand, Saito et al. [18] reported that chick embryonic cells could grow without transfer- rin in 10-100 “M Fe?*- or Fe**-ion, suggesting an existence of unknown iron-uptake mechanism in the chick embryonic cells. We confirmed that the scleral fibroblasts grow even in the condition of less than 0.2 ~M of Fe**-ion (data not shown). So, if such iron-uptake mechanism without trans- ferrin actually operated in the scleral fibroblasts, it should be more enhanced. Anatomically, the scleral fibroblasts are derived from perichondrial tissue, and we recently found the scleral fibroblasts possessed a capacity to dif- ferentiate into cartilage in soft agar [20]. It is of Cell Proliferation without Transferrin 755 interest if such nutritional characteristics as iron- uptake without soluble transferrin are specific for precartilagenous perichondrial cells, because they manifest an adaptive proliferation and differentia- tion in either skeletal remodeling during develop- ment or fracture healing [21] under restricted vascular environment. ACKNOWLEDGMENTS The authors are indebted to Dr. Makoto J. Tabata in our laboratory for technical advices in hybridoma pro- duction, and also to Dr. Akira Kawahara for his gener- ous advices. The work was supported by a Grant-in-Aid for Scientific Research to K.W. (No. 03833023; No. 03304009) from the Ministry of Education, Science and Culture, Japan. REFERENCES 1 Neuman, R. E. and Tytell, A. A. (1961) Iron replacement of lactalysate and embryo extract in growth of cell culture. Proc. Soc. Exp. Biol. Med., 107: 876-880. 2 Ozawa, E. (1989) Transferrin as a muscle trophic factor. Rev. Physiol. Biochem. 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(1978) A better cell line for making hybridomas secreting specific antibodies. Nature, 276: 269-270. Galfre, G., Home, S. C., Milstein, C., Butcher, B. W. and Howard, J. C. (1977) Antibodies to major histocompatibility antigen produced by hybrid cell lines. Nature, 266: 550-552. Kawahara, A., Ishikawa, S. and Amano, M. (1985) In vitro growth of adult amphibian (Xenopus laevis) hepatocytes and characterization of hepatocyte- proliferating activity in homologous serum. Exp. Cell Res., 159: 344-352. li, I., Kimura, I. and Ozawa, E. (1982) A myo- trophic protein from chick embryo extract: its purification, identity to transferrin, and indispensa- bility for avian myogenesis. Dev. Biol., 94: 366-377. Shimo-Oka, T., Hagiwara, Y. and Ozawa, E. (1986) Class specificity of transferrin as a muscle trophic factor. J. Cell. Physiol., 126: 341-351. Saito, K., Hagiwara, Y., Hasegawa, T. and Ozawa, E. (1982) Indispensability of iron for the growth of cultured chick cells. Develop. Growth and Differ., 24: 571-580. Rose, T. M., Plowman, G. D., Teplow, D. B., Dreyer, W. J., Hellstrom, K. E. and Brown, J. P. (1986) Primary structure of human melanoma- associated antigen p 97 (melanotransferrin) deduced from the mRNA sequence. Proc. Natl. Acad. Sci. USA., 83: 1261-1265. Watanabe, K., Yagi, K., Ohya, Y. and Kimata, K. (1992) Scleral fibroblasts of the chick embryo diffe- rentiate into chondrocytes in soft-agar culture. In Vitro Cell. Dev. Biol. 28A: (in press) Rosen, V. and Thies, R. S. (1992) The BMP proteins in bone formation and repair. Trends Genet., 8: 97-102. aye + BT) nui AH ar —— i - nei Ht cote pele one? ry, ae H lve aorresmamiyy aevcadind), acriboriel Senne alihe he aad twig” nf © dbs t “Ail toTetcorlep yn ay | 1) ANE oAy y ot RP H 7 he -, ‘Ae § bh gaa - 4 Ero rove anah) Te eget til ou nye as 2% ifiaeprie Ye bat May ‘ " reoahy b . ‘ p Avy Siete WN waa MY, chee ieg eben ; a Neral ate I = ' a) ‘endo at ; ae oe: Ele Hy ‘a Obi i ( » ee eT ree alts eorhyrh aie 0 rere eT Yoriety Lt oy) dane hae ro _ i bas hi a #). oe vith re ‘ mn writ aoe ees Ae) pe Mi : moins? i iw Ani a ae rid i ey f Testi ne “ ice i aity il ij ~ hon 4 : , ‘ 1 (1h) tp Tentyay fr n i Tine? et ‘ A een A : | ertey etna Fis pitied Menderes eke Vie hb ted en le i) Oe, we al i) Ce Ar Yanai arr" f buadat ly eee ; shu derah eee ea « 123 ey | ~~— Young gastropod stage —> Metamorphosis Fic. 9. Symbiotic relationship and the rate of survival of the gastropods. VP*: Veligers with planulae, VP: Veligers without planulae, GP*: young gas- tropods with planulae, GP: young gastropods with- out planulae. Ordinate: length of each line (i.e. numeral on each line) indicates numbers of veligers or gastropods. Abscissa: stage of the gastropod. a (Veligers without planulae) and GP (young gas- tropods without planulae) was estimated as 89% [(77% + 100%) /2] and 52% [(73% +30%)/2], re- spectively. If the calculation is simply done at the last observation (i.e. the right-most line of Fig. 9), the rate of survival of gastropods without hydroids is about 7% [4/(14+37+4) x 100]. DISCUSSION In the laboratory symbiosis took place between planula and late veliger stage respectively. The ability of planulae to go through metamorphosis appears to be maintained for about 20 days and then gradually disappears. This in turn implies that suppression of metamorphosis also lasts for this period provided a stimulation for triggering metamorphosis is not given. As for stimulation, it has been reported that in Hydractinia echinata the stimulus causing initiation of metamorphosis emanates from certain marine bacteria [2]. The stimulus for Cytaeis sp. may also be a chemical factor, because the planulae of Cytaeis sp. always settle at the base of the siphon of N. livescens. It is considered that a receptive system for a metamorpnosis-triggering factor must exist in planulae. It has been reported that neural dif- 764 C. INOUE AND Y. KAKINUMA ferentiation begins in the planular ectoderm 24 hours after fertilization and continues throughout larval development [3, 4]. One reason explaining the inability of 60-day planulae to attach is consi- dered to be a lack of swimming ability which reduces the chance for planulae to encounter the gastropods. It is also possible that in 60 day planulae a degeneration of the neural system re- duces their ability to perceive a stimulation factor. As mentioned before, a few planulae differenti- ated into a polyp when cultured in a petri dish. It is considered that metamorphosis of Cytaeis. sp. can be triggered by other factors, and once triggered, metamorphosis seems to progress without any further exogenous stimulation. However, in the field we have never seen a colony of Cytaeis sp. except on the surface of the N. livescens. The larvae of N. livescens clearly offer a substra- tum on which the planulae adhere and meta- morphose. Since the planulae and the juvenile gastropod display specific behavior as soon as a planulae and a juvenile gastropod encounter each other, and since the position of planula settlement is always at the base of the siphon, we can specu- late that the gastropods secrete some planula- attacting substance from the base of the siphon, to which the palnula reacts. On the other hand, the hydrozoan planula may secrete some factor to make the gastropod motionless. Although one trial was not enough to determine the survival rate, the rate of survival of gastropods that are attached by planula tends to be higher than that of gastropods without planulae. There- fore the adhesion of planula may provide some adequate condition for the gastropodan physiology as Hirai and Kakinuma previously reported for the adult sage [1, 5]. Among the collected gastropods, the percentage of gastropods without hydrozoa is fairly high (about 70%). And much higher than calculated from the laboratory trials (finally about 7%). Therefore, in the laboratory, symbiosis may be preferable for the gastropod particularly in the juvenile stage. Since in the field the breeding seasons of the two species coincide with each other, symbiosis be- tween the two species must take place at the larval stage, although the gastropods can survive without hydrozoa. Whether the loose symbiosis between these species inclines to a more strict relationship or declines towards dissolution requires long term observation. For the hydroids, however, the cur- rent relation seems to be necessary to survive and to diverse in the field. ACKNOWLEDGMENTS We thanks Dr. H. Shirai (Okayama University) for critical reading of the manuscript, Messrs. M. Tabata and Y. Ikimori (Kagoshima Marine Park) for help in collect- ing the gastropods, and Dr. J. Komen and Dr. Cocket (National Institute for Basic Biology) for reading the manuscript. REFERENCES 1 Hirai, E. and Kakinuma, Y. (1971) On symbiotic relations between differentiation of a hydrozoan Cytaeis uchidae with a gastropod Niotha livescens. Bull. Mar. Biol. Stat. Asamushi, 14: 65-77. 2 Spindler, K. D. and Muller, W. A. (1972) Induction of metamorphosis by bacteria and by a lithium-pulse in the larvae of Hydractinia echinata (Hydrozoa). Wilhelm Roux’ Archiv 169: 271-280. 3 Martin, V. J. (1988) Development of nerve cells in hydrozoan planulae: I. Differentiation of ganglionic cells. Biol. Bull. 174: 319-329. 4 Martin, V. J. (1988) Development of nerve cells in hydrozoan planulae: II. Examination of sensory cell differentiation using electron microscopy and im- munocytochemistry. Biol. Bull. 175: 65-78. 5 Hirai, E. and Kakinuma, Y. (1973) Differentiation and symbiosis in two hydrozoans. Publications of the Seto Marine Biological Laboratory, 20: 257-273. ZOOLOGICAL SCIENCE 9: 765-773 (1992) Effects of Olfactory Tract Section on Brain GnRH Distribution, Plasma Gonadotropin Levels, and Gonadal Stage in Goldfish Maxito Kopayasui!, MAsSAFUMI AMANO!, YoOSHIHISA HASEGAWA2 Koicut Okuzawa? and Katsumi Arpa! ‘Department of Fisheries, Faculty of Agriculture, University of Tokyo, Bunkyo, Tokyo 113, *Department of Obstetrics and Gynecology, Gunma University, School of Medicine, Maebashi, Gunma 321-16, and *Inland Station, National Research Institute of Aquaculture, Tamaki, Watarai, Mie 519-04, Japan ABSTRACT— Goldfish in early sexual recrudescence were olfactory tract-sectioned (OTX) or sham- operated in order to examine the distribution of brain gonadotropin releasing-hormone (GnRH) originating from the terminal nerve (IN), and the involvement of GnRH in reproduction. Fish were kept at 20°C, and one month following surgery, brain contents of two types of GnRHs, salmon type (sGnRH) and chicken-II type (CGnRH-II), were measured by specific radioimmunoassays. Plasma gonadotropin (GTH) levels and the stages of gonadal development were also determined. After OTX, sGnRH contents showed a marked decrease in all brain areas (telencephalon, hypothalamus, optic tectum-thalamus, cerebellum, medulla oblongata) except in the olfactory bulbs, whereas cGnRH-II contents in the brain and the pituitary gland showed no clear changes. Pituitary sGnRH contents and plasma GTH levels showed slight decreases in OTX males, but not in females. In spite of large decreases in brain sGnRH contents, gonadal development in OTX fish was as active as that in sham-operated fish with the progression of spermatogenesis in males and vitellogenesis in females. These results suggest that most of the sGnRH in the brain originates from the TN, and that TN-GnRH is © 1992 Zoological Society of Japan not essential to gonadal development in goldfish. INTRODUCTION Gonadotropin releasing-hormone (GnRH) is a decapeptide which regulates reproductive activi- ties, such as the stimulation of gonadotropin (GTH) release from the pituitary gland [1] and the potentiation of sexual behavior [2]. Recent studies have shown that more than one type of GnRH exists in the brain of teleosts, and most of the species examined possess salmon type (sGnRH) and chicken-II type (CGnRH-II) [3]. Immunocyto- chemical studies revealed that major populations of GnRH-producing cells in teleosts are localized in the preoptic area, the terminal nerve (TN, homologous to the nucleus olfactoretinalis), the midbrain tegmentum, and the nucleus lateralis tubelis [4-12], although particular GnRH type was Accepted April 19, 1992 Received February 14, 1992 not necessarily examined in all the cases, in con- text of the antibodies used in each study. Among these GnRH populations, the GnRH system in the preoptic area is considered to be morphologically equivalent to that of the pre- optico-infundibular pathway of other vertebrates [13]. The GnRH in this system is presumed to function as a hypophysiotropic hormone in the stimulation of GTH release [7]. However, direct evidence of the release of GnRH from this system has not been obtained in teleosts. Since teleosts lack the hypothalamo-pituitary portal system, the measurement of GnRH near its site of release is quite difficult. Many studies on the TN system in teleosts have been conducted regarding morphological aspects [14], but the physiological role of the system is not fully understood. Demski and Northcutt [15] suggested that the TN system mediates the be- havioral response to sex pheromones in goldfish. 766 M. KosayAsut, M. AMANo et al. However, this possibility now seems less probable, since it has been demonstrated that chemosensory responses to sex pheromones in the goldfish are mediated by the medial olfactory system instead of the TN system [16]. Although the TN system is known to produce GnRH, it is not clear whether GnRH originating from the TN is involved in gonadal maturation through GTH secretion. There are some studies on changes in brain GnRH contents in relation to gonadal maturation. However, the results of these studies are not consistent. In caribe colorado, Pygocentrus notatus [17] and masu_ salmon, Oncorhynchus masou [18], brain GnRH contents (sGnRH in masu salmon) were high when gonads were mature, but no clear correlation was observed between brain GnRH contents and gonadal maturity in eel, Anguilla anguilla [19], goldfish, Carassius auratus [20] and rainbow trout, Oncorhynchus mykiss [21]. In brown trout, Salmo trutta [22], a correlation was observed between pituitary GnRH contents and plasma GTH levels. These studies measured brain and pituitary GnRH contents without considering the origin of GnRH fibers, and moreover, most of these studies were conducted using radioimmunoassay (RIA) whose antibodies were not specific to different types of teleost GnRH. In the goldfish, two types of GnRH, sGnRH and cGnRH-II, were chromatographically and im- munologically identified in the brain [23]. Major populations of GnRH-producing cells were found in the IN, preoptic area, midbrain tegmentum, and nucleus lateralis tubelis by immunocyto- chemical studies [4, 7]. Since the olfactory bulbs of goldfish are of the pedunculated type with elon- gated olfactory tracts, we can easily block the axonal transport of GnRH from the TN to other brain regions by sectioning the olfactory tracts. In the present study, the effects of olfactory tract section were studied in goldfish in order to ex- amine the brain distribution of GnRH of the TN. Brain GnRH was measured by RIAs using highly specific antibodies to sGnRH and cGnRH-II [21]. Plasma gonadotropin (GTH) and gonadal de- velopmental stages were also determined in order to examine whether TN-GnRH was involved in gonadal development. MATERIALS AND METHODS Goldfish Goldfish were obtained from a commercial source and kept in stock tanks under natural condition. Fish weighing 30-40 g were used for the experiment. Experimental Protocol Fish in early sexual recrudcence were olfactory tract-sectioned (male N=13; female, N=7) or sham-operated (male, N=13; female, N=5) on October 19. Initial body weights of each group were as follows (mean+SEM g): male; sham 34.9 +0.8, OTX 33.7+0.8: female; sham 32.1+1.8, OTX 32.4+1.9. The experiment was originally designed to use males since the involvement of olfactory system in reproduction has been reported mostly in male goldfish [15, 24, 25]. Female groups were supplemtned as an additional part of the experiment, although sample size was small. For olfactory tract section, four-sided flap was cut in the frontal bone using a disc saw. The first cut was made aligned with the center of the iris. If this cut were made more posteriorly, damage to the pineal gland would be likely to occur. The second cut, parallel to the first, was made just behind the nares. The third and fourth cuts, parallel to the longitudinal axis of the fish, con- nected to the first and the second cuts. After removal of a square flap of the frontal bone, fat and cranial fluid were gently wiped out with paper. The olfactory tracts were bilaterally sectioned at two places with Wecker’s scissors (Fig. 1), and the resultant sections were removed to prevent regen- eration of the olfactory tract fibers (OTX fish). The cavity resulting from the operation was filled with gelatin sponge (Spongel, Yamanouchi Phar- maceutical Co.). A sham operation was per- formed in the same manner without cutting the olfactory tracts (sham-operated fish). After the operation, fish were kept in 25 ppm oxyteracyclin (Sigma) overnight, and then trans- ferred to a 60 liter experimental tank (males and females in the same tank) which was kept at 20°C under ISL (lights on at 0500 hr). Fish were fed with commercial trout pellets once a day. Fish Brain GnRH in OTX Goldfish 767 were sampled on November 20. After being anesthetized with 0.02% tricaine methansulfonate, their body weights were measured. Blood samples were taken from caudal vasculature with a hepari- nized needle and syringe. Fish were sacrificed by decapitation, and the brain and pituitary glands were collected. The brains were dissected into six parts (olfactory bulbs, telencephalon, hypothala- mus, optic tectum-thalamus, cerebellum, medulla oblongata) as shown in Fig. 1. The tissues were frozen on dry ice after being weighed. The gonads were dissected out and weighed to determine the gonadosomatic index (GSI). For histological observation, the gonads were fixed with Bouin’s solution and embedded in paraffin. Sections were stained with hematoxylin and eosin. Ovarian developmental stages were identified according to the classification by Yamamoto and Yamazaki [26]. PIT Fic. 1. Schematic diagram of a sagittal section of goldfish brain showing sites of severance of the olfactory tracts (arrows) and disection of the brain for the determination of GnRH. OLT, olfactory tracts; OB, olfactory bulbs; TEL, telencephalon, including preoptic area; OT-THAL, optic tectum- thalamus, including midbrain tegmentum; CER, cerebellum; MED, medulla oblongata; PIT, pituitary. RIA Salmon GnRH and cGnRH-II were measured by specific RIAs as described by Okuzawa et al. [21]. The cross-reactivity of cGnRH-II in the sGnRH RIA (antibody to sGnRH, Lot No. 2) was 1.58%, and the cross-reactivity of sGnRH in the cGnRH-II RIA (antibody to cGnRH-II, Lot R-E) was 0.051%. Measurement of GnRH in the goldfish brain by the RIA was validated by examing parallelism of the displacement curves of goldfish brain extract. High performance liquid chromatography (HPLC) analysis followed by RIA was also employed for the validation of the RIA. To test parallelism, goldfish brain tissue was homogenized by sonica- tion in 0.1 N HCl. The homogenate was centri- fuged at 10,000 xg for 30 min at 4°C. The super- natant was frozen, lyophilized, and reconstituted in assay buffer. The supernatant obtained after centrifuging again at 10,000 g for 30 min at 4°C was used for the GnRH RIAs. The brain extract of goldfish produced displacement curves which Salmon GnRH RIA 100 Dilution of brain extract _———_—— el Brain extract = 50 Salmon GnRH {e) gl a 0 9.8 39 156 625 (pg/ml) Chicken GnRH-Il RIA 100 Dilution of brain extract Brain extract SS = 501 Chicken GnRH-II a oO 9.8 39 156 625 (pg/ml) Fic. 2. Top. Competition curves for salmon GnRH and brain extract of goldfish in the salmon GnRH RIA. Bottom. Competition curves for chicken GnRH-II and brain extract of goldfish in the chicken GnRH-II RIA. The scale for dilution of brain extract indi- cates a twofold serial dilution. Each point repre- sents the mean of duplicate determinations. 768 M. Kopayasui, M. AMANO et al. were parallel to sGnRH and cGnRH-II standards in respective RIAs (Fig. 2). For the measurement of brain GnRH contents of the experimental fish, the brains were extracted in the same method as for the paralellism test. For HPLC analysis, the brain was extracted as described by Okuzawa et al. [21]. The extract was injected through a 2 ml-injection loop onto a TSK- ODS 80TM column (0.4625 cm, Tosoh Co. Ltd., Tokyo, Japan). A Hitachi high performance liquid chromatography (Model 655 A) was pro- grammed for a flow rate of 1 ml/min. The extract was applied at the beginning of a 10-min isocratic period of 17% acetonitrile (CH3CN) in 0.1% trifluoroacetic acid; CH3CN was then increased to 27% over a 10-min period. GnRHs were eluted under isocratic conditions at 27% CN3CN. Frac- tions (1 ml) were collected and aliquots were assayed for GnRH. HPLC analysis revealed that goldfish brain contained a peptide chromato- graphically and immunologically identical to sGnRH and a peptide identical to cGnRH-II (Fig. 3). Although there was some immunoreactive materials which were chromatographycally diffe- rent from sGnRH and cGNRH-II in the goldfish brain, their amount was considered negligible, and therefore it was not necessary to separate sGnRH and cGnRH-II by HPLC prior to the RIAs. Plasma GTH levels were measured using silver carp GTH RIA as described by Kobayashi et al. [27]. Purified silver carp GTH (Lot SS) was used as a standard and for iodination [28], and an antibody (Lot No. 1) raised against the silver carp GTH (Lot SS) was used for the RIA [29]. Valida- tion of the RIA system for use in goldfish was achieved by obtaining parallelism for a serial dilu- tion of plasma samples collected from ovulated females. Recoveries at three different doses, high, middle, and low, were 95.2, 102.2, and 115.4%, respectively. The GIH measured in this study is considered to be equivalent to GTH II according to Kawauchi’s designation [30] by physicochemical characters of the silver carp GTH [28], an im- munocytochemical study of goldfish pituitary GTH cells using the silver carp GTH antibody (Lot No. 3) [29], and plasma profiles of GTH during ovula- tion in goldfish measured by the silver carp GTH RIA [31]. Salmon GnRH RIA Salmon Chicken-ll 4 > 8 5 3 3 3 = S z= Chicken GnRH-II RIA H 5 I I Elution time (min) Fic. 3. Reverse-phase HPLC of goldfish brain extract followed by salmon GnRH RIA (top) and chicken GnRH-II RIA (bottom). Arrows indicate the elu- tion time of synthetic chicken GnRH-II and salmon GnRH. The mobile phase was CH3CN containing 0.1% TFA. Statistics Statistical analysis of the results were done by Student’s t-test or Cochran-Cox method. RESULTS One month following surgery, the experimental fish exhibited gains in body weight; and there were no significant differences in final body weights between sham-operated and OTX fish (mean+ SEM g): male; sham 42.6+0.7, OTX 43.7+1.4; female; sham 41.8+2.8, OTX 44.4+2.2 (Fig. 4). After OTX, sGnRH contents in OTX fish of both sexes showed a marked decrease (P<0.01) in all brain areas (telencephalon, hypothalamus, optictectum-thalamus, cerebellum, medulla oblon- gata) except in the olfactory bulbs (Fig. 5 and 6). Chicken GnRH-II contents in OTX fish showed a tendency toward decrease, but only the decrease in hypothalamus of males was significant (P<0.05). Brain GnRH in OTX Goldfish 769 Male Female Body weight 60 y g 40 i) 20 S A S N 13 13 7 GSI 4 3 & 2 if S A S N 12 13 5 7 Plasma GTH 20 = * ro.) Cc — 10 S S 0 N 12 12 BY Fic. 4. Effects of olfactory tract section on body weight, gonadosomatic index (GSI), and plasma levels of gonadotropin (GTH) in goldfish. S, sham- operated fish; O, olfactory tract-sectioned fish. Each column represents the mean and SEM. Level of significance; *, P<0.05. Salmon GnRH contents in the olfactory bulbs showed no significant difference between sham- operated and OTX fish in both sexes. Chicken GnRH-II contents of the olfactory bulbs were mostly low and no clear change was detected after the operation. Pituitary sGnRH contents showed decreased levels in OTX males (P<0.05), but not in females. Pituitary cGnRH-II contents showed no significant changes after the operation in both males and females. Plasma GTH levels in OTX males were slightly lower (P<0.05) than those in sham-operated males, but no significant difference was observed in females (Fig. 4). There was no significant differences in GSI values between OTX and sham-operated fish in both sexes (Fig.4). Histological observation showed that under conditions which are favorable for gonadal maturation in goldfish (20°C, 15L) both OTX and sham-operated fish had developing gonads regardless of the difference in brain GnRH contents. All males had spermatogenic testes and lobules filled with spermatozoa. The oocytes of the ovaries in all females had advanced to the primary yolk stage except one OTX fish which had the ovaries with secondary yolk stage oocytes. DISCUSSION The brain GnRH distribution and contents in sham-operated goldfish showed a pattern similar to those of goldfish reported in other studies [20, 23] and those of salmonid fishes [18, 21]: Salmon GnRH was distributed in a larger amount in the olfactory bulbs, telencephalon, hypothalamus, and the pituitary gland than in other regions, whereas cGnRH-II was distributed widely throughout the brain with the highest concentration in the medulla oblongata. The difference between goldfish and salmonid fishes is that the goldfish pituitary con- tains cGnRH-II, which is absent in salmonid pituitaries [12, 18, 21]. After OTX, brain sGnRH contents showed a marked derease in all brain areas except in the olfactory bulbs. These results suggest that sGnRH in the goldfish brain mostly originates from the TN. That is, sGnRH contents in various brain regions reflect sGnRH in fibers which are trans- ported from the TN cell bodies in the olfactory bulbs. Immunocytochemical studies in other spe- cies support this hypothesis. In the catfish, Clarias batrachus [9] and the dwarf gourami, Colisa lalia [11] GnRH fibers from the TN send projections to various regions of brain parts. Electrical lesion of the TN cells in gourami caused disappearance of most of immunoreactive fibers in the brain leaving GnRH cells and fibers in preoptic area intact [32]. Furthermore, it has been also demonstrated by 770 GnRH (ng/mg tissue) M. KopayasHi, M. AMANO et al. OB TEL HYP OT CER MED PIT THAL Salmon GnRH 70 60 150 50 40 100 30 as 20 50 10 0 s O Chicken GnRH-II Fic.5. Effects of olfactory tract section on salmon GnRH and chicken GnRH-II concentrations in discrete brain areas in male goldfish. OB, olfactory bulbs; TEL, telencephalon, including preoptic area; OT-THAL, optic tectum-thalamus, including midbrain tegmentum; CER, cerebellum; MED, medulla oblongata; PIT, pituitary. S, sham-operated fish (N=12); O, olfactory tract-sectioned fish (N=13). ND, Nondetectable. Each column represents the mean and SEM. Level of significance: *, P<0.05; **, P<0.01; ***, P<0.001. GnRH (ng/mg tissue) OB TEL HYP OT CER MED PIT THAL Salmon GnRH 40 100 30 15 20 10 50 10 5 * * * * * * * * 0 0 0 Ss oO Ss Oo Ss Oo s O Ss Oo s Oo Chicken GnRH-II Fic. 6. Effects of olfactory tract section on salmon GnRH and chicken GnRH-II concentrations in discrete brain areas in female goldfish. Abbreviations and symbols as in Fig. 5. S, N=5; O, N=7. Brain GnRH in OTX Goldfish 771 intracellular staining in gourami that a single TN- GnRH cell projects to all brain regions where GnRH fibers has been demonstrated but not to the pituitary [33]. Other immunocytochemical studies are also suggestive of the existence of projections of GnRH fibers from the TN to other brain regions [5, 7]. It seems likely that a common anatomical feature in teleosts is projection by the TN system of some type of GnRH to various regions of the brain, although the physiological function of such GnRH remains to be elucidated. It is not clear from the present study whether TN cells of goldfish produce cGnRH-II. Chicken GnRH-II contents were mostly undetectable in the olfactory bulbs and showed no clear changes after OTX in the other brain areas except in the hypothalamus of males. In masu salmon, cell bodies of cGnRH-II neurons were localized in the midbrain tegmentum, and only a few fibers were found in the olfactory bulbs [12]. Small amounts of cGnRH-II in the goldfish olfactorty bulbs mea- sured by RIA may be provided by fibers from other regions of the brain, such as the midbrain tegmentum. To confirm the anatomical distribu- tion of cell bodies and fibers of sGnRH and cGnRH-II immunocytochemical approach is required. Although OTX caused a marked decrease in brain sGnRH contents, no gonadal regession was observed both in males and females in contrast to the example of hypophysectomized female goldfish [34]. There was no difference in the stages of gonadal development between OTX and sham- operated fish. Under conditions which enhance gonadal maturation of goldfish, fish of both groups showed active gonadal development which would not normally occur during non-spawning period. These results suggest that TN-sGnRH is not essen- tial to gonadal development in goldfish. Although abnormal changes were seen in the gonads after lesion of nucleus olfactoretinalis in platyfish, Xiphophorus maculatus [8], reasons for different responses to a similar treatment between the two species is unclear. OTX males showed decreased levels of pituitary sGnRH which may be responsible for the decrease in plasma GTH levels. There are some possible interpretations for this decrease, such as blockade neurons, an of a direct innervation of sGnRH fibers from the TN to the pituitary gland, or a removal of stimula- tion to sGnRH neurons which send fibers to the pituitary, etc. 17a,20-Dihydroxy-4-pregnen-3- one, one of the goldfish sex pheromones, is known to stimulate GTH secretion in males during spawn- ing [35]. If female goldfish release sex pheromones which stimulate basal GTH release in males, the decrease in the pituitary sGnRH contents and plasma GTH levels in OTX males may be partly explained by inability in receiving sex pheromones since OTX fish were anosmic in the present study. However, a decrease in pituitary sGnRH contents and plasma GTH levels to the extent observed in this study did not seem to affect basal testicular development. As well as the gonadal stages, no differences were observed in body weight between OTX and sham-operated fish. Reduction of the number of feeding acts (bites directed toward gravel and artificial vegetation) after OTX is reported in goldfish [24], but the actual feeding of the OTX fish in the present study did not seem to be impaired by OTX, since both sham-operated and OTX fish gained weight similarly during the one- month experimental period. It is known in the goldfish that GnRH stimulates growth hormone release from the pituitary gland [36]. The results of present study suggest that GnRH in the TN is not likely to be involved in the regulation of growth hormone secretion. Neuromodulatory effects are suggested as one of physiological func- tions of TN-GnRH in teleosts [11, 33], but what aspects of brain activities are modulated by the TN-GnRH should be elucidated. Although there are some studies on changes in brain GnRH contents in relation to gonadal maturation, a clear correlation between brain GnRH content and gonadal maturity has not al- ways been observed [17-22]. Since the present study suggests that most of sGnRH contents in the brain reflect those in fibers originating from TN cell bodies, changes in GnRH contents measured in those studies may reflect the activities of GnRH producing-cells in the TN system. In fact in masu salmon, sGnRH contents of most of discrete brain areas showed seasonal changes parallel to those of the olfactory bulbs [18], where cell bodies of 772 sGnRH neurons shown by immunocytochemistry to be abundant [12]. More recently, Suzuki et al. [37] revealed by in situ hybridization techniques that the intensity of hybridization signals were greater in the olfactory neurons and the olfactory bulbs than in the hypothalamic area in masu salmon. These results combined with the present study suggest that changes in the contents of GnRH attributed to the TN system seem to mask changes in the hypothalamic GnRH which are considred to regulate GTH release. Since the axonal transport of GnRH from the TN system to other brain areas can be easily blocked by OTX in goldfish, OTX fish appear to be good models for studying relationship between gonadal maturation and GnRH of hypothalamic origin. In summary, the present study suggests that most of the sGnRH in the brain originates from the TN system, and that this GnRH is not essential to gonadal maturation in goldfish. The physio- logical functions of the TN system and sGnRH of TN origin remain unknown. The origin of brain cGnRH-II and its functions should also be eluci- dated. Presently, we are further examining the involvement of YTN-sGnRH in vitellogenesis, ovulation, and sex behavior, and using specific antisera, the distribution of sGnRH and cGnRH-II neurons are being immunocytochemically studied. ACKNOWLEDGMENTS We thank Dr. Yoshitaka Oka, Zoological Institute, University of Tokyo, for his critical reading of the manuscript. This study was supported in part by Grants- in-Aid for Scientific Research from the Ministry of Education, Science, and Culture and by a Grant-in-Aid (Bio Media Program 91-II-2-5 from the Ministry of Agriculture, Forestry, and Fisheries of Japan. REFERENCES 1 Fink, G. 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(1989) The duality of teleost gonadotro- pins. Fish Physiol. Biochem., 7: 29-38. Kobayashi, M., Aida, K. and Hanyu, I. (1987) Hormone changes during ovulation and effects of steroid hormones on plasma gonadotropin levels and ovulation in goldfish. Gen. Comp. Endocrinol., 67: 24-32. Yamamoto, N., Oka, Y., Amano, M., Aida, K., Hasegawa, Y. and Kawashima, S. (1991) Investiga- tion of brain GnRH systems in the dwarf gourami: immunohistochemistry and radioimmunoassay. Proc. Japan Soc. Comp. Endocrinol., 6: 46. Oka, Y. (1991) Intracellular recording and staining of terminal nerve-GnRH cells in a fish brain in vitro, Soc. Neurosci. Abstr., 17: 1335. Yamazaki, F. (1965) Endocrinological studies on the reproduction of the female goldfish, Carassius auratus 1L., with special reference to the function of the pituitary gland. Mem. Fac. Fish. Hokkaido Univ., 13: 1-64. Dulka, J. D., Stacey, N. E., Sorensen, P. W. and Van Der Kraak, G. J. (1987) A steroid sex pher- omone synchronizes male-female spawning readi- ness in goldfish. Nature, 325: 251-253. Peter, R. E., Habibi, H. R., Chang, J. P., Nahor- niak, C. S., Yu, K. L., Huang, Y. P. and Marchant, T. A. (1989) Actions of gonadotropin-releasing hormone (GnRH) in the goldfish. Progress in clinic- al and biological research, 342: 393-398. Suzuki, M., Hyodo, S., Kobayashi, M., Aida, K. and Urano, A. (1992) Chaaracterization and local- ization of mRNA encoding the salmon-type go- nadotrophin-releasing hormone precursor of the masu salmon. J. Mol. Endocrinol., in press. erred oli it inary Of cthnerpartes “4 ! S—— Ti hah heteieat ENT epee Ay te SHELA aye hs Boe ; OD VA Athen oie aii ral phage sheen) ara tired ‘eteranle ted) 4 ’ A"s } Opry belt «1%, as vita. Be Y. ave = , iis 4ese }Aeeaere line a ; ‘awnin ® i o=lby re yf gern Hen) wr e i dy eae | qa , ik th qian . cowry! «A! >a). ei + ' i ' ) he Oe Gap ett rien? ly. haceulge, ary io swted? Heath jonny Si “tem ft read ft Am 7 ] Im Jeareetee aa rh 07a baeols Moline roller i> ieibuds + 4 il 4 a) tts tee pea ae , “ be (ber em eunteel o overeat Herd do lhinae ZOOLOGICAL SCIENCE 9: 775-784 (1992) Antagonism of Prolactin and Growth Hormone: Impact on Seawater Adaptation in Two Salmonids, Salmo trutta and Oncorhynchus mykiss STEFFEN S. MADSEN!” and Howarp A. BERN* ‘Institute of Biology, Odense University, Campusvej 55, DK-5230 Odense M, Denmark; *Department of Integrative Biology, Cancer Research Laboratory and Bodega Marine Laboratory, University of California, Berkeley, California 94720, U.S.A. ABSTRACT—The effect of simultaneously-injected prolactin and growth hormone on short-term adaptation to seawater was investigated in two salmonid teleosts, Salmo trutta and Oncorhynchus mykiss. Freshwater-adapted fish were given 5—6 injections of prolactin, growth hormone or combina- tions of the two hormones on alternate days and subsequently challenged by transfer to seawater. Major plasma ions, osmolarity, muscle water content and gill Nat, K*-ATPase activity were measured. In both species, increased plasma ions and decreased muscle water were observed 2-3 days after transfer to seawater. Growth hormone significantly reduced post-transfer changes in plasma ions and muscle water, whereas prolactin injections alone had no effect on either freshwater-adapted or seawater- challenged fish. However, when prolactin was injected simultanously with growth hormone, prolactin significantly abolished the seawater-adaptive effect of growth hormone in a dose-related manner. Prolactin also significantly reduced the growth hormone-induced stimulation of gill Na*, K ‘-ATPase activity. We conclude that prolactin is antagonistic to growth hormone during seawater adaptation and © 1992 Zoological Society of Japan development of hypoosmoregulatory mechanisms in salmonids. INTRODUCTION Despite a close similarity in the primary struc- tures of prolactin (PRL) and growth hormone (GH), there is much evidence to suggest an antagonistic relationship during development of hypoosmoregulatory mechanisms in salmonids. Plasma PRL and GH levels are normally inversely related during both smoltification [1, 2] and sea- water (SW) adaptation [1, 3-7], with PRL levels decreasing and GH levels increasing in both events. PRL is essential for freshwater (FW) survival and osmoregulation in several euryhaline teleosts [8-10]. The importance of PRL in FW adaptation of salmonids is, however, less clear, as hypophysectomized fish survive well in FW with little or no ionic disequilibrium [11-13]. PRL causes hypernatremia when injected into FW- and Accepted April 19, 1992 Received March 7, 1992 SW-adapted teleosts including salmonids (see [10], [14], [15]). Furthermore, PRL decreases Cl~ excretion by opercular membrane chloride cells [16] and reduces chloride cell size in seawater- adapted tilapia, Oreochromis mossambicus [17]. The importance of GH during the early phase of SW adaptation and in smoltification is well estab- lished for several salmonids including sea trout, Salmo trutta, and rainbow trout, Oncorhynchus mykiss [18-22]. GH has a hypoosmoregulatory effect, stimulating gill chloride cell proliferation and Na‘, K*-ATPase activity in vivo [20-22], but not in vitro [23]. Osmoregulatory effects of GH in nonsalmonid fishes have not yet been reported. As the above evidence suggests that GH and PRL may act antagonistically during SW adapta- tion, the present study aimed to examine their interaction following simultaneous injection into intact fish. The impact of hormone manipulation was studied for a period before and during the early phase of SW adaptation. Major plasma ion levels, muscle water content and gill Na‘, K*- 7716 S. S. MADSEN AND H. A. BERN ATPase activity were measured. MATERIALS AND METHODS Food was withheld from all fish used in the following experiments beginning two days before and lasting throughout each experiment. Fish were briefly anesthetized in 0.01% phenoxyetha- nol prior to tagging, injection and sampling. Experiment 1 Fish and protocol Immature sea trout (Salmo trutta, age 1+, mixed sexes, mean weight 66.1+ 1.9g, N=160) were obtained in October 1989 from a commercial trout farm in Egtved, Jutland, Denmark, where they had been reared under natural photoperiod and water temperatures. The fish were brought to the Boegebjerggaard Marine Laboratory (Odense University), separated into four groups by fin clipping, and acclimated to the experimental conditions in fresh water (FW; 10°C, 12:12 L:D artificial photoperiod). After two weeks of acclimation (=day 0), fish in the four groups were injected intraperitoneally according to body weight with saline, ovine growth hormone (NIADDK-oGH-15: 2 g/g), ovine pro- lactin (NIADDK-oPRL-19: 2 ug/g) or combined oGH-+o0PRL (2 ug/g each). Hormones were dis- solved in saline (0.9% NaCl, 0.5% bovine serum albumin, pH 9.0) prior to injection, and the injec- tion volume was 2 yl/g. Hormone doses were based on those used in previous studies [17, 21, 22]. During the following 14 days, all groups were given five additional injections with the above dosages (on days 2, 4, 7, 9 and 11). Three days after the last injection (day 14), subgroups from each group were directly transferred to 25 ppt natural seawater (SW; 10°C), where they were held for 3 days or until sampling. Sampling and analyses Subgroups of eight fish were sampled from each group 3 days after the last injection in FW, and 2 and 3 days after transfer to SW. Eight additional fish were sampled from the control group on day 0 before the first injection. Blood was drawn from the caudal vessels using heparinized syringes and centrifuged at 5000Xg; the plasma was frozen (—20°C) until analysed. After bleeding, the fish were decapitated, and a piece of paraxial muscle was dissected and weighed for determination of muscle water content. Plasma sodium and potassium were determined by flame photometry (Instrumentation Laboratory 243), and plasma magnesium and total calcium analysed by atomic absorption spectrophotometry (Perkin Elmer 2380, Mountain View, CA). Plasma chloride was determined by coulometric titration (Radiometer CMT 10, Copenhagen). Muscle water content was determined as weight loss after drying at 105°C until constant weight was attained (48 hours). Experiment 2 Fish and protocol Steelhead trout (Oncorhyn- chus mykiss, age 0+, mixed sexes, mean weight 71.5+1.4 g, N=130) used in this experiment were obtained from the California Department of Fish and Game Warm Springs Hatchery in November 1991. They were transported to the Bodega Marine Laboratory and acclimated in outdoor raceways for 2 weeks in freshwater prior to experi- mentation (12-13°C). During that period the fish were randomly separated into 6 groups and tagged using visible implants in the clear postocular tissue. On days 0, 2, 4, 6 and 8, fish in the six groups were injected intraperitoneally with one of the following hormone doses/combinations: Con: saline; GH: 2 ug/g oGH; PRL: 2 ug/g oPRL; GH +PRLI1: 2 ug/g oGH+0.2 u/g oPRL; GH+ PRL2: 2 ug/g oGH+1 ug/g oPRL; GH+PRL3: 2 ug/g OGH+2 ug/g oPRL. Saline, hormones and injection volumes were the same as described in experiment 1. One day after the last injection (day 9) sub- groups were transferred directly to 32 ppt natural SW (12°C) and held for two days. Sampling and analyses Eight to twelve fish were sampled from the treatment groups on day 9 in FW and again on day 2 after transfer to SW. The sampling procedure was the same as in experi- ment 1, except that gill filaments were also sam- pled. Filaments were placed in sucrose-EDTA- imidazole buffer (SEI; 300mM sucrose, 20 mM EDTA, 50 mM imidazole, pH 7.3), frozen on dry PRL-GH Antagonism in Salmonid Osmoregulation 7717 ice and stored at —80°C until analyzed for Na‘, K*-ATPase activity. Plasma osmolarity was analyzed on a micro vapor pressure osmometer (Wescor, 5100C, Logan, UT). Muscle samples were used to deter- mine muscle water content. Gill filaments were analyzed for Nat, K*-ATPase activity using a method slightly modified from McCormick and Bern [24]. Gill tissue was thawed and homog- enized in SEI buffer with 0.1% sodium deoxycho- late and 10 mM mercaptoethanol using a handheld glass homogenizer. The homogenate was centri- fuged at 5000 g for 30 sec, and the supernatant was kept on ice until analysed for Na‘, K*- ATPase (within 30-60 minutes). Ten microliters of gill homogenate were added to 990 yl assay mixture (50 mM imidazole, 1 U/ml L-lactic dehydrogenase, 2.5 U/ml pyruvate kina- se, 2mM _ phosphoenolpyruvate, 0.05mM {- NADH, 0.5mM ATP, 0.4mM KCN, 45 mM NaCl, 2.5 mM MgCh, 10 mM KCl, pH 7.5). After mixing, the decrease in absorbance at 340 nm was recorded over 6 min at 25°C in a Beckman spec- trophotometer (DU 70; Fullerton, CA). A second cuvette containing 0.5 mM ouabain was run simul- taneously, and the difference in rate of NADH oxidation between assays with or without ouabain was used to calculate specific Na*, K*-ATPase activity. Protein content of the gill homogenate was analyzed according to Lowry et al. [25], and enzyme activity was expressed as micromoles of ADP hydrolyzed per mg of protein per hour. Statistics Statistical differences among groups were analyzed using CRISP software (CRUNCH interactive statistical package, San Fransisco, CA). One-way ANOVA was performed and followed by Student-Newman-Keuls multicomparison _ test. Significant differences are reported if the confi- dence level was >95%. RESULTS Experiment 1 The results obtained in Experiment 1 are summarized in Figure 1. Within each treatment group, changes in each of the major plasma ions followed a similar pattern after transfer to SW. Control, PRL-treated and GH+PRL-treated fish all showed a major increase in plasma ions on day 2 and a stabilization on day 3. The only significant difference among these groups was in plasma Mg** on day 3 in SW (Fig. 1D), when the GH+ PRL-treated group showed a lower value than the control group. In contrast, the GH-treated fish showed less deviation from FW values than did all other treatment groups transferred to SW. In this group, the posttransfer increase was significantly less than that seen in controls for plasma Na‘, Cl, Mg’? and total Ca and less than that in the GH+ PRL-treated for plasma Na* and Cl-. Further- more, plasma ion levels had returned to FW values on day 3 after transfer. Changes in muscle water (Fig. 1F) were inverse- ly related to changes in plasma ions, with dehydra- tion taking place on day 2 in SW. GH- and GH+ PRL-treated fish were less dehydrated than con- trols, with GH-+PRL-treated fish losing more muscle water than GH-injected fish. Experiment 2 Plasma osmolarity increased in all groups after SW transfer (Fig. 2A). However, the magnitude of the increase varied among the groups and showed the following order: Con=PRL>GH+ PRL3 >GH+PRL2>GH+PRL1>GH. Changes in muscle water (Fig. 2B) were inversely related to changes in plasma osmolarity after SW transfer and the order of the degree of dehydration was PRL >Con >GH+ PRL3 >GH+ PRL2>GH +PRL1I=GH. There was a significant negative correlation between plasma osmolarity and muscle water in SW-challenged fish (r7=0.9361, P< 0.01). The effect of hormone treatment on gill Na‘, K*-ATPase activity is shown in Figure 3. PRL significantly decreased and GH significantly in- creased gill ATPase activity. None of the com- bined treatment groups showed enzyme activities significantly different from the control group; nevertheless, the higher doses of PRL inhibited gill Na‘, K*-ATPase activity more effectively than the lower dose. Regression analyses showed that there was a significant correlation between pretransfer gill 778 Plasma Nat (mmol/|) Plasma Cl” (mmol/|) Plasma Kt (mmol/|) 200 180 160 140 120 100 200 180 160 140 120 100 6.0 5.0 4.0 3.0 2.0 SW transfer i | b SW transfer C oD » AC SW transfer Plasma Mg** (mmol/!) Plasma Ca (mmol/l) Muscle water (%) 5.0 4.0 3.0 2.0 1.0 3.0 2.0 1.0 S. S. MADSEN AND H. A. BERN SW transfer \ 44 SW transfer a — SW transfer PRL-GH Antagonism in Salmonid Osmoregulation 779 400 [_] control IN) 2 wg GH + 02 wg PRL/g 2ug GH/g MM 2 ug GH + 1 ug PRL/g fX] 2 ug PRL/g FA 2 ug GH + 2 ug PRL/g 370 340 310 Osmolarity (mOsm/|) 280 250 Ix} = 75 noe fa OS Xi] FW groups after 5 injections 48 hr in SW Fic. 2. Effect of various hormone treatments on plasma osmolarity (A) and muscle water content (B) in O. mykiss in FW and 48 hr after transfer to SW. Fish were given 5 injections of each dose and combination of PRL and GH in FW and transferred to 32 ppt SW one day after the last injection. Values are mean+SEM of 8-12 fish. Values with shared symbols are not significantly different (P<0.05). Fic. 1. Changes in plasma sodium (A), chloride (B), potassium (C), magnesium (D), and total calcium (E) and in muscle water content (F) in S. trutta given 6 injections of saline (O), 2 ug/g oRPL (©), 2 ug/g oGH (A) or 2 pg/¢g oPRL +2 ug/g oGH (@) and subsequently transferred to 25 ppt SW two days after the last injection. Values are mean+SEM of 8 fish. a, Significantly different (P<0.05) from the control group; b, significantly different from the GH+PRL-treated group. 780 = L] control oO 2 ug GH/g E 8 E] 2 ug PRL/g O = at 6 ® a ao 4 - < 7 Mew D < = J = 0 Fic. Osmolarity (mOsm/l) Muscle water content (%) S. S. MADSEN AND H. A. BERN a ist 2 ug GH + 0.2 ug PRL/g 2 ug GH + 1 wg PRL/g 2 ug GH + 2 ug PRL/g FW groups dea 5 injections 3. Effect of various doses and combinations of PRL and GH on gill Na* , K*-ATPase activity in O. mykiss. Fish were given 5 injections in FW and sampled 1 day after the last injection. Values are mean +SEM of 8-10 fish. Values with shared symbols are not significantly different (P>0.05). 400 375 350 Zo 325 79.0 78.0 77.0 ag 76.0 Geri Ctr tae ia TURE Oe Le eel ep anoee aeal (0) 2.0 4.0 6.0 8.0 Na*,K*-ATPase (umol/mg/hr) Na‘, K*-ATPase activity and both posttransfer plasma osmolarity (Fig. 4A: r>=0.7637, P<0.05) and muscle water content (Fig. 4B: r>=0.6652, P <0.05). DISCUSSION GH treatment was used to induce osmoregula- tory changes comparable to those expected during smolting. Both experiments confirmed the SW- adaptive effect of GH reported earlier for S. trutta and O. mykiss [19, 21, 22] as well as for other salmonids [18, 26]. The mechanism responsible has been shown to involve stimulation of branchial chloride cell development and Na‘, K*-ATPase activity in vivo [20-22], but not in vitro [23]. However, more studies are needed to clarify the exact mechanisms. The ion-retaining effect of PRL, well established Fic. 4. Regression analyses of pretransfer gill Na“, K*-ATPase activity and plasma osmolarity (A) and muscle water content (B) in O. mykiss 48 hr after SW transfer. Analyses are based on mean values (+ SEM, N=8-12) from hormone-manipulated fish in Experiment 2 (see Fig. 2). A: r°-=—0.7637, P< 0.05; B: r= —0.6652, P<0.05. PRL-GH Antagonism in Salmonid Osmoregulation 781 and developed into a bioassay for PRL bioactivity in several euryhaline teleosts [27-31], remains controversial in salmonids [31]. Generally, PRL seems to play a minor role in FW osmoregulation in salmonids compared with that seen in other teleosts. For instance, PRL treatment in the present study did not have any effect on ionic balance in FW-acclimated fish, and hypophysecto- mized (Hx) O. mykiss and O. kisutch survive well in FW with only minor ion losses [11-13]. Also, salmonid pituitary PRL secretion in vitro is not altered by changes in extracellular osmolarity in the physiological range as occurs in other species [32, 33]. One study by Oduleye [34], however, indicated that brown trout depends on pituitary factors for FW survival. In contrast to the clear effect of GH, pretreat- ment with PRL had no effect on iono-osmotic regulation during the subsequent early phase of SW adaptation in either species used. By compari- son, only a minimal hypernatremic effect after 3 injections of 2.5 ug/g chum salmon PRL (sPRL) into O. mykiss was seen by Bolton et al. ({[19]—see also Hirano [14]). Any difference in the results reported could arise from differences in sampling time, the use of mammalian vs. teleost PRL [29- 31] and size of fish used. In most salmonids, a hyperosmoregulatory (i.e., anti-SW-adaptive) effect becomes evident when PRL is injected into either SW-adapted fish or into fish simultaneously injected with GH. Hasegawa et al. [31] reported that both sPRL (0.5 ug/g) and oPRL (10 ug/g) induced hypernatremia in O. mykiss adapted to 50% SW. Similarly, S. Vester- vang and S. S. Madsen (unpublished) found that oPRL (2 ug/g) induced hypernatremia and re- duced muscle water content when injected into O. mykiss adapted to 28 ppt SW but not when fish were adapted to 20 ppt SW. Hasegawa et al. [31], however, did not find any effect of sPRL or oPRL on ion levels in SW-adapted O. keta fry, suggesting that either interspecific differences in sensitivity may exist or that the effect was compensated for by high levels of SW-adaptive hormones (such as GH). In our studies, PRL-GH antagonism was evident when the two hormones were coinjected. PRL then inhibited the SW-adaptive effect of GH in a dose-related manner, with the effect of GH almost abolished when the two hormones were injected at equal doses. Increasing doses of GH may antagonize PRL in a similar manner. How- ever, in a converse experiment conducted at a different time of year (March instead of Novem- ber), evidence for the ability of PRL to antagonize the effect of GH in O. mykiss, although occa- sionally indicated, could only be considered equivocal over all (S. S. Madsen, unpublished). The endocrine effects on hypoosmoregulatory performance are well correlated with the effects on gill Nat, K*-ATPase activity (Fig. 5A, B), sug- gesting a causal relationship. GH stimulated gill Nat, K*-ATPase in Exp. 2, whereas PRL has a small but significant inhibitory effect when injected alone, but in particular when coinjected with GH. This may be viewed as a “FW-adaptive” effect of PRL, since lower enzyme levels are normally associated with FW adaptation in salmonids and most other teleosts. The effect is in accordance with a similar inhibitory effect of PRL on gill Na‘, K*-ATPase in SW-adapted Chelon labrosus [35], Hx FW-transferred Anguilla japonica [36], Hx FW-adapted Fundulus heteroclitus [37] and SW- adapted Morone saxatilis (S. S. Madsen, in prep). Interestingly, in SW-adapted O. mossambicus, PRL had no effect on gill Na*, K*-ATPase [17, 38], even though it reduced average chloride cell size [17] and decreased opercular membrane Cl~ excretion rate [16]. Additional osmoregulatory effects of PRL that may antagonize SW adaptation include reduced osmotic permeability of the gill epithelium [39], reduced intestinal water and ion transport rates [40], and increased glomerular filtration rate in vitro in O. mykiss (B. Dunne and C. Rankin, personal comm.). There are as yet no reports on the effects of GH on these targets. During “normal” development of hypo- osmoregulatory mechanisms (e.g., in smolting and/or SW adaptation), PRL levels are decreasing while GH levels are increasing [1—7, 41], suggest- ing that endogenous release and turnover of PRL and GH may be inversely controlled during these processes. Therefore, our results imply that any development-associated increase in PRL levels during a hypoosmoregulatory phase of the salmo- nid life cycle may be critical and incompatible with hypoosmoregulation due to PRL’s antagonism of 782 S. S. MADSEN AND H. A. BERN GH. Increased PRL levels reported during sexual maturation in SW [14, 42-44] could ultimately cause osmoregulatory maladaptation to SW. Stress-associated elevations in PRL levels during smolting [45] may be deleterious to smolt quality and subsequent performance in SW. Finally, the effect of experimentally administered hormones may potentially be antagonized by endogenous hormones. The mechanism for the observed antagonism remains unclear from our in vivo experiments. Competition between PRL and GH at the receptor level is unlikely to be a major factor, since dis- placement studies in various salmonid tissues show that the GH receptor has only 1-10% of the affinity for PRL that it has for GH [46-48]. Furthermore, each hormone has its own specific receptor in several osmoregulatory organs in sal- monids [36-49]. Although hypothetical at this moment, it is possible that other endocrine path- ways may be involved in the observed antagonism. Insulin-like growth factor-I and “synlactin” are possible hepatic mediators or synergists for GH and PRL, according to the somatomedin and “syn- lactin” hypotheses, respectively [50, 51]. Recent research in our laboratory has indicated that IGF-I may stimulate gill Na*, K*-ATPase in O. kisutch in vitro, thereby supporting the suggestion of McCormick ef al. [52] that IGF-I is the mediator for the osmoregulatory action of GH in salmonids. In conclusion, our study shows that the SW- adaptive effect of exogenous GH may be abolished by increasing PRL levels, thus adding another facet to the multihormonal control of osmoregula- tion in salmonids. ACKNOWLEDGMENTS S.S.M. was a postdoctoral fellow of the Carlsberg Foundation (Denmark) for part of this study. We are grateful to Drs. S. D. McCormick and R. S. Nishioka for their reviews of the manuscript and to H. Ahm (Odense), A. Corrales and J. S. Endersen (Berkeley) for their help with the analytical work. NIH and the National Pituitary Program (Baltimore, U.S.A.) pro- vided the ovine prolactin and growth hormone used in this study, and Zenyaku Kogyo Co. of Tokyo provided research support. This work is a result of research sponsored in part by NOOA, National Sea Grant Col- lege Program, Department of Commerce, under grant number NA89AA-D-SG138, project number R/F-117, through the California Sea Grant College, and in part by the California State Resources Agency. The U.S. Gov- ernment is authorized to reproduce and distribute for governmental purposes. REFERENCES 1 Prunet, P., Boeuf, G., Bolton, J. P. and Young, G. (1989) Smoltification and seawater adaptation in atlantic salmon (Salmo salar): Plasma prolactin, growth hormone, and thyroid hormone. Gen. Comp. Endocrinol., 74: 355-364. 2 Young, G. Bjérnsson, B. T., Prunet, P., Lin, R. J. and Bern, H. A. (1989) Smoltification and seawater adaptation in coho salmon (Oncorhynchus kisutch): Plasma prolactin, growth hormone, thyroid hor- mones, and cortisol. Gen. Comp. Endocrinol., 74: 346-354. 3 Prunet, P., Boeuf, G. and Houdebine, L. M. 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BERN Atlantic salmon (Salmo salar) during sexual matura- tion. Aquaculture, 95: 169-179. Avella, M., Schreck, C. B. and Prunet, P. (1991) Plasma prolactin and cortisol concentrations of stressed coho salmon, Oncorhynchus kisutch, in fresh water or salt water. Gen. Comp. Endocrinol., 81: 21-27. Gray, E. S., Young, G. and Bern, H. A. (1990) Radioreceptor assay for growth hormone in coho salmon (Oncorhynchus kisutch) and its application to the study of stunting. J. Exp. Zool., 256: 290- 296. Yao, K., Niu, P. D., Le Gac, F. and LeBail, P.-Y. (1991) Presence of specific growth hormone binding sites in rainbow troup (Oncorhynchus mykiss) tis- sues: Characterization of the hepatic receptor. Gen. Comp. Endocrinol., 81: 72-82. Sakamoto, T. and Hirano, T. (1991) Growth hor- mone receptors in the liver and osmoregulatory organs of rainbow trout: characterization and dyna- mics during adaptation to seawater. J. Endocrinol., 130: 425-433. McKeown, B. A., Fargher, R. C. and van Over- beeke, A. P. 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ZOOLOGICAL SCIENCE 9: 785-789 (1992) © 1992 Zoological Society of Japan Effects of Ca Concentrations in Culture Medium on the Release of Calcitonin from Incubated Ultimobranchial Glands of the Bullfrog, Rana catesbeiana Hiroyuki Kal-yA, JUNKO OKUYAMA, TAKASHI ISHIJIMA, YUICHI SASAYAMA, Hipext YosHizAwa! and Cuitaru OGcuro Department of Biology, Faculty of Science, Toyama University, Toyama 930, and ‘Department of Oral Anatomy II, Matsumoto Dental College, Shiojiri, Nagano 399-07, Japan ABSTRACT—Calcitonin released from ultimobranchial glands incubated in culture media having different Ca concentrations was determined by rat bioassay. In the first incubation of 30 min, a large quantity of calcitonin was released into various culture media such as normal medium, and high Ca media which have 2 times, 3 times and 6 times Ca concentrations compared to the normal medium. In this term, there was no significant difference in the quantity of calcitonin released into the media among those groups. However, during 48 hr following the first incubation, in the Ca 2 times medium, calcitonin quantity released into the medium was maximum. In the Ca 3 or 6 times medium, the amount of calcitonin released was less than that of Ca 2 times medium. These results imply that in the bullfrog, secretion of calcitonin from ultimobranchial glands in vivo may be induced by a suitable rise in the serum Ca concentration. INTRODUCTION Calcitonin is secreted from thyroid glands in mammals or from ultimobranchial glands in non- mammals. It has been well known that in mam- mals, calcitonin is secreted against the rise of blood Ca levels. In pigs and rabbits, blood calcitonin level is increased when the thyroid gland is per- fused with high Ca solution [1-3]. Furthermore, under in vitro condition, calcitonin secretion from the thyroid gland of pigs is accelerated by moder- ate elevation of Ca concentrations in culture medium [4]. In birds, when the ultimobranchial gland is perfused with high Ca solution, blood calcitonin level is increased [5, 6]. Incubated avian ultimobranchial glands also show the same reac- tions as in mammals [7, 8]. These facts imply that in higher vertebrates, the rise of blood Ca level is one of the factors which accelerate calcitonin secretion. On the other hand, it has been reported that in Accepted May 20, 1992 Received March 16, 1992 bony fishes such as trouts and eels, rise of blood Ca level does not cause calcitonin secretion from ultimobranchial glands [9, 10]. Therefore, it is suggested that mechanisms which trigger calcitonin secretion may be different between higher verte- brates and lower ones. In the present study, effects of Ca concentra- tions in culture medium on calcitonin release from incubated ultimobranchial glands of the bullfrog, Rana catesbeiana, were examined by rat bioassay. MATERIALS AND METHODS Male bullfrogs (body weight, 200-300 g) were purchased from commercial source. One pair of ultimobranchial glands found near the glottical sphincter was dissected out carefully under the binocular microscope, and was immediately put into an incubation chamber (Lab-Tek 4804, Nunc Inc.) which contained 1 ml of amphibian Ringer’s solutions with different Ca concentrations as de- scribed below. The ultimobranchial glands were kept at room temperature for 30 min (the first incubation). Then, the ultimobranchial glands 786 were transferred into a separate chamber which contained the same kind of incubation medium, and incubated for 24 hr at 25°C (the second in- cubation). After that, those were again displaced into a distinct chamber with the same kind of the solution, and incubated furthermore for 24 hr at 25°C (the third incubation). Each of the culture media obtained at each incubation time, which included released calcitonin, was lyophilized and frozenly stocked —50°C until use. Three sets of incubation chambers were pre- pared according to the incubation period, such as: 30 min (the first incubation), 24 hr (the second incubation), and next 24 hr (the third incubation). Each set included four culture media having dif- ferent Ca concentrations as follows: the normal amphibian Ringer’s solution, which served as the standard incubation medium, was composed of NaCl 6.50 g/l, KCl 0.14g/l, CaCl, 0.12 g/], NaHCO; 0.20 g/1l, glucose 1 g/l; the other three were composed of Ringer solutions that contained 2, 3, and 6 times amount of Ca of the standard medium (Ca 2X, Ca 3X, and Ca 6X incubation medium, respectively). Bullfrog calcitonin released from ultimobran- chial glands into incubation medium was detected by rat bioassay [11]. Each of the pooled lyophi- lized samples which were obtained after incubating 3 pairs of ultimobranchial glands separately, was adjusted to 0.4 ml by saline solution (0.9% NaCl) and administered to a rat. As a control, the saline solution was administered. Furthermore, salmon calcitonin (Novabiochem Inc.) (10, 25 and 100 mU) was administered as a calcitonin standard for comparison with samples. Blood was samples just before medium administration and at 0.5, 1, 2, and 3 hr after. Serum Ca concentrations in rats were determined by atomic absorption spectropho- tometry (Hitachi-Zeeman 180-70 type). In the present study, changes in serum Ca levels were exhibited as decline rates from the initial Ca level. Furthermore, the areas which were lower than the initial level were determined to examine the dura- tion of hypocalcemic effect of the culture medium, and were exhibited as serum Ca graphs. Student’s f-test was applied to evaluate the data. Fic. H. Kari-ya, J. Okuyama et al. Oro a fe) N iS) serum Ca 4%, we We \ -30 QO Of 1 2 3 time(hr) O vs 0 = oe Te =| (3) serum Ca 4%, tn (e) il * {2} 9 -20 ue a tC SO TOS A 3 time(hr) Oro —_ Wea = a pe) N Ss) serum Ca 4 ¥, as \ ron S os TES a 2 3 time(hr) 1. Time courses (0-3 hr) of serum Ca concentra- tion fall (delta % from the initial levels) in rats after administrations of the first incubation (30 min) medium (A) and the second incubation (24 hr) medium (B) and the third incubation (24 hr) medium (C). Vertical bar shows mean+SE. Each symbol means normal Ca medium (4), Ca 2 times medium (@), Ca 3 times midium (™), Ca 6 times medium (*) and saline solution (©). The numbers in parentheses mean number of rats used. Signif- icantly different from the value of normal Ca medium: * P<0.05, ** P<0.005, *** P<0.001. Ca Effect on CT Release in vitro in Frog 787 RESULTS When incubation media were administered to rats, serum Ca levels were decreased. Decline patterns by the first, the second and the third incubation media having different Ca concentra- tions are exhibited in Figure 1A, B, C, respective- ly. In Figure 2, decline patterns are shown when salmon calcitonin (10, 25 and 100 mU) was admi- nistered as a calcitonin standard. In Figure 3A, areas (cm’) declined by the admi- nistration of normal Ca Ringer (1 x), Ca 2 times medium (2x), Ca 3 times medium (3X), Ca 6 times medium (6X), salmon calcitonin (sCT: 10, 25 and 100 mU) and saline solution as a control, which were lower than the initial Ca level during 0-3 hr in each of the incubation time (the first culture, the second culture and the third culture), are shown in histograms. Among the areas made by administration of 4 kinds of the first incubation media, there was no significant difference, when 153 150 A 100 7 yh ac (cm?) serum Ca 4% ND oe) =e= Oo | JA us (ft SC OOISNE D) 2 time(hr) Fic. 2. Time courses (0-3 hr) of serum Ca concentra- KEE ! 1 1 1 1 1 i} i] 1 ' 1 200 I I I ! ! ! * 1 1 100 | 1 1 i] if ! 1 I ; a ! 0 tion fall (delta % from the initial levels) in rats after administrations of salmon calcitonin 10 mU (@), 25 mU (4) and 100 mU (™). The numbers in parenthe- ses mean number of rats used. * P<0.05, ** P< 0.01. B 300 (cm?) Saline 10 25 100 1X 2X 3X 6X 1X 2X 3X 6X 1X 2X 3X 6X 1X 2X 3X 6X sCT (ml) Ist culture Qnd culture 3rd culture Total Fic. 3A. Histograms showing areas (cm7), which are lower than the initial Ca level in Figure 1 and 2, declined by administrations of saline solution, salmon calcitonin (sCT: 10, 25 and 100 mU), normal Ca Ringer (1x), Ca 2 times medium (2x), Ca 3 times medium (3X) and Ca 6 times medium (6X) in each incubation term of the first incubation (1st culture), the second incubation (2nd culture) and the third incubation (3rd culture). Significantly different from 1 x area in each incubation time, but the value was calculated after subtraction of saline area: * P <0.05, ** P<0.005, *** P<0.001. Fic. 3B. Histograms showing total areas (cm)), which include all areas during 48.5 hr declined by adiminstrations of normal Ca Ringer (1x), Ca 2 times medium (2X), Ca 3 times medium (3x) and Ca 6 times medium (6x). 788 H. Kat-ya, J. Okuyama et al. they were compared after the subtraction of the area by the saline administration. The calcitonin released in this incubation time was in large quan- tities for its short term. Those amounts were approximately compared to 30-72% of salmon calcitonin 10 mU judging from the area. However, among the second incubation media, the area made by Ca 2X medium was the largest (P< 0.001 to the normal Ringer), which corresponded to 118% of salmon calcitonin 25 mU. The area by Ca 3X medium was also significantly larger than that by saline control (P<0.05). When the third in- cubation media were administered, the area made by Ca 2x medium was also the largest (P<0.005 to the normal Ringer), which was comparable with 87% of salmon calcitonin 25mU. Furthermore, the areas by Ca 3X and Ca 6X media were also larger than that by the saline control (P<0.05). In Figure 3B, the total areas, which include all areas declined during 48.5 hr, are exhibited in histo- grams. The area made by Ca 2 medium was 1.9 times larger than the normal Ringer. The area made by Ca 3X medium was 1.4 times larger than the normal Ringer. The area by Ca 6X was only 1.1 times larger. DISCUSSION It has been reported that in the incubated thy- roid gland of the pig, secretion of calcitonin into the medium was concentrated in the first 15 min period [4]. In the present study, it was known that also in the bullfrog, release of large quantity of calcitonin from the incubated ultimobranchial gland occurred in the first 30 min incubation time. It has been reported that in the leopard frog, Rana pipiens, ultimobranchial glands are innervated, and that the secretion of calcitonin is suppressed by the nervous system [12]. Therefore, it is possible that also in the bullfrog, under in vitro condition, suppressive control of the nervous sys- tem for ultimobranchial secretion was eliminated. We reported previously that in the ultimobran- chialectomized bullfrog tadpoles kept in high Ca water (20mg/100 ml) for 48 hr, a conspicuous hypercalcemia was observed [13]. In that experi- ment, Ca concentration of the environmental wa- ter was about 3 time that of the serum Ca level (the average value of the serum Ca before the treat- ment was 7.4mg/100 ml). Final serum Ca value was 14.3 mg/100 ml which was 1.9 times that of the initial value. However, the serum Ca levels of the sham-operated group did not show any increases. This fact suggests that in bullfrog tadpoles, the ultimobranchial glands can secrete calcitonin effec- tively against at least 2 times rise of the serum Ca concentrations. Furthermore, when 0.5 ml of ex- tremely high Ca water (200 mg/100 ml) was in- fused in the intestine of the ultimobranchialecto- mized bullfrog tadpoles, the serum Ca concentra- tions was raised 1.6 times that of the sham-control group at 24hr later [14]. These observations suggest that in the ultimobranchialectomized bull- frog tadpoles, serum Ca level does not elevate more than twice of the normal level at least for a short term, even if any treatments are done. Therefore, also in the adult bullfrog, serum Ca level may not elevate more than twice of the normal level. In mammals, it has been known that secretion rate of calcitonin is directly and linearly related to the actual increment in plasma Ca level [1-3]. On the other hand, ultimobranchial glands of teleosts do not respond to rises of serum Ca level [9, 10]. In the persent study using adult bullfrog, it was the Ca 2X medium that was most effective on releasing calcitonin from incubated ultimobranchial glands. Therefore, calcitonin secretion from ultimobranchial glands may be re- lated to the rise of serum calcium levels, to some extent in bullfrogs. The Ca 3x medium and Ca 6 x medium were also effective, but less potent than Ca 2x medium. In anuran amphibians, delicate control system of calcitonin secretion as in mam- mals may not have been established. REFERENCES 1 Care, A. D., Cooper, C. W., Duncan, T. and Orimo, H. (1968) A study of thyrocalcitonin secre- tion by direct measurement of in vivo secretion rates in pigs. Endocrinology, 83: 161-169. 2 Lee, M. R., Deftos, L. J. and Potts, J. T. Jr. (1969) Control of secretion of thyrocalcitonin in the rabbit as evaluated by radioimmunoassay. Endocrinology, 84: 36-40. 3 Cooper, C. W., Deftos, L. J. and Potts, J. T. Jr. (1971) Direct measurement of in vivo secretion on pig thyrocalcitonin by radioimmunoassay. Endocri- Ca Effect on CT Release in vitro in Frog 789 nology, 86: 747-754. Bell, N. H. (1975) Further studies on the regulation of calcitonin release in vitro. Horm. Metab. Res., 7: 77-83. Ziegler, R., Telib, M. and Preiffer, E. F. (1968) The secretion of calcitonin by perfused ultimobran- chial gland of the hen. Horm. Metab. Res., 1: 39- 40. Boelkins, J. N. and Kenny A. D. (1973) Plasma calcitonin levels in Japanese quail. Endocrinology, 92: 1754-1760. Feinblatt, J. D. and Raisz, L. G. (1971) Secretion of thyrocalcitonin in organ culture. Endocrinology, 88: 797-804. Feinblatt, J. D., Raisz, L. G. and Kenny, A. D. (1973) Secretion of avian ultimobranchial calcitonin in organ culture. Endocrinology, 93: 277-284. Ross, B. A. and Deftos, L. J. (1976) Calcitonin secretion in vitro II. Regulation effects enteric mammalian polypeptide hormone on the trout C- cell cultures. Endocrinology, 98: 1284-1288. 10 11 12 13 14 Shiraki, M., Hasegawa, S., Hirano, T. and Orimo, H. (1982) Calcitonin secretagogues in the eel. In “Comparative Endocrinology of Calcium Regula- tion” Ed. by C. Oguro and P. K. T. Pang. Japan Sci. Soc. Press, Tokyo, pp. 125-133. Oguro, C. Nagai, K., Tarui, H. and Sasayama, Y. (1980) Hypocalcemic factor in the ultimobranchial gland of the frog, Rana rugosa. Comp. Biochem. Physiol., 68: 95-97. Robertson, D. R. (1968) The ultimobranchial body in Rana pipiens. Z. Zellforsh., 90: 273-288. Sasayama, Y. and Oguro, C. (1976) Effects of ultimobranchialectomy on calcium and sodium con- centrations of serum and coelomic fluid in bullfrog tadpoles under high calcium and high sodium en- vironment. Comp. Biochem. Physiol., 55A: 35-37. Sasayama, Y. and Oguro, C. (1985) The role of the ultimobranchial glands on Ca balance in bullfrog tadpoles. In “Current Trends in Comparative En- docrinology” Ed. B. Loft and W. H. Holmes. Hong Kong Univ. Press, Hong Kong, pp. 837-838. ii iW “ihe oe Ae EE at Ta i ¥ berth pene ret heave ek) PLE: e A NEA: RPM BRD AAD, ey Rea wi 1 Fi by ue via rf oh Sh : ; ‘yw ‘ / th iay ; rose] Pensa F si) a7 ac ate tet ‘ bul tira ny eh 5 ef ‘4 i 7 : rita : P = n : L a/ y i eRe er on ua ac : Ae wi \2 vidi vad 7 1 i - wits tt Atte wT AD are a a UL RD A vovlat* hs AEVO totter Jolog pelle crawl | i wash Bebe a wb uf hed , - Crone cere TES, el Patt ae! 4 ; tet bs wu peer t Corny een Le i i eras Bi | " ’ ' iv 4 : t | la © h “ x a i j 4 - ’ § i ‘ ( : ; ‘aan “ ; \ i ‘ ay ry ral HW" igus, ayer nat ‘sa ZOOLOGICAL SCIENCE 9: 791-802 (1992) © 1992 Zoological Society of Japan Effects of Chronic Treatment with Chlorpromazine on the Aging of Hypothalamo-Pituitary-Ovarian Axis in the Rat Yuta Kopayasut'”, Sencuiro KAwAsHima!, SuMIO TAKAHASHI? and KatsuMI WAKABAYASHI* ‘Zoological Institute, Faculty of Science, University of Tokyo, Tokyo 113, Department of Pharmacology, Shimane Medical University, Izumo 693, 3Department of Biology, Faculty of Science, Okayama University, Okayama 700, and *Institute of Endocrinology, Gunma University, Maebashi 371, Japan ABSTRACT— Effects of chlorpromazine (CPZ) on the age-related changes of the estrous cycle and the glial histology in the hypothalamus were studied in female Wistar rats. CPZ (3.0 or 5.5 mg/day/rat) was orally administered for 6 months beginning at 8 months of age. About 28% of rats given no CPZ treatment showed prolonged estrus at 13-14 months of age, while about 41% of rats was cycling. In contrast, none of high-dose CPZ-treated rats of the same age was in a prolonged estrous state and the difference was statistically significant. The ovarian weight was heavier and the number of Graafian follicles in the ovary was less in CPZ-treated rats. In the hypothalamic arcuate nucleus, CPZ inhibited the increase in the number of astrocytic granules. These results indicate that CPZ treatment effectively delayed the occurrence of some age-related changes of the ovarian function and the hypothalamus in the rat. INTRODUCTION Chlorpromazine (CPZ) is a common antipsycho- tic drug showing complex biological effects [1]. Although many patients are given CPZ for years, the reports on the effect of long-term administra- tion of CPZ on aging processes are a few [2]. Reduction of the accumulation of lipofuscin pig- ments during aging in neurons of the nucleus reticularis gigantocellularis by long-term oral administration of CPZ in C57BL/10 female mice was observed [3]. In addition, the effect of CPZ on neurons of rat cerebral cortex in primary culture was reported by Ohtani and Kawashima [4]. They observed that CPZ was effective in reducing the accumulation of lipofuscin pigments in cultured neurons. The lipofuscin pigments accumulate in various tissues during aging and they are regarded Accepted May 28, 1992 Received April 8, 1992 Reprint requests should be addressed to: Dr. S. Kawa- shima, Zoological Institute, Faculty ef Science, Universi- ty of Tokyo, Tokyo 113, Japan. as one of the parameters of aging [5, 6]. These results led us to propose a working hypothesis that CPZ affects some aging processes in the brain. The age-related increase in the astrocytic and microglial activities in the hypothalamic arcuate nucleus in female rats was reported [7, 8] and it is suggested that the age-related increase in the hypothalamic gliosis may be closely related with the deterioration of hypothalamic function. Simi- larly, an increase in the number of reactive micro- glia (microglia containing more than five granules) and the number of astrocytic granules in the arcu- ate nucleus with aging in the rat and mouse was reported [9]. The decline in reproductive function in female rats with aging has been observed as the loss of regular estrous cycles and the manifestation of irregular cycles, usually characterized by pro- longed vaginal estrus at middle ages [10-14]. The age-related deviation of the estrous cycle was suggested to be dependent on the hypothalamus (for review, see 13). The present paper will deal with the effects of 792 Y. KosBAyAsHI, S. KAWASHIMA et al. chronic administration of CPZ on the occurrence of prolonged vaginal estrus and the microglial and astrocytic histology in the arcuate nucleus in female rats during aging. MATERIALS AND METHODS Experimental design One hundred fifty female rats of the Wistar strain at 4 weeks of age were purchased from Nihon Rat Co. Ltd. (Urawa, Japan). Upon arri- val, they were housed 5 each in an aluminum cage lined with wood shavings at 22°C under 12 hr-light (6:00-18:00) and 12hr-dark cycle, and were given rat chow (CA-1, Clea Japan Inc., Tokyo) and tap water ad libitum. These animals generally live to a maximum of 30 months of age. From 31 weeks of age, the commercial chow was replaced by hand-made chow containing no CPZ. CPZ administration in hand-made chow began in some rats at 32 weeks of age. From 56 weeks of age, commercial chow was supplied again. The procedures to prepare hand-made chow were as follows: The chow powder (CA-1) was kneaded with tap water equivalent in weight to 90% of the chow powder with or without CPZ (Contomin, Yoshitomi Pharmaceutical Ltd., Osa- ka) and chopped into cubes of about 15 mmx 15 mm 30mm. The cubes were dried in an oven about 10 hr at 80°C to 60% of the original weight. Rats were divided into the following five groups; (1) High dose CPZ-treated group. Forty rats were given chow containing 0.35 mg of CPZ per g CA-1 powder. (2) Low dose CPZ-treated group. Forty rats were given chow containing 0.175 mg CPZ/g. (3-1) Food restricted group to the level of the food intake of high-dose CPZ-treated group. Twenty rats were given the corresponding restricted amount of hand-made chow containing no CPZ. (3-2) Food restricted group to the level of the food intake of low dose CPZ-treated group. Twenty rats were given the corresponding restricted amount of hand-made chow containing no CPZ. (3-3) Control ad libitum feeding group. Hand- made chow containing no CPZ was given ad libitum to thirty rats. Food restricted groups were prepared because the food intake of CPZ-treated groups was less than that of control group of ad libitum feeding. The consumption of food per day in each cage of CPZ-treated groups was recorded once a week and the same mean daily amount of chow without CPZ was given to the corresponding food restricted group in the next week. In high dose and low dose CPZ-treated groups, the aver- age uptake of CPZ was 5.5 mg/day/rat and 3.0 mg/day/rat, respectively. During 41st week of age, water consumption in each cage was mea- sured daily. Vaginal smears were recorded during 25-27, 40-42, 53-55 and 60-62 weeks of age daily at 15 :00-17:00. Animals for plasma hormone determination and ovarian histology were killed by decapitation at 56, 63 and 68 weeks of age between 11:00 and 14:00. The plasma was separated from the trunk blood and kept frozen at —20°C until radioimmunoassay for luteinizing hormone (LH) and _ prolactin (PRL). The ovaries and uterus were weighed, and the ovaries were fixed in Bouin’s fluid. Histological observation of ovaries Paraffin sections of the right and left ovaries were cut at 10 ~m thickness, and one each section at the first, second and third quarters of the serial sections, i.e. six sections per animal, was stained with hematoxylin and eosin. The numbers of Graafian follicles and corpora lutea larger than 500 ym in diameter were counted. Histological observation of microglial and astrocy- tic granules At 59 weeks of age, five control rats (four persistent estrous and one cycling rats), five low dose CPZ-treated rats (one persistent estrous and four cycling rats) and five high dose CPZ-treated cycling rats were used for this study. For cycling rats those at estrus were used. They were anesthe- tized by i.p. injection of nembutal and perfused with Lock’s solution from the heart ventricle, followed by perfusion with 0.12 M phosphate buf- fer containing 1% glutaraldehyde and 1% para- formaldehyde. The hypothalami were dissected out and fixed in 0.1 M cacodylate buffer containing 1% glutaraldehyde and 1% paraformaldehyde for 4hr at 4°C. Then the hypothalami were postfixed Effects of Chlorpromazine on Rat Aging in 1% OsO, in the same buffer for 2 hr and embedded in epon. The ovaries were fixed in Bouin’s fluid for histological observations. The microglial and astrocytic changes were quantified according to the method described by Brawer et al. [7]. Briefly, coronal semithin sections 1 ym thick were cut at the mid-level of the arcuate nucleus (4-6 sections). These semithin sections were stained with toluidin blue and were observed under a light microscope. The microglia were identified as relatively small ellipsoidal cells con- taining toluidin blue-positive granules. The astro- cytes are small cells generally stained pale with less frequent granules. Four sections from the ipsi- lateral arcuate nucleus in each rat were used for measurements, and the number of reactive micro- glia (the cell containing more than five granules) and the number of granules in the astrocyte were counted. Radioimmunoassay Plasma concentrations of LH and PRL in rats at 56 and 63 weeks of age were determined with radioimmunoassay kits, NIADDK rat LH-I-5, anti-rat LH S-4, rat LH RP-1 (0.03 NIH unit/mg), rat PRL-I-3, anti-rat PRL S-6 and rat PRL PR-1 (11 IU/mg) provided by Rat Pituitary Hormone Distribution Program, NIADDK, National Insti- tutes of Health, Bethesda, Md. The assay results for LH and PRL were expressed as mg of NIH-rat- LH-S, (=NIH unit), and mIU, respectively. Statistics The differences in body weight, food intake and water intake were tested by analysis of variance (ANOVA). Tissue or organ weights, results of vaginal smear and blood LH and PRL concentra- tions were compared by Mann-Whitney’s U-test, as the distribution of the data did not show appa- rent “normal distribution”. Concerning the results of histological observation of the ovaries, the numbers of follicles and large corpora lutea were compared by Mann-Whitney’s U-test, and intersti- tial tissue hypertrophy was compared using Fisher’s exact probability test as the results were non-parametric. The numbers of reactive micro- glia and astrocytic granules were tested by ANO- VA and Duncan’s multiple range test. 793 RESULTS Body weight, food intake and water intake The number of death by 56 weeks of age and/or apparently unhealthy rats at 56 weeks was as follows; (1) high dose CPZ-treated group, 4; (2) low dose CPZ-treated group, 1; (3-1) food res- tricted group for high dose CPZ group, 0; (3-2) food restricted group for low dose CPZ group, 3; (3-3) ad libitum feeding group, 2. There was no significant difference among groups (Fisher’s exact probability test). Changes in body weight during experiments are shown in Figure 1. The body weight temporarily decreased in all groups after changing commercial chow to hand-made chow, and then, it began to increase. The inhibition of body weight gain in CPZ-treated groups was dose-dependent (Fig. 1, Table 1). After the withdrawal of CPZ from the chow at 56 weeks of age, the rate of body weight gain recovered to the level of ad libitum feeding group (Fig. 1). The body weights of food res- tricted groups were smaller than those of the corresponding CPZ-treated groups and ad libitum feeding group, although consumed food amount 450) ++ Ad lib feeding (19) °-—- CPZlowdose (19) *-—s €PZ high dose (19) a Body weight (g) 300° —_ 25 30 35 40 45 50 55 60 65 Age (weeks) Fic. 1. Changes in body weight by long-term chlorpro- mazine (CPZ) administration. Hand-made chow containing 0.175 mg of CPZ per g chow powder was given to low dose group and that containing 0.35 mg/g, to high dose group of rats from 32 to 55 weeks of age. The period of CPZ administration is shown as solid column in abscissa. The numbers in pa- rentheses indicate the numbers of rats. * P<0.05 compared with the control ad libitum feeding group (ANOVA). 794 Y. KOBAYASHI, S. KAWASHIMA et al. TaBLe 1. Effects of chlorpromazine (CPZ) on body weight, food intake and water intake Caer Body weight Body weight Food intake Water intake P (g) gain (g) (g/day/rat) (ml/day/rat) Ad libitum feeding group 398+9 (28) 47 +3 (28) 18.6+0.3 (6) 28.8+1.6 (6) low dose CPZ group Bw aeY) (39) suiae se (GY) 17.4+0.3* (8) 24.7+0.6 (8) high dose CPZ group 362+7** (36) 10+3** (36) IS). 7/sE0).3'* (() 19.0+0.6** (8) Food restricted groups ie to low dose group 368+8* (17) 8+6** (17) 17.5+0.3* (4) 31.54+2.2* (4) to high dose group 352+7** (20) 6+3** (20) 16.0+0.3** (4) 28.44+1.17* (4) Mean+S.E. Numbers in parentheses indicate the number of animals. Body weight at 55 weeks of age. Body weight gain during 32 and 55 weeks of age. Food intake shows the mean during 32 and 55 weeks of age. Water intake at 41 weeks of age. * P<0.05, ** P<0.01 compared with ad libitum feeding group (ANOVA). # P<0.05, ** P<0.01 compared with corresponding CPZ-treated group. was adjusted to be equal to that of CPZ-treated groups (Table 1). The difference in body weight gain between low dose CPZ group and matched food restricted group was statistically significant (Table 1). The food intake in CPZ-treated groups was significantly and dose-dependently decreased. The water intake in high dose CPZ-treated group was significantly less than that in ad libitum feeding group, while the water intake in low dose CPZ- treated group and food restricted groups was not significantly different from that in ad libitum feed- ing group. Water intake in food restricted groups was significantly greater than that in matched CPZ-treated groups. Estrous cycle, ovarian histology and plasma LH and PRL concentrations In the gonadal functions of control rats, there were no significant differences between ad libitum feeding group and food restricted groups. There- Fic. 2. Effects of chlorpromazine (CPZ) adiministra- tion on frequency distribution of the total number of proestrous and estrous days (left) and the number of estrous cycles (right) in young rats at 25-27 weeks of age (top panel) and rats at 40 to 42 weeks of age (lower three panels). Arrow indicates the median of the distribution. n=number of rats. The frequency distribution of high dose CPZ-treated group was significantly different from that of the control (P< 0.05) (U-test). fore, data of ad libitum feeding group and food restricted groups were combined and served as the control in the following analyses. At 25-27 weeks of age, regular 4- or 5-day estrous cycles were observed (Fig. 2). At 40-42 weeks of age, a few control rats showed prolonged % Young n=30 Control n=65 CPZ low dose n=39 CPZ high dose n=36 6 2 4 6 8101214161820 135 7 9111315171921 Number of days in proestrus and estrus during 3-week period 01234567 Number of estrous cycles during 3-week period Effects of Chlorpromazine on Rat Aging estrus (Fig. 2). Some CPZ-treated rats showed estrous cycle with relatively long diestrous phase and a few rats showed prolonged diestrus. The median of the number of proestrous plus estrous days of the control group at 42—42 weeks was not significantly different from those of rats at 25-27 weeks and of high dose CPZ-treated group. The frequency distribution of prostrous and estrous days of high dose CPZ-treated group was signi- ficantly different from that of the control. The number of estrous cycles during a 3-week period was about 5 in the young and high dose CPZ- treated groups and it was about 4 in the control at 40—42 weeks of age (P<0.05, vs. young group). Fic. 3. Effects of long-term chlorpromazine (CPZ) administration on frequency distribution of the total number of proestrous and estrous days (left) and the number of estrous cycle (right) in rats at 53 to 55 weeks of age. Arrow indicates the median of the distribution. n=number of rats. The frequency distribution of the control group was significantly different from that of high dose CPZ-treated group (P<0.01) and that of low dose CPZ-treated group (P<0.02). The frequency distribution of low dose CPZ-treated group was significantly different from that of high dose CPZ-treated group (P<0.01). The frequency distribution of the control group was significantly different from that at 40 to 42 weeks of age shown in Fig. 2 (P<0.01) (U-test). 795 The distribution of vaginal smear pattern at 53- 55 weeks of age is shown in Figure3. In the control group about 28% of rats showed prolonged estrus, resulting in a mass of distribution locating toward the right. Another mass was present in the % Control n=65 30 u \/ 20 10 0 CPZlowdose n=39 v 30 y 1| 20 10 CPZ high dose n=36 Vv 0 0 2 4 6 810121416 18 20 01234567 135 7 9111315171921 Number of days in proestrus and estrus during 3-week period Number of estrous cycles during 3-week period TaBLE2. Effects of chlorpromazine (CPZ) administration (from 32 weeks of age to 55 weeks of age) on some organ weights and plasma LH and prolactin (PRL) concentrations at 56 weeks of age (upper part), and changes in these parameters at 63 weeks of age, 7 weeks after the cessation of CPZ treatment (lower part) Control low dose CPZ group high dose CPZ group At 56 weeks of age Ovarian weight (mg/100 g BW) 2.8) 38 IY) (18) A oY SEAS) (10) 28.5+1.0* (9) Uterine weight (mg/100 g BW) 191+19 (18) 152+ 16 (10) 146+ 13 (9) LH concentration (mg S,/ml) 0.88 + 0.33 (18) 0.39+0.06 (10) 0.51+0.11 (9) PRL concentration (mIU/ml) 3.22 +0.99 (18) 1.96 +0.66 (10) 2.49 + 0.74 (9) At 63 weeks of age Ovarian weight (mg/100 g BW) WD) ae MF (19) 23.2+2.4% (10) ipo rial (10) Uterine weight (mg/100 g BW) 192+ 14 (19) 185+ 12 (10) 173+10 (10) LH concentration (mg S,/ml) 0.52+0.11 (19) 0.44 +0.09 (10) 0.57+1.10 (10) PRL concentration (mIU/ml) 3.32 £0.66 (19) 2.15+0.46 (10) 1.80+0.28 (10) Mean+S.E. BW; body weight. Numbers in parentheses indicate the number of rats. * P<0.05; compared with control group, * P<0.05, compared with the values at 56 weeks of age (U-test). 796 Y. KoBAYASHI, S. KAWASHIMA et al. middle showing cyclic estrous pattern (about 41%). The number of estrous cycles of the control group showed a large variation indicating irregular cycle of some animals. In contrast, many rats showed cyclic estrous pattern with a fragmentary mass at the right in low dose CPZ-treated group. In high dose CPZ-treated group, the median of distribution of proestrous and estrous days was located in the middle with a small mass at the left showing a prolonged diestrous phase. The fre- quency distribution was significantly different among the three groups. The frequency distribu- tion of the control group at 53-55 weeks of age was significantly different from that at 40-42 week of age (Fig.3 vs. Fig.2). The difference in the frequency distribution of CPZ-treated groups be- tween 40-42 and 53-55 weeks was statistically not significant. Some organ weights and plasma levels of LH and PRL in three groups at 56 weeks of age are shown in Table 2. The ovarian weight was signi- ficantly heavier in high dose CPZ-treated group than the control group. The low dose CPZ-treated group showed the same tendency. The uterine weight was lighter, and LH and PRL levels were lower in CPZ-treated groups than the control group, although these differences were statistically not significant. TaBLeE 3. Summary of histological observations of the ovaries of rats given chlorpromazine (CPZ) Follicles ale Number of follicles (>500 ~m) Total nue 0-2.0 2.1-4.0 4.1-7.0 CLES Control ay) 16 6 26 CPZ low 10 7 3 20 CPZ high** 10 4 3 17 Corpora lutea Number of corpora lutea (>500 um) Total number Group ¥ n sre of rats Control 5 3 18 26 CPZ low y, 0 18 20 CPZ high 0 16 17 Interstitial tissue Interstitial tissue hypertrophy Total number Group 2 ak a of rats Control 15 3 8 26 CPZ low 13 5 2 20 CPZ high 14 2 1 17 *) Number of rats. observation of hypothalamus) Follicles Ovaries were obtained at 56 weeks (Table 2) and at 59 weeks (for the Number of follicles per section is shown. ** P<0.01 compared with the control by U-test. Corpora (—) indicates no C.L. in 6 sections. lutea (C.L.) (+) indicates less than one C.L. per section. (+) indicates more than one C.L. per section. Interstitial (—) indicates no hypertrophy. tissues (+) indicates slight hypertrophy. (+) indicates marked hypertrophy. Effects of Chlorpromazine on Rat Aging 797 When control rats were divided into subgroups, one showing prolonged estrus (more than 18 days were in estrus or proestrus out of 21 days) and the other showing regular estrous cycles (8-13 days of estrus or proestrus out of 21 days), the ovarian weight of the former was significantly lighter than the latter (prolonged estrus, 15.2+2.2 mg/100 g body weight, n=7; regular cycle, 28.2+2.1 mg/ 100 g body weight, n=7; P<0.01). The uterine weight was significantly heavier (prolonged estrus, 226+23 mg/100 g body weight; regular cycle, 126 +17 mg/100 g body weight; P<0.01) in prolonged estrus group than regular cycle group. The LH level was greater in the former group (prolonged estrus, 1.61+0.80 mg S,/ml; regular cycle, less than detectable level of 0.25 mg S;/ml; P<0.01 by Fisher’s exact probability test). The PRL level was greater (prolonged estrus, 5.6+2.3 mIU/ml; regu- lar cycle, 1.5+0.5 mIU/ml) in prolonged estrus group than regular cycle group, however, the difference was not significant. In CPZ-treated rat subgroups, the results were similar to those in the control subgroups. The ovaries of rats showing regular estrous cycles in three groups contained some follicles and corpora lutea, and the hypertrophy of interstitial tissue was not observed in these ovaries. On the contrary, the ovaries of rats showing prolonged estrus in the three groups contained numerous follicles but no or few large corpora lutea, and showed the hypertrophy of interstitial tissue. Sum- mary of ovarian histology at 56 and 59 weeks of age is shown in Table 3. In the ovaries of 85% of control rats, more than two large follicles per section were observed. In the ovaries of 59% of high dose CPZ-treated group rats and 50% of low dose CPZ-treated rats, less than two follicles per section were present. The difference in the num- ber of follicles between the control and high dose CPZ-treated group was statistically significant. On the other hand, less than one large corpora lutea per section was observed in the ovaries of about 30% of control rats, and in those ovaries the hypertrophy of interstitial tissue was apparent. In 94% of the ovaries of high dose CPZ-treated rats and 90% of them of low dose CPZ-treated rats, more than one large corpora lutea per section was present. Difference in the number of large corpora lutea was not statistically significant. Seven weeks after the cessation of CPZ treat- ment the same parameters were studied. The occurrence of estrous and proestrous days during a 3-week vaginal smear test at 60-62 weeks of age is shown in Figure 4. In CPZ-treated groups, the incidence of rats showing prolonged estrus became greater as compared with that at 53-55 weeks of age, and there was no significant difference from the value of the control group. The shift of the frequency distribution was significant in the high and low dose CPZ-treated groups at 60-62 weeks of age compared with that of the same animals at % Control n=39 fo y } 10 0 — CPZlowdose n=19 | 30 | i? 20} 1 CPZ highdose n=19 30 | ' ' | Vi 20 | 10 j 0 0 2 4 6 8101214161820 135 7 9111315171921 Number of days in proestrus and estrus during 3-week period 01234567 Number of estrous cycles during 3-week period Fic. 4. Effects of the cessation of chlorpromazine (CPZ) administration on frequency distribution of the total number of proestrous and estrous days (left) and the number of estrous cycle (right) in rats at 60 to 62 weeks of age. Solid arrow indicates the median of the distribution. n=number of rats. The difference in the frequency distribution of the con- trol group and CPZ-treated group was not statisti- cally significant. The median of the frequency distribution of high and low dose CPZ-treated groups became significantly larger than those (broken arrows) of the identical groups at 53 to 55 weeks of age (P<0.01) (U-test). 798 53-55 weeks of age. The ovarian weight of CPZ-treated rats at 63 weeks of age was not significantly different from that of the control (Table 2). The ovaries reduced in weight seven weeks after the cessation of CPZ treatment compared with that during CPZ treat- ment in low dose CPZ-treated group (Table 2). Plasma levels of LH and PRL at 63 weeks of age were similar to those at 56 weeks of age (Table 2). Observations of ovarian histology revealed no significant differences among the three groups at 63 weeks of age, although the number of follicles in high dose CPZ-treated group tended to be smaller than the other two groups (Table 4). The increase in the number of follicles in low dose CPZ-treated group and the increase in the inci- dence of interstitial tissue hypertrophy in high dose CPZ-treated group were significant after the cessa- Y. KoBAyYAsHI, S. KAWASHIMA et al. tion of CPZ treatment compared with that during treatment (Tables 3 and 4). Vaginal smear pattern, organ weights and ova- rian histology in rats killed at 68 weeks of age (9 high dose CPZ-treated, 9 low dose CPZ-treated and 20 control rats; data not shown) were similar among the three groups and were almost the same compared with those at 63 weeks. The number of reactive microglia and astrocytic granules Figure 5 shows an example of reactive microglia and astrocytic granules in the hypothalamic arcu- ate nucleus of a control rat. The effects of CPZ on the arcuate glial histology are presented in Table 5. In the number of reactive microglia, no signi- ficant differences were detected among the three groups, although the number of reactive microglia TABLE 4. Summary of histological observations of the ovaries of rats 7 weeks after the cessation of chlorpromazine (CPZ) treatment Follicles Gat Number of follicles (>500 «m) Total Aono 0-2.0 2.1-4.0 4.1-7.0 of rats Control 6» 7 6 19 CPZ low* 2 4 ti CPZ high A i rm Corpora lutea Number of corpora lutea (>500 «m) Total number GrONp a u ie of rats Control 1 3 15 19 CPZ low 1 8 10 CPZ high 0 2 8 10 Interstitial tissue Interstitial tissue hypertrophy Totalbwumbee Group i of rats = ae + Control 12 2 5) 19 CPZ low 4 4 2 10 CPZ high* 4 3 3 10 @) Number of rats. # P<0.05, compared with the score during CPZ administration (Table 3) by U-test (follicles) or Fisher’s exact probability test (interstitial tissue). Follicles; Number of follicles in a section is shown. interstitial tissue, refer to Table 3. For indices of corpora lutea and Effects of Chlorpromazine on Rat Aging 799 Fic. 5. Mid-level of hypothalamic arcuate nucleus of a control rat. Toluidin blue preparation. Microglia (arrows) and astrocytic granules (arrow heads) are visible. Lipofuscin-like granules are present in the neurons (N). Scale: 5 um. TaBLE5. Glial histology in the hypothalamic arcu- ate nucleus in chlorpromazine (CPZ)-treated rats killed at 59 weeks of age Number Number of Number of Group of reactive astrocytic rats microglia® granules Control 5 7.6+1.1 SERS Ie CPZ low 5) 7.0+1.9 ADS seas CPZ high 5 3.2+0.6 Aide Mean+S.E. a; Number of microglia containing more than five granules in four semithin sections. b; Total number of granules in astrocytes in four sections. ** P<(0.01, among groups (ANOVA). ## P<(.01, difference from the control (Duncan’s multiple range test). tended to decrease in high dose CPZ-treated group. On the other hand, the ANOVA clearly showed a singificant effect of CPZ treatment on the number of astrocytic granules. The numbers of astrocytic granules in low and high dose CPZ- treated groups were significantly less than that in the controls (P<0.01 in both comparisons). DISCUSSION Reduction in body weight gain by chronic CPZ treatment in rats in the present study agrees with the previous report using mice [3]. A significant decreases in food intake may be the main reason for the reduction in the rate of body weight increase. Although food intake was manipulated to be the same by food restriction, body weight gain was significantly less in food restricted control rats than CPZ-treated rats. We have already reported that 5 months treatment with high dose of CPZ reduced the ambulatory activity in open field test in 13-month-old rats [15]. Such a change in behavior in CPZ-treated rats may correlate to the energy metabolism and may result in larger body weight gain in CPZ-treated groups than food in- take-matched control rats. Food restriction increases longevity [16-18]. This is the reason why we prepared a food res- tricted group for each dose group. However, there was no difference between ad libitum feeding group and food restricted groups as concerns the parameters of gonadal function in the present study. Therefore, it is considered that the effects of CPZ on gonadal function observed in the pre- sent study were not the consequences of the reduc- tion in food intake and body weight gain. Previous studies restricted food to 40 to 60% of ad libitum intake [17-18]. In the present study, the reduction of food intake by low and high doses CPZ-treated groups were to 94 and 84% of ad libitum intake, respectively. Such a small reduction of food intake may have no influence on aging. Samorajski and Rolsten [3] could not detect any change in water intake by CPZ treatment. The present results disagree with theirs. On the other hand, water intake of food restricted groups was almost the same as that of ad libitum feeding group, indicating that the reduction in water intake by CPZ treatment is mediated not through the reduction of food intake. Vaginal smear records showed that the adminis- tration of CPZ was effective for the maintenance of regular estrous cycle, when prolonged estrous State was predominant in age-matched control rats. Ovarian weight and the number of follicles in high dose CPZ-treated rats were significantly diffe- rent from those in the control, due to the differ- ence in the proportion of subtypes of gonadal function between the two groups. Any differences on some other organ weights but ovaries and on ovarian histology were not observed between CPZ-treated rats showing estrous cycle and control 800 Y. Kopayasui, S. KAWASHIMA et al. cyclic rats, indicating normal gonadal function in the cyclic CPZ-treated rats. The blood hormone levels, uterine weight and ovarian histology did not conflict with this interpretation. At 63 weeks of age, 7 weeks after the cessation of CPZ adminis- tration, regular estrous cycles disappeared in most CPZ-treated rats. In our separate study, high dose of CPZ chow was administered for 10 days to 12-month-old rats showing persistent estrus, but any recovery to regular estrous cycles was not observed (unpub- lished observation). The maintenance of estrous cycle by CPZ seems to be related with long-term administration of this agent from younger ages. Aschheim [12, 19] demonstrated that the trans- plantation of ovaries from young rats into ovar- iectomized aged animals which had been in persis- tent estrous state did not result in reinitiation of estrous cycles, and he suggested that the origin for the loss of estrous cycle in aged rats was not at the ovarian level. Electrical stimulation of the preop- tic area of aged rats restored resproductive func- tion [20]. Maintenance of cyclic cornification of the vaginal epithelium was observed in young adult female rats given heterochronic transplantation of the pituitary and the ovary from aged female rats. These results indicate that the age-related devia- tion of the estrous cycle was not dependent on the ovary but on the hypothalamus. The hypothalamus or higher central nervous system is important as the action site of CPZ in the present study. CPZ treatment decreased the accu- mulation of astrocytic granules compared with controls. The increase in the glial activities in microglia and astrocytes with aging was reported [9]. In a preliminary study, we also found the age-related increase in glial activities. In Wistar/ Tw strain maintained in our laboratories, the num- ber of reactive microglia in the arcuate nucleus in 19-21-month-old females showing persistent estrus (33.8+3.5 cells/4 sections, n=6) was signi- ficantly greater (P<0.001) than that in 5-month- old females at estrus (5.6+0.3 cells/4 sections, n= 5). Similarly, the number of astrocytic qranules in the arcuate nucleus in aged females (871+43 granules/4 sections, n=7) is significantly higher (P <0.001) than that in young females (215+25 granules/4 sections, n=5). Similar changes were reported in other brain areas: for example, the increase in the numerical density of hypertrophied astrocytes in the hippocampus in rats with aging [22]. The reduction in astrocytic granules by CPZ treatment in the present study may have some relevance to the delay in gonadal aging. CPZ treatment reduced the accumulation of lipofuscin pigments in the nucleus reticularis giagantocellularis of mice [3]. They interpreted the effect of CPZ on its action on the biomem- brane properties. It is suggested that the forma- tion of lipofuscin was related with peroxidation of lipids in biomembranes [6]. In support of this view, some anti-oxidants or free-radical scavengers have been reported to reduce the accumulation of lipofuscin pigments and show other anti-aging acti- vities [23-26]. The anti-oxidative activity or effects of CPZ on biomembrane properties may have some relation with the present anti-aging effects of CPZ. Acute administration of CPZ (25 mg/kg body weight) stimulates PRL release from the pituitary due to its action as a dopamine antagonist [1, 27— 29]. Inhibition of gonadal function by enhanced PRL release and the occurrence of some pro- longed diestrous rats in CPZ-treated group may be correlated with the PRL release activity of CPZ. However, in the present study, blood PRL level was somewhat lower in CPZ-treated rats at 56 weeks of age. Measurement of blood PRL level in CPZ-treated rats at earlier period, especially dur- ing prolonged diestrous state, was necessary to clarify the correlation. The possibility that other effects of CPZ, such as a non-competitive acetyl- choline blocking action [30] on the central nervous system, caused the delay of aging in the hypothala- mo-hypophysial-gonadal system cannot be ex- cluded. To conclude, delay of the occurrence of some age-related changes of the ovarian function and the hypothalamic glial histology by chronic CPZ treatment in the rat was evident. ACKNOWLEDGMENTS The authors wish to express their gratitudes to Dr. S. Raiti and the Pituitary Hormone Distribution Program, the National Institute of Arthritis, Diabetes and Diges- Effects of Chlorpromazine on Rat Aging tive and Kidney Diseases (NIADDK) and Dr. A. F. Parlow, Pituitary Hormones/ Antisera Center, Harbor- UCLA Medical Center for kind supply of RIA kits. 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(1989) Striatal dopamine receptor occupancy during and following withdrawal from neuroleptic treatment: correlative evaluation by positron emission tomography and plasma prolactin levels. Psychopha- rmacology., 99: 463-472. Changeux, J.-P., Pinset, C. and Ribera, A. B. (1986) Effects of chlorpromazine and phencyclidine on mouse C2 acetylcholine receptor kinetics. J. Physiol. , 378: 497-513. ZOOLOGICAL SCIENCE 9: 803-809 (1992) Differential Effects of Testosterone and 5a-Dihydrotestosterone on Growth in Mouse Submandibular Gland KAZUHIKO SAWADA and TETSuO NOUMURA Department of Regulation Biology, Faculty of Science, Saitama University, Urawa, Japan ABSTRACT—Testosterone and 5a-dihydrotestosterone (DHT) were compared their morphometrical effects on the mouse submandibular gland in relation to the developmental profiles of their serum levels. In male mice, serum levels of both testosterone and DHT were dramatically increased between days 20 and 30, when sexual difference of the submandibular gland occurred morphometrically. In 30-day-old males, serum testosterone and DHT levels were 2.9+0.59 ng/ml and 1.0+0.21 ng/ml, respectively (3:1 in the ratio). Daily injection of testosterone and DHT for 1-20 days starting on day 20 induced morphometrical changes in the glands of neonatally-castrated mice. The glandular weight gain was caused only in males by treatment with testosterone for 10-20 days, but in both sexes by DHT for 7-20 days. The relative occupied area (ROA) of the granular convoluted tubules (GCT) in castrated mice of both sexes were increased by treatment with testosterone for 10-20 days, but by DHT for 7-20 days. The mitotic rates of GCT were increased only in males by treatment with testosterone for 10-13 days, but in both sexes by DHT for 2-7 days. The present study suggests that androgenic effects of these two androgens on growth in the mouse submandibular gland are different, being more affected by DHT than by testosterone, and also that © 1992 Zoological Society of Japan androgen responsiveness of the gland is sexually different, being higher in male than in female. INTRODUCTION In rodents, the male submandibular gland is larger and has more complex morphology than the female one. The glandular contents of biologically active polypeptides including nerve growth factor, epidermal growth factor, renin and proteases, are higher in the male than in the female, being responsive to androgens [1-7]. By histological, ultrastructural and morphometrical studies, both sexes in the mice experience a similar mor- phogenesis of the mouse gland during perinatal development, and then the sexual difference arises at 3-4 weeks of age, when the granular convoluted tubules (GCT) grows more rapidly in the male than in the female [8-11]. The masculine develop- ment of GCT in the gland is caused by androgens, testosterone and 5a-dihydrotestosterone (DHT), but not by estradiol-17 [11]. In a completely androgen-independent state (neonatally-castration Accepted June 2, 1992 Received April 8, 1992 and androgen-insensitive Tfm mutation), the gland displays the feminine development [11, 12]. The present study was aimed to compare the effects of testosterone and DHT on morphometric- al changes in the mouse submandibular gland in relation to the developmental profiles of their circulating levels. MATERIALS AND METHODS Animals CD-1 mice were obtained from Charles River Japan Co. and maintained by randomly mating in our laboratory. The animals were given a com- mercial diet (CRF-1: Charles River Japan Co.) and tap water ad libitum and were kept at 23+1°C under 12 hr artificial illumination (from 8:00 to 20:00). Hormone assay In mice of both sexes on days 20, 30 and 90, blood sample was collected from the tail vein 804 K. SAWADA AND T. Noumura under the ether anesthesia. The serum was sepa- rated and stored at —20°C until assayed. Serum levels of testosterone and 5a-dihydrotestosterone were separately determined by a radioimmunoas- say. The inter- and intraassay variations of testos- terone were 11.5% and 5.2%, and those of DHT were 12.4% and 5%, respectively. Individual values were the mean of the duplicate determina- tions and were expressed as nanogram per millili- ter of serum. Treatments of animals and histological procudure Both male and female mice were castrated on day 0 and were subcutaneously given daily treat- ments with testosterone (10 or 100 ug), DHT (10 or 100 ug) or sesame oil vehicle alone (0.1 ml) for 1, 2, 4, 7, 10, 13, 16 and 20 days starting on day 20. Mice were killed on days 21, 22, 24, 27, 30, 33 36 and 40, respectively. All animals were sub- cutaneously given a single injection of colchicine (5 pg/g body weight) dissolved in 0.9% NaCl, 5 hr before they were killed to arrest cell division at metaphase. Submandibular glands were weight and fixed in Bouin’s solution, embedded in par- affin and sectioned at 8 wm. Sections were stained with Delafield’s hematoxylin and eosin. Morphometrical Methods The sectional figures of submandibular glands in the microscopic enlargements (x 400) were traced by the camera lucida, and the areas cf GCT were measured by the picture analyzer (Logitec K-510, Kantou Denshi Co., Japan) connected with a microcomputer (NEC PC-9801DA). The areas were expressed as percentages of total area (the relative occupied area, ROA). Cell height of GCT was measured in randomly chosen five sections. Mitotic Activities Cells in division per 5S00-1,000 cells were counted in the GCT of the glands. Mitotic rate was estimated by counting the cell at metaphase per 5 hr in 100 cells. Statistical Analysis Data were statistically analyzed by Student’s t-test or by Cochran-Cox test. RESULTS Serum levels of testosterone and DHT in mice On day 20, serum testosterone levels showed no sexual difference statistically, but were slightly higher in males than in females (0.35 +0.08 ng/ml and 0.23+0.01 ng/ml). In male mice, serum tes- tosterone levels dramatically raised on day 30 and attained adult levels (Fig. 1A). Similarly, serum DHT levels were slightly higher in males than in females on day 20 (0.45+0.10 ng/ml and 0.11+ 0.01 ng/ml) and then the DHT levels in males dramatically increased to adult levels by day 30 (Fig. 1B). At 30 and 90 days of ages, serum levels of testosterone were 3-fold higher in males and 2-fold higher in females than those of DHT, re- spectively (Fig. 1A and 1B). testosterone A serum levels (ng/ml) ~~ IN) (et) ag > 20 30 90 age in days Fic. 1. Serum levels of testosterone and DHT in mice. a: P<0.05, b: P<0.01 vs age-matched females (Cochran-Cox test). Androgens and Mouse Submandibular Gland 805 male A female 150 testosterone e oil ©1049 ia} 10049 100 50 weight of the glands (mg) 0) 10 20 0 10 20 treatment periods (days) Fic. 2. Change in the weight of the submandibular glands of neonatally-castrated mice, treated with testosterone (A, C) and with DHT (B, D) for 1-20 days from day 20. a: P<0.05, b: P<0.02, c: P<0.01, d: P<0.002, e: P<0.001 (Student’s ¢-test). male i female 60 testosterone c @ oil ° 10u9 40; © 100yu9 ~ ° 20 ~— i— © © — S D AS abe = . \ 1% Loi ’ ral ri ' re » +) Oe ss cts ante a - oa \ 4 . — a, o 1 ss : 18) ZOOLOGICAL SCIENCE 9: 811-821 (1992) © 1992 Zoological Society of Japan Functional Morphology of Feeding Apparatus of the Cookie-Cutter Shark, /sistius brasiliensis (Elasmobranchii, Dalatiinae) SHIGERU SHIRAI’ and KAzuHIRO NAKAYA2 ‘Department of Zoology, University Museum, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113, and ? Laboratory of Marine Zoology, Faculty of Fisheries, Hokkaido University, 3-1-1, Minato, Hakodate 041, Japan ABSTRACT— External morphology, skeletons, and muscles related to the peculiar feeding mechanism of Isistius brasiliensis are described and compared with other squaloid sharks. On the feeding function of this shark, a few characteristic conditions have been emphasized (e.g., fleshy oral margins, scoop-like lower jaw with a sharp tooth row, and developed basihyal and throat muscle). Serious morphological differences are, however, found in the whole of neurocranium, viscerocranium, shoulder girdle, and related musculature. These characteristic conditions are not suitable for the clutching-cutting feeding mechanism of typical squaloids but should effectively work on scooping the flesh of preys, making a strong oral vacuum, and rotating the body itself. These functions explain the cause of a crater-like wound on fish and cetaceans, and /sistius must apply them not only on large preys but on small fish and squids. INTRODUCTION The cookie-cutter shark, Isistius brasiliensis (Quoy et Gaimard), is a pelagic species of world- wide distribution throughout tropical and temper- ate waters. Among squaloid members, it is rather small reaching a size of 50cm TL. Recently this shark attracted many oceanological and fishery scientists because of its curious feeding habit re- vealed by Jones [1]. Isistius brasiliensis often feeds by biting chunks out of teleostean swimmers (e.¢., dolphinfish, marlin, tuna, albacore, and wahoo), cetaceans, seals, and large pelagic sharks including the megacasmids [1-5]. This small shark gives these preys a crater-like wound, which has been thought to be caused by the bacteria, parasites, or cyclostomes [6]. The purpose of this unusual feeding habit is ambiguous, because I[sistius’s sto- mach contents are found largely occupied with pelagic squids, as large as the body of this shark in most specimens, small fishes including Vinciguerria (Photichthyidae), and crustaceans [1, 7, 8]. In its Accepted April 13, 1992 Received February 14, 1992 feeding mechanism, we do not still understand enough; Jones [1] referred to only a few character- istic structures, which will be of help to scoop the flesh of prey and to induce the strong oral vacuum. The purpose of the present paper is to describe anatomical features of the feeding apparatus of Isistius brasiliensis in detail and to compare with those of closely related groups. Also, we discuss these functions on the morphological basis. MATERIALS AND METHODS Examination of skeletons and muscles of Jsistius brasiliensis was based on one male and four females. Several dalatiine species and other squaloids were dissected for comparison. Collec- tion information of these specimens is listed below. Study materials are deposited in HUMZ (Labora- tory of Marine Zoology, Faculty of Fisheries, Hokkaido University) and FSFL (Far Seas Fisher- ies Research Laboratory, Shimizu, Japan). Defini- tion of the Dalatiinae follows Compagno [5]. Dalatiinae TIsistius brasiliensis: one male—HUMZ 119287, 425mm TL; four females—HUMZ 69173, 364 812 S. SHIRAI AND K. NAKAYA mm TL, HUMZ 86585, 480mm TL, HUMZ 87120, 438 mm TL, HUMZ 89900, 516 mm TL. Squaliolus aliae: HUMZ 119285, male, 185 mm TL, S. laticaudus : two females—HUMZ 74972, 250 mm TL, HUMZ 74974, 245 mm TL. Euprotomicrus bispinatus: HUMZ 90413, male, 197 mm TL. Dalatias licha: two males—HUMZ 74603, 390 mm TL, HUMZ 95229, 621 mm TL. Other squaloids Deania quadrispinosum: FSFL-EE422, female, 325 mm TL. Etmopterus lucifer: HUMZ 90230, male, 337 mm TL. Oxynotus bruniensis: HUMZ 91383, male, 545 mm TL. Somniosus microcephalus : female, 1,425 mm TL. Squalus acanthias : HUMZ 87733, male, 495 mm TL. Zameus squamulosus : HUMZ 75872, male, 526 mm TL. HUMZ 112816, Figure drawings were made in the camera lucida image with dissected specimens stained by alcian blue. Anatomical terminology mainly follows Daniel [9] and Edgeworth [10]. Abbreviations used in figures are as follows: alc +—anterior upper labial cartilage am —adductor mandibulae ams —adductor m. superficialis bb —basibranchial bh —basihyal btp —basitrabecular process cb —ceratobranchial ch —ceratohyal chd —constrictor hyoideus dorsalis chy —constrictor hyoideus ventralis co —coracoid coa —coraco-arcuales cob —coraco-branchialis cod —constrictor dorsalis coh _—coraco-hyoideus es —eye stalk fbc —articular fossa of basihyal with ceratohyal fopp —foramen for ophthalmicus profundus gco —genio-coracoideus gcob —f-slip of genio-coracoideus gm —gum of upper jaw gop —groove for orbital process hb —hypobranchial hm —hyomandibula hmf —hyomandibular fossa hyp —hypaxial body muscle im —intermandibularis iow —interorbital wall jhm —yjoint cartilage of hyomandibula If —lip fold Ihc —ligamentum hyomandibulo-hyoideum lb —levator labialis llc —lower labial cartilage Imc —ligamentum mandibulo-hyoideum Ipc —lip cover ma —mandibula mfl mandibular flap mk —mandibular knob of mandibula op —orbital process opd —oral pocket pap —palatine plate of palatoquadrate pem —pericardial membrane ple —posterior upper labial cartilage potp —postotic process pmg —posterior mouth groove pnf —posterior nasal fenestra pq —palatoquadrate proc —preoral cleft prop—preoral pouch qup —dquadrate plate of palatoquadrate slc —fleshy sack of posterior upper labial car- tilage so —suborbitalis sos —subotic shelf II —(foramen for) optic nerve DESCRIPTION In this section, external and internal characters of Isistius brasiliensis, which appear to accommo- date its feeding mechanism directly or indirectly, are described and compared with the other mem- bers of Dalatiinae. The condition of the latter (or the other squaloid sharks if necessary) is noted in braces. Anatomy of Cookie-Cutter Shark 813 Fic. 1. Mouth structure of squaloids. A) ventral view of Jsistius brasiliensis (the lip cover is deleted in right half); B) ventral view of same when jaws and lips are protruded showing the exposed gum of upper jaw and the lip sack for labial cartilages; C) lateral view of same; D) ventral view of Squaliolus aliae (the lip cover is deleted in right half); E) ventral view of Deania quadrispinosum. Scales are 10 mm. Externals (Fig. 1) The mouth of Jsistius (Fig. 1A-C) resembles that of other dalatiines (Fig. 1D) in having a large cutaneous lip cover (Ipc) and a thick and naked lip fold (If) at the mouth corner. The lip cover is a skin continuing from the upper lip near the jaw symphysis to the posterior mouth groove (pmg), concealing a deep oral pocket (opk) and preoral pouch (prop); non-dalatiine squaloids have no lip cover, and the preoral pouch is exposed as the preoral cleft (proc; Fig. 1E). The lip fold is supported by a joint of the posterior upper and lower labial cartilages (plc, llc); in non-dalatiine squaloids (Fig. 1E), the lip fold also conceals another piece of labial cartilage (alc). The pos- terior mouth groove of Jsistius is deep and extends posteriorly rearward to the half of distance from the corner of mouth to the first gill opening as in Squaliolus and Euprotomicrus. In Isistius, the lip fold is stout with the smooth surface and conceals well-developed labial carti- lages {lip fold is more or less lamellated, and labial cartilages are somewhat weak in other dalatiines (Fig. 1D)}. A gum in the upper jaw (gm) is considerably thickened, and a fleshy sack for the upper labial cartilages (slc) is made by the upper lip {such a bulky gum and the fleshy sack are absent}. Skeleton In the neurocranium of Isistius (Fig. 2A-B), the nasal capsule is somewhat reduced, but the orbit and otic capsule are well-expanded, with many modifications, e.g., an unusual opening of nasal capsule (pnf) at the subnasal fenestra, unchon- drified interorbital wall around the foramen op- ticum (II), no eye stalk (es), and no subotic shelf (sos). Two conditions related to the jaw suspen- sion are that: (1) the basitrabecular process (btp) is 814 S. SHIRAI AND K. NAKAYA (A) hmf fopp Sos Fic. 2. Neurocrania of two dalatiines. A-B) lateral (A) and ventral views (B) of Isistius brasiliensis; C-D) lateral (C) and ventral views (D) of Squaliolus laticaudus. Scales are 10 mm. expanded laterally to form a shelf below the pos- terior part of orbit {the basitrabecular process is only a small bulge supporting the orbital process of palatoquadrate (Fig. 2C-D)}; and (2) the postotic process (potp) is developed to form the dorsopos- terior ridge of a deep hyomandibular fossa (hmf) |the hyomandibular fossa and postotic process are not so developed}. The mandibular arch of Isistius (Fig. 3A-B, 4A- B) is characterized by fairly reduced upper and voluminous lower jaws, and it is noticeably dif- ferent from that of other squaloids in the following respects: (1) the upper teeth are wholly inside the lower teeth when mouth is closed, and then they are not engaged each other {upper and lower teeth are engaged each other at least in part (Fig. 3D)|; (2) the palatoquadrate (pq) is composed of two thin and soft pieces, the anterior one (palatine plate: pap) is capable to warp dorsally, and the posterior one (quadrate plate: qup) have a minute orbital process (op) at its anterior end for the loose palatoquadrate-neurocranium articulation {the palatoquadrate is a single and stout cartilage with a higher orbital process, and the palatoquadrate- neurocranium articulation is firmer}; (3) the man- dibula (ma; Meckelian cartilage) is thick and scoop-like, not curved laterally at the posterior end {the posterior end of mandibula is somewhat curved laterally to form a fossa for the adductor mandibulae (am)|; (4) the mandibula has a flexi- ble, weakly chondrified plate (mandibular flap: mfl) at its posteroventral edge {absent}; and (5) the ligamentum mandibulo-hyoideum (Imc) is very tough, inserted onto the posterior margin of man- dibula near the joint between the hyomandibula and ceratohyal {this ligament is weaker and in- serted onto the medial surface of the mandibula below the mandibular knob (mk) somewhat sepa- rated from the hyomandibula-ceratohyal joint}. The jaw dentition of Jsistius essentially resem- bles that of the dalatiines in having the dignathic heterodonty (Fig. 3); upper teeth are lanceolate arranging quincuncially and the lower teeth are with a large plate-like crown forming an interlock- ing tooth blade. In Jsistius, the lower tooth rows almost extend to the point of jaw joint (articula- tion of upper and lower jaws) to form a semicircu- lar saw as a fringe of the scoop-like lower jaw {the most posterior lower tooth is moderately separate from the jaw joint{. The lower teeth are enor- mously large and have a triangular, fully erected cusp without a regular serration {lower teeth have a more or less inclined cusp with or without serration|. In contrast, upper teeth are weak, and Anatomy of Cookie-Cutter Shark 815 (} \ 1 1 | i \ Fic. 3. A) Neurocranium and mandibular and hyoid arches of Isistius brasiliensis showing jaw suspension (the hyomandibula is drawn from dorsal view), and lateral view of hyoid arch as inset (right upper); B) dorsal view of mandibular arch of Jsistius brasiliensis; C) same as (A) in Squaliolus laticaudus; D) same as (B) in Squaliolus laticaudus. Scales are 10 mm. 816 S. SHIRAI AND K. NAKAYA the functional tooth rows are not supported by the palatoquadrate in part |upper tooth rows are entirely supported by the palatoquadrate}. The hyoid arch (Figs. 3A, 5A) of Jsistius is also modified considerably. The hyomandibula (hm), like Dalatias, is short, flat, and directed laterally, not inclined posteriorly {the hyomandibula is somewhat longer, compressed, and directed pos- tero-latero-ventrally (Fig. 3C)|. The proximal ter- minus of hyomandibula is bifurcate, and its pos- teroventral condyle is composed of a separate cartilaginous piece (jhm) {it is also slightly bifur- cate, but without a separate cartilage}. The distal terminus of hyomandibula has a small expansion to receive the mandibular knob of mandibula (mk) at its anterior edge and a simple depression for the articulation with the ceratohyal at its posterior edge. Thus, the hyomandibula-ceratohyal arti- culation is somewhat behind the mandibula- hyomandibula articulation {this feature is also seen in Dalatias, but not so separate as in Isistius ; the distal terminus of the hyomandibula is distinct- ly bifurcate for the articulation with the ceratohy- al, and the hyomandibula-ceratohyal articulation is very close to the mandibular knob}. The ceratohy- nem ———— Fic. 4. Head musculature of two dalatiines. al (ch) of Jsistius is very stout, connected with the hyomandibula by a thick ligament (ligamentum hyomandibulo-hyoideum: lhc); this ligament arises from the basicranium below the hyomandibular fossa, covering the posterodorsal surface of the hyomandibula, and it is inserted on the anterodor- sal surface of ceratohyal {the ceratohyal is also developed, but such a ligament is absent except Dalatias|. The large quadrangular basihyal (bh) has a simple concavity (fbc) at its posteroventral surface to receive the anterior terminus of the ceratohyal (Fig. 5A) |the basihyal is smaller, generally trapezoid in shape, and the fossa for articulation with the ceratohyal is situated on its posterolateral margin (Fig. SB-C)}. Isistius has strongly reduced branchial arches, and the basibranchial copula (hypobranchials plus basibranchials) is almot absent (Fig. SA). The first ceratobranchial (cb1) is not articulated with the basihyal, situated far posterior from it |in Dalatias, the ceratobranchial-basihyal articulation is also absent, but the first ceratobranchial is supported by the hypobranchial as a transverse bar (hb1: Fig. 5C); in other squaloids, the first ceratobranchial is connected with the basihyal tightly or loosely (Fig. AX Mi \ \Y wit A-B) lateral (A) and ventral views (B) of J/sistius brasiliensis; C-D) lateral (C) and ventral views (D) of Squaliolus laticaudus (B and D: mandibula is removed in upper half, above double solid line). Scales are 10 mm. Anatomy of Cookie-Cutter Shark 817 5B)}. Jsistius is unique in having an elongate separate hypobranchial in the fifth arch (hb5), to which the large, paddle-shaped ceratobranchial (cb5) is loosely connected |the fifth arch does not have a discrete hypobranchial}. The gill raker is completely absent {present in most other squaloids} . The shoulder girdle (Fig. 4B) of Jsistius is char- acterized by the fragile and curiously curved cora- coid (co). It courses medially for a short distance, turns anteriorly at a right angle, and extends anteroventrally to form a peculiar hairpin turn at the symphysis that forms the pericardial floor |the coracoid bar is a stout transverse bar, and this is not strongly curved (Fig. 4D)}. Musculature The mandibular muscles (Fig. 4A-B) are also modified in Jsistius as follows: (1) the adductor mandibulae (am) is very reduced, and its insertion onto mandibula is restricted slightly below the jaw joint via tough tendinous tissue {the adductor mandibulae is housed at the whole posterior half of mandible (Fig. 4C)}; (2) the thick suborbitalis (so) is inserted onto the tendinous tissue of the adduc- tor mandibulae {the suborbitalis is inserted onto the anterior part of mandibula with (Dalatias) or Fic. 5. Basal portion of hyoid and branchial arches with the coraco-branchiales of three dalatiines. brasiliensis (left—ventral view showing the coraco-branchialis 1; right—ventral view showing the coraco- branchialis 5; B) Squaliolus laticaudus (ventral view); C) Dalatias licha (ventral view). Scales are 10 mm. without (Euprotomicrus and Squaliolus) a tendon for insertion (Fig. 4C-D)}; (3) the adductor m. superficialis (ams) is completely separate from the adductor mandibulae, arising from the outer sur- face of the mandibula {this muscle is usually a subdivision of the adductor mandibulae, situated on the otic process of palatoquadrate}; (4) a unique thin muscle, the levator labialis (IIb), arises from the otic process of palatoquadrate and is inserted on the medial edge of the posterior mouth groove {absent}; and (5) the anterior and pos- terior extremities of the constrictor hyoideus dor- salis (chd) are inserted on the mandibula | the ante- rior edge of this muscle is inserted onto the pala- toquadrate, not reaching the mandibula}. The hypobranchial longitudinal muscles, occu- pying the space below the oral cavity and pharynx, are noticeably modified in Jsistius. The genio- coracoideus (gco) is thin and broad, tapering for- ward, and originates from the ventral surface of the hypaxial muscle below the origin of pectoral fin (Fig. 4B). Another slip of muscle (gcob), arising from the anterior curved surface of the coracoid and inserted onto the mandibula with the genio- coracoideus, is present (genio-coracoideus 2) {the genio-coracoideus is an unpaired, narrow muscle bundle that arises from coracoid symphysis or A) Isistius 818 S. SHIRAI AND K. NAKAYA pericardial membrane, and the genio-coracoideus B is absent (Fig. 4D)}. The rectus cervicis, often subdivided into the anterior (=coraco-hyoideus: coh) and posterior halves (=coraco-arcuales: coa), is expanded well and is directly connected with the hypaxial muscle posteriorly {the rectus cervicis arises from the coracoid, and is not directly connected with the hypaxial muscle}. The coraco- branchiales (cob) are simplified, composed of two slips (Fig. 5A); one (cobl) is a wide and flat, arising from the pericardial membrane and in- serted on the basihyal; the other (cob5) originates on the dorsal side of the fascia of coraco-arcuales, and its insertion is on the fifth hypobranchial and ceratobranchial {the coraco-branchiales are com- posed of five slips (Fig. 5B); their origins are the fascia of rectus cervicis (first) and the pericardial membrane or the anterior surface of coracoid (others), and insertions are, from front, the basihyal, the ceratobranchial and hypobranchial of the second, third, fourth, and fifth arches; Dalatias (Fig. 5C) also has two separate coraco-branchiales like Jsistius, but the posterior slip arises from the coracoid}. DISCUSSION We can often find crater wounds on the surface of tunas and swordfishes in a fish market (Fig. 6). It is round or oval with the size of 5 cm by 7 cm at the most. Jones [1] discussed the probability of the relation between this wound and the feeding mechanism of Jsistius. His arguments on the feeding function of this shark are summarized as follows: (1) Isistius gives such a wound by pushing its lower tooth row against the body of prey and then scooping the flesh. In his experiment with a dead fish, the scoop-like lower teeth of Jsistius can produce a crescentic wound, which is very similar in size and shape to that often observed in tunas caused by the incomplete attack. (2) Isistius can make a strong oral vacuum when the tongue (basihyal) is pulled back by the volumi- nous throat muscle (rectus cervicis). Concurrent- ly, the shark closes its spiracles and protrudes lips completely around the mouth. (3) The body rotation should be facilitated by the Fic. 6. Crater wounds on sides of tunas and swordfish at the Kesennuma Fish Market, Miyagi prefecture, Japan (A-B, Tunnus alalunga; C, Xiphias gladius). A wound in (C) penetrates into the body cavity. Each wound is 5 to 7 cm in a major axis. (B and C: photo by Toshiro Chiba). drag of water when J/sistius heads on the prey as expected in many examples. At the next moment, the body of Jsistius would rotate around the point of attachment until the shark is oriented in the same direction as the prey. The crater wound is then completed. Morphological features of this shark can explain these functions as mentioned below. On the first hypothesis, Jones [1] noticed the peculiarity of dentition in Jsistius. This shark shares a typical dignathic heterodonty with many other squaloids, which should work for the clutch- ing-cutting-type feeding habit [11]. Squaloid sharks (Figs. 3C, 4C) generally hold food items by the teeth of both jaws, and cut them by sharp lower teeth. Developed adductor mandibulae plays the main role in this function. In Jsistius (Figs. 3A-B, 4A-B), upper and lower teeth cannot be engaged each other. Upper and lower jaws are ill-balanced in size, and the palatoquadrate is Anatomy of Cookie-Cutter Shark 819 subdivided into the anterior and posterior halves. The adductor mandibulae is small, not widely inserted on the lateral surface of lower jaw. At the collision with a prey, the lower jaw is given a strong shock (reaction of the collision), but it must keep the appropriate angle to penetrate the flesh with the lower teeth. The massive suborbital muscle, with the reduced adductor mandibulae, appears to support this function. In contrast with the tough lower teeth, the role of upper teeth seems rather moderate. Jones [1] observed several scratches produced by Isistius’s upper teeth. These are opposite to a crescentic wound caused by lower teeth, at barely inside the crater if this attack were complete. The flexible upper jaw might be bent at the middle portion to make the oral area greater at the collision. Upper teeth are thus effectively anchored on the prey to prevent the shark body whisked off during the gouging. Such unique jaw morphology is apparently not suited to clutch and masticate foods. The scoop- like tooth row of the lower jaw along the ventral surface of body thus appears more appropriately designed to gouge out the flesh of prey as sug- gested by Jones [1]. Jsistius usually feeds small pelagic fish and squids [1, 7, 8], and this scooping function also should occur for these small preys. Second, Jones [1] explained that the strong oral vacuum is induced by the function of a large basihyal, the developed rectus cervicis, spiracles, and lips. Thickened labial fold and gum in the upper jaw and the flexible upper jaw must be useful to fit the oral margin to the shape of food. We found the unusually curved shoulder girdle, and this allows the rectus cervicis directly contacts with the thick hypaxial muscle. The oral vacuum will be thus induced by the contraction of whole ventral muscle of body. We also noticed that the basal portion of branchial arches is incomparably reduced, especially in the separation between the first ceratobranchial and basihyal. This must be concerned with stronger and smoother retraction of the basihyal. Isisttus has another mechanism to make the stronger oral vacuum with ingenuity (Fig. 7). In most shark groups, the lower jaw is lowered main- ly by the function of the whole hyoid arch and muscles inserted onto the basihyal (rectus cervicis and coraco-branchialis 1). As shown in Figure 7C, Fic. 7. Diagrams of the retraction of basihyal and movements of the associated parts of (A-B) Jsistius and (C-D) Squaliolus (upper—lateral view; lower—dorsal view). Black dots show the following points: /, the ligamentous connection between ceratohyal and mandibula; 2, the articulation between mandibular knob and hyomandibula; 3, the hyomandibula-ceratohyal articulation; and 4, the basihyal-ceratohyal articulation. 820 S. SHIRAI AND K. NAKAYA the ligamentum mandibulo-hyoideum is usually short and fastens the ceratohyal and mandibular rigidly (point /) somewhat below the hyomandibu- la-ceratohyal articulation (point 3). The point 3 is the center of rotation of the ceratohyal, and it is very near the center of rotation of the mandibula (point 2). When the basihyal is retracted (Fig. 7D), therefore, the mandibula will be lowered at the almost same degree as the rotation of ceratohy- al, and the oral cavity does not expand so strongly. In Isistius (Fig. 7A), the point 3 is characteristical- ly posterior to the point 2 by the width of hyoman- dibula. The ligamentum mandibulo-hyoideum (/) joins the posterior margin of mandibula with the ceratohyal near the hyomandibula-ceratohyal ar- ticulation (3). The anterior part of ceratohyal thus can be somewhat free from the mandibula. Moreover, the basihyal has a semi-globate fossa for the articulation with ceratohyal (point 4), and it gives the flexibility to the basihyal-ceratohyal When the basihayl is pulled back (Fig. 7B), the anterior end of the ceratohyal can swing posterointernally like man’s wrist when elbows are spread out. Therefore, the basihyal can move backward more strongly than the lowering of mandibula, and the oral cavity is expected to be more enlarged than the other squaloids. The bigger the oral cavity is, the stronger the oral vacuum should be induced. The shape and size of mandibula and basihyal clearly show that Isistius essentially has a larger oral cavity. The movement of ceratohyal mentioned above will serve to expand it further. In addition, the mod- ification of the hyomandibula should be also re- lated with this function. This cartilage is directed posterolaterally when the mouth is closed and has a separate cartilaginous piece at the proximal end. The hyomandibular fossa on the otic capsule is well developed. These structures probably make the hyomandibula lower more strongly to enlarge the oral cavity. On the third hypothesis (mechanism of body rotation), Jones [1] explained that /sistius utilizes the locomotion of prey and the drag of water for gouging. It is reasonable for the fast swimmers. However, I/sistius attacks the slower fishes and cetaceans, and even the dead fishes. Almost all the wounds on tunas and swordfishes we observed articulation. were so fresh with bleeding that these wounds would have been given after they were restrained by the fishing gear (long line) or dead on hooks (Fig. 6). The body rotation of Jsistius should be thus more spontaneous than Jones considered. The body itself must be twisted by the differential contraction of right and left body muscles. We consider that these three basic functions for feeding occurs in a very moment. This small shark stabs the prey with the sharp lower teeth at the collision. Immediately after this, the ventral body muscle must be suddenly contracted to pull the basihyal back and the oral vacuum is induced in order to stick the mouth to the prey. The contrac- tion of body muscle should simultaneously create a twisting moment of the body itself with or without the help of the drag of water. A half turn of body probably makes a complete crater-like wound. Two dwarf dalatiine shark genera, Euprotomi- crus and Squaliolus, have the similar habitat and outlook to Jsistius, and these were often compared with each other [8, 12, 13]. The feeding mecha- nisms of Euprotomicrus and Squaliolus, however, should be the typical squaloid-type (clutching- cutting type) [11], because their feeding apparatus is essentially same as that of squaloids. Isistius rather share some characteristic conditions in the feeding apparatus with another dalatiine shark, Dalatias licha, i.e., the hyomandibula-ceratohyal articulation somewhat behind the mandibular knob, the first ceratobranchial widely apart from the basihyal, and reduced coraco-branchiales com- posed of two muscle slips. However, Dalatias’s palato-quadrate, arrangement of upper and lower tooth rows, basihyal, and related musculature are well similar to those of other squaloids, and it is unlikely that it takes same feeding manner as Isistius does. Two other rare dalatiine sharks, Euprotomicroides and Heteroscymnoides, whose feeding apparatus is hardly known, cannot scoop out the flesh of fish either because of their typical dalatiine dentition [5, 14]. A culprit of the crater wound is thus clearly identified, the cookie-cutter shark. ACKNOWLEDGMENTS We would like to thank Kunio Amaoka (Laboratory of Anatomy of Cookie-Cutter Shark 821 Marine Zoology, Faculty of Fisheries, Hokkaido Uni- versity) who gave us considerable support and sugges- tions during this study. We are also indebted to Stuart G. Poss (Gulf Coast Research Laboratory Museum, Ocean Springs) for constructive reading of the manuscript. Toshiro Chiba (Kesennuma City) kindly took photo- graphs of crater wounds. Keiichi Matsuura, Richard Rosenblatt, “H. J.” Walker, Jr., Yasunori Sakurai, Shigeyuki Kawahara, and Harumi Yamada provided specimens for comparative studies. REFERENCES 1 Jones, E. C. (1971) Isistius brasiliensis, a squaloid shark, the probable of crater wounds on fishes and cetaceans. Fish. Bull., 69: 791-798. 2 Van Utrecht, W. L. (1959) Wounds and scars on the skin of the common porpoise, Phocaena phocaena (L.). Mammalia, 23: 100-122, pls. 6-9. 3 Baker, A. N. 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(1967) External and internal characters, horizontal and vertical distribution, luminescence, and food of the dwarf pelagic shark, Euprotomicrus bispinatus. Bull. Scripps Inst. Oceanogr., 10: i-vi+ 1-64, pls. 1-8. Daniel, J. F. (1934) The elasmobranch fishes. 3rd ed. Univ. California Press., Berkeley, California, xii +332 pp. Edgeworth, F. H. (1935) The cranial muscles of vertebrates. Cambridge Univ. Press, Cambridge, vill+493 pp., 841 figs. Cappetta, H. (1987) Handbook of Paleoichthyolo- gy. Volume 3B. Chondrichthyes II. Mesozoic and Cenozoic Elasmobranchii. Gustav Fischer Verlag, Stuttgart, i11+193 pp. Hubbs, C. L. and McHugh, J. L. (1951) Rela- tionships of the pelagic shark Euprotomicrus bispi- natus, with description of a specimen from off California. Proc. Calif. Acad. Sci., 27: 159-176. Seigel, J. A. (1978) Revision of the dalatiid shark genus Squaliolus: anatomy, systematics, ecology. Copeia, 1978: 602-614. Stehmann, M. and Krefft, G. (1988) Results of the research cruises of FRV “Walther Herwig” to South America. LXVIII. Complementary redescription of the dalatiine shark Euprotomicroides zantedeschia Hulley and Penrith, 1966 (Chondrichthyes, Squali- dae), based on a second record from the Western South Atlantic. Arch. FischWiss., 39: 1-30. pitt att ital ow nee i Widfey Get bh: hpi jite- ts ang pn He Sat! eon! eavolia ) Vile bes Hist é heey ade ; I Me i ie mer ee ‘ i Tite 4 iia 4 ye 1AtL) ah AGT EL av jung witadind reas ‘on tye.” wit) vara WN Oe aeeneacon 5 tines) Hab ONAN ed ee wba 4 "i fli: rey i an bi ane “Arn arin padsonderd neh A ee ee pg catate wile eel baal A Oe ee ey ea Layee. .. i aid 4 ae ae (nia y me ii} hi s ¥ {ij thi teddy Ww is iyy PNAS jm a He Hey Fi hiithe PRP? al if ide OF “ohhh FY fi bie siete ah yay wt), (tore VaR ag A, ilurbertyles), Ls obsiah (ade BY bby Miter” tt (yen epee pie a fhe 7 4 (ov 1 i apes Hi ya % ai nas caaikoorat al spats bev A, yee ae 18 4s iy aL AL { ie on A ‘alaill haley Aah i MAR . ih ae yi iee ph iu "Gite dbo ued eet PENS nat be Tag : veered SS come’ rt Limity ie Diwan web «roar aaa ancy dey, ahh en a Pega he, iA satay. yarns. ges) min ‘ muy tt | “ie re ear j \ eui\ 7 ai, 7 $ soa boy vr Py i | YS he | how cane hi Nae wept bangle every: ? —_ ZOOLOGICAL SCIENCE 9: 823-834 (1992) Neuropeptide Y Innervation of Cerebral Arteries in Microchiropteran Bats Korcut ANpbo! and Suser ARAI* ‘Biological Laboratory, Liberal Arts, Kyushu Sangyo Universtiy, Matsukadai, Higashi-ku, Fukuoka, 813, and *Department of Oral Anatomy, Kyushu Dental College, Manazuru, Kokura-Kita, Kitakyushu, 803, Japan ABSTRACT— The distribution and origin of neuropeptide Y (NPY) immunoreactive nerves in the walls of the cerebral arteries were investigated in two microchiropteran species. The majority of cerebral perivascular NPY nerves originated in the sympathetic internal carotid nerve emanating from the superior cervical ganglion. The NPY nerves ran in the carotid canal, intermingled with parasympathetic axons containing vasoactive intestinal polypeptide (VIP), and reached the circle of Willis along the internal carotid artery. In addition, a part of cerebrovascular NPY nerves entered the cranial cavity along the internal ethmoidal and vertebral arteries. The presence of NPY nerves with VIP but not with tyrosine hydroxylase, a key enzyme for noradrenaline synthesis, seems to indicate that some of the cerebral perivascular NPY nerves are not of sympathetic in nature. The supply of NPY nerves to the cerebral arterial tree of the small bat was the richest among mammalian species studies up to date. Compared with the general mammalian pattern, NPY nerve supply was much more prominent in the vertebrobasilar system, particularly along the walls of the posterior cerebral and basilar arteries, than in © 1992 Zoological Society of Japan the internal carotid system. INTRODUCTION Many histochemical studies have demonstrated dual innervation of cerebrovascular bed by sym- pathetic noradrenergic and parasympathetic cho- linergic nerves in a variety of mammalian species [1-4]. Besides these two classical populations of cerebrovascular nerves, recent advance in im- munohistochemical techniques revealed that cere- bral vessels are innervated by populations of neurons containing various biogenic active pep- tides, such as neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), substance P (SP), and calcitonine gene-related peptide (CGRP) [5, 6]. Of these neuropeptide-containing nerves, NPY nerves have been identified to be largely sym- pathetic in nature (coexistence of NPY with norad- renaline (NA) in the same cerebral perivascular nerves) and mainly originate from the superior cervical ganglion (SCG) [7-10]. Pharmacological- ly, NPY is known to exert a potent vasoconstrictor Accepted January 6, 1992 Received June 4, 1992 effect on cerebral arteries, with a long lasting action that is resistant to a-adrenoceptor and cho- linergic receptor blockages [11-13]. NPY may also potentiates prejunctionally the vasoconstrictor effect of noradrenergic nerves [10, 12]. Thus, it has now been emphasized that NPY, as well as other neuropeptides (VIP and SP for vasodilator, and CGRP for vasoconstrictor), participates in the regulation of the cerebral circulation as a neuro- transmitter or neuromodulator [14-16]. The above-mentioned morphological and pharmacolo- gical evidence concerning the cerebrovasular pep- tidergic innervation is issued mostly from labora- tory mammals, especially from the rat, cat and guinea pig. As a general angioarchitecture for mammalian cerebral circulation, the internal carotid and ver- tebral arteries (ICA, VA) enter the cranial cavity through the carotid and vertebral canals, respec- tively, and send many branches to the brain. The branches arising from the ICA are distributed to most parts of cerebrum and diencephalon, forming the anterior part of cerebral arterial tree, the internal carotid system (ICS). The right and left 824 K. ANDO AND S. Aral VA joint together to construct the basilar artery (BA) on the midline of medulla oblongata. The branches from the VA and BA supply the brain area caudal to the caudal part of cerebrum, form- ing the posterior part of the arterial tree, the vertebrobasilar system (VBS). Rostrally, the right and left ICS are jointed to each other in front of the optic chiasm by the anterior communicating artery. Caudally, the right and left terminal bran- ches of the BA are jointed ipsilaterally to the intracranial part of the internal carotid artery, the cerebral carotid artery (CCA), by the posterior communicating artery, or by the posterior ramus. Thus, the ICS and VBS connect with each other at the base of brain to build up the circle of Willis. In microchiropteran species, the cerebral vascu- lar system shows structural difference from the basic mammalian pattern [17-20]: the major cere- bral arteries of the ICS are so fine as to be almost non-functional, while the arteries of the VBS are markedly well-developed. Consequently, blood supply to the brain is predominantly supplied by the VBS. Furthermore, the heart rate and body temperature of small bats strikingly increase or decrease in flying and hibernating periods [19]. Such special behaviour and ecology of these mam- mals should cause the seasonal change of physiolo- gical conditions in their brains, and may also involve cerebral blood flow that is unusual in other mammals. Therefore, it is a matter of interest to explore the neurogenic mechanisms by which the bat cerebral vascular bed is regulated, and to ACA anterior cerebral artery ACA1 rostral part of the anterior cerebral artery ACA2 caudal part of the anterior cerebral artery AR anterior ramus BA basilar artery C cochlea CA common carotid artery CC carotid canal ECA external carotid artery CCA cerebral carotid artery IAA internal acoustic artery ICA internal carotid artery ICS internal carotid system internal ethmoidal artery middle cerebral artery OC optic chiasm posterior cerebral artery proximal part of the posterior cerebral artery compare them with those of other mammals. Such a comparative study provides new insights that allow further comprehension of the neuronal in- fluence on the mammalian cerebral circulation. The aim of the present study was to investigate the distribution, origin and pathway of NPY nerves surrounding the cerebral arteries of the bent- winged bat and the greater horseshoe bat, and to correlate them with those of noradrenergic and VIP nerves using double immunostaining. MATERIALS AND METHODS Tissue preparation Four greater horseshoe bats (Rhinolophus fer- rumequinum nippon) and eight bent-winged bats (Miniopterus schreibersii fuliginosus) were used in this study. The indivuduals examined were of either sex; all were adults. The animals were anesthetized with ethyl ether and perfused through the left ventricle of the heart with ice-cold Ringer’s solution, followed by 50 ml of Zamboni’s fixative [21]. The major cerebral arteries, the internal carotid and vertebral arteries outside the cranial cavity, and the superior cervical ganglia were rapidly dissected out, and postfixed in the same fixative for 18h at 4°C. They were washed with 70% ethanol, dehydrated in a graded ethanol concentration, and placed in cold 0.1 M phosphate buffer (PB, pH 7.4). The specimens were then immersed in PB containing 10% and 20% sucrose PCA2 distal part of the posterior cerebral artery PR posterior ramus S stapes SA stapedial: artery SCG superior cervical ganglion SCA superior cerebellar artery SICN sympathetic internal carotid nerve TB terminal branch of the basilar artery VA vertebral artery VAI rostral part of the vertebral artery VA2 caudal part of the vertebral artery VBS vertebrobasilar system I rhiencephalon II cerebrum Ill cerebellum IV medulla oblongata Vv diencephalon Cerebrovascular NPY Innervation in Bats 825 for 2 days each at 4°C. For whole-mount prepara- tion, the cerebral arteries, and the extracranial internal carotid and vertebral arteries were put in 0.1M phosphate-buffered saline (PBS, pH7.2), and stripped of pia mater or outer adventitial connective tissue. The nerve bundles accompany- ing the two extracranial arteries were also dis- sected and carefully stripped of their perineural connective tissue. They were then stored in PBS for 1-24h at 4°C. For sectioning, the superior cervical ganglion and small blocks of the brain parenchyma containing intracerebral vessels were soaked with 10% gelatine PB for 1-2 h in vacuo at 38—40°C after the treatment with sucrose PB. The gelatine-embedded samples were quickly frozen in isopentane or acetone chilled with dry ice, sec- tioned at 20 um thickness in a cryostat, and stored in PBS at 4°C. Immunohistochemical protocol Whole-mount preparations and free-floating sections were processed for immunohistochemistry by avidin-biotin immunoperoxidase [22] or indirect immunofiuorescence [23] techniques. Before the antigen-antibody reaction of peroxidase immuno- histochemistry, the specimens were treated for 1 h at room temperature with PB containing 0.1% hydrogen peroxide to inhibit endogenous perox- idase in tissues, and washed with PBS containing 0.3% Triton X-100 (PBST). The specimens were incubated for three days at 4°C in polyclonal rabbit NPY antiserum (Code no. 06246; Cambridge Re- search Biochemicals Lts., Harston, England) at a dilution of 1:500, followed by washing in cold PBST. For peroxidase immunohistochemistry, the specimens were incubated with biotinylated anti- rabbit immunoglobulin G (IgG) and then with rabbit avidin-biotin-peroxidase complex (Vectas- tain kit, Vector Lab., England) for 1 h each time at room temperature. They were stained with 3,3’- diaminobenzidine for 5-10 min [24], mounted on glass slides coated with chrome-alum gelatin, and examined under a light microscope. For im- munofluorescence staining, the whole-mounts and sections treated with the NPY antiserum were incubated for 1-2h at 37°C in sheep anti-rabbit IgG conjugated with fluorescein isothiocyanate (FITC) (Cappel, PA) at a dilution of 1:100. They were then washed with PBS, mounted in glycerine- PB mixture (1:1), and examined in a fluorescence microscope. To identify the location of NPY and two other neuronal substances (NA, VIP) in the same speci- men, double-immunostaining was carried out with primary antisera raised in different species and second antisera labelled with different fluorochromes. Tissues were first incubated with rabbit NPY antiserum and then with sheep anti- rabbit IgG conjugated with FITC. Next, they were exposed to monoclonal mouse antiserum against tyrosine hydroxylase (TH), a key enzyme for NA synthesis, (Code no. 10968620-01; Boehringer Mannheim, W. Germany) at a dilution of 1:1 (40 vg/ml), or polyclonal guinea pig antiserum against VIP (Code no. M-8701-1, Milab, Malmo, Sweden) at a dilution of 1:1250, and then to rabbit anti- mouse (Amersham, England) or goat anti-guinea pig IgG (E-Y Laboratories, INC, San Mateo, CA) conjugated with Texas Red (TR) at a dilution of 1:200. Tissues were mounted in the PB-glycerine, and viewed under a fluorescence microscope. Coexistence of NPY and TH or VIP was estab- lished by switching between the filter combinations for FITC-and TR-induced fluorescence. To check the specificity of NPY, TH or VIP immunoreactivity, the following control experi- ments were performed: (1) incubation with the serum from non-immunized rabbit, mouse or guinea pig as primary antiserum; (2) incubation omitting the primary antiserum in the first step of immunohistochemical procedures; (3) incubation with primary antisera preabsorbed with the corres- poinding peptide (more than 10 g/ml diluted antiserum) for 24 h at 4°C. When the tissues were treated in one of the three ways, no specific immunoreactivity was found in neurons. RESULTS Course of the nerve bundles accompanying the internal carotid artery within the tympanic cavity In the greater horseshoe bat and the bent- winged bat, the ICA and the sympathetic nerve bundle accompanying it (the sympathetic internal carotid nerve (SICN) emanating from the SCG) 826 K. ANDO AND S. ARAI (Rostral side) (Caudal side) 1 Fic. 1. Course of the internal carotid artery, sympathe- tic internal carotid nerve, and microganglion- containing nerves in the typmanic cavity of the bent-wingted bat. Asterisks indicate microganglia in the nerve bundle accompanying the sympathetic internal carotid nerve. 2A eu air 2B entered the tympanic cavity immediately after their branching. They then ran rostro-medially on the surface of the cochlea in close association with each other, and reached the cranial cavity through the carotid canal (Fig. 1). In the bent-winged bat, another delicate nerve bundle, with two to three microganglia, accompanied the tympanic part of the SICN at the level where the ICA on the cochlea gave off a branch to the stapes, and then ran towards the carotid canal. Arterial Supply of the brain The cerebral arterial systems of the greater horseshoe and bent-winged bats were basically identical to each other. The middle and anterior cerebral arteries (MCA, ACA), and the internal ethmoidal artery (IEA) belonging to the ICS were distributed only to the ventral surface of cerebrum (Fig. 2A). Compared to this, the posterior cere- bral artery (PCA) arising from the VBS ran caudo- rostrally within the longitudinal cerebral fissure to reach as far as the rhiencephalon, and during its course sent numerous branches to most parts of the medial, dorsal, and lateral surfaces and paren- chyma of cerebrum (Figs. 2BC). 2c Fics. 2A, B, C. The arterial supply to the brain of the greater horseshoe bat, from ventral (A), dorsal (B), and midsaggital (C) aspects. Cerebrovascular NPY Innervation in Bats 827 Immunohistochemistry In the two microchiropteran species studied, most of cell bodies in the SCG was immunoreac- tive for NPY (Fig. 3A), and no VIP immunoreac- tivity was found in this sympathetic ganglion (Fig. 3B). The tympanic and carotid parts of the ICA had a scarce supply of NPY nerves, and the SICN accompanying these cranial parts of the ICA ex- pressed intense NPY immunoreactivity over its entire length (Figs. 4, 5). In the bent-winged bat, two to three microganglia in the nerve bundle accompanying the tympanic part of the SICN comprised a large number of cell bodies with VIP Fics. 3A, B. Fluorescence photomicrographs of cross-sections of the superior cervical ganglion of the greater horseshoe bat. Double immuno-fluorescent staining for NPY (A) and VIP (B). 150. Fics. 4, 5. Photomicrographs of the internal carotid artery of the bent-winged bat showing NPY immunoreactivity in the sympathetic internal carotid nerve (arrows). Tympanic (4) and carotid (5) regions. X67. Fics. 6,7. Fluorescence photomicrographs of whole-mounts showing VIP (6) and NPY (7) immunoreactivity in the microganglia (asterisks) in the nerve bundle accompanying the sympathetic internal carotic nerve at the rostral part of the typmpanic cavity of the bent-winged bat. Fig. 6x 100; Fig. 7<200. 828 K. ANDO AND S. Aral Cerebrovascular NPY Innervation in Bats 829 (40-S0 cells per ganglion) as reported previously [25], but had no cell bodies with detectable level of NPY (Figs. 6, 7). Such microganglia were not detected in any parts of the tympanic cavity of the greater horseshoe bat. No appreciable difference in the density of NPY nerves was found between the corresponding ma- jor cerebral arteries of two bats. As was the case in the carotid part of the ICA, the CCA had a very poor supply of NPY nerves. However, several thick fibre bundles with NPY, which also exhibited VIP immunoreactivity, reached the circle of Willis through the CCA (Fig. 8), and provided abundant NPY fibres to the major arteries of both the ICS and VBS. NPY axons from these fibre bundles mainly extended in a caudal direction along the posterior ramus, so that the supply of NPY nerves was much more prominent in the major arteries of the VBS as compared to those of the ICS (Table 1, Figs. 8-13). The immunoreactive nerves were particularly rich along the walls of the PCA to BA, formed complicated meshworks which were orga- nized mainly from thin varicose fibres (Figs. 11, 12). The distal PCA, the major branches of the BA, such as the superior cerebellar and internal acoustic arteries, and the rostral part of the VA were also furnished with well developed networks of NPY nerves (Figs. 11-13). The VA just after entering the cranial cavity was pooly supplied with NPY nerve fibres, but one or two fibre bundles with NPY were present on its wall, and ascended TABLE 1. bent-winged bat towards the BA (Fig. 14). As to the ICS, the walls of the anterior ramus to the caudal part of the ACA were invested with a rich or moderate num- ber of NPY nerves (Fig. 8A), but the immunoreac- tive nerves became distinctly sparse towards the rostral part of the ACA, the MCA, and the IEA (Figs. 9, 10). In addition, one or two thin fibre bundles showing weak NPY immunoreactivity were seen to run along the IEA (Fig. 10), and their fibre branches could be traced to the confluence of the ACA and MCA (Fig. 10). There were no ganglionic structures with NPY along the walls of the whole lengths of the extracranial ICA and VA, and of the major cerebral arteries at all parts of the small bat brain (see Fig. 2). Nearly all of the small branches from the major cerebral arteries of the VBS were supplied with NPY nerves (Fig. 15). Similarly, NPY fibres penetrating into the brain parenchyma via the pial arteries could often be observed in the walls of intracerebral arteries and arterioles of this arterial system, especially those of the PCA (Fig. 16). After the immunoreactive nerves entered the small pial or intraparenchymal branches via the large pial branches, they rapidly decreased in number, and only a few fibres ran spirally or parallel to the axis of arteriolar branches. Double immunostainig for NPY and TH showed that most of the cerebral perivascular NPY nerves on the BA were immunoreactive to TH. Some of them showed no immunoreactivity to TH (Fig. Relative density of NPY nerves in the major cerebral arteries of the greater horseshoe bat and ICS Artery IEA ACAI MCA ACA2 AR CCA PR PCA1PCA2 SCA TA BA VBS IAA VAI VA2 Density 2 2 2a weeS 3 ls 2 5 4 4 5 Sm S4. Prd 2 1, very few fibres; 2, few fibres; 3, a moderate number of fibres; 4, numerous fibres; 5, very numerous fibres. Fics. 8A, B. Fluorescence photomicrographs of the cerebral carotid artery of the greater horseshoe bat with double immuno-fluorescent staining for NPY (A) and VIP (B). Arrows indicate the thick fibre bundles with both NPY and VIP immunoreactivities. 100. Fics. 9-14. Photomicrographs of whole-mounts showing NPY innervation of the major cerebral arteries of the greater horseshoe bat. Fig. 9. Middle and anterior cerebral arteries. 67. Fig. 10. Anterior cerebral and internal ethmoidal arteries. 100. Fig. 11. Posterior ramus, posterior cerebral and superior cerebellar arteries and terminal branch of basilar artery. X67. Fig. 12. Basilar and internal acoustic arteries (BA, IAA). X67. Fig. 13. Confluence of the basilar and vertebral arteries. x67. Fig. 14 Vertebral artery just behind the entrance of the cranial cavity. < 100. 830 K. ANDO AND S. ARaAI iksd=} Fics. 15, 16. Whole-mounts of pial arterial and arteriolar branches of the posterior cerebral artery (15) and sections of its small branches perforating into the cerebral cortex (16) of the greater horseshoe bat. Arrowheads indicate NPY fibers. Fig. 15, 100; Fig. 16, «200. Fics. 17A, B. Fluorescence photomicrographs of the basilar artery of the greater horseshoe bat with double immuno-fluorescent staining for TH (A) and NPY (B). Arrowheads indicate NPY nerve fibres without TH immunoreactivity. 200. Fics. 18A, B. Fluorescence photomicrographs of a branch from the posterior cerebral artery of the greater horseshoe bat with double immuno-fluorescent staining for NPY (A) and VIP (B). Arrowheads indicate the axon with NPY immunoreactivity, but not with VIP immunoreactivity. Arrows indicate the axon with both NPY and VIP immunoreactivities. 200. 17). By double immunostaining for NPY and VIP, VIP immunoreactivity, while others were im- it was evident that some of the cerebral perivascu- _uunoreactive to VIP (Fig. 18). lar NPY axons with terminal appearance had no Cerebrovascular NPY Innervation in Bats 831 DISCUSSION There is conclusive evidence that cerebral peri- vascular noradrenergic and NPY nerves enter the cranial cavity along the ICA, IEA, and VA, and mainly come from the ICA [26]. The main origin of these two types of nerves supplying the cerebral arterial tree via the above three arteries is the SCG [7-10]. This neuronal projection seems to be held the small bats as well: the SICN emanating from the SCG, which expresses intense NPY im- munoreactivity as well as formaldehyde-induced fluorescence for NA, reaches the circle of Willis along the CCA as several fibre bundles. Fur- thermore, the presence of one or two fibre bundles with NPY lying on or along the IEA and the VA is highly suggestive that cerebral perivascular NPY nerves also travel along these two arteries. The present study demonstrated the existence of many VIP axons within NPY fibre bundles on the wall of the CCA of the two microchiropteran species. Since no cell bodies with VIP are found in their SCG, these VIP fibres seem to be of non- sympathetic. In the rat, it has recently been confirmed that cerebral perivascular VIP and cho- linergic nerves are derived from the three distinct cranial parasympathetic ganglia of facial or glos- sopharyngeal nerves, the sphenopalatine ganglion, the internal carotid miniganglia, and the otic gang- lion, and that the major source for cerebrovascular parasympathetic VIP and cholinergic nerves is the sphenopalatine ganglion [27-31]. In the bent- winged bat, Ando [26] demonstrated the presence of microganglia with many VIP and cholinergic cell bodies in the nerve bundle running in close asso- ciation to the tympanic part of the SICN. These microganglia do not emit NA fluorescence, so they are interpreted as being homolgous with the inter- nal carotid mini-ganglia located near the carotid part of the ICA in the rat [32-34] and the monkey [35, 36]. The microganglion-containing nerve bun- dle and the SICN are intermingled with each other to form a nerve bundle consisting of NA fluores- cent and non-fluorescent axons near the carotid canal, and some fibre bundles arising from this nerve bundle enter the cranial cavity along the ICA. Accordingly, it is most likely that in the bent-winged bat, the VIP axons within fibre bun- dles on the CCA, if not all, originate from cell bodies in these parasympathetic microganglia. However, no existence of VIP microganglia in any parts of the tympanic cavity of the greater horseshoe bat indicates that the VIP axons con- tained in the corresponding fibre bundles of this small bat must have their origin at other local or major cranial parasympathetic ganglia. Nerves containing NPY but not NA have been disclosed in the rat VBS [37] after removal of the SCG. Double staining immunohistochemistry combined with retrograde tracing techniques has corroborated in the rat that cerebral perivascular NPY nerves showing no TH immunoreactivity are mostly immunoreactive for VIP, and originate from the cell bodies with both NPY and VIP of the sphenopalatine ganglion, the internal carotid mini- ganglia, and the otic ganglion [34]. The appear- ance of NPY nerves without TH and those with VIP in the pial arteries has also been noticed in small bats in the present study, suggesting that some of the NPY nerves surrounding the bat cerebral arteries are of non-sympathetic in nature. The contribution of neurons from the microganglia in the tympanic cavity of the bent-winged bat to the cerebrovascular NPY innervation seems to be ruled out, because no NPY immunoreactivity has been ascertained within this ganglia. The possibil- ity that neurons with NPY are present in the sphenopalatine and otic ganglia of small bats, and project from there to the cerebral vessels, is still open. The most characteristic feature of cerebrovascu- lar NPY innervation in small bats is that the density of nerves is distinctly much high in the VBS than in the ICS, especially throughout the walls of the PCA to BA. This pattern is quite different when compared to the general pattern of cerebro- vascular NPY innervation reported for the rat [38], gerbil [39], ginea pig [40], cat [13], and man [41]: the nerves are less densely distributed along the VBS than along the ICS, with the lowest supply in the PCA. Thus, the VBS of small bats has the richest supply of NPY nerves among the mammals that have been reported up to now. The predomi- nantly caudal innervation by cerebral perivascular NPY nerves in small bats results in the preferential caudal distribution of NPY axons from the fibre 832 K. ANDO AND S. Aral bundles on the CCA. This is in contrast to the projection of NPY nerves to the rat cerebral arterial system from the same neuronal pathway [26]. The innervation density of NPY nerves in the respective major cerebral arteries of the bats is virtually equal to that of noradrenergic, cho- linergic, and VIP nerves [20, 25], and does not correspond to the SP innervation which is mainly confined to the BA and VA, and to the CGRP innervation which is very sparse in the greater horseshoe bat and lacking in the bent-winged bat [42, 43]. There is much evidence concerning the innerva- tion of small pial and intracerebral arteries by sympathetic noradrenergic nerves [e.g., 1, 44]. NPY nerve fibres of peripheral origin have also been demonstrated to associate with intracerebral arteries and arterioles in the cat [10], whereas peripheral cholinergic and VIP innervation has not yet been precisely substantiated for the brain parenchymal arteries in laboratory mammals. In our previous studies on the bent-winged bat, the innervation of intraparenchymal arteries and arter- ioles not only by sympathetic noradrenergic nerves, but also by cholinergic and VIP nerves arising from a peripheral origin has been frequent- ly encountered in the brain areas where the PCA gives off many branches [20, 25]. The same was true for the NPY innervation of these small brain vessles in the present study. To date, no information on the functional in- volvement of NPY that is contained in sympathetic and parasympathetic nerves has been published for the bat cerebral circulation. However, in addition to the predominant development of the VBS in small bats, the very rich NPY innervation focused on the bat VBS must be considered in relation to the vasomotor actions essential for the functioning of this major arterial system in supplying blood to their brains during the active and hibernating seasons. In laboratory mammals, the effects of NPY on the cerebral circulation, non-adrenergic and non-cholinergic vasoconstriction, and modula- tion for the vasoconstriction induced by NA, have been well documented by pharmacological experi- ments [11-13]. Therefore, it is possible to predict that the VBS in small bats may have an important role in the regulation of local and systemic cerebral blood flow in correspondence with the changes of brain metabolic activity characteristic of these mammals, as a result of such direct and indirect vasoconstrictor effects of NPY nerves. 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Kajikawa, H., Inagawa, T. and Ishikawa, S. (1973) Studies on adrenergic innervation of the cerebral blood vessels using a histochemical fluorescent method. Hiroshima J. Mes. Sci., 22: 169-180. Schon, F., Ghatei, M., Allen, J. M., Mudlderry, P. K., Kelly, J. S. and Bloom, S. R. (1985) The effect of sympathectomy on calcitonin gene-related pep- tide levels in rat trigeminovascular system. Brain Res., 348: 197-200. ZOOLOGICAL SCIENCE 9: 835-842 (1992) © 1992 Zoological Society of Japan Familial Association, Nymphal Development and Population Density in the Australian Giant Burrowing Cockroach, Macropanesthia rhinoceros (Blattaria: Blaberidae) TADAO MATSUMOTO Department of Biology, College of Arts and Sciences, The University of Tokyo, Tokyo 153, Japan ABSTRACT—The Australian giant burrowing cockroach Macropanesthia rhinoceros was studied with respect to population density, familial association and nymphal development. These, and other field observations, were made in the eucalypt open woodlands of northeastern Queensland at intervals from October 1987 to October 1989. Three hundred one nest burrows were excavated in 16 plots (each 8 m X 4m), and 329 adults and lone nymphs (old and middle age) were examined. Thirty-two families, which consisted of a group of young nymphs together with either an adult pair or an adult female were found in and around the plots from Nov. 1987 to Mar. 1988. The cockroaches form family groups in the early spring and close adult female-offspring relationships persist for about half a year. The nymphs reach the sixth or seventh instar by the autumn. Then they disperse from their natal burrows to make their own. INTRODUCTION The cockroaches (Blattaria) are of special in- terest among the presocial insects because they are closely related to the Isoptera (termites), all of whose members are eusocial [1]. The ovovivipar- ous Blaberidae are frequently subsocial. Roth and Willis [2] described the familial associations of 15 species of cockroaches, mostly ovoviviparous, that probably brood their young. Schal et al. [3] discus- sed the reproductive tactics of cockroach females and males. Brood care is generally seen in aggregations of newly-hatched offspring around their mother, the mother to some extent facilitat- ing the nymphal aggregation by remaining im- mobile for varying periods of time [4]. In addition, some species dwelling inside wood, and feeding on it, have a monogamous family life. The link between adults and nymphs in the wood-feeding cockroaches Cryptocercus (Cryptocercidae) and Salganea (Blaberidae, Panesthiinae) is long last- ing, and can last the whole nymphal life [5-8]. The appearance of a monogamous family structure in wood-feeding cockroaches and termites is a true Accepted June 4, 1992 Received April 4, 1992 convergence [9, 10]. The giant burrowing cockroach Macropanesthia rhinoceros is an ovoviviparous blaberid that dis- plays brood care and is the largest and bulkiest blattarid in Australia [11]. The body length of the largest adult male reaches about 8 cm and the live weight reaches 30g. However, little information has been available to date on the ecology in the field. Only Day [12] notes as follows: “Little is known of the life history of Macropanesthia, but some details have been supplied by Mr. W. A. Henson. The roaches are infrequently observed during the dry season from March to October. They burrow quite deeply, about two feet below the surface of sandy soil in stands of cypress pine (Callitris sp.). They make a nest of dead leaves, grass roots, etc., frequently among the pine roots. The young nymphs rarely appear above ground, but following rain the adults burrow to the surface, especially at night”. The present paper deals with familial associa- tions, development of nymphs, population density and other field observations of the giant burrowing cockroach, M. rhinoceros in the eucalypt open woodlands of northeastern Queensland. Studies of the distribution pattern of nests and the material cycles (carbon and nitrogen) in ecosystem medi- 836 ated by the cockroach will be published elsewhere. MATERIALS AND METHODS The giant burrowing cockroach belongs to the Panesthiinae most species of which are found primarily in the Indo-Malayan and Australian re- gions [11]. In the Australian continent, the sub- family is largely restricted to the eastern part including species which live in and feed on dead wood in rain forests (e.g. Panesthia and Ancaudel- lia) and species which inhabit underground bur- rows in open woodlands or grasslands. (e.g. Mac- ropanesthia and Geoscapheus). Almost all of these taxa are found in Queensland but are comparative- ly poorly represented in other states. M. rhi- T. Matsumoto noceros occurs in rather dry areas around the tropical rain forests of northeastern Queensland from Cooktown to Rockhampton along the Great Dividing Range [11]. The study sites are located in the open eucalypt woodlands near Smith Creek about 12 km south- west of a town, Mount Garnet (17.41S, 145.07E, altitude 680m), and near Uramo, about 10 km east of the town in northeastern Queensland. The forest and soil types of Smith Creek and Uramo are not different. The soil is sandy and not developed, the thickness of the whole (A) layer being about 10cm of which litter (Ao) layer is under lcm. The dry season is from May to November and wet season is from December to April and these directly influence the water con- TasLe 1. Number of nest burrows, cockroaches and social units per plot of 32 m* (8 mx4m) in 16 plots 2) . . Plot Plies gait Season, Ne. a No. of cockroaches Social unit No. of survey (temp.) nests Male Female Pair?) Family” 1 Smith 23-28 Oct. 87 Spring 28 15 15 1 1 2 Uramo 32— 2 Nov. (29°C) 18 7 12 0 5 3 Uramo 13-15 Dec. ’87 Summer 20 7 14 1 4 4 Smith 16-18 Dec. (31°C) 29 12 20 3 1 5 Smith 12-13 Mar. 88 Autumn 14 8 0 0 6 Smith 14. Mar. (26°C) 11 4 8 1 0 7 Uramo 15-16 Mar. 18 13 10 3 3 8 Uramo 17. Mar. 26 14 17 3) 2 9 Smith 15-17 Jul. ’89 Winter 13 6 9 2 0 10 Uramo 17-19 Jul. (21°C) 21 11 13 3} 0 11 Uramo 20 Jul. 9 4 5 0 0 12 Smith 21-22 Jul. 16 7 10 1 0 13 Uramo 20- 2 Oct. ’89 Spring 21 11 11 1 0 14 Smith 3— 4 Oct. (26°C) 17 5 14 2 0 15 Uramo 5— 7 Oct. 30 17 14 1 0 16 Smith 8 Oct. 10 5 5) 0 0 Total 301 146 183 24 16 (in average) (18.8) (9.1) (11.4) (1.5) (1.0) ') Mean temperature in the bottom of nest burrows. season did not vary more than the 1 C precision allowed by the thermometer. be calculated. The temperature observed in all nests during the same Thus no standard deviation could ?) These figures include the number of adults, sub-adults and large nymphs, not include young numphs under sixth instar. *») Adult pairs (male and female) » Families consiting of a group of young mymphs together with an adult pair, and families with an adult female and young numphs. Familial Association of Macropanesthia 837 tent of the soil. The sandy soil is compacted in the dry season and is soft but not sticky in the wet season. The largest tree measured was about 30 cm in diameter and ca. 25m high. Grasses are common on the woodland floor and used for pasture in rainy season. Litter accumulation was 263 g/m? near Smith Creek (n= 11, leaf litter= 124 +76 g, small branch litter=139+82 g) and was 126 g/m? near Uramo (n=11, leaf litter =68 +32 g, small branch litter=58 +52 g). The field studies and collections were made on five occasions from October 1987 to October 1989 (Table 1). Sixteen plots (each 8 m x 4 m) and some areas around the plots were investigated. The surface of the sandy soil in plots was removed to a depth of about 10 cm using a scoop, and then, the entrance holes of burrows were mapped. A trench about 50cm wide and 50cm deep was then dug carefully along the nest burrow using shovel for the heavy work, and a trowel for the finer work. The burrows descend about 40cm deep in a broad curve and have a small chamber at the bottom where the adults live and rear their nymphs. We opened 301 nest burrows in and around the plots and examined the cockroaches, food storages, feces, predators and sometimes guests in the bur- rows. These items were dried for about 5 days before being weighed. Some samples of insects and eggs were kept in 80% alcohol. Eggs in ovaries and brood pouches were studied by dissec- tion of females in July and October 1989. Nest temperature was measured by inserting a mercury thermometer into the terminal portion of nest prior to excavating it. The thermometer was calibrated in 1°C intervals, and all observations were made during the day. RESULTS Nest burrows Figure 1 shows a schematic presentation of a nest burrow with an adult pair, nymphs and a predator (centipede) in spring. The size of the nest burrow is about 1 m long and 40 cm deep, and it descends at about a 20° angle. The semi-circular cross-section of the burrow is 4-15 cm in diameter and has a plastered wall. The entrance is con- Fic. 1. Schematic diagram of a nest burrow of the Australian giant cockroach Macropanesthia rhi- noceros with an adult pair, young nymphs and a predator (centiped) in spring. Leaf litters are trans- ported by adults from ground surface at night. The tough adult external skeleton may protect against centipede attack. (drawn by Y. Ohira) cealed in raised loose dirt in the dry season. Two examples of the distribution of nest burrows in the plots No. 4 and No. 8 are shown in Figure 2. Most nest burrows are more or less curved. In a few cases (e.g., Nest No. 30 in Plot No. 4), the burrow was in the form of a spiral. I did not observe the case in which two burrows are connected. The distribution pattern of nest burrows is almost uni- form. About 10% to 50% of burrows in a quadrat appeared to be abandoned. In those burrows, fungi were usually found growing on the remaining feces and food. The cockroaches prefer litter on the ground as food, which contains dead leaves, woods and grasses. A cockroach grasps the food in its mandibles and transports it walking astride of it. Foraging activity apparently takes place mostly at night in the rainy season. We believe the cock- roaches play an important role in litter turnover (unpublished). Population density Table 1 shows the abundance of nest burrows, the number of individuals per plot and the sociality 838 T. MATsuMOoTO Plot 4, Smith Creek Fic. 2. Two examples of the distribution of nest bur- rows in the plots No. 4 and No. 8. Most net burrows are more or less curved. The distribution pattern of nest burrows is almost uniform. Connection be- tween two nest burrows is rare. Several (approx. 10%) of burrows in a plot appeared to be aban- doned. of groups. In the plot (No. 4) in which nest burrows were most abundant, there were 29 bur- rows and 32 large cockroaches. No cases were seen in which three or more adults occured together in a burrow. In each plot, a mean of there was 19 burrows, and 1.5 pairs, 1.01 families were observed, 9.1 males and 11.4 females live in bur- rows. Table 2 summaries the composition of the groups found in the same burrows and shows the total number of individuals including young nymphs which were found with adult pair or adult females. One hundred forty seven adults, 179 non-adults (sub-adults and old nymphs) and 321 young nymphs were collected from 16 plots. The sex ratios of adults and non-adults were slightly lower than 0.5 (males: females=1:1). The sex ratio of young nymphs was not measured. Familial composition Thirty two families consisting of a group of young nymphs together with an adult pair or an adult female were observed in and around the plots from Nov. 1987 to Mar. 1988 (Table 3). Nymphs are housed in the deepest portions of the burrows (Fig. 1). Table 4 summarizes number of eggs in ovaries or the brood sac, and the brood sized of families. In late October on 1987 (late spring), eleven families were observed with an TABLE 2. Composition of the groups collected in 16 plots and number of cockroaches per two or four plots Plots Plots Plots Plots Plots No. 1, 2 No. 3, 4 No. 5-8 No. 9-12 No. 13-16 CREO 23-2 Nov. 12-18 Dec. 12-17 Mar. 15-22 Jul. 3028) Octane 1987 1987 1988 1989 1989 Single (adult @) 0 2 1 ‘ 9 14 26 Single (non-adult ¢) 19 12 28 13 20 92 Single (adlult $) 1 2 11 21 20 55 Single (non-adult $) 19 23 15 10 20 87 Pair (1$+1) 1 4 9 6 4 24 Family ( +nymphs) 4 5 5 0 0 14 Family (% 2 +nymphs) 2 0 0 0 0 2 No. of young nymphs* 157 89 75 0 0 321 Total no. of indv.** 206 141 153 65 82 647 Sex ratio ($/7+¢) 0.45 0.35 0.49 0.46 0.44 * Total number of nymphs consisting of a group with an adult pair or an adult female ** adults and non-adults not including young nymphs TaBLE 3. Family compositions (Adult and young nymphs) of 32 colonies of M. rhinoceros in and around the plots Familial Association of Macropanesthia 839 Number of nymphs Date of Presence of Suey adult(s) (instar) (number) 23 Oct. °87 2 Ist instar 29 2 Ist instar 13 30 Oct. 2, 8 lst instar 16 2 Ist instar 17 31 Oct. 9. Ist instar 32 9 Ist instar 32 @ Ist instar 28 Q Ist instar 20 2 Nov. 2 Ist instar 29 4 Nov. Oo, # Ist instar 24 9, 8 Ist instar 22 13 Dec. °87 Oo 2, 3rd instars 20 @ 2, 3rd instars 15 Q 3, 4th instars 13 15 Dec. @ 2, 3rd instars D2 a 3rd instar 21 17 Dec. @ 2, 3rd instars 11 19 Dec. g& 3rd instar 24 2 3rd instar 17 2 3, 4th instars 19 - 2 3, 4th instars 18 @ 3, 4th instars 7 15 Mar. ’88 2 5, 6th instars 19 g 5, 6th instars 17 g 5, 6th instars 12 17 Mar. @ 5, 6th instars 11 o@ 6th instar 17 18 Mar. g 5, 6th instars 18 @ 5, 6th instars 16 Q 5, 6th instars 13 g@ 5, 6th instars 10 2 6th instar 15 average brood size of 22.8. Interestingly, in only four of thirty two cases were adult males M. rhinoceros found in familial associations with nymphs and females. These nests were excavated in the spring of 1987. In all other cases (n=28), only adult females were found in familial associa- tions with nymphs. In middle December of 1987 (summer), nymphs had reached third or fourth instar and brood size decreased to a mean of cockroaches 17.0. No adult males were observed in family groups in the summer. In middle March on 1988 (autumn and rainy season), nymphs had reached the fifth or sixth instar and the mean brood size was 14.8. In late July of 1989 (winter), no families with young nymphs were observed. M. rhinoceros is a ovoviviparous cockroach and new nymphs emerge from the brood sac of female in spring. All eggs in the ovaries or sacs of each adult female were removed and weighed as a group in July 1989. The number of eggs in ovaries is 23.8 on average as shown in Table 4. The body length of pre-emergent nymph in brood sac is about lcm. The newly-emerged cockroaches might appeared first in middle October (late spring). Growth of nymphs The new nymphs eat leaf litter stored in nest burrow by the adults. All data on the size of young nymphs living in nest burrows with adults in late October 1987, mid December 1987 and mid March 1988 are presented in Figure 3. Nymphs seem to grow synchronously, and disperse from their nest burrow in the sixth instar, after which they live a solitary existence until they become adults. The body length of adults is about 7-8cm. The size distribution of pronotum widths of old nymphs and adults living in a solitary life or forming a pair are TaBLE 4. Number of eggs in ovaries or brood sac, and brood size of Macropanesthia rhinoceros Date No. of female Age of rood! Se of surve SEAS with offsprings offsprings y Pune Pants Mean+S.D. Range 23 Oct.—4 Nov. ’87 Spring 11 1st instar 23.8+6.7 13-32 13-19 Dec. °87 Summer 11 3—4th instar 17.0+5.1 7-24 12-18 Mar. ’88 Autumn 10 5—6th instar 14.8+3.1 10-19 15-22 Jul. °89 Winter 16 Eggs in ovary 23.8+5.3 13-33 20 Sep.—8 Oct. °89 Spring 17 Eggs in brood sac 22.8+4.6 14-29 840 T. MaATsuMoTO middle Mar. '88 (fall) No. of individuals Width of pronotum (mm) Fic. 3. The size distribution of pronotum widths of young nymphs living in nest burrows with adults on late Oct. ’87, middle Dec. ’87 and middle Mar. ’88. Nymphs seems to grow synchronously. Instars are denoted by Roman numerals. 22 204 a 18 Female = 16 3 14 > 5 12 =eH0) © 8 (2) ome 4 2 P 0 IAA 14 16 18 20 22 24 26 28 30 32 34 22 20: Single b 18 &2 Family we 16 Pair 3 14 2 12 mo} & = (e) fe} z ONPROAWDS 14 16 i) iS) (e%) aS 18 20 22 24 26 28 30 Width of pronotum (mm) Fic. 4a, b. The size distribution of pronotum widths of old nymphs and adults living solitarily, in pairs, and in familial associations. A few large nymphs and some adults form pairs, and some adults have young numphs in their nest burrows. Data from nest burrows not censused in plots at Smith Creek and Uramo is included in these figures. A little old- nymphs and some adults make pair life, and some adults have young nymphs in their nest burrows. Instars are denoted by Roman numerals. shown in Figure 4a, b. I estimated how many instars the cockroaches have in nymphal stage on the basis of the size distribution of pronotum widths in Figure 3 and 4a, b. Sexual dimorphism of M. rhinoceros is not apparent until the fifth or sixth instar. After this point the differences in pronatal shape become clear. A total 597 of predispersal nymphs and 141 free-living nymphs from 16 plots and adjacents areas were investigated. Nine peaks can be recog- nized from Figures 3 and 4. The cockroach prob- ably has nine instars in the nymphal stage. Nymphs live a solitary life from the seventh to ninth instar, or in a few cases from the sixth instar stage. Some adults form pairs which have young nymphs in the nest burrows as shown Figure 4a, b. DISCUSSION Gautier et al. [10] discussed the relationship between ecology and social behavior in cock- roaches, and stated the importance of research for correlations between habitat parameters and social characteristics. Cockroaches may use any one or a combination of the following proximate and evolu- tionary defensive tactics: concealment, evasive be- haviors, protective or aposemantic colouration, chemical defence, disturbance sound production, and fighting [3]. Species of Pycnoscelus, Areniva- ga, Epilampra, Geoscapheus, Blaberus, Hyporhic- noda, Eublaberus, and Brysotria cited in Schal et al. [3] burrow into the substratum during the inactive period, or in response to disturbance. I consider that Macropanesthia use apparently the burrowing behavior for defensive and protective tactics against natural enemy and severe climate. Why are the adults of M. rhinoceros so large, and why do they have a long-lasting family life? To answer these questions from ecological point of view, natural enemies comprise the most impor- tant among many habitat parameters. Field observations indicated that the main natural ene- mies are centipedes and large spiders. Both anim- als were observed frequently in the field, and I observed a large centipede (Ethomostigmus sp., body length is about 18 cm) feeding on a solitaly nymph in a nest burrow. Centipedes can not eat adult cockroaches, because the tough adult exter- nal skeleton protects cockroaches against cen- tipede attack. This observation was confirmed in a preliminary manner with a laboratory experiment. Large spiders (Theraphosidae, body length is ab- out 4.5cm) were found several times in nest Familial Association of Macropanesthia 841 burrows where no cockroach was found. It is possible that nymphs were eaten by the spiders. It is unlikely that the spiders prey on adult M. rhinoceros, because these insects are larger and more heavily armored than the spiders. During early nymphal stages, when natural enemies in- vade a nest burrow, adult cockroaches defend themselves and their offspring. Adults may be able to block a nest burrow against the enemy by using its robust body (Fig. 1). I have seen many large ditches about 20cm deep on the surface of ground in the field; possibly resulting wild pigs or marsupials (bandicoots?) which were foraging for the cockroaches in shallow burrow, but detail are unknown. Obviously the shape of M. rhinoceros has adapted for life in dry sandy areas. The cockroach has a smooth, flattened body, finger-like spines on front legs (reminiscent of those of a mole), a shovel-shaped pronotum, and completely lacks wings. These aspects of body shape may represent adaptations for digging in sandy soil. Day [12] studied on internal anatomy and histology of this cockroach in comparison with a relatively small one, Blattella germanica (weight, approximately 0.06 g) and concluded that gravity, diffusion,.and the area available for secretion and absorption are not factors limiting the size of M. rhinoceros, and that the specializations observed in M. rhinoceros are related to its burrowing habits and food. Some males of Macropanesthia were observed to live with a female and first instar nymphs in early spring (Fig. 4a). This is similar to the monoga- mous family life observed in xylophagous species such as Cryptocercus punctulatus, Salganea taiwanensis and S. esakii [6-8]. But the familial association of M. rhinoceros does not continue for long, in that all males leave the families when nymphs have reached to the second instar. It is possible that the males search for other females for mating. The growth of nymphs is synchronously as shown in Figure 3. This could be due to the fairly prolonged dry period in this locality, which limits the timing of reproduction. And the steady en- vironmental condition in deep burrow is also im- portant. I observed that the temperature of bur- row is warmer than ambient temperature in the early morning on mid July in 1989 (in mid-winter, 21°C:7 °C), and that of the burrow is cooler than the ambient temperature in the afternoon on mid December in 1987 (in mid-summer, 31°C :44°C). It can be said that the deep burrows buffer the roaches from severe temperature change. There- fore, the nymphs can grow synchronously. However the factors triggering the dispersal of middle-aged nymphs from their mother’s nest re- main unclear. Roth [11, 13-15] divided the Panesthiinae into five tribes: Panesthiini, Ancaudelini, Salganeini, Caepariini and Geoscapheini. The first four tribes all live in and feed on dead wood. All species in the Geoscapheini, which includes Macropanesthia, live in burrows and feed on litter from the ground surface. On the basis of the differences in repro- ductive behavior observed by Rugg and Rose [16] and in morphology and habits as noted by Roth [11, 15], Rugg and Rose [17] believe that the Geoscapheini represent a sister-group to the other tribes and should be ranked as a subfamily. I consider the Geoscapheini, which is composed of four genera (Macropanesthia, Parapanesthia, Geoscapheus and Neogeoscapheus), derived from the ancestor of a xylophagous cockroach similar to the rain forest Panesthiini, which became secon- darily adapted for savannah life. The familial bond of Geoscapheini with adult and offspring in the ground were strengthend at the same time, when savannah life was adopted. ACKNOWLEDGMENTS The author wishes to express his hearty thanks to Dr. R. W. Taylor (Division of Entomology, CSIRO) and Prof. Y. It6 (Nagoya University) for valuable advice during the whole course of this study, and to Prof. R. H. Crozier (La Trobe University) and Dr. A. C. Messer (University of Shizuoka) for critical reading of the manu- script. Thanks are also extended to Mr. B. Brotherton in Mount Garnet, and to Drs. H. A. Rose and D. Rugg (University of Sydney) for giving us information on the study sites and Macropanesthia, and to Drs. K. Masuko and Y. Hirono and Messrs. S. Ichitani and Y. Obata (University of Tokyo) for assisting field studies. This work was supported by Grant-in-Aid for International Scientific Research Program (No. 01041029) and in part for Scientific Research (Nos. 01540543, 02454004, 03269102) from the Ministry of Education, Science and Culture of Japan. 842 REFERENCES Eickwort G. C. (1981) Presocial Insects. In “Social Insects”. Ed. by H. R. Hermann, Academic Press, New York, Vol. II, pp. 199-280. Roth, L. M. and Willis, E. R. (1960) The biotic associations of cockroaches. Smithsonian Misc. Coll., 141: 1-470. Schal, C., Gautier, J. Y. and Bell, W. J. (1984) Behavioral ecology of cockroaches. Biol. Rev. Camb. Philos. Soc., 59: 209-254. Liechti, P. M. and Bell, W. J. (1975) Brooding behavior of the Cuban burrowing cockroach Brysot- ria fumigata. Insectes Soc. 22: 35-46. Cleveland, L. R., Hall, S. R., Sanders, E. P. and Collier, J. (1934) The wood feeding roach Cryp- tocercus, its protozoa and the symbiosis between protozoa and roach. Mem. Am. Acad. Arts. Sci., 17: 185-342. Seelinger, G. and Seelinger, U. (1983) On the social organization, alarm and fighting in the primitive cockroach Cryptocercus punctulatus Scudder. Z. Tierpsychol., 61: 315-333. Nalepa, C. A. (1984) Colony composition, proto- zoan transfer and some life history characteristics of the wood cockroach Cryptocercus punctulatus Scud- der (Dictyoptera: Cryptocercidae). Behav. Ecol. Sociobiol., 14: 237-279. Matsumoto, T. (1987) Colony compositions of the subsocial wood-feeding cockroaches, Salganea taiwanensis and S. esakii Roth (Blattaria, Panes- thiinae). In “Chemistry and Biology of Social In- sects”. Ed. by J. Eder and H. Rembold, Verlag Peperny, Munchen, p. 394. Deleporte, P. (1985) Phylogenese et organisations sociales chez les Blattes (Dyctioptera, Blattaria). 10 16 T. MATSUMOTO Actes. Coll. Insecte Soc., 2: 27-33. Gautier, J. P., Deleporte, P. and Rivault, C. (1988) Relationships between ecology and social behavior in cockroaches. In “The Ecology of Social Be- havior”. Ed. by C. N. Slobodchikoff, Academic Press, London, pp. 335-351. Roth, L. M. (1977) A taxonomic revision of the Panesthiinae of the world. I. The Panesthiinae of Australia (Dictyoptera: Blattaria: Blaberidae). Aust. J. Zool. Suppl. Ser., 48: 1-112. Day, M. F. (1950) The histology of a very large insect, Macropanesthia rinocerus Sauss. (Blattidae). Aust. J. Sci. Res. Ser., 3: 61-75. Roth, L. M. (1979a) A taxonomic revision of the Panesthiinae of the world. II. The genera Salganea Stal, Microdina Kirby and Caeparia Stal (Dictyop- tera: Blattaria: Blaberidae). Aust. J. Zool. Suppl. Ser., 69: 1-201. Roth, L. M. (1979b) A taxonomic revision of the Panesthiinae of the world. III. The genera Panesthia Serville and Miopanesthia Saussure (Dictyoptera: Blattaria: Blaberidae). Aust. J. Zool. Suppl. Ser., 74: 1-276. Roth, L. M. (1982) A taxnonomic revision of the Panesthiinae of the world. I1V. The genus Ancaudel- lia Shaw, with additions to Parts I-III, and a general discussion of distribution and relationships of the components of the subfamily (Dctyoptera: Blattaria: Blaberidae). Aust. J. Zool. Suppl. Ser., 82: 1-142. Rugg, D. and Rose, H. A. (1984a) Reproductive biology of some Australian cockroaches (Blattodea: Blaberidae). J. Aust. ent. Soc., 23: 113-117. Rugg, D. and Rose, H. A. (1984b) The taxonomic significance of reproductive behavior in some Au- stralian cockroaches (Blattodea: Blaberidae). J. Aust. ent. Soc., 23: 118. ZOOLOGICAL SCIENCE 9: 843-857 (1992) © 1992 Zoological Society of Japan Notes on the Phylogeny of Various Taxa of the Orthorrhaphous Brachycera (Insecta: Diptera) AKIRA NAGATOMI Entomological Laboratory, Faculty of Agriculture, Kagoshima University, Kagoshima 890, Japan ABSTRACT— This paper pursues the recognition and significance of synapomorphic characters for various taxa, and supplements Woodley (1989) on the interpretation of the phylogeny and classification of the orthorrhaphous Brachycera. The most plausible phylogenetic relationships of the orthorrhaphous Brachycera are given at the present state of my knowledge. INTRODUCTION Grateful acknowledgment is made to Woodley [1] for his elaborate treatment of the phylogeny and classification of the orthorrhaphous Brachycera, in an acceptable, yet conservative, way, apart from the use of the names Stra- tiomyomorpha, Xylophagomorpha, Tabanomor- pha and Muscomorpha (including Asiloidea etc.). I now wish to discuss various problems arising from Woodley’s treatment and furthermore to express my firm beliefs on the true situation with a hope that my views (which at present I regard as beyond doubt) may in their turn be modified by some future workers. For essential data and illustrations related to this paper, other than Woodley, see the following literature: Colless and McAlpine [2]; Krivosheina [3]; Mackerras and Fuller [4]; McAlpine, Peter- son, Shewel, Teskey, Vockeroth and Wood [5]; Nagatomi [6-11]; Nagatomi and Iwata [12, 13]; Nagatomi, Saigusa, Nagatomi and Lyneborg [14- 16]; Stuckenberg [17]; Teskey [18, 19]; Tsacas [20]. From my present knowledge, the most plausible phylogenetic relationships of the orthorrhaphous Brachycera are shown in Figs. 1-3. Accepted April 19, 1992 Received March 12, 1992 GEOLOGICAL AGE OF XYLOPHAGOIDEA AND TABANOIDEA Within the Tabanoidea s. lat., the Stratiomyidae and Tabanidae (which have large numbers of genera and species) diverge from the central stock consisting of the Xylophagidae s. lat. and Rha- gionidae s. lat. I have a strong impression that the most ancestral extant genera of the Tabanoidea s. lat. are as follows: Exeretonevra (Australia and Tasmania), Heterostomus (Chile), Pseudoerinna (= Bequaertomyia) (N. America and Japan), and Glutops (N. America, Siberia and Japan). The first two belong to the Xylophgidae s. lat. and the last two to the Rhagionidae s. lat. The Xylophagoidea is certainly older phylogenet- ically (or more closely related to the nematocerous ancestor) than the Tabanoidea in the following more plesiomorphic character states: larval head strongly sclerotized and non-retractile; female cer- cus 2-segmented and segment 1 simple (not dilated posterolaterally or posteroventrally); tibial spur formula 1:2:2 (or rarely 2:2:2) without excep- tion in the Xylophagidae s. lat. (from which the Pantophthalmidae is excluded). It may be said that the Xylophagoidea shows a stronger decline in the number of the genera and species than do the Tabanoidea and Stra- tiomyoidea. In the Rhagionidae a number of the Jurassic fossil records are known and adult female mandi- bles are present in the Tabanoidea, but these 844 A. NAGATOMI Stratiomyoidea Xylophagoidea Tabanoidea Stratiomyidae S Xy lomyidae Nemestrinoidea Pantophthalmidae Asiloidea Rachiceridae 1 x Xy lophagidae Vermileonidae zs Coenomyiidae Apsilocephalidae Empidoidea Exeretonevridae r-T14 Cyclorrhapha Heterostomidae Pelecorhynchidae sO - Stratiomyoidea Rhagonidae . Pantophthalmidae Athericidae .Xylophagidae s. lat. -—___ Tabanidae Rhagionidae s. lat. Nemestrinidae wl Athericidae Acroceridae Tabanidae — — Hilarimorphidae Nemestrinoidea Bombyliidae Bombyliidae et Hilarimorphidae Therevidae ne other Asiloidea Scenopinidae -——— Mydidae —— Apioceridae Asilidae Vermileonidae t Apsilocephalidae Empididae an Dolichopodidae Cyclorrhapha Fics. 1-3. Possible phylogenetic relationships of the orthorrhaphous Brachycera. 2, Simplified from Fig. 1; 3, modified from Fig. 2 (Pantophthalmidae is treated as the sister group of Stratiomyoidea, and Empidoidea ete. is omitted). A, Asiloidea; E, Empidoidea; N, Nemestrinoidea; r, Rhagionidae s. lat.; S, Stratiomyoidea; T, Tabanoidea; Tl, Tabanoidea, s, lat.; X, Xylophagoidea; x, Xylophagidae s. lat. features are lacking in the Xylophgoidea. How- ever, these two factors seems to be less significant in the determination of geological age, because the fossil records are so incomplete throughout the Diptera and the presence of adult female madibles is so sporadic throughout extant Nematocera and orthorrhaphous Brachycera. Phylogeny of Orthorrhaphous Brachycera 845 SYSTEMATIC POSITION Pantophthalmidae The Pantophthalmidae, which was tentatively placed in the Xylophagoidea by Nagatomi [9] and Woodley [1], has been relegated to the Stra- tiomyoidea by Mackerras and Fuller [4] and Kri- vosheina [3]. The Pantophthalmidae resemble the Stra- tiomyidae + Xylomyidae in having the larval head without paired metachephalic rods and are similar to the Stratiomyidae (not the Stratiomyidae+ Xylomyidae) in having the female cerci separated from each other. The Pantophthalmidae are also more similar in the structures of the male genitalia to the Stratiomyoidea than to the Xylophgoidea (see Nagatomi [11]). Woodley (p. 1376) wrote, “Pupation within a puparium formed from the larval integument is unique to the Stratiomyomorpha [Stratiomyidae + Xylomyidae] within the more primitive Brachycera and is clearly apomorphic. ------ In my opinion, this character is the most obvious and conclusive syn- apomorphy linking two families of brachycerous flies.” The absence of this conspicuous feature certainly excludes Pantophthalmidae from the Stratiomyoidea. Woodley (p. 1376) also wrote as to the position of Pantophthalmidae, “Further evidence is neces- sary before their relationships to other primitive Brachycera can be elucidated.” In any case, the Pantophthalmidae seems to be intermediate in position between the Xylophagoidea and Stra- tiomyoidea and should be relegated to one of them. Exeretonevridae (Exeretonevra) and Rhagionidae (Austroleptis) Colless and McAlpine ([2] p.755) wrote, “Typical soil-dwelling xylophagid larvae (see e.g., [5]) have been found in Tasmania (M. Williams, pers. comm.), but no adults have been reared. It seems likely that they will be found to belong to some genus currently placed in another family. Atherimorpha and Exeretonevra, currently in Rha- gionidae and Nemestrinidae respectively, seems likely candidates. In any case, we propose on general grounds to transfer to latter genus to this family [=Xylophagidae]. Despite some resembl- ance to Nemestrinidae in wing venation, Exere- tonevra is clearly misplaced there; but Nagatomi’s (1977) erection of a monotypic family seems to us premature. Adults of Exeretonevra occur in high- land areas from northern N. S. W. to Tas., and may be locally common on vegetaion near streams. Their flight is weak and slow, and they appear reluctant to fly.” It is unlikely that Atherimorpha, which is more closely related to Rhagio than to Chrysopilus (see Nagatomi [10]; Nagatomi and Nagatomi [21]), has typical xylophagid-type larva. It is almost certain that the larvae in question belong to Exeretonevra. If so, Exeretonevra would prove itself to be a member of the Xylophgidae s. lat. Austroleptis differs from the members of Rha- gionidae by the combination of following ple- siomorphic and apomorphic character states: face flat and shallow dish-like (plesiomorphic); female sternum 9 present (plesiomorphic); female cercus 1-segmented (apomorphic). Colless and McAlpine ((22] p. 701; [2] p. 753) wrote, “Austroleptis rhyphoides has been reared from rotting wood.” Xylophagid larvae differ markedly from rhagionid larvae. It is probable that the larva of Austroleptis does not fall into the xylophagid type. If so, Austroleptis would be most closely related to the Rhagionidae within extant families of the lower Brachycera, granting that Austroleptis is treated as representing an inde- pendent family. Pelecorhynchidae (Glutops) Glutops was placed in the Pelecorhynchidae (Teskey [18, 23]; Woodley), in the Rhagionidae (Nagatomi [8, 9]) or in the Glutopidae (Kri- vosheina [24, 3]). Various views on the phylogenetic relationships between the families of Tabanoidea (Pelecorhyn- chidae, Rhagionidae, Athericidae, Tabanidae or Glutopidae) are shown in Figs. 4-6. Stuckenberg [17] and Nagatomi [9] considered the larval mandible with a poison canal as one of the synapomorphic characters for the Tabanidae + Athericidae. Woodley took this character as the evidence which supports the monophyly of Glu- 846 A. NAGATOMI Pelecorhynchidae Rhagionidae (including Glutops) Athericidae 4 Tabanidae Rhagionidae Pelecorhynchidae (including Glutops) Athericidae 5 Vawciebibes Fics. 4-7. Krivosheina [3]; 7, prepared for the present paper. tops (as the representative of Pelecorhynchidae) + Athericidae + Tabanidae. However, it can be im- agined that the character state above is due to either convergence or symplesiomorphy. Krivosheina [24] erected a monotypic family Glutopidae and later, Krivosheina [3], interpreted its phylogenetic position as shown in Fig. 6 on the following basis: “The tracheal branches of the larvae are fused posteriorly and the caudal spira- cles are small, point-like in Tabanidae and Gluto- pidae or are lacking in Athericidae” (see figs. 8-13 in Krivosheina [3]). However, this similarity is undoubtedly due to the result of convergence in adapting to aquatic life. I am convinced that Pelecorhynchus and Glutops are more closely related phylogenetically to the Rhagionidae than to the Athericidae + Tabanidae. The definitive synapomorphic character for Pele- corhynchus + Glutops + Pseudoerinna+ Rhagioni- dae is as follows: segment 1 of the female cercus is dilated posterolaterally or posteroventrally, although in genera of the subfamily Spaniinae, the bases of the cerci are more widely separated and the posterolateral part of segment 1 is usually not dilated, undoubtedly due to the result of secondary development or reduction (see figs. 12-25 in Naga- tomi and Iwata [12]). Thus, judgment varies with the supposed syn- apomorphic character adopted such as (1) larval mandible with poison canal, (2) caudal spiracles small and point-like, and (3) segment 1 of female cercus dilated posterolaterally. Character (3) can- .Glutopidae Athericidae Tabanidae 6 Pelecorhynchidae Rhnagionidae Pelecorhynchidae (including Glutops) ——— Rhagionidae Athericidae 7 Tabanidae Proposed phylogenetic relationships of Tabanoidea. 4, After Nagatomi [9]; 5, after Woodley [1]; 6, after not be rejected, because various features of Glu- tops are so similar in both adult and larval stages to those of the Pelecorhynchidae + Rhagionidae and are so sharply different from those of the Atherici- dae+Tabanidae. The Athericidae+Tabanidae has definitive synapomorphic characters which are absent in the Pelecorhynchidae and Rhagionidae and which are discussed in the forthcoming sec- tion. Mackerras and Fuller ([4] p. 29) concluded that “Thus, on its larval characters alone, Pelecorhyn- chus would be classified without hesitation as a Rhagionid, and the question of its relationship with the Tabanidae or the Stratiomyoid families [including Xylophagidae s. lat.] would hardly arise.” For the basis of this fundamental conclu- sion, see Mackerras and Fuller ([4] p. 28-29). It must be confirmed whether or not Pelecorhynchus has the larval mandible with a poison canal, which is present in Glutops, Athericidae and Tabanidae. Mackerras and Fuller ({4] p. 29) wrote of the larva: “Pelecorhynchus has no well-marked relationships with the Tabanidae, differing from all known Tabanids in being rigid and incapable of contrac- tion, and in the absence of pseudopods striations, and rugose plates. Also the mouthparts of Taba- nus are highly specialized and have no oral aper- ture, the food being taken in through the mandibu- lar pore.” Stuckenberg ([17] p. 669) wrote, “In Rhagio the distal portion of the mandible has an internal channel but this does not open to the exterior Phylogeny of Orthorrhaphous Brachycera 847 apically, and there are no poison glands (Roberts, 1969: 385).” Regrettably, the nature of this chan- nel is unknown to me. Clearly, further studies should be made on the mandibular pore in the Tabanoidea. Concerning the family position of Glutops, I must change my previous idea (Nagatomi (8-11, 25]), admitting that the similarity of the larval general body form between Glutops and Pele- corhynchus is not due to convergence or symple- siomorphy but is due to synapomorphy. It seems to me in the present instance that the synapomor- phy is the strongest probability in this case. Here, I must transfer Glutops from the Rhagionidae to the Pelecorhynchidae, following Teskey [18, 23] and Woodley [1], though I believe that the sister- group of Pelecorhynchidae is not the Athericidae +Tabanidae but the Rhagionidae (see Fig. 7). Vermileonidae Nagatomi ef al. [15] offer a hypothesis that the Vermileonidae is a sister group of the Apsi- locephalidae + Empidoidea+ Cyclorrhapha on the following basis: the structure of the larval head; the shape of antennal segment 3 and style; the isolated position of the Vermileonidae within the taxa of the Tabanoidea s. lat. Tsacas [20] studied in detail the structure and function of the larval head and mouthparts in Rhagio scolopaceus, Chrysopilus auratus and Ver- mileo vermileo, and concluded that “Un fait est dés maintenant acquis, c’est que les Rhagionidae présentent des affinités dune part par les Rha- gioninae avec les Tabanidae et, d’autre part, par les Vermileoninae avec les Asilidae et Empidi- formia.” I believe there are difficulties which rule against the above hypothesis. (1) There are some common characters in the Stratiomyidae, Xylomyidae, Pantophthalmidae and Vermileonidae as follows: no dorsal and ven- tral plates enclosing aedeagus; larval head without metacephalic rods. (2) The paired metacephalic rods (which are absent in the Vermileonidae) are present in the Empidoidea. (3) Vermileonidae differs considerably from Apsilocephalidae, Empidoidea, and Cyclorrhapha in many external characteristics. (4) The male genitalia of Apsilocephalidae, which resemble those of Asilidae, differ so much from those of Vermilionidae. These points are now further elaborated. (1) In Vermileonidae, the larval head is com- pletely retractile within the thorax; the female cercus (which may be 2-segmentee as a basic plan) is unique in shape and autapomorphic for this family; and so on. The monophyly of Stratio- myoidea-+ Pantophthalmidae + Vermileonidae is a weak possibility. (2) The paired metachphalic rods arose inde- pendently in the Asiloidea (where the rod is not paired but single) and Empidoidea, respectively, as discussed by Woodley (pp. 1386, 1388, 1391). (3) The Vermilenonidae is situated near the orthorrhaphous Brachycera stem and has many plesiomorphic characters. (4) The transition in evolutionary line of the male genitalia is not definite among the Ver- mileonidae, Apsilocephalidae and Empidoidea, although well-developed and more complicated surstyli in the Apsilocephalidae show such transi- tion towards the Empidoidea. However, discrep- ancy in the structure of the male genitalia between Vermileonidae and Apsilocephalidae may be due to the fact that these two taxa are relics of antiquity and distantly related to each other. I realize some of these arguments may be re- garded as subjective. Nemestrinoidea Verrall [26], Malloch [27], Colless and McAI- pine [22, 2], etc. placed the Nemestrinidae and Acroceridae in the Tabanoidea s. lat. and Bomby- liidae in the Asiloidea. In the Nemestrinidae and Acroceridae, the pulvilliform empodium is present and the macrosetae on the body are absent. These two plesiomorphic character states seem to be generally underestimated in the present day under- standing of the phylogeny of the orthorrhaphous Brachycera. The presence or absence of charac- ters mentioned above often varies with genus or family within the same family or superfamily, that is, (1) the presence of pulvilliform empodium in some Asilidae (see fig. 4 in Oldroyd [28]), some Empididae and some Dolichopodidae; (2) the ab- 848 A. NAGATOMI sence of macrosetae in the Scenopinidae, Mydi- dae, Hilarimorphidae and some Bombyliidae; (3) the presence of macrosetae in Atherimorpha (Rha- gionidae) from South America; (4) the absence of the empodium in some Acroceridae. However, (1), (3) and (4) above undoubtedly occurred secondarily. So, these two character states should be reassessed. If they are significant to some degree in elucidating phylogeny, the treatment by several workers mentioned above should be re- vived. Woodley placed the Nemestrinoidea in his Mus- comorpha (Nemestrinoidea-+ Asiloidea [including Bombyliidae]+ Empidoidea+Muscoidea) on the follwing three bases: (1) Antennal flagellum with only four (or fewer) flagellomeres; (2) Tibial spurs lost; (3) Female cerci one-segmented. However, these three characters very often occurred second- arily in many taxa of Tabanoidea s. lat., and it is possible that they became entirely so in the Nemestrinidae and Acroceridae within the Taba- noidea s. lat. No definite synapomorphic character occurs for the Tabanoidea + Nemestrinoidea and the position of Nemestrinoidea is vague, but I prefer to place the Nemestrinoidea in the Tabanoidea s. lat. based on negative evidence which is discussed again in the forthcoming section. Bombyliidae Which of the Nemestrinoidea and Asiloidea is the nearer relative of the Bombyliidae? The Bombyliidae is related to the Nemestrinoidea by having the following characters: larval head with- out metacephalic rod; larva parasitic, with hyper- metamorphosis. On the other hand, the Bomby- liidae resembles the Asiloidea in having the follow- ing apomorphic characters: larval posterior spira- cles located on the apparent penultimate abdomi- nal segment and more widely separated from each other; empodium bristle-like; body very often with macrosetae; female abdomen very often with acan- thophorites. Woodley (p. 1385) wrote, “First instar bomby- lid larvae are active, whereas later instars are grub-like after they have located and infested their host. The only other Brachycera outside of the Muscoidea | =Cyclorrhapha] that are parasitic are the Nemestrinoidea, treated above. The two groups have apparently evolved along these lines convergently, as other evidence suggests the place- ment of the bombyliidae in the Asiloidea. ------ the first instar larvae (of Bombyliidae) bear a resem- blance to those of Therevidae and Scenopinidae, especially in having long lateral setae on the thorax and a small, exerted head capsule (Verrall 1909: fig. 64). +--+ I therefore conclude that parasitism and hypermetamorphosis are autapomorphic for the Bombyliidae.” I have been privileged to see an unpublished manuscript by D. K. Yeates and M. E. Irwin, who write, “Thus it is possible that the most plesiomor- phic larvae in the Bombyliidae (e.g., Glabellula of the subfamily Mythicomyiinae; see Andersson [29]) are predatory and lack hypermetamorpho- sis.” The immature stages are unknown in the Hilar- imorphidae which is thought to be the nearest relative of Bombyliidae. Furhter studies are des- perately needed on the larval stages of primitive Bombyliidae. At present, I would like to place the Bomby- liidae in the Asiloidea, following Woodley. After all, the absence of a pulvilliform empodium has led to the placement of Bombyliidae in the Asiloidea. SYNAPOMORPHIC OR AUTAPOMORPHIC CHARACTER Tabanoidea s. lat. (including Nemestrinoidea) No definite autapomorphic character state is found for the Tabanoidea s. lat. “Accordingly, present knowledge does not provide support for the Tabanomorpha sensu Hennig [=Tabanoidea s. lat.] as being a monophyletic taxon” (Woodley, p. 1373): The component families are united with one another by having the following plesiomorphic characters: pulvilliform empodium; no mac- rosetae; no acanthophorites. Except for the Nemestrinoidea, most Stra- tiomyidae and some Rhagionidae, the component families have bare tibial spurs apparently derived from the nematocerous ancestor. Nevertheless, bare tibial spurs would become synapomorphic for Phylogeny of Orthorrhaphous Brachycera 849 the Tabanoidea s. lat. in relation to the Nema- tocera, if pilose tibial spurs are a basic plan for the Nematocera. However, the presence or absence of pile on spurs may possibly be too slight for cladistic significance. Except for the Nemestrinoidea, Athericidae, Rachiceridae, many Rhagionidae, many Stra- tiomyidae, and some Vermileonidae, the antennal flagellum is 8-segmented. This character, which is definitely a basic plan for the Tabanoidea s. lat., is plesiomorphic in relation to the Asiloidea, Empi- doidea, and Cyclorrhapha but synapomorphic for the Tabanoidea s. lat. in relation to the Nema- tocera. Stratiomyoidea+ Xylophagoidea Woodley (p. 1375) wrote, “:-- there is at present no conclusive evidence that the three families [Stratiomyidae, Xylomyidae and Xylophagidae s. lat.] form a monophyletic group.” Indeed, it is so. The two superfaimilies are united with each other by the following plesiomrphic character states: larval head strongly sclerotized and non- retractile; female cercus 2-segmented, with seg- ment 1 simple (not dilated posterolaterally or posteroventrally). The non-retractile larval head of Stratiomyoidea + Xylophagoidea differs from that of the Nema- tocera by the head capsule elongated posteriorly into thorax (Woodley, p. 1372), and in this respect it becomes synapomorphic character for the Stra- tiomyoidea+ Xylophagoidea in relation to the Nematocera, although a pair of metacephalic rods are present in the Xylophagidae s. lat. but absent in the Stratiomyidae, Xylomyidae and Pantoph- thalmidae, and a non-retractile head is also seen in the Therevidae+Scenopinidae within the Asi- loidea. Except for the Xylomyidae and several Stra- tiomyidae, the clypeus is “flattened and shallow dish-like (=plate like), i.e., with the margin more or less turned up and continued around below the palpus and proboscis (=FP)” (Nagatomi [30] p. 397), as an undoubted basic plan for the Stra- tiomyoidea+ Xylophagoidea. I still believe that this character is derived from the nematocerous ancestor. Nevertheless, the detail of this character may possibly be different from that of Nema- tocera. If so, FP-face would be synapomorphic in relation to the Nematocera or even autoapomor- phic for the Stratiomyoidea+ Xylophagoidea. Comparative studies throughout the Nematocera are necessary to establish the validity of this hypothesis. For the monophyly of the Stratiomyoidea+ Xylophagoidea, there is one piece of circumstan- tial evidence, i.e., the presence of intermediate forms, namely, the Beridinae, Chiromyzinae, Parhadrestiinae, Xylomyidae and Pantophthalmi- dae that link the specialized Stratiomyidae with the Xylophagidae s. lat. Tabanoidea+ Nemestrinoidea There is one common character for the Taba- noidea+ Nemestrinoidea, i.e., the larval head is completely retractile within the thorax and has no metacephalic rod, although the Vermileonidae and Bombyliidae share these characters. The Tabanoidea and Nemestrinoidea (as well as some primitive Bombyliidae) are linked by the following plesiomorphic character state: larval posterior spiracles are present on the last abdomin- al segment and are more closely located to each other. | Woodley (p. 1384) wrote, “All larvae of Asi- loidea [including most of the Bombyliidae] have the posterior spiracle located in the apparent penultimate segment of the abdomen, directed more or less laterally (figs. 37.23, 38.11, 40.28, 42.76-77). This character state is not found in the Nemestrinoidea or in the other three infraorders of Brachycera. In the Scenopinidae and Therevidae, a more derived state is found, in which the pos- terior spiracle is apparently in the antepenultimate abdominal segment.” For the systematic position of the Vermileoni- dae and Bombyliidae, see preceding section. Rachiceridae + Xylophagidae There is one character state for the Rachiceridae +Xylophagidae, i.e., the cerci are fused for almost the whole length of segment 1, tergum 9 is longer than wide, and tergum 10 is absent or reduced to a pair of indistinct sclerites (see figs. 4— 6 in Nagatomi and Iwata [12]; tergum 10 is well developed in the Coenomyiidae and Heterostomi- 850 A. NAGATOMI dae but less developed or possibly absent in the Exeretonevridae). This character state will be- come autapomorphic, when commonly seen. Judging from illustrations (figs. 7-8 in Webb [32]), the female cerci of Rachicerus obscuripennis are widely separated. The examination of more spe- cies is needed in this respect. There are some common adult characters in the Rachiceridae and Xylophagidae, i.e., vein Rs ends at or very near the wing tip; alula is practically absent (margion of alula straight or nearly so); eyes in both sexes are widely separated. However, none of them can possibly be defined as a synapo- morphic character. If monophyly of the Rachiceridae + Xylophagi- dae is true, synapomorphic characters may be seen in the larval stage. James [31] prepared a key to genera of the Xylophagidae s. lat. based on the larva as follows: “Head capsule at least three times as long as broad. Thoracic segment with sclerotized plates lorsallhy (tyes. 5 113))) conesseossrocosonoonoce ensescquasceaae ual eed ES ee Rachicerus and Xylophagus Head capsule not more than twice as long as broad. Thoracic segment not sclerotized dorsally (ir UID) Secesonbgaandscusondsoddoneoouconuass Coenomyia.” Two character states in the key will be synapo- morphic or autapomorphic for the Rachiceridae + Xylophagidae beyond doubt, when commonly seen. The two characteristics quoted under Coenomyia are applicable to Arthropeas sibirica (see Krivosheina [33]) and Dialysis fasciventris (see Webb and Lisowski [34]) of the Coenomy- lidae. There are some conspicuous autapomorphic characters for the members of Rachiceridae, i.e. The antennal flagellum is over 10-segmented and either pectinate or serrate; apical portion of the aedeagus consists of 2 tubes in vertical plane and there are no dorsal and ventral plates enclosing the apical portion of aedeagus. There is also a possible autapomorphic character, i.e., the dorsal part of larval prothorax has an assemblage of sclerotized amoeba-shaped dots (see figs. 13-14 in James [31]. For the members of Xylophagidae, there is an evident autapomorphic character, i.e., the last antennal segment is not pointed but rounded. This character is unique within those Tabanoidea hav- ing 8-segmented antennal flagellum. A possible autapomorphic character is also seen: the sclero- tized dorsal plates on the larval thorax are exten- sive in area and not consisting of amoeba-shaped dots (see fig. 12 in James [31]). Coenomyiidae There is one clear autapomorphic character throughout the genera of Coenomyiidae, 1.e., the mid-ventral part of the fused gonocoxites has a large desclerotized patch which is transversely elongate and which is distinct from the posterior margin of the fused gonocoxite (very often except- ing mid-posterior channel leading to the posterior margin). This peculiar membranous patch dis- appears in Dialysis kesseli and Napemyia illinoen- sis (after Webb [35, 36]) certainly due to secondary reduction. Apart form the state of apomorphy or ple- siomorphy, the male genitalia of Coenomytidae differ strikingly in many respects from those of other taxa of Xylophagidae s. lat., namely, Rachiceride, Xylophagidae, Exeretonevridae and Heterostomidae, but resemble those of Rhagioni- dae and Glutops (Pelecorhynchidae) of Taba- noidea (see Nagatomi [11] p. 155 who wrote, “the genera of Coenomyiidae, which are almost identic- al with one another in male genitalia, may easily be separated from each genus of Rhagionidae, which may be characteristic in the details of male geni- talia.”). The palpus is 1-segmented in the Coenomyiidae except for some individuals of Arthropeas species with 2 segments (Nagatomi [37]). The 1- segmented palpus is nuique within the Xylophagi- dae s. lat. The larval anal segment and the sclerotized dorsal plate (surrounding the posterior spiracles) may possibly be relatively larger in the Coenomy- iidae than in the Rachiceridae and Xylophagidae (see figs. 11-12 in James [31]). If it is constantly recognized, this character state will become syn- apomorphic for the Coenomyiidae. The genera of Coenomyiidae are easily sepa- rated from those of other families which resemble the former in appearance (see Nagatomi [6-8}]). Nevertheless, to find a synapomorphic character is Phylogeny of Orthorrhaphous Brachycera 851 unexpectedly difficult for the Coenomyiidae. One reason is certainly that the state of apomorphy or plesiomorphy is hard to determine in the structure of male genitalia and the details of wing venation. However, the principal reason should be attribut- able to a certain type of primitiveness possessed by the Coenomyiidae. Some primitive characters must be specialezed in order to survive for a long time, but the Coenomyiidae has achieved this without a strong specialization. Exeretonevridae + Heterostomidae There are two characteristics: ocellar triangle much longer than wide; upper part of clypeus protruded forward to some extent. Unless the states are the result of convergence, these two character states become synapomorphic for the Exeretonevridae + Heterostomidae, which differ also from the Coenomyiidae by having two ple- siomorphic features: palpus 2-segmented; meta- pleuron bare. The following are autapomorphic characters for the Exeretonevridae: “vein R>,3 curved upward at apical portion but ending far beyond apex of R;; a crossvein present between veins R>,3 and R4+Rs; directly arising from discal cell and vein Rs ending distinctly before wing tip; vein M, ending before and M> ending far beyond wing tip; vein M3 meeting with vein M, before wing margin; poste- rior callus (near metapleuron) with a flat elevation which is elliptical, wider than long and minute pilose; abdominal tergum 1 with a pair of bare, elliptical, flat, caudal callosities which are wider than long” (Nagatomi [9]). On the other hand, only one autapomorphic character is seen for the Heterostomidae: the second palpal segment is much widened. This character state 1s also seen in the Xylophagidae, where it probably evolved independently. The monophyly of the Exeretonevridae+ Heterostomidae + Coenomyiidae is uncertain, be- cause no definite synapomorphic character has been detected. Only through the general appear- ance, may it be said that the Exeretonevridae + Heterostomidae is more closely related to the Coenomyiidae than to the Rachiceridae+ Xylophagidae. Pelecorhychidae + Rhagionidae As discussed in the preceding section, there is one definite autapomorphic character for the Pele- corhynchidae + Rhagionidae, that is, segment 1 of the female cercus is dilated popsterolaterally, although this dilation disappears in Austroleptis (Austroleptinae), Ptiolina and Spania (Spaniinae). For the Pelecorhynchidae, consisting of Pele- corhynchus and Glutops, there is an apparent autapomorphic character, i.e., “larva with stout spines laterally on labrum and apically on maxilla” (Woodley p. 1381) (see Mackerras and Fuller [4]; Teskey [18]). Woodley (p. 1380) wrote, “Glutops and Pseudoerinna are more similar in general appear- ance to Rhagionidae than is Pelecorhynchus, but, based on the extreme similarity of the general body form of larvae of Pelecorhynchus and Glu- tops (Teskey 1970) and on the difference between these and known rhagionid larvae, the two are likely closely related. The larvae are not known for Pseudoerinna.” | quite agree with his statement quoted above, to which some supplementary notes are here appended. For Glutops-+ Pseudoerinna+ Pelecorhynchus, there is apparent synapomorphic characters in female terminalia, 1.e., segment 1 of cercus situ- ated near base of sternum 10; tergum 8 including anterior membranous part much longer than wide; tergum 10 absent or reduced to a pair of indistinct sclerites. The female terminalia of Glutops and Pseudoerinna differ from those of Pelecorhynchus in two ways: segment 1 of the cercus has a strong posterolateral process and tergum 8 is more exten- sively sclerotized, showing the apparent monophy- ly of Glutops and Pseudoerinna. The female terminalia of Rhagionidae are broadly divided into the generalized and special- ized forms, of which the former has the following characters: segment 1 of cercus dilated postero- laterally and situated near apex of sternum 10; tergum 10 well developed. On the other hand, the specialized forms are given below: Austroleptis (Austroleptinae): cercus 1-seg- mented, not dilated posterolaterally, and situated near base of sternum 10; tergum 10 reduced to a pair of small sclerites. Ptiolina and Spania (Spa- 852 A. NAGATOMI niinae): cercus not dilated posterolaterally; paired cerci more widely separated at bases; tergum 10 short in Ptiolina and absent in Spania. Spaniopsis (Spaniinae), and Atherimorpha (Rhagioninae) (af- ter Mackerras and Fuller ([4] p. 15): segment 1 of cercus more widely separated (at least in Spaniop- sis), with a distinct inner section but dilated poste- rolaterally; tergum 10 well developed in Atheri- morpha and absent in Spaniopsis. For a discussion of the female terminalia of the lower Brachycera, see Nagatomi and Iwata [12, 13]. Pelecorhynchus is very similar to Glutops, Pseudoerinna and many genera of Rhagionidae in the structure of female terminalia, but differs markedly from the latter in the structure of the male genitalia (see Mackerras and Fuller [4]; Nagatomi [11]). The external adult characters of Pelecorhynchus are also conspicuously different from those of Glutops, Pseudoerinna and all known genera of Rhagionidae by having the following autapomor- phic characters: cheek (below palpus) with a large bulbous protuberance which has many long erect hairs and which varies in size and shape with sex and species; palpus shorter, broadly cylindrical, not tapering toward apex, and with an apical pit or with a transverse section; 2nd submarginal cell shorter and much wider; (see Nagatomi [9]). Glutops differs from Pseudoerinna in the follow- ing apomorphic characters: tibial spurs 0:2:1 (not 1:2:2); side of face and facial swelling large and produced forward. The apomorphic characters of Pseudoerinna in relation to Glutops are as follows: metapleuron (except lower border) wholly pilose (not bare); 2nd submarginal cell shorter and wider. See Nagatomi and Saigusa [38] and Nagatomi [10]. In short, the synapomorphic characters for the Pelecorhynchidae including Pseudoerinna (whose larva is unknown) and Glutops are as follows: “larva with stout spines laterally on labrum and apically on maxilla” (Woodley) already quoted; larval body long (in relation to the Rhagionidae), smooth and without creeping welts; three features in female terminalia already mentioned. No definite synapomorphic character has been found for the Rhagionidae, as long as the Pele- corhynchidae is treated as an independent family. I believe that several genera of Rhagionidae, whose larvae are unknown, are not so different in the immature stage from Rhagio, Chrysopilus, Symphoromyia (Rhagioninae) and Ptiolina (Spa- niinae), whose larvae have a common character, that is, “terminal segment with lobes or tubercles of various form surrounding spiracles” (see figs. 16, 18-19, 20-21 in James and Turner [39]), and the spiracular disc is concave or cleft. This charac- ter state may be unique for the Rhagionidae within the Tabanoidea. Athericidae + Tabanidae Stuckenberg [17] first established the monophyly of the Athericidae + Tabanidae. The most reliable synapomorphic character for the Athericidae+Tabanidae is the presence of long aedeagal tines which are also seen independ- ently in the genus Bolbomyia of Rhagionidae (see Nagatomi [11]). For the origin of aedeagal tines, see Nagatomi [40] who discussed the homologies of several characters along and behind the aedeagus in the lower Brachycera. The male genitalia of Dasyomma, the most primitive genus of Athericidae, are very similar to those of Tabanidae. Nagatomi ({11] p. 101) wrote: “Tergum 9 not divided into a pair of sclerites and its anterior margin not concave ......... Athericidae Tergum 9 divided or not divided into a pair of sclerites, and in the latter case its anterior margin deeply concave:...s...2:.-2o2.009. eee Tabanidae.” Nagatomi ([{11] p. 145) also wrote, “It appears that the posterior part of aedeagus is present or longer in Tabanidae but absent or shorter in Athericidae.” The similarity of female terminalia between Athericidae and Tabanidae may also represent the monophyly of these two families, in which the ovipositor is not telescoped; cercus 1-segmented, generally roughly as wide as long; tergum 8 much smaller than tergum 7; terga 7-8 much wider than long; intersegmental membrane between terga 7-8 short or hardly visible (see Iwata and Nagatomi [41] and Nagatomi and Iwata [12]). Nagatomi and Iwata [12] wrote, “Athericidae are very similar in shape of female terminalia to Pangoniinae of Tabanidae and are difficult to distinguish from the latter. But in Athericidae Phylogeny of Orthorrhaphous Brachycera 853 tergum 9 is larger than in usual Pangoniinae, and mid-distal part to genital furca protrudes forward (toward base of abdomen), allthough in shape of genital furca some Pangoniinae resemble some- what Atrichops.” It is still inconceivable to me that the Pele- corhynchidae (including Glutops and Pseudoerin- na) is the sister group of Athericidae + Tabanidae, because the male and female genitalia and the larval body of the former differ so much in many respects from those of the latter. Vermileonidae+ Apsilocephalidae + Empidoidea+ Cyclorrhapha There is one synapomorphic character for the above taxa, 1.e., “antennal segment 3 is rounded, triangular or pyriform, and the style is needle-like or straight, tapering apically and pointed” (see figs. 1-46 in Nagatomi et al. [15]). However, there are sO many deviations from the typical scheme and it is difficult to define the shape of antennal segment 3 and style precisely. Nevertheless, I believe that this character state is common throughout the above taxa as a basic plan. The larval head of Vermileonidae resembles that of some Empidoidea (see Tsacas [20]; Teskey [19]). Unfortunately, the immature stages of Apsi- locephalidae are unknown. The monophyly of the Apsilocephalidae + Empidoidea is uncetain, because no definite syn- apomorphic character has been found. The sister group of Apsilocephalidae is probably the Empi- doidea+ Cyclorrhapha. For the diagnosis of Apsi- locephalidae, see Nagatomi et al. [14, 16]. For the monophyly of the above taxa, there is one piece of circumstantial evidence, i.e., there is a big mrphological gap, on the whole between the Asiloidea and Empidoidea, apart from the pres- ence of acanthophorites which has a scattered distribution in the Empidoidea. DISCUSSION Presence or absence of metacephalic rod The larval characters discussed in this paper are rearranged below. (1) Head strongly sclerotized and not com- pletely retractile within thorax: Stratiomyoidea, Xylophagoidea, Therevidae and Scenopinidae; plesiomorphic in relation to the Tabanoidea, Ver- mileonidae, Nemestrinoidea, Empidoidea and many Asiloidea. (2) Head completely retractile within thorax: Tabanoidea, Vermileonidae, Nemestrinoidea, Asiloidea (except for Therevidae and Scenopini- dae) and Empidoidea; apomorphic in relation to Nematocera, Stratiomyoidea, Xylophagoidea and some Asiloidea. (3) Head with paired metacephalic rods: Xylophagoidea (except for the Pantophthalmi- dae), Empidoidea and some Asilidae. (4) Head with a single median metacephalic rod: Therevidae, Scenopinidae, Mydidae, Apioceridae and most Asilidae. (5) Head without metacephalic rod: Stra- tiomyoidea, Pantophthalmidae, Tabanoidea, Ver- mileonidae, Nemestrinoidea and Bombyliidae. (6) Posterior spiracles closer together on the last abdominal segment: Stratiomyoidea, Pantoph- thalmidae, Xylophagoidea, Tabanoidea, Ver- mileonidae, Nemestrinoidea and Empidoidea; ple- siomorphic in relation to the Asiloidea. (7) Posterior spiracles more widely separated from each other on the apparent penultimate abdominal segment: Asilidae, Mydidae, Apiocer- idae and most Bomyliidae; apomorphic in relation to other orthorrhaphous Brachycera (except for Therevidae and Scenopinidae). (8) Posterior spiracles more widely separated from each other on antepenultimate abdominal segment: Therevidae and Scenopinidae; apomor- phic in relation to other Asiloidea. It remains doubtful to me whether the presence of paired metacephalic rods in the Xylophagidae s. lat. is apomorphic or plesiomorphic in relation to the Stratiomyoidea, Pantophthalmidae, Taba- noidea, Vermileonidae, Nemestrinoidea and Bomyliidae. The metacephalic rod is absent in the Nematocera but present in the Xylophagidae s. lat., Asiloidea (except for Bombyliidae), and Empidoidea. It seems therefore that the ancestor of the orthrrhaphous Brachycera had the metacephalic rod. When apomorphic, presence of the metacephal- ic rod evolved independently in the Asiloidea 854 A. NAGATOMI (except for Bombyliidae) and Empidoidea respec- tively. When plesiomorphic, loss of the metacephalic rod occurred independently in the Stratiomyoidea+ Pantophthalmidae, Vermileoni- dae, Tabanoidea+Nemestrinoidea, and Bomby- liidae respectively. Either of these two cases is equally conceivable. Krivosheina [3] suggested monophyly of the Stratiomyoidea + Pantophthalmidae + Tabanoidea +Nemestrinoidea+Bombyliidae or that of the Xylophagoidea-+ Asiloidea (except for Bomby- liidae) according to the absence or presence of the metacephalic rod. The above hypothesis is not deniable, but the various factors discussed in this paper weaken the argument for it in the present state of our knowledge. Apomorhy or plesiomorphy in some characters of male genitalia Nagatomi ({11] p. 154) wrote, “(1) The tergum 9 is strongly arched in the genera Rachicerus, Xylophagus, Exeretonevra, and Pelecorhynchus but rather flat in Pantophthalmus, Coenomyia et al., Heterostomus, Rhagio et al. etc., and among the former four genera (2) the postero-lateral part of tergum 9 is developed ventro-inwardly into a plate or flap in Rachicerus and Pelecorhynchus but not in Xylophagus and Exeretonevra. It is ques- tionable whether these two characters are ple- siomorphic or are developed in each group of the lower Brachycera independently of the phylogene- tic relationship. The former view seems to be more probable.” In the last sentence quoted above, read “latter” for “former”. The arched tergum 9 and well-developed surstyli are beyond doubt apomorphic characters. These two characters are distributed in the Xylophagidae s. lat. and the Pelecorhynchidae, and their inde- pendent development was considered to be less probable in Nagatomi [11]. The genera Rachi- cerus, Exeretonevra and Pelecorhynchus are each markedly specialized in some external characters as already discussed in this paper. It is better to refrain from further comments on the various structures of the male genitalia with regard to the apomorhy or plesiomorphy, because of the difficulty of assessment. Two types in primitive creatures A number of archaic creatures must specialize to a certain or extreme degree in order to survive long, while not a few ancient living things survive without strong specialization. Thus, there are two forms in the Xylophagidae s. lat. and Rhagionidae s. lat. which constitute the stem or base of the phylogenetic tree in the orthorrhaphous Brachy- cera. Specialized form: Rachiceridae (the antennal flagellum is markedly specialized); Exeretonevri- dae (wing venation); Pelecorhynchus of Pele- corhynchidae (cheek, palpus, and 2nd submarginal cell). Generalized form: Coenomyiidae; Heterostomi- dae; Glutops and Pseudoerinna of Pelecorhynchi- dae; Rhagionidae. As a matter of course, to find a synapomorphic character is very difficult in the generalized form. Phenetic taxonomy and cladistic taxonomy The Nematocera and the orthorrhaphous Brachycera each seems to be not monophyletic but paraphyletic, and many higher taxa of the insects may remain as paraphyletic ones. When the principle of cladistic taxonomy is applied strictly, some taxa can become unnatural instead. For instance the sister group of the snakes and lizards is the crocodiles+birds, and that of the orang-utan is gorilla+chimpanzee+man. The oldest ancestor of the birds would greatly resemble the crocodiles. Even if the monphyly of the Vermileonidae + Apsilocephalidae + Empidoidea + Cyclorrhapha was recognized as true, the Empidoidea et al. would still be placed in the orthorrhaphous Brachycera, because the Cyclorrhapha has devi- ated so far from the Empidoidea etc. Validity of small families There are many small families, namely, Rachiceridae, Xylophagidae, Coenomyiidae, Heterostomidae, Exeretonevridae and Pele- corhynchidae, of which the first five belong to the Xylophagidae s. lat. and the last to the Rhagioni- dae s. lat. All of them could be reduced to Phylogeny of Orthorrhaphous Brachycera 855 subfamily status and in that case, Pelecorhynchi- nae and Glutopinae (or plus Pseudoerinniinae) of the family Rhagionidae may be recognized. For explanatory convenience, family status is given to the taxa above in this paper. In any case, the choice between “family” and “subfamily” will be determined by personal preference for the taxa above. On the other hand, the Vermileonidae and Apsilocephalidae should each be treated as an independent family, because they are so isolated from each other and from other taxa of the orthor- thaphous Brachycera. The validity of the Hilarimorphidae as an inde- pendent family is left for the future. ADDITIONS TO NAGATOMI [25] Some notes are added to Nagatomi [25] on the history of some families of Deptera, chiefly those of the lower Brachycera. Canthyloscelidae The genera Canthyloscelis and Hyperoscelis (Canthyloscelidae) and Synneuron (Synneuridae) have the following common characters: “head cap- sule greatly reduced and membranous, with mouthparts unsclerotized and indistinct” (Wood and Borkent [42]). Wood and Borkent ([42] p. 1354) wrote, “Hut- son [43] contended that the Scatopsidae are de- rived from Synneuron as a terminal branch of one of four lineages making up the Synneuridae plus Canthyloscelidae. If his contention is so, the head capusule of the larva must have been lost at least twice (thrice if the larva of Exiliscelis also lacks a fully formed head capsule), or else a fully formed head capsule must have been reacquired in the Scatopsidae. We consider both of these possibili- ties unlikely and have abandoned Hutson’s clado- gram (his fig. 26) in favor of a sister-group rela- tionship between the Scatopsidae and the Syn- neuridae [= Canthyloscelis + Hyperoscelis + Exilis- celis + Synneuron].” Pelecorhynchidae Daniels [44] described 2 new species of Pele- corhynchus from Victoria and Western Australia (see also Daniels [45]). These 2 species were accidentally omitted in Nagatomi [46, 25]. Nagatomi ((25] p. 14) wrote, “The genus Glu- tops was placed in the Pelecorhynchidae by several North American workers. If this treatment is correct, Glutops would be a more plesiomorphic northern-hemisphere sister-group of Pelecorhyn- chus. However, the phylogenetic position of Glu- tops is not necessarily established and will be discussed in a separate paper.” In the present instance, I have come to believe that Glutops and Pseudoerinna (= Bequaertomyia) are more closely related phylogenetically to Pele- corhynchus than to other genera of Rhagionidae s. lat., following Woodley and Teskey [18, 23]. Presence or absence of multiple species groups within one genus, each of which stretches over Australia and South America On p. 29 in Nagatomi [25], add the figure 3 in Hennig [47]. CONCLUDING REMARKS (1) The Xylophagidae s. lat. seems to be older in Origin than the Rhagionidae s. lat. (2) The systematic position of various taxa is judged as follows: Pantophthalmidae: either Stra- tiomyoidea or Xylophagoidea; Exeretonevra: Xylophagidae s. lat.; Austroleptis: Rhagionidae or close relative of Rhagionidae; Glutops: Pele- corhynchidae; Vermileonidae: sister group of Apsil- ocephalidae + Empidoidea+Cyclorrhapha; Nem- estrinoidea: sister group of Tabanoidea; Bombyl- iidae: Asiloidea. (3) There are some definite autapomorphic or synapomorphic characters for Coenomyiidae, Pelecorhynchidae + Rhagionidae, and Athericidae + Tabanidae, respectively. (4) There are some possible synapomorphic characters for Rachiceridae+ Xylophagidae, Ex- eretonevridae + Heterostomidae, and Vermileoni- dae + Apsilocephalidae + Empidoidea+ Cyclor- rhapha, respectively. (5) There is one case of circumstantial evi- dence for monophyly of Stratiomyoidea+ Xylophagoidea. (6) Various problems are discussed as follows: 856 A. NAGATOMI presence or absence of metacephalic rod; apomor- phy or plesiomorophy in some characters of male genitalia; two types in primitive creatures; phene- tic taxonomy and cladistic texonomy; validity of small families in the orthorrhaphous Brachycera. (7) Some notes are added to Nagatomi [25]. (8) Figures 1-3 show the most plausible phy- logenetic relationships of the orthorrhaphous Brachycera. ACKNOWLEDGMENTS This paper is dedicated to Dr. Kintaro Baba (in Niigata Pref.) for his interest, encouragement and help which greatly facilitated its preparation. My heart-felt thanks are also expressed to Dr. Leif Lyneborg (Zoological Museum, Copenhagen), Professor Toyohei Saigusa (Kyushu University, Fukuoka), Mr. K. G. V. Smith (formerly British Museum [Natural His- tory], London), Dr. Brian R. Stuckenberg (Natal Museum, Pietermaritzburg, South Africa) and Dr. H. J. Teskey (formerly Biosystematic Research Institute, Agriculture Canada, Ottawa) for their long-term help in many ways. Mr. Smith has also kindly checked the English of this manuscript. I have learned much from the unpublished manuscript on larval stages of primitive Bombyliidae by Dr. D. K. Yeates (American Museum of Natural History, New York) and Dr. M. E. Irwin (University of Illinois, Champaign) to whom I am much indebted. A part of this paper was read at the 19th International Congress of Entomology (Beijing, China) under the chairmanship of Dr. G. C. D. Griffiths (University of Alberta, Edmon- ton) to whom I am also deeply indebted. REFERENCES 1 Woodley, N. E. (1989) Phylogeny and classification of the “orthorrhaphous” Brachycera. In “Manual of Nearctic Diptera, Vol.3” Coord. by J. F. McAlpine and D. M. Wood. Research Branch Agriculture Canada, Monograph No. 32. pp. 1371-1395. 2 Colless, D. H. and McAlpine, D. K. (1991) 39. Diptera. In “The insects of Autralia, Vol. 2. Second ed.” Ed. by Division of Entomolgy, CSIRO. Mel- bourne University Press. pp. 717-786. 3 Krivosheina, N. P. 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(1984) Male genitalia of the lower Brachycera (Diptera). Beitr. Ent., 34: 99-157. Nagatomi, A. and Iwata, K. (1976) Female termina- lia of lower Brachycera (Diptera), I. Beitr. Ent., 26: 5-47. Nagatomi, A. and Iwata, K. (1978) Female termina- lia of lower Brachycera (Diptera), II. Beitr. Ent., 28: 263-293. Nagatomi, A., Saigusa, T., Nagatomi, H. and Lyne- borg, L. (1991) Apsilocephalidae, a new family of the orthorrhaphous Brachycera (Insecta, Diptera). Zool. Sci., 8: 579-591. Nagatomi, A., Saigusa, T., Nagatomi, H. and Lyne- borg, L. (1991) The systematic position of the Apsilocephalidae, Rhagionempididae, Protempidi- dae, Hilarimorphidae, Vermileonidae and some genera of Bombyliidae (Insecta, Diptera). Zool. Sci., 8: 593-607. Nagatomi, A., Saigusa, T., Nagatomi, H. and Lyne- borg, L. (1991) The genitalia of the Apsilocephali- dae (Diptera) Jpn. J. Ent., 59: 409-423. Stuckenberg, B. R. (1973) The Athericidae, a new family in the lower Brachycera (Diptera). Ann. 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Melbourne University Press. pp. 656-740. Teskey, H. J. (1981) Pelecorhynchidae. In “Manual of Nearctic Diptera, Vol. 1.” Coord. by J. F. McAI- pine, B. V. Peterson, G. E. Shewll, H. J. Teskey, J. R. Vockeroth and D. M. Wood. Research Branch Agriculture Canada, Monograph No. 27. pp. 459- 461. Krivosheina, N. P. (1971) The family Glutopidae, fam. n. and its position in the system of Diptera Brachycera Orthorrhapha. Ent. Obozr., 50: 681- 694. (In Russian) Nagatomi, A. (1991) History of some families of Diptera, chiefly those of the lower Brachycera (In- secta: Diptera). Bull. Biogeogr. Soc. Japan, 46: 21- Bie Verrall, G. H. (1909) British flies, Vol. 5. London. 780 pp. Malloch, J. R. (1917) A preliminary classification of Diptera exclusive of Puparia, based on larval and pupal characters, with keys to imagines in certain families, Part I. Bull. Ill. Lab. Nat. Hist., 12: 161- 409. Oldroyd, H. (1972) Two robber flies (Diptera: Asilidae) of unusual structure. J. nat. Hist., 6: 635- 642. 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Wash., 85: 691-697. 35 36 37 38 39 40 41 42 43 45 46 47 857 Webb, D. W. (1978) A revision of the Nearctic genus Dialysis (Diptera: Rhagionidae). J. Kansas Ent. Soc., 51: 405-431. Webb, D. W. (1983) A new genus and species of Nearctic Coenomyiid (Diptera: Coenomyiidae). Proc. Entomol. Soc. Wash., 85: 822-825. Nagatomi, A. (1987) Taxonomic notes on Coenomyiidae (Insecta: Diptera). Zool. Sci., 4: 711-720. Nagatomi, A. and Saigusa, T. (1970) The Coenomyiidae of Japan (Diptera). Mem. Fac. Agric. Kagoshima Univ., 7: 257-292. James, M. T. and Turner, W. J. (1981) Rhagioni- dae. In “Manual of Nearctic Diptera, Vol. 1.” Coord. by J. F. McAlpine, B. V. Peterson, G. E. Shewell, H. J. Teskey, J. R. Vockeroth and D. M. Wood. Research Branch Agriculture Canada, Monograph No. 27. pp. 483-488. Nagatomi, A. (1985) Redescription of Heterosto- mus curvipalpis (Diptera, Heterostomidae) and some notes on my paper of the male genitalia of the lower Brachycera (Diptera). Kontyi, Tokyo, 53: 699-710. Iwata, K. and Nagatomi, A. (1976) Female termina- lia of Tabanidae (Diptera) from Japan. Jpn. J. Sanit. Zool., 27: 83-89. Wood, D. M. and Borkent, A. (1989) Phylogeny and classification of the Nematocera. In “Manual of Nearctic Diptera, Vol. 3.” Coord. by J. F. McAlpine and D. M. Wood. Research Branch Agriculture Canada, Monograph No. 32. pp. 1333-1370. Hutson, A. M. (1977) A revision of the families Synneuridae and Canthyloscelidae (Diptera). Bull. Br. Mus. nat. Hist. (Ent.), 35: 65-100. Daniels, G. (1977) Two new species of Pelecorhyn- chus Macquart (Diptera: Pelecorhynchidae), with the first record of the family from Western Austral- ia. Aust. Entomol. Mag., 4: 73-75. Daniels, G. (1989) 27. Family Pelecorhynchidae. In “Catalog of the Diptera of the Australasian and Oceanian Regions.” Ed. by N. L. Evenhuis, Bishop Museum Special Publication 86. pp. 275-276. Nagatomi, A. (1982) Geographical distribution of the lower Brachycera (Diptera). Pac. Insects, 24: 139-150. Hennig, W. (1960) Die Dipteren-Fauna von Neuseeland als systematisches und tiergeographis- ches Problem. Beitr. Ent., 10: 221-329. (translated by Wygodzinsky, P., 1966, The Diptera fauna of New Zealand as a problem in systematics and zoogeogrphy, Pac. Ins. Monog., 9: 1-81). ratty y \ 5 TPE VRS #8. ta r ¢ Fy i Dame sc WOMPCL Bhat a aaoneb pp tile ert nie wer j shirts 1) aan i (ins pmo: " ¥ 4 y it yet FI i ae Ra? handed inandha™: ee re ee “peav rat fe sivas tc ‘anatel CHa? ny = epson ae r eit Tot Na ike tah dee oid Leah: pelt te Rtn apien: CEL eed ha ae Sire APMIS \ Wisieo inal : Fs wit tigi’ f VE = ni oT, eli 4 nas bY CNY pera Sdeg rs amatptous éanmrA Hendler neivanalt Be ~ SPR na ioe Mk) be choy meee) pat wit TD a nde CTE gah all> yin Prat, afiene Fk M rat 4 ers hepa vi eae lege ati inant of ants tht CEE eed ha oN tS fat mY _ ; Ud a? 4ghe) ihe cet ae ars TL Reel Yo) eo eet rere hatewilahl 168 fees boo Pe eek PT He) TE (% fae erp lis ee ee MR (i $ I 7 - ual A mire P ; : fh ‘ > t i -) i 4 y ; Ts4 \ or aT = " r L 2ewnhont Uw tants Siew op ae tiv esse) ewcpee ig lee al 7 Dre Ae i) a a nn ar eee | dal jose POLLED co lan tp lately AN 4 ; cchyetye Be)» caeapllly toed tiniee et f oT Wie JAR Sit ole ae “pe » Hn fo -UhpOvagyerdin 2a Ey > iLahiags L Rs , ig hal ual omer - ayoy? Gat at agent inom’ Ls Ma tert ri wy Ratt tan: tw Ce A ae are ¥ . , er * | ayy atl obertieinaet 21 a in es o) Lebo ected. at iting ; SP hone rg out ai 4 { ; “He 7 ' : ; Or) emer rey dd: ‘peels ariel Oh mea: iA HAM gS F : wie we) ie alia eee |: = | i j A) Goalie eae A 2.) Meyers) Lal Me jl ley 2 " im prt rt ue. \ yo tenet) A oe ines BAe ( b} cocci pele ale We bed y m ‘anon +4 sellivetiag ; waa a) ‘ pl 4 fq a i, ist ot aN éi i (Ni nt hoethoete Aap, Aoi ZOOLOGICAL SCIENCE 9: 859-874 (1992) © 1992 Zoological Society of Japan Misophriopsis okinawensis sp. nov. (Crustacea: Copepoda) from Hyperbenthic Waters off Okinawa, South Japan, with Definitions of Related Genera Misophria Boeck, 1864 and Stygomisophria gen. nov. Susumu Ontsuka!, Rony Huys’, GEorrrEY A. BoxsHALl” and Tatsunort It6° ‘Fisheries Laboratory, Hiroshima University, Takehara, Hiroshima 725, Japan, "Department of Zoology, The Natural History Museum, Cromwell Road, London SW7 5BD, England, and 3The Seto Marine Biological Laboratory, Kyoto University, Wakayama 649-22, Japan ABSTRACT—A new misophrioid copepod Misophriopsis okinawensis (Crustacea) is reported from Kume Island, Okinawa, South Japan. A full description of the new misophrioid is presented, together with supplementary notes on the males of Misophria pallida Boeck, 1864. Consideration of the phylogeny of the new species led to a reassessment of the affinities between all the named species of Misophria Boeck, 1864 and Misophriopsis Boxshall, 1983. A new genus, Stygomisophira, is recognised based on Misophria kororiensis Boxshall et Iliffe, 1987. Diagnoses are presented of all three genera. INTRODUCTION During taxonomic and phylogenetic studies of the marine hyperbenthic copepod fauna of Japan by one of the authors (SO) (e.g., [1, 2]), a new species of Misophriopsis Boxshall, 1983 (Copepo- da: Misophrioida) was collected at a depth of ca. 170 m off Kume Island, Okinawa, South Japan. The new species is described in detail in the present paper. The misophrioid copepods were infested by a new small crustacean ectoparasite, whose description is given in another paper by the same authors as the first record of the subclass Tantulocarida in the North Pacific region (see [3]). The genus Misophriopsis comprised two sepcies, namely, the type speices, M. dichotoma Boxshall, 1983 [4] and an undescribed new species referred to as Misophriopsis sp. nov. by Boxshall [5] and by Huys and Boxshall [6]. The original diagnosis of Misophriopsis emphasized the presence of a dis- crete 1-segmented endopod on the female fifth leg Accepted May 28, 1992 Received January 31, 1992 " To whom all correspondence should be addressed. 3 Deceased on April 8th, 1990 (see [4]) but the discovery of a similar discrete endopod in female Misophria pallida Boeck, 1864 by Huys and Boxshall [6] makes a reexamination of the validity by these two genera necessary. The group of species currently placed in Misophria comprises the type species, M. pallida, plus two additional species, M. kororiensis Boxshall and Iliffe, 1987 [7] and M. sinensis Boxshall, 1990 [8]. Distinct genera can be recognized within this com- plex of species by reference, in particular, to the segmentation of the antennules, the form of the seminal receptacle of the female, and the structure and armature of the fifth and sixth legs of both sexes. Based on these characters, new differential diagnoses of Misophriopsis, Misophria, and a new genus accommodating M. kororiensis are pre- sented here. The male of Misophria pallida Boeck, 1864 is also redescribed in order to redefine these three genera. One of the authors, Tatsunori Ito deceased suddenly on April 8, 1990 before he finished describing the new host misophrioid copepod and the new parasitic tantulocaridan. This study is dedicated to the late Dr. T. It6 by the first three authors (SO, RH, GAB), in honor of his excellent 860 S. OntsuKA, R. Huys et al. taxonomic and phylogenetic works on maxillopo- dans. MATERIALS AND METHODS The new misophrioid copepods were collected with an originally designed sledge-net (mouth area: 1450 mm X 326 mm; mesh size: 0.33 mm) at a depth of about 167 m off Kume Island, Okinawa, South Japan (26°17.9'N, 126°54.2’E) on 23 May 1989 (local time: 0824-0858). The sledge-net was towed along the bottom at a speed of 2 knots by the T/RV Toyoshio-maru of Hiroshima University for ca. 5 minutes while the depth was measured by a remote-sensing system (Furuno-denki, Color net recorder CN-8). The specimens were fixed in 10% neutralized formalin/sea-water immediately after collection. The new misophrioids were examined with differential interference microscopes (Leitz Dialux 20; Nikon Optiphot) and a scanning elec- tron microscope (JEOL JST-T20). All types of the new species of Misophrioida are deposited in the collections of The Natural History Museum, London. Misophria pallida was redescribed based on three adult males collected from deep sediment of an unspecified locality in Norway (Zoologisk Museum, Oslo (G. O. Sars collection), reg. no. F20793). Redefinition of Misophriopsis Boxshall, 1983 and description of Misophriopsis okinawensis sp. nov. Family Misophriidae Brady Genus Misophriopsis Boxshall, 1983 Diagnosis. Female antennule 18-segmented; male antennule 12-segmented, with aesthetasc on segment I. Seminal receptacle produced trans- versely. Fifth legs without intercoxal sclerite in both sexes; female 5th leg with undivided pro- topod, endopod a small unisetose lobe, free or fused to protopod, proximal exopod segment un- armed, distal exopod segment with 1 seta on each side of apical spine; male Sth leg with undivided protopod, unisetose free endopod and_ 3- segmented exopod, lst exopod segment unarmed, 2nd with 1 inner seta, 3rd with 1 inner seta and 1 seta on each side of apical spine; 6th legs with 1 long seta and 2 short spines in female, with 1 spine and 2 setae in male. Type species. shall, 1983. Remarks. Remarks on the genus Misophriop- sis is presented after the description of the new species described below. Misophriopsis dichotoma Box- Misophriopsis okinawensis sp. nov. (Figs. 1-9) Types. All types collected from near sandy bottom (ca. 167m in depth) off Kume Island, Okinawa, Japan, and deposited in The Natural History Museum, London. Holotype: adult female, dissected and mounted on glass slides, BM (NH) 1991. 151. Paratypes: two adult females and two adult males, dissected and mounted on glass slides, BM (NH) 1991. 152-155; 25 adult females and two adult males, whole specimens, BM (NH) 1991. 156-182; 15 adult females, whole specimens, BH (NH) 1991. 376-390. Body length. Female: mean+standard devia- tion=0.73+0.03 mm (range =0.66—0.79 mm, number examined=28). Male: 0.54+0.01 mm (r =0.52-0.55 mm, n=5). Description. Female (holotype: Figs. 1-A~F, 6-A~D; paratype: Figs. 2~5). Body (Fig. 1-A) compact, 0.78mm in length; prosome oval in dorsal view, about 3 times longer than urosome (Fig. 1-A, B). First pedigerous somite entirely concealed beneath carapace-like expansion from posterior end of maxilliped-bearing somite. Third and 4th pedigerous somites produced posteriorly. Urosome (Figs. 1-B, 2-C) 5-segmented; 5th pedigerous somite posterolaterally produced into acute process on both sides; genital and 1st abdo- minal somites fused to form genital double-somite; original subdivision marked by difference in width between anterior and posterior parts of double- somite; pair of minute copulatory pores located in a small transverse median slit on mid-ventral sur- face of double-somite, each of which leading to a common, transversely produced seminal recepta- cle; pair of slit-like genital pores (Figs. 2-C, 3-C) located ventrolaterally on double-somite and co- New Misophrioid Copepod from Okinawa 861 0.1mm C-E Fic. 1. Misophriopsis okinawensis sp. nov. Female (holotype). A. Habitus, dorsal view; B. Urosome, dorsal view; C. Antennule; D. Antenna; E. Basal two exopod segments of antenna; F. Distal exopod segment of antenna. Male (paratype). G. Habitus, dorsal view. vered by operculum derived from leg 6, armature consisting of 2 spinous processes and 1 seta; 4th somite completely concealed beneath 3rd; anal somite fringed with minute prominences dorsopos- teriorly. Caudal ramus (Figs. 3-D, 4-A) wider than long, bearing 1 dorsal (VII in Fig. 4-A), 1 anterolateral accessory (I), 1 anterolateral (II), 1 posterolateral (III), 1 terminal accessory (VI) and 862 S. Outsuka, R. Huys et al. Fic. 2. Misophriopsis okinawensis sp. nov. Female (paratype). A. Ventral view of oral area and rostrum; B. Lateral view of oral area and rostrum; C. Legs 5 and 6 and genital double-somite, dotted area indicates seminal receptacle. A,: Antennule; R.: Rostrum; L.: Labrum; P.: Paragnath; M.: Mandible; IMS.: Intermaxillary swelling. 2 stout, terminal spinulose setae (IV and V). _ pair of sensilla near its pointed tip. Naupliar eye Rostrum (Fig. 2-A, B) partly fused with labrum, absent. Labrum (Fig. 2A, B) covered with numer- posteroventrally produced, and furnished with a ous minute spinules, bearing row of fine hairs near 863 New Misophrioid Copepod from Okinawa —— { Pith, 4 Hl pS ANY We x aa ———— LE Fic. 3. Misophriopsis okinawensis sp. nov. Female (paratype). A. Mandibular gnathobase; B. Mandibular palp; C Leg 6 and genital and copulatory pores on genital double-somite, lateral view; D. Caudal ramus, ventral view. its posterior margin and pair of short prominences on both sides of posterior end. Pair of paragnaths (Fig. 2-A, B) located posterior to end of labrum, ornamented with fine spinules on tip; intermaxil- lary swelling (Fig. 2-A, B) arising from mid- ventral surface, with row of minute spinules along posterior margin. Antennule (Fig. 1-C) 18-segmented. Segmental distribution of armature elements compiled in Table 1. Antenna (Fig. 1-D~F): coxa bearing row 864 S. OntsuKA, R. Huys et al. yA SEs > 5 DS PASS Zl 7, LT, Fic. 4. of fine setules near anterior margin; basis with 2 apical setae of unequal lengths and patch of fine spinules on posterior surface. Endopod 3- segmented; segment 1 bearing 2 small, subdistal inner setae of unequal lengths and patch of minute Misophriopsis okinawensis sp. nov. Female (paratype). A. Caudal ramus, lateral view; B. Maxillule. spinules on posterior surface; segment 2 with 2 lateral and 3 distal setae along inner margin; segment 3 bearing 6 long setae and short seta apically. Exopod 6-segmented; setal formula as follows: 0, 2, 1, 1, 1, 3. Mandibular gnathobase New Misophrioid Copepod from Okinawa 865 OO UE Te BAG WG Z ul y CE Cas ad a ieee SSS lle Lp LF 4 Lz of << SI r Fic. 5. Misophriopsis okinawensis sp. nov. Female (paratype). A. Maxilla, setae of allobasis and endpod omitted; B. Maxillary allobasis and endopod; C. Maxilliped; D. Distal segment of maxillipedal endopod. (Fig. 3-A) with 5 multicusped teeth, 3 serrate blades and 1 spiniform seta; ornamentation of fine spinules and relatively long setules present on anterior surface near palp. Mandibular palp (Fig. 3-B) biramous; basis bearing patches of fine spi- nules and inner medial seta. Endopod 2- segmented; proximal segment with inner subter- minal seta; distal segment bearing 1 medium 866 S. OutsukA, R. Huys et al. SSS SK Fic. 6. Misophriopsis okinawensis sp. nov. Female (holotype). surface; C. Leg 3, anterior surface; D. Leg 4, anterior surface. length, 1 short and 6 long setae. Exopod 5- segmented; setal formula as follows: 1, 1, 1, 1, 2. Maxillule (Fig. 4-B): praecoxal arthrite with 7 strong spines and 8 setae, 2 of which arising from anterior surface; coxal endite bearing 1 thick se- trate seta and 5 pinnate setae distally; proximal basal endite with 4 apical setae, distal basal endite armed with 4 setae; epipodite of coxa with 7 setae of unequal lengths. Endopod comprising single compound segment, representing fused Ist to 3rd A. Leg 1, anterior surface; B. Leg 2, anterior segments; armature divided into groups of 3 inner medial, 3 inner subterminal and 6 terminal setae representing original segmental elements. Exopod 1-segmented, bearing 3 inner lateral and 5 terminal setae and relatively long setules along inner and outer margins. Maxilla (Fig. 5-A, B): praecoxa partly fused with coxa, with 2 endites, proximal armed with 6 setae, distal with 3 setae; coxa with 2 endites and outer patch of fine spinules, each endite with 3 distal setae; allobasis (Fig. 5-B) New Misophrioid Copepod from Okinawa 867 Fic. 7. Misophriopsis okinawensis sp. nov. Male (paratype). A. Antennule; B. Antennulary segments 3 to 7, dorsal view; C. Antennulary segment 3 to 7, ventral view; D. Leg 5, anterior surface; E. Leg 6, anterior surface. derived from fusion of basis and 1st endopodal segment; produced into strong curved claw and bearing 6 setae (3 of which are derived from 1st endopodal segment); free endopod indistictly 3- segmented, with setal formula 2, 2, 4. Maxilliped (Fig. 5-C, D) having 4 endites on syncoxa with setal formula 0, 1, 3, 2; row of long spinules along outer medial margin; proximal patch of minute spinules and irregular rows of small spinules near bases of setae on middle and subdistal endites; 868 S. OntsuKA, R. Huys et al. oad Fic. 8. Misophriopsis okinawensis sp. nov. SEM micrographs of female. A. Prosome, dorsal view; B. Urosome, dorsal view; C. Cephalosome, lateral view; D. Epicuticular ornamentation on cephalosome. Scale bars=100 «m (A, C); 50 um (B, D). basis with 3 spinulose inner setae medially and row of long spinules proximally, fringed with short spinules along posterior half of inner margin; free endopod 5-segmented; setal formula: 2, 2, 2, 2, 5. The armature formula of legs 1 to 4 is shown in Table 2 and compared with two other congeneric species, Misophriopsis dichotoma and M. sinensis (Boxshall, 1990) comb. nov. Leg 1 (Fig. 6-A): coxa with relatively long spinules near base of plumose inner distal seta; intercoxal sclerite with spinulose row along distal margin; basis bearing row of spinules on anterior surface, and its medial New Misophrioid Copepod from Okinawa 869 - Z TSS Fic. 9. Misophriopsis okinawensis sp. nov. SEM micrographs of female. A. Labrum, pore indicated by an arrow; B. Leg 6. Scale bars=50 um (A); 10 um (B). distal margin expanded into lamellar plate; inner distal spine on basis reaching beyond distal angle of 1st endopod segment; outer distal angle of 2nd endopod segment bifid; outer margins of Ist and 2nd exopod segments lamellar, fringed with mi- nute serrations. Leg 2 (Fig. 6-B): inner distal margin of basis produced into acute tip; its medial distal end expanding into fan-like structure with serrate inner margin; inner distal angle of 2nd endopod segment irregularly produced; inner dis- tal angles of Ist and 2nd exopod segments pro- duced into blunt process. Leg 3 (Fig. 6-C) similar to leg 2, but basis bearing outer medial seta instead of spine and 3rd exopod segment with 5 inner setae. Leg 4 (Fig. 6-D) covered with numerous spinules, in particular, on posterior surface; inner distal end of basis not produced as in legs 2 and 3; intercoxal sclerite relatively small compared with in preceding legs. Leg 5 (Fig. 2-C) indistinctly biramous; protopod comprising fused coxa and basis; inner distal seta on inner process represent- ing endopod partly separated from basis by pro- ximal constriction; outer basal seta present. Pro- topod fringed by minute spinules along inner mar- gin. Exopod 2-segmented; proximal exopod seg- ment unarmed; distal segment laterally and medially bearing fine spinules, and distally serrate spine, inner small and outer medium-length setae. Leg 6 (Figs. 2-C, 3-C) represented by genital oper- culum bearing outer seta on low cylindrical process and 2 small blunt processes. Male (paratypes). Body (Fig. 1-F) 0.54 mm in length, much smaller than in female. Urosome 6-segmented; Sth pedigerous somite with acute posterolateral angles; genital somite large; 3rd abdominal somite small, largely concealed beneath 2nd. Both (Fig. 7-A~C) 12- segmented; geniculate with geniculation between 10th (XIX-XX) and 11th (XXI-XXII]) segments; fusion pattern and distribution of armature ele- ments shown in Table 1. Leg 5 (Fig. 7-D) bira- mous; compound protopodal segment fringed by minute spinules along inner margin, and bearing 1 seta derived from basis; endopod represented by small subcircular segment, bearing small seta dis- tally. Exopod 3-segmented; proximal segment antennules 870 S. OuntsuKA, R. Huys et al. TABLE 1. pallida. numeral. Armatures of antennules of Misophriopsis okinawensis sp. nov. and Misophria Number of antennulary segment of ancestral copepod represented by Roman Number of setae on the segment represented by Arabic numeral: a+b= (number of anterior setae)+(number of posterior setae). ae: Aesthetasc; proc: Spinous process derived from modification of setal element M. okinawensis (female) I 1 II-VII 12 VIII 2 IX-XII 8 XIll 2 XIV 2 XV 2 XVI 2+ae XVII 2 XVIII 2+ae XIX D xX 2 XXI 2+ae XXII 1 XXIII 1 XXIV 1+1 XXV 1+1-+ae XXVI-XXVIII 7+ae M. okinawensis (male) I 1+ae II-VI 9+2ae VII 2+ae VIII 2 IX-XIII 10+ 2ae XIV 2+ae XV-XVI 4+ae XVII 2 XVIII 2+ae XIX-XX 2+ proc XXI-XXIII 3+ ae XXIV-XXVIII 11+2ae unarmed; middle segment with thick, plumose seta at distal inner angle; apical segment with 1 inner seta and 1 seta on each side of apical serrate spine. Leg 6 (Fig. 7-E) forming opercular plate overlying genital opening, bearing large serrate inner spine and 2 plumose setae. Body surface observed with SEM. The body surface of Misophriopsis okinawensis is almost M. pallida (female) I 1 II-VI 14 IX-XII 8 XI 2 XIV 2 XV 2 XVI 2+ae XVII 2 XVIII 2+ae XIX 2 XX 2 XXI 2+ ae XXII 1 XXIll 1 XXIV 1+1 XXV 1+1+ae XXVI-XXVIII 7+ae M. pallida (male) I 1 II-VI 9+2ae Vil 2+ ae VII 2 IX-XIII 10+ ae XIV 2+ae XV 2 XVI 2+ae XVII 2 XVII 2+ae XIX-XX 2+ 2proc XXI-XXIIl 3-+ae+ proc XXIV-XXVIII 11+2ae entirely covered with shallow, epicuticular lamel- lae except for the anterior half of the dorsal cephalic shield which has a smooth surface with a number of hair-like sensilla and small pores but no lamellae (Fig. 8-A, C, D). The lamellae are per- pendicular to the surface on the prosome (Fig. 8-D), whereas they incline posteriorly on the uro- some (Fig. 8-B). Cone organs lacking on the New Misophrioid Copepod from Okinawa 871 TaBLeE2. Seta and spine formula of legs 1 to 4 of Misophriopsis okinawensis sp. nov., M. dichotoma and M. sinensis comb. nov. Seta represented by Arabic numeral, and spine by Roman numeral. o=outer border of segment; t=terminal border of segment; i=inner border of segment Coxa Basis Endopod Exopod 1 2 3 1 2 3 (0) i (0) 1 ©) i (0) i (0) 1 (0) i (0) 1 0 t i Misophriopsis okinawensis sp. nov. Leg 1 (Ds A wile Th, Mul Gg mam ied anh 2 Leg 2 ge tile) anil Mae Veh fade Oa Leg 3 CRemelane i MOCO” yt Poimeks Leg 4 (Eee eee TONe Os teen !2 M. dichotoma’ Loo 1) CRN i ce es ee Leg 2 0 1 1 0 0 1 0 2 M. sinensis comb. nov.” Leg 1 0 1 1 I @) 1 0 2 Leg 2 0 1 1 0 0 1 0 2 1 2 3 I 1 I Ill It 3 2 3 I 1 I 1 Ill I 4 1 2 3 I 1 I 1 Ill I 5 1 2 2 I 1 I 1 Ill I 5 1 2 3 I 1 I 1 Ill I 1 2 3 I 1 I 1 Ill I 5 1 2 3 I 1 I 1 Ill I 4 1 2 3 I 1 I 1 Ill I 5 " Armature formula of legs 3 and 4 the same as in M. okinawensis but the third endopod segments of legs 3 and 4 missing. 2 Armature formula of legs 3 and 4 the same as in M. okinawensis. lateral side of cephalosome (Fig. 8-C) as in Benth- omisophria palliata Sars, 1909 and Misophriopsis dichotoma Boxshall, 1983 [4, 6]. The urosome is covered with lamellae except for the anal somite, as in other misophrioids (Fig. 8-B). The fifth pedigerous somite has a relatively large pore on the dorsomedial surface; the genital somite, anter- ior and posterior dorsomedial pores; the 1st abdo- minal somite, posterior medial pore dorsally; the fourth somite, anterior pore dorsomedially. The labrum is densely covered with ornamenta- tion consisting of dentate scales separated by open smooth areas (Fig. 9-A). Fig. 9-A shows that the rostrum and the labrum are partly separate from each other. A pore located medially on the anterior part of labrum is arrowed in Fig. 9-A. Leg 6 of the female (Fig. 9-B) is armed with a plumose seta on a low cylindrical process, a small spine, and smooth and serrate processes. Etymology. The specific name “okinawensis” is derived from the type locality, Okinawa Prefec- ture. Remarks. Misophria sinensis from Hong Kong is known only from the female but conforms to this new generic diagnosis of Misophriopsis in the 18-segmented antennule and the absence of any armature from the proximal exopod segment of the fifth legs. It is here transferred to Misophriop- sis. The nature of the seminal receptacle of M. sinensis could not be confirmed in the only avail- able specimen. The genus Misophriopsis now comprises the type species plus M. sinensis (Box- shall, 1990) comb. nov. and M. okinawensis sp. nov. These species differ in the armature of legs 1 and 2 (see Table 2): M. okinawensis and M. dicho- toma have a spine on the outer border of the basis while M. sinensis possesses a seta in this position; the numbers of setae along the inner margin of the third exopod segments of legs 1 and 2 are more reduced in M. okinawensis than in M. dichotoma and M. sinensis. The undescribed Misophriopsis species from the North Atlantic off the coast of Norway, mentioned by Boxshall [5] and illustrated in part by Huys and Boxshall [6] differs significantly from the new concept of Misophriopsis. Its systematic position will be considered in a separate account after it has been fully described. Misophria kororiensis, de- scribed from a subtidal anchialine cave in Palau [7], cannot be placed in either Misophria or Mis- 872 S. Outsuka, R. Huys et al. ophriopsis. Accordingly a new genus is proposed to accommodate M. kororiensis. The differential diagnosis of the new genus is given below. Redefinition of Misophria Boeck, 1864 and establishment of Stygomisophria gen. nov., with description of male Misophria pallida Boeck, 1864 Genus Misophria Boeck, 1864 Diagnosis. Female antennule 17-segmented; male antennule 13-segmented, without aesthetasc on segment I. Seminal receptacle not transversely produced. Fifth legs without intercoxal sclerite in both sexes; female 5th leg with undivided pro- topod, endopod a small bisetose free lobe, proxim- al exopod segment with outer seta, distal exopod segment with 1 seta on each side of apical spine; male Sth leg with bisetose endopod fused to undi- vided protopod, exopod as in female; 6th legs armed with 1 long seta and 2 short spines in female, with 2 setae in male. Type species. Misophria pallida Boeck, 1864, monotypy. Remarks. The genus Misophria is disting- uished from the genus Misophriopsis in: (1) the numbers of antennulary segments of both sexes; (2) the presence or absence of an aesthetasc on antennulary segment I; (3) the shape of seminal receptacle; (4) the armature elements of legs 5 and 6 of both sexes. Misophria pallida Boeck, 1864 (Fig. 10) Both sexes of Misophria pallida were described by Sars [9] but the description contains insufficient detail for a full comparison to be made with the other known species currently attributed to Mis- ophria (see [7, 8]). Huys and Boxshall [6] illus- trated some features of M. pallida, including the female antennule, the rami of the mandibular palp, the female fifth leg and the female genital region including the sixth legs. Supplementary descriptions of the antennules and the fifth and sixth legs of the males are presented below in order to compare the male of M. pallida with that of Misophriopsis okinawensis sp. nov. Male antennule (Fig. 10-A) 13-segmented, seg- mental fusion pattern and setation pattern summa- rized in Table 1; compound segments II-VI and IX-XIII without any incomplete sutures marking fusion planes. Segments XV and XVI partly fused along posterior surface; sheath present on segment XV (arrowed in Fig. 10-B) but with anteroventral margin of sheath integrated into segment leaving shallow transverse furrow (arrowed in Fig. 10-C). Geniculation in neocopepodan position between compound segments XIX-XX and XXI-XXIII. Male 5th legs (Fig. 10-D) separate, intercoxal sclerite lacking; leg comprising compound pro- topodal segment bearing outer basal seta and 2 setae on inner distal margin representing endopod armature; endopod completely incorporated into compound protopodal segment, with no vestige of suture remaining; exopod 2-segmented; proximal segment with seta on outer margin, distal segment produced into spinous process at outer distal angle, distal margin armed with long outer seta, bilaterally serrate spine and shorter, naked, inner angle seta (Fig. 10-D). All segments with surface and marginal ornamentation of fine spinules. Male 6th leg (Fig. 10-E): a transverse plate closing off genital aperture on each side; armed with 2 setae and ornamented with spinular row along free margin. Stygomisophria gen. nov. Diagnosis. Female antennule 18-segmented. Seminal receptacle not produced transversely. Female 5th leg with intercoxal sclerite and sepa- rate coxa and basis, endopod represented by | seta on inner distal margin of basis, proximal exopod segment with outer seta, distal exopod segment with 1 seta on each side of apical spine; female 6th leg with 1 long seta and 1 small spine. Male unknown. Type species. Misophria kororiensis Boxshall et Iliffe, 1987, monotypy. The new genus is distinguished from both Misophria and Misophriopsis by the retention of an intercoxal sclerite and separate coxa and basis in the female fifth leg. It also differs from Misophria in segmentation of the female antennule Remarks. 873 New Misophrioid Copepod from Okinawa 0.05 mm A o.05mm B,C Fic. 10. Misophria pallida Boeck, 1864. Male. A. Rostrum and antennule, geniculation indicated by an arrowhead; B. Antennulary segments 2 to 8, dorsal surface, distal margin of sheath on segment 7 indicated by arrowheads, aesthetascs omitted; C. Antennulary segments 2 to 8, ventral surface, distal margin of sheath on segment 7 indicated by arrowheads; D. Leg 5, anterior surface; E. Leg 6, anterior surface. 874 and from Misophriopsis in the presence of an outer seta on the proximal exopod segment of the female fifth leg. ACKNOWLEDGMENTS We express our sincere thanks to the captain and crew of the T/RV Toyoshio-maru of Hiroshima University for cooperation at sea. Thanks are due to Prof. E. Harada of the Seto Marine Biological Laboratory for his permission to observe the specimens of the present new misophrioid copepod which the late Dr. T. Ito had been examining just before his death. This work was in part supported by grants from the Research Institute of Marine Inverte- brates and the Nissan Science Foundation to one of the authors (SO). REFERENCES 1 Ohtsuka, S. and Mitsuzumi, C. (1990) A new asym- metrical near-bottom calanoid copepod, Paramis- ophria platysoma, with observations of its in- tegumental organs, behavior and in-situ feeding habit. Bull. Plankton Soc. Japan, 36: 87-101. 2 Ohtsuka, S., Fosshagen, A. and Go, A. (1991) The hyperbenthic calanoid copepod Paramisophria from Okinawa, South Japan. Zool. Sci., 8: 793-804. 3 S. OutTsuKA, R. Huys et al. Huys, R., Ohtsuka, S., Boxshall, G. A. and Ito, T. (1992) Itoitantulus misophricola gen. et sp. nov.: first record of Tantulocarida (Crustacea: Maxillopoda) in the North Pacific region. Zool. Sci., 9: 877-888. Boxshall, G. A. (1983) Three new genera of mis- ophrioid copepods from the near-bottom plankton community in the North Atlantic Ocean. Bull. Br. Mus. Nat. Hist. (Zool.), 44: 103-124. Boxshall, G. A. (1989) Colonization of inland marine caves by misophrioid copepods. J. Zool. Lond., 219: 521-526. Huys, R. and Boxshall, G. A. (1991) Copepod Evolution, The Ray Society, London, 468 pp. Boxshall, G. A. and Iliffe, T. M. (1987) Three new genera and five new species of misophrioid copepods (Crustacea) from anchialine caves on Indo-West Pacific and North Atlantic islands. Zool. J. Linnean Soc., 91: 223-252. Boxshall, G. A. (1990) A new species of Misophria (Copepoda: Misophrioida) from Hong Kong. In “Proceedings of the Second International Marine Biological Workshop: The Marine Flora and Fauna of Hong Kong and Southern China, Hong Kong, 1986”. Ed. by B. Morton, Hong Kong University Press, Hong Kong, pp. 515-522. Sars, G. O. (1903) An account of the Crustacea of Norway V. Copepoda Harpacticoida. Bergen Museum, pp. 1-28. ZOOLOGICAL SCIENCE 9: 875-886 (1992) © 1992 Zoological Society of Japan Itoitantulus misophricola gen. et sp. nov.: First Record of Tantulocarida (Crustacea: Maxillopoda) in the North Pacific Region Rony Huys!, Susumu OntsukA*, GEOFFREY A. BOXSHALL! and Tatsunort IT6° 'Department of Zoology, The Natural History Museum, Cromwell Road, London SW7 SBD, England, * Fisheries Laboratory, Hiroshima University, Takehara, Hiroshima 725, Japan, and 3The Seto Marine Biological Laboratory, Kyoto University, Wakayama 649-22, Japan ABSTRACT—A new tantulocaridan, Itoitantulus misophricola Huys, Ohtsuka et Boxshall gen. et sp. nov., (Crustacea) ectoparasitic on a hyperbenthic misophrioid copepod, Misophriopsis okinawensis Ohtsuka, Huys, Boxshall et It6, 1992, is described from Kume Island, Okinawa, South Japan. This is the first record of Tantulocarida from the North Pacific region. The new tantulocaridan is placed in the Deoterthridae on account of the 1-segmented abdomen, the absence of a rostrum in the tantulus larva, the segmentation of the rami of the thoracopods, and the position of the expanded trunk sac in the male. The new genus can be distinguished from other deoterthrid genera by the absence of a lobate endite of thoracopod 1 and the presence of the dorsally directed, recurved spine on the apex of the sixth thoracopod in the tantulus larva. INTRODUCTION The Tantulocarida was established as a new class of the subphylum Crustacea by Boxshall and Lin- coln [1]. The recent phylogenetic analysis by Boxshall and Huys [2] revealed that the Tantulo- carida is a taxon within the class Maxillopoda and closely related to the Thecostraca, Branchiura and Ostracoda in having an unpaired penis derived from the seventh thoracic legs of the adult male. The maxillopodan affinities of the Tantulocarida were supported by Huys’ [3] new interpretation of the trunk tagmosis of tantulocarids. All known species of the Tantulocarida are ectoparasitic on crustacean hosts, including harpacticoid copepods, myodocopid ostracods, asellote isopods, cuma- ceans, and tanaidaceans. More than 20 species have been described from the subtidal to the abyssal depth zones in the North and South Atlan- tic, the Indian and the South Pacific Oceans [1- Accepted May 28, 1992 Received January 31, 1992 2 To whom all correspondence should be addressed. 3 Deceased on April 8th, 1990 15]. The hypothetical life cycle of the Tantulocar- ida was presented by Boxshall and Lincoln [8] and Huys [3]. It is characterized by extreme abbrevia- tion: tantulocaridans have a single larval stage which attaches to a crustacean host, the female then sloughs its pedigerous and abdominal somites and forms a sac-like trunk containing eggs behind the cephalic shield, whereas the male differentiates within the expanded larval thoracic somites and is released as a free-swimming stage with six pairs of powerful thoracic legs for swimming but no mouth- parts. A new species of the Tantulocarida infested a new species of the Misophrioida collected from the near-bottom off Kume Island, Okinawa, South Japan (see [16]). This is the first record of a tantulocaridan in the North Pacific Ocean and the first of a tantulocaridan parasitic on misophrioid copepods. The present paper describes immature females and males, and the tantulus larva of the new tantulocaridan from Okinawa. One of the authors, Tatsunori It6 deceased suddenly on April 8, 1990 at the age of 45 before completion of this study. The first three authors 876 R. Huys, S. OutsuKA et al. (RH, SO, GAB), therefore, determined on car- rying out his intention and dedicate this study to the late Dr. T. Ito. MATERIALS AND METHODS The host misophrioid copepod Misophriopsis okinawensis Ohtsuka, Huys, Boxshall et It6, 1992 [16] was collected at a depth of about 167 m off Kume Island, Okinawa, South Japan (26°17.9'N, 126°54.2°E) on 23 May 1989 (local time: 0824— 0858). The sampling gears and techniques and laboratory procedures were described in detail in Ohtsuka et al. [16]. All types of the new species of the Tantulocarida are deposited in the collections of The Natural History Museum, London. Family Deoterthridae Boxshall et Lincoln Genus /toitantulus Huys, Ohtsuka et Boxshall gen. nov. Diagnosis. Yantulus larva comprising cepha- lon, 6 pedigerous somites and 2-segmented uro- some. First thoracic tergite largely concealed beneath posterior margin of dorsal cephalic shield. Cephalic shield tapering anteriorly, giving a pointed appearance; ornamentation consisting of 4 anterolateral, 2 dorsal and 6 posterior pairs of pores and 2 distinct longitudinal lamellae. Cepha- lic stylet straight. Thoracopods 2 to 5 with well developed endite, thoracopods 1 and 6 without endite. Protopods of thoracopods 1 to 5 undi- vided; exopods apparently 2-segmented with 2 (leg 1), 4 (legs 2-4) or 5 setae (leg 5). Setae of legs 2 to 5 on distal exopod segment, leg 1 with 1 seta on each segment. Endopod apparentlyl-segmented, lacking setae in leg 1 or armed with 2 setae (legs 2— 5). Thoracopod 6 without distinct rami, armed with 2 setae apically, the medial seta very strong and curved. Posterior urosomal somite ornamented with 5 incomplete transverse lamellae and an array of spinous processes around posterior margin. Caudal rami armed with 2 long, distinctly geniculate setae and a shorter simple seta. Trunk sac containing adult male formed posterior to 6th thoracic tergite. Male cephalothorax with 4 pairs of aesthetascs anteriorly. Penis slightly recurved. Caudal rami with 3 setae. Adult female with scar formed after larval trunk sloughed located some distance posterior to ventral margin of head. Ecto- parasitic on a misophrioid copepod Misophriopsis okinawensis Ohtsuka, Huys, Boxshall et It6, 1992. Type species. Itoitantulus misophricola Huys, Ohtsuka et Boxshall gen. et sp. nov. Etymology. The generic name “J/toitantulus” is named in honor of the late Dr. Tatsunori It6, and derived from his family name, It6 and the Latin tantulus, which forms part of the name Tantulo- carida. Itoitantulus misophricola Huys, Ohtsuka et Boxshall gen. et sp. nov. (Figs. 1-7) Types. All types infesting Misophriopsis oki- nawensis Ohtsuka, Huys, Boxshall et It6, 1992 collected from the sandy bottom (ca. 167m in depth) off Kume Island, Okinawa, South Japan, and deposited in the Natural History Museum, London. Holotype: tantulus larva attached on antennary basis of M. okinawensis. BM (NH) 1991. 183. Paratypes: 3 tantulus larvae, 2 early stage males, 4 early stage females, BM (NH) 1991. 184-192. Description. Tantulus larva (holotype). Body comprising cephalon covered by dorsal shield, 6 pedigerous somites and 2-segmented urosome (Fig. 1-A, B). Total body length 156 ~m, mea- sured from tip of cephalon to posterior margin of caudal rami. Cephalon tapering markedly towards oral disc; posterior margin of dorsal shield slightly convex. Shield about 2.1 times longer than wide (78 um X37 um); 1 pair of longitudinal epicuticu- lar lamellae extending almost to base of oral disc; short transverse lamellae present near rear margin. Rostrum absent. Oral disc about 13 ~m in dia- meter, located anteroventrally at frontal margin of head; disc bearing 1| pair of filament-like structures laterally, originating beneath the outer sheath. Pore pattern comprising 4 anterolateral pairs, 1 of which with sensillum, 2 dorsal pairs and 6 postero- lateral pairs, 2 of which with sensillum. Ventral surface lacking ornamentation. Cephalic stylet visible through integument, about 36 ~m long, more or less straight, without lateral barbs, base hollow. Little internal structure discernible. Tantulocaridan from the North Pacific 877 Fic. 1. Jtoitantulus misophricola gen. et sp. nov. Tantulus larva (holotype). A. Habitus, dorsal view, tantulus larva attached to antennary basis of the host Misophriopsis okinawensis. B. Habitus, lateral view; C. Thoracic somite 1, anterior view; D. Abdomen, dorsal view. 878 R. Huys, S. OHTSUKA et al. | | olo2in A-D | Fic. 2. Itoitantulus misophricola gen. et sp. nov. Tantulus larva (holotype). A. Thoracopod 1; B. Thoracopod 2; C. Thoracopod 5; D. Thoracopod 6, seventh thoracic somite and abdomen, ventral view. Tantulocaridan from the North Pacific 879 Fic. 3. Jtoitantulus misophricola gen. et sp. nov. Male contained within trunk sac formed by the preceding tantulus stage (paratypes). A. Early stage male attached to second exopodal segment of antenna of Misophriopsis okinawensis (adult female); B. Early stage male attached to antennule of M. okinawensis (copepodid V female); C. Early stage male more developed than the stage illustrated in A, enlargement of B, lateral view; D. Same, dorsal view; E. Lateral view of head of early stage male attached to antennulary segment of the host M. okinawensis, showing internal anatomy. 880 R. Huys, S. OnTSUKA et al. Fic. 4. Itoitantulus misophricola gen. et sp. nov. Early stage male (A) and female (B-D) (paratypes). A. Recurved penis (indicated by an arrowhead) and abdomen of early stage male (see Fig. 3-C), lateral view; B. Early stage female attached to antennulary segment of Misophriopsis okinawensis, dorsal view; C. Early stage female, lateral view, scar indicated by an arrowhead; D. Early stage female more developed than the stage illustrated in C, lateral view, scar indicated by an arrowhead. Tantulocaridan from the North Pacific 881 Fic. 5. Itoitantulus misophricola gen. et sp. nov. SEM micrographs of tantulus larva. A. Habitus, lateral view, attached to antenna of Misophriopsis okinawensis; B. Isolated head attached to antenna of host, dorsolateral view; C. Isolated head attached to antennule of host, dorsal view; D. Isolated head, posteroventral view, sensilla indicated by large arrows, pores with collar by small arrows. Scale bars=10 um. Thoracic somites 1 to 6 each with well developed tergite, that of 1st somite (Fig. 1-C) small, largely concealed beneath posterior rim of cephalic shield; 3rd to 5th tergites ornamented with 4 longitudinal lamellae. Each somite with well developed tho- racopod. Thoracopod 1 (Fig. 2-A) comprising un- divided protopod, 2-segmented exopod and 1- segmented endopod; protopod without endite, armed with single medial spine; endopod with bifid tip; exopod with short lateral seta on Ist segment and long apical seta on 2nd segment. Thoracopods 2 to 4 (Fig. 2-B) comprising undivided protopod bearing endite armed with 2 coupling spines, 2- segmented exopod and 1-segmented endopod; ex- opod with 2 long and 2 medium length setae on distal segment; endopod with bifid tip, armed with 2 setae arising from common base located laterally on proximal swollen part of ramus. Thoracopod 5 (Fig. 2-C) as in thoracopods 2 to 4 but with 2 long and 3 short setae on exopod. Sixth thoracopod (Fig. 2-D) with undivided protopod bearing 2 cou- pling spines medially; 2 apical setae representing rami, lateral seta slender and straight, powerful medial seta strongly recurved dorsally. Urosome (Figs. 1-D, 2-D) 32 um in length, com- prising small 7th thoracic somite and large ab- dominal somite. Seventh thoracic somite un- ornamented. Abdominal somite 1.5 times longer than wide (25 ~m X16 ~m); ornamented with 5 transverse epicuticular lamellae, 2 of which are 882 R. Huys, S. OntTsuKA et al. Fic. 6. Itoitantulus misophricola gen. et sp. nov. SEM micrographs of tantulus larva. A. Oral disc, dorsal view; B. Oral disc, dorsolateral view, folded anterior part of cephalic shield indicated by an arrow; C. Cephalic shield of isolated head, posterodorsal view, dorsal pores indicated by arrows; D. Dorsal pores indicated by arrows. Scale bars=10 um (A, C); 1 xm (B, D). incomplete ventrally; posterior margin with 3 pairs of spinous processes ventrally, decreasing in size away from midline. Caudal rami not clearly delim- ited from somite; bearing 2 pairs of large setae, each with transverse flexure plane proximally and a complex, sheathed geniculation more distally; 3rd pair of simple setae present, arising dorsal to main caudal setae, either side of operculum-like structure with finely spinulated free margin. Male metamorphosis (paratypes). Adult male formed in reflexed position within expanded trunk sac of preceding tantulus larva (Fig. 3-A~D). Tantulocaridan from the North Pacific 883 : be fis) j FG Fic. 7. Jtoitantulus misophricola gen. et sp. nov. SEM micrographs of tantulus larva. A. Thoracic somites and urosome, lateral view; B. Thoracic somites, enlargement of A, tergopleural grooves indicated by arrows; C. Thoracopod 6 and urosome, enlargement of A, lateral view; D. Thoracopods 1 to 4. Scale bars=10 um (A, B, D); 1 um (C). Trunk sac located dorsally, posterior to 6th tho- racic tergite. Head, rest of thorax and urosome remaining unchanged from preceding stage; 1st tergite beneath cephalic shiled. Metamorphosing male supplied with nutrients via tissue connection (umbilical cord) with larval head (Fig. 3-E). Male at early stage in material available; thoracopods 1-6 present, setose, details not visible; cepha- 884 R. Huys, S. Ontsuka et al. lothorax with 4 pairs of aesthetascs (Fig. 3-C). Penis recurved at early stage of development (arrowed in Fig. 4-A). Caudal ramus armed with 3 short spiniform elements (Fig. 4-A). Adult female (paratypes). Early stages of adult female showing scar formed by sloughing of larval trunk (arrowed in Fig. 4-C, D). Larval head unchanged from preceding stage (Fig. 4-B~D). Trunk sac contents undifferentiated at this early stage. Etymology. The specific name “misophricola” is derived from the host misophrioid copepod, and the Lation colere, meaning to inhabit. SEM observations of tantulus larvae of Itoitantulus misophricola Huys, Ohtsuka et Boxshall gen. et sp. nov. Tantulus larvae of Jtoitantulus misophricola were also examined with SEM (Figs. 5~7). The attachment sites of the examined tantulus larvae were on proximal segments of the antennules (Fig. 5-C) and the basis and rami of the antennae (Fig. 5-A, B) of the host misophrioid, Misophriopsis okinawensis. Isolated cephalic shields without thoracic somites and abdomen were often found remaining on the host (Fig. 5-B~D), probably due to damage during collection. The oral disc are shown in Figs. 5-C and 6-A, B. The oval oral disc is ca. 8 wm in length and ca. 5 um in width. The cephalic shield has numerous integumental pores and sensilla and has complex epicuticular orna- mentation. The integumental organs are basically distributed symmetrically except for two dorsome- dial pores with a complex opening. The posterior margin of head (Fig. 5-D) has two pairs of fine sensilla (indicated by large arrows) and a pair of relatively large pores with collar (indicated by small arrows). The collar seems to function as an operculum of the pore, because some of pores are closed by the collars (see Figs. 5-D, 6-C), but this may be an artifact of the preparation process for SEM. Such integumental organs are hitherto unknown in any other tantulocaridan species [1- 15]. The dorsal surface of head is entirely covered with fine mesh-like epicuticular ridges (Fig. 6-C, D), while the ventral surface has no epicuticular ridges such as on the dorsal surface but is smooth (Fig. 5-D). A pair of longitudinal lamellar plates on the dorsomedial surface extends from near the oral disc to in front of the pores with a complex opening (Figs. 5-C, 6-C). These complicated epicuticular structures on the cephalic shield are also found in tantulus larvae of other genera, Campyloxiphos, Coralliotantulus and Apho- tocentor, but not in Microdajus [3, 10, 12, 14, 15]. Thoracic somites lack mesh-like epicuticular ornamentation (Fig. 7-A, B), but each possesses a transverse lamellar ridge dorsally, and a pair of pleurae laterally separated by a deep tergopleural groove (Fig. 7-B, indicated by an arrow). Such deep tergopleural grooves on thoracic somites are also found in Doryphallophora harrisoni (Boxshall and Lincoln, 1987) [8]. The urosome is depicted in Fig. 7-C. The joint is between the seventh thoracic and first abdominal somites. The seventh thoracic somite is not ornamented. The abdomen is partly covered with small epicuticular prominences as in thoracopod 6. The caudal ramus is ornamented with serrated processes on the posterior end. Thoracopods 1 to 4 and thoracopod 6 are shown in Fig. 7-D and -C, respectively. The outermost setae on apical exopod segments of thoracopods 1 to 4 are clearly found to be serrate. The curved, outer spiniform seta on thoracopod 6 bears minute spinules proximally and medially, and the lateral seta is also serrate. DISCUSSION The new tantulocaridan definitely belongs to the family Deoterthridae redefined by Huys [15], on the basis of 2-segmented urosome in the tantulus larvae, the absence of a rostrum from the tantulus, the segmentation of the rami of thoracopods, and the position of the expanded trunk sac in the male behind the sixth thoracic tergite. The Deoterthri- dae currently comprises six genera, Deoterthron Bradford et Hewitt, 1980, Boreotantulus Huys et Boxshall, 1988, Dicrotrichura Huys, 1989, Apho- tocentor Huys, 1990, Campyloxiphos Huys, 1990 and Coralliotantulus Huys, 1990 [3, 15]. The new genus can be distinguished from all of these by the absence of a lobate endite from thoracopod 1. Another distinctive feature is the presence of the Tantulocaridan from the North Pacific 885 dorsally-directed, recurved spine on the apex of the sixth thoracopod in the tantulus larva. This is not found in any other tantulocaridans although the form of the sixth leg of Dicrotrichura is similar. Within the Deoterthridae the affinities of [toitantu- lus clearly lie with Dicrotrichura, with which it shares the modified caudal setae, an unarmed endopod on thoracopod 1 and a recurved medial seta on the sixth thoracopod (see [13]). Itoitantulus misophricola is the first occurrence of the Tantulocarida in the North Pacific Ocean, although six species have hitherto been recorded from the shallow- to deep-seas around Tasmania, New Zealand and Peru [3]. As already shown by Huys [3], tantulocaridans are widely distributed from high latitudes to subtropical regions in both northern and southern hemispheres. The discov- ery of Coralliotantulus coomansi Huys, 1990 from the Great Barrier Reef [14] might have suggested a new locality of tantulocaridans in the subtropical regions in Japanese waters. Tantulocaridans are usually found parasitic on shallow- and deep-water epibenthic crustaceans but rarely on hyperbenthic or pelagic crustaceans except for the new tantulo- caridans. This may suggest the epibenthic origin of ancestral tantulocaridans. This is the first record of tantulocaridans parasit- ic On misophrioid copepods. The Deoterthridae currently accommodates seven species, four of which parasitized harpacticoid copepods, one parasitized ostracods and the rest were found free in sediment before attachment to hosts (see [3]). The present new tantulocaridan infested a variety of body parts of the host misophrioid copepods, namely, the antennules, antennae, mandibular palps, maxillipeds, and urosomes, but was most usually found attached to the antennae. Four deoterthrids parasitic on harpacticoids, namely, Deoterthron lincolni (Boxshall, 1988), Boreotantu- lus kunzi Huys et Boxshall, 1988, Campyloxiphos dineti Huys, 1990, and Coralliotantulus coomansi Huys, 1990 were attached to cephalosomes, uro- somal somites, swimming legs and caudal setae of their hosts [9, 11, 14, 15]. Deoterthron dentatum Bradford et Hewitt, 1980 was found attached to a seta on the antennary exopod of ostracod [6, 15]. Within the Deoterthridae, only the present new tantulocaridan and D. dentatum have been found infesting the cephalic appendages of their hosts. ACKNOWLEDGMENTS We express our sincere thanks to the captain and crew of the T/RV Toyoshio-maru of Hiroshima University for cooperation at sea. We are also thankful to Prof. E. Harada of the Seto Marine Biological Laboratory for permitting us to observe the specimens of the present new tantulocaridan which the late Dr. T. It6 was describ- ing just before his death. This work was in part sup- ported by grants from the Research Institute of Marine Invertebrates and the Nissan Science Foundation to one of the authors (SO). REFERENCES 1 Boxshall, G. A. and Lincoln, R. J. (1983) Tantulo- carida, a new class of Crustacea ectoparasitic on other crustaceans. J. Crust. Biol., 3: 1-16. 2 Boxshall, G. A. and Huys, R. (1989) New tantulo- carid, Stygotantulus stocki, parasitic on harpacticoid copepods, with an analysis of the phylogenetic rela- tionships within the Maxillopoda. J. Crust. Biol., 9: 126-140. 3 Huys, R. (1991) Tantulocarida (Crustacea: Maxillo- poda): A new taxon from the temporary meioben- thos. P. S. Z. N. I.: Mar. Ecol., 12: 1-34. 4 Greve, L. (1965) A new epicaridean from western Norway, parasitie on Tanaidacea. Sarsia, 20: 15-19. 5 Becker, K.-H. (1975) Basipodella harpacticola n. gen., n. sp. (Crustacea, Copepoda). Helgolander Wiss. Meeresunters., 27: 96-100. 6 Bradford, J. M. and Hewitt, G. C. (1980) A new maxillopodan crustacean, parasitic on a myodocopid ostracod. Crustaceana, 38: 67-72. 7 Lincoln, R. J. and Boxshall, G. A. (1983) A new species of Deoterthron (Crustacea: Tantulocarida) ectoparasitic on a deep-sea asellote from New Zea- land. J. Nat. Hist., 17: 881-889. 8 Boxshall, G. A. and Lincoln, R. J. (1987) The life cycle of the Tantulocarida (Crustacea). Phil. Trans. R. Soc. Lond. B315: 267-303. 9 Boxshall, G. A. (1988) A new genus of tantulocar- idan (Crustacea: Tantulocarida) parasitic on a har- pacticoid copepod from Tasmania. Bull. Br. Mus. Nat. Hist. (Zool.), 54: 271-274. 10 Grygier, M. J. and Sieg, J. (1988) Microdajus (Crustacea: Tantulocarida) parasitic on an Antarctic tanaidancean, and a range extension of M. langi Greve. J. Nat. Hist., 22: 1495-1505. 11 MHluys, R. and Boxshall, G. A. (1988) A new genus and species of tantulocaridan (Crustancea: Tantulo- carida) parasitic on a harpacticoid copepod from the Skagerrak. Sarsia 73: 205-211. 12 13 14 886 Boxshall, G. A., Huys, R. and Lincoln, R. J. (1989) A new species of Microdajus (Crustacea: Tantulo- carida) parasitic on a tanaid in the northeastern Atlantic, with observations on M. langi Greve. Syst. Parasit., 14: 17-30. Huys, R. (1989) Dicrotrichura tricincta gen. et spec. nov.: A new tantulocaridan (Crustacea: Maxillopo- da) from the Mediterranean deep sea off Corsica. Bijdr. Dierk., 59: 243-249. Huys, R. (1990) Coralliotantulus coomansi gen. et sp. n.: first record of a tantulocaridan (Crustacea: Maxillipoda) from shallow subtidal sands in tropical 15 16 R. Huys, S. OuntsuKa et al. waters. Stygologia, 5: 183-198. Huys, R. (1990) Campyloxiphos dineti gen. et spec. nov. from off Namibia and a redefinition of the Deoterthridae Boxshall & Lincoln (Crustacea: Tan- tulocarida). J. Nat. Hist., 24: 415-432. Ohtsuka, S. Huys, R., Boxshall, G. A. and Ito, T. (1992) Misophriopsis okinawensis sp. nov. (Crus- tacea: Copepoda) from hyperbenthic waters off Oki- nawa, South Japan, with definitions of related genera Misophria Boeck, 1864 and Stygomisophria gen. nov. Zool. Sci., 9: 861-876. ZOOLOGICAL SCIENCE 9: 887-889 (1992) [COMMUNICATION] © 1992 Zoological Society of Japan Effect of Monochromatic Illumination of the Brain on the Phototactic Behavior of Orb Weaving Spiders, Argiope amoena and Nephila clavata SHIGEKI YAMASHITA Biological Laboratory, Kyushu Institute of Design, Shiobaru, Fukuoka 815, Japan ABSTRACT—The effect of monochromatic light ap- plied to the brain on the phototactic behavior of tethered orb weaving spiders was examined. It is suggested that cerebral photosensitive neurons which have a maximum sensitivity at about 440 nm play a role in phototactic behavior. INTRODUCTION The existence of photosensitive neurons in the brain of orb weaving spiders has been revealed electrophysiologically [1-3] and behaviorally [4, 5]. Yamashita and Tuji [4] examined the phototac- tic behavior of tethered orb weaving spiders, Argiope and Nephila, while they walked on a y-maze globe. On a dark background, the spiders tended to turn away from light given to the eyes. On a light background, however, the spiders tended to turn towards the light. When the brain was illuminated through the cuticle covering it with a small light spot, the spiders also tended to turn towards the light given to the eyes. Blue light given to the brain had a marked effect on the phototactic respone, but not green light. Yamashi- ta and Tuji [4] concluded that cerebral phtosensi- tive neurons control phototactic behavior, and suggested that these neurons had maximum sensi- tivity in the blue region. In the present study, I examined the efficiency of monochromatic light given to the brain on phototactic behavior. Accepted June 5, 1992 Received April 30, 1992 MATERIALS AND METHODS Female orb weaving spiders, Argiope amoena and Nephila clavata, were collected in open fields. The methods for these experiments were similar to those described previously [4]. In brief, the spider was held rigidly in space and was given a y-maze globe to hold (Fig. 1). For illumination of the eyes, two light beams emitted from two tungsten filament lamps were focused onto the ends of two light guides. The exits of the light guides were LG to left eyes toright eyes = Fic. 1. The spider is fixed in space by a quartz light guide and given a y-maze globe to hold. LG, light guide. 888 S. YAMASHITA aimed horizontally at the right eyes from 45° clockwise and at the left eyes from 45° anticlock- wise to the body axis of the spider, respectively. The intensity of the light was about 30 lux at the eyes. For monochromatic light stimulation of the brain, a light beam emitted by a 500 W Xenon arc lamp was passed through interference filters and focused onto one end of a quartz light guide of 2 mm in diameter. The other end of the light guide was joined onto the central part of the prosoma just above the brain (Fig. 1). The intensity of the light given to the brain (I,,) is referred to as relative quantum flux. Maximum relative intensity (Ip,=1.0) corresponded to about 10'* quanta/ cm*-sec at the output of the light guide. In most cases, spiders made 100 turns at y-arms for each test (e.g. each point on Figure 2); 50 during stimulation of the left eyes and 50 during stimulation of the right eyes. Prior to the start of each test run, spiders were adapted to each test condition for 5-10 min. Usually, three or four tests were performed on a single spider. The turning reaction (TR) was defined as P—N/P+N, where P is the number of turns at y-arms directed towards the light given to the eyes, and N is the number of turns directed away from the light. +0.2 Turning reaction -0.4 _——————E—— ee 0 0.2 0.4 0.6 0.8 1.0 Illumination of brain, Ipr Fic. 2. Effects of illumination of the brain by 360, 440 and 520nm light on phototactic response. The turning reactions (TR=P—N/P+N) are plotted against the relative intensity of light given to the brain (1,,). RESULTS AND DISCUSSION The effects of intensity of 360, 440 and 520 nm light given to the brain on the phototactic response are shown in Figure 2. The three curves were obtained from three different spiders. When the intensity of light given to the brain (I,,) increases from 0 to 1.0, the turning reactions (TRs) increase by 0.54 from —0.36 to +0.18 for 440 nm, by 0.28 from —0.42 to —0.14 for 360 nm and by 0.08 from —0.28 to —0.20 for 520nm light. Cerebral illumination by 440 nm light changes TR from negative to positive but dose not do so in the cases of 360 and 520 nm light, at least, in the region of the intensity used in the present study. It is clear that blue light has a marked effect on phtotactic response, as was reported by Yamashita and Tuji [4]. The efficiency of monochromatic light given to the brain was defined, for convenience, as the difference between TR at I,,=1.0 and TR at I,,= 0. Spectral efficiencies for 1-3 wavelengths were determined for each spider. The average spectral efficiency curve obtained from 18 spiders is shown in Figure 3. The curve has a single peak at 440 nm. The mean value of TRs at I,,=0 for these spiders was —0.38+0.10 (SD). The cerebral photosensi- tive neurons reported by Yamashita and Tateda [1] had a maximum sensitivity at 420-440 nm, sug- gesting that these neurons control the phototactic behavior of the spiders. 0.6 o bh Efficiency i) N 440 480 520 560 Wavelength, nm 360 400 Fic. 3. Average spectral efficiency curve. See text. Vertical lines indicate standard deviation. ACKNOWLEDGMENTS This research was supported in part by Grant 03640608 from the Ministry of Education of Japan. Phototactic Behavior of Spiders 889 REFERENCES 1 Yamashita, S. and Tateda, H. (1983) J. Comp. Physiol., 150: 467-472. 2 Yamashita, S. (1985) In “Neurobiology of Arach- 3 4 5 nids”. Ed. by F. G. Barth, Springer-Verlag, Berlin- Heidelberg-New York-Tokyo, pp. 103-117. Yamashita, S. (1990) Vision Res., 30: 817-821. Yamashita, S. and Tuji, R. (1987) Zool. Sci., 4: 23- 30. Yamashita, S. (1987) Zool. Sci., 4: 31-35. Tari ve iV jars ? he a, nye Vy yyy be ce ayy waco wre obi“: vid | mab C aye AO A, colapeninp Ree w yal Win ck materi hie ae bg ne bite} ‘ ha, 40S, FATE Ke ivev th ¥ yf coding ae hep qeecle pri met a we Lely mi ; Tari { Aa i ; ' Lk ‘ i , : ey s : Ai i Z * ; vem? iH saa har 4 eae Fae % itd Sele are ipsa in Fi obtaining Sell thine frowt, fa i, Aira bi ni; WiF tH) (rnitive tend ue a =a) ann H gah me . at 7M} he 1145 (ty ond ali (Ber wo a Hin) Aad yh, rive oa mathe aCayyece: We Mie Se nirted cn Tah ae trabe Yeu tots Aitinte je | eri cogt erate pee Y chart e ae eye ny I if: oS TY, TL PT c i ere Savy LY Onl \ er - an earereed pee ret pd Megha heya eh) J, ZOOLOGICAL SCIENCE 9: 891-895 (1992) [COMMUNICATION] © 1992 Zoological Society of Japan Isolation of Intact Yolk Spheres of Fish Embryos, Which Contain the Majority of Lysosomal Acid Phosphatase Responsible for Yolk Phosphoprotein Metabolism MasAHirRO MuraKamt., Icuiro Iucui and KENJIRO YAMAGAMI- Life Science Institute, Sophia University, 7-1 Kioicho, Chiyoda-ku, Tokyo 102, Japan ABSTRACT — Intact yolk spheres (membrane-bounded yolk masses) completely devoid of yolk cytoplasmic layer (YCL), yolk syncytial layer (YSL) and a blastoderm could be isolated from mid-blastulae or early gastrulae of the teleost, Oryzias latipes. Most of soluble yolk proteins and acid phosphatase in a whole embryo were found in the isolated yolk sphere. More than 80% of the acid phosphatase in the yolk sphere were sensitive to tarta- rate, probably belonging to lysosomal acid phosphatase. Considering that the acid phosphatase in the early embryos of this fish is responsible for dephosphorylation of yolk phosphoproteins [1-4], the present result strong- ly suggests that yolk phosphoproteins are dephosphory- lated in the yolk sphere itself independently of the surrounding YCL, YSL and blastoderm. INTRODUCTION In oviparous vertebrates, yolk phosphoproteins provide the embryo with amino acids (or peptides) and phosphates as nutrients. Recently. we iden- tified two phosvitins and two lipovitellins in egg yolk of the fish, Oryzias latipes [4, 5]. A remark- able degradation of these phosphoproteins occurs during early phase of development from blastulae (Day 1 embryos) to optic vesicle stage embryos (Day 2 embryos) and the degradation is considered Accepted May 19, 1992 Received April 14, 1992 ' Present address: Division of Radiation Hazards, National Institute of Radiological Sciences, Chiba 263, Japan * To whom correspondence should be addressed. to be closely associated with dephosphorylation by acid phosphatase and proteolysis by some sub- cellular structure-associated cathepsin-like pro- tease [1-4]. On the other hand, an important role of periblast, or yolk syncytial layer (YSL) [6], in yolk nutrition is conjectured [7]. In Oryzias latipes embryos, total amount of soluble yolk proteins decreases markedly after Day 3 instead of before Day 2 of development [4], and electron micros- copy reveals that various-sized yolk masses are endocytosed actively by YSL only after Day 3 (Murakami et al., unpublished). Thus, the dephos- phorylation of phosphoproteins probably occurs independently of YSL. The role of yolk sphere (a membrane-bounded whole yolk mass) in yolk digestion in fish has scarcely been analyzed, as intact yolk spheres have never been isolated to be analyzed. In the course of our Studies on egg yolk proteins, we worked out a practical method of isolation of intact yolk spheres completely devoid of YSL and yolk cytoplasmic layer (YCL) from blastulae of Oryzias latipes, and found that the yolk spheres contained the majority of lysosomal acid phosphatase present in whole embryos. This method would facilitate a further analysis of yolk metabolism in fish embryos. MATERIALS AND METHODS Embryos Outbred fish of orange red variety of Oryzias latipes were fed on Tetrafin in order to 892 M. Murakami, I. IucHI AND K. YAMAGAMI obtain naturally fertilized eggs as described else- where [4]. Mid-blastulae and early gastrulae (Day 1 embryos) were exclusively employed as mate- rials. Preparation of partially purified hatching en- zyme A mixture of partially purified high and low choriolytic enzymes (HCE and LCE) was obtained from the hatching liquid following the Step 1 of the purification procedure of HCE [8]. The 0.02 M Tris-HCI-0.3 M NaCl (pH 8.5) eluate (CM-0.3 enzyme) which showed a high activity of egg envelope (chorion)-digestion was frozen and stored until use. The CM-0.3 enzyme (OD2g9= 0.16) was mixed with an equal volume of 0.02 M Tris: HCl-0.25 M sucrose (pH 7.2) and the mixture was employed as a hatching enzyme solution. Isolation of yolk sphere. About 50 mid-bla- stulae or early gastrulae were incubated in 1 ml of the hatching enzyme solution on 2% agar plate at 30°C for about 1hr. Partially digested egg en- velopes (chorions) could be easily removed by a pair of sharp tweezers. The dechorionated embryos were then transferred to 0.02M Tris- HCI-0.25 M sucrose-0.1 M ethylenediamine tetra- acetate (EDTA) (pH7.2), and incubated at 30°C for 1 hr. Somewhat loosened YCL could be peeled off, together with YSL and a blastoderm, by sharp tweezers. A yolk sphere surrounded by a fragile membrane could be washed by transferring carefully to a buffered 0.25 M sucrose containing no EDTA. Electron microscopy Specimens were fixed in ice-cold 4% paraformaldehyde-3% glutaraldehyde- 0.19% picric acid-0.2M_ sucrose-0.1M _ phos- phate buffer (pH 7.4) for 3 hr. The fixed speci- mens were washed with 0.2 M phosphate buffer- 0.4M sucrose (pH 7.4) for 15 min three times. Post-fixation was performed with 1.33% OsO,-0.2 M sucrose-0.1 M phosphate buffer (pH 7.4) at 5°C for 2.5 hr. After washing with 0.4 M sucrose-0.2 M phosphate buffer (pH 7.4) for 15 min two times, the fixed specimens were dehydrated through an alcohol series, followed by treatment with pro- pylene oxide and embedded with Spurr embedding resin (Polyscience Inc., Warrington). The sections were cut at 0.1 ~m, stained with lead citrate and uranyl acetate and observed with a Hitachi H300 transmission electron microscope. Polyacrylamide gel electrophoresis Whole embryos or isolated yolk spheres were homoge- nized in ice-cold 0.02 M Tris-HCI-0.25 M sucrose (pH 7.2) and the homogenates were centrifuged at 14000 g at 4°C for 10 min to prepare the extracts. Polyacrylamide gel disc electrophoresis (disc PAGE) using 7% gel (pH 8.0) was performed following the method of Williams and Reisfeld [9]. Proteins were stained with Coomassie brilliant blue and acid phosphatase was detected by the method of Gabriel [10]; After electrophoresis, the gels were treated with 0.1 M acetate buffer (pH 5.0) at 4°C for 15min and incubated in the reaction/staining medium of 0.04M _ Veronal- acetate buffer containing 0.01% sodium a- naphthyl phosphate as substrate, 0.16% rosanilin and 0.16% NaNO; (pH 5.0) at 4°C overnight. Acid phosphatase activity Acid phosphatase activity was determined following the method of Bessey et al. [11] by using p-nitrophenyl! phosphate (Acid Phospha-B Test, wako Pure Chemicals, Tokyo) as substrate. Whole embryos or isolated yolk spheres were extracted with ice-cold 0.02 M Tris-HCl-0.25 M sucrose (pH7.2). One tenth milliliter of the extracts, equivalent to 1-10 embryos, was incubated with 0.5 ml of 0.25% p-nitrophenyl phosphate-0.05 M citrate buffer (pH 4.9) at 30°C min. After the reaction was stopped by adding 5 ml of 0.05 M NaOH, the absorbance of the mixture at 405nm was measured. Acid phosphatase activities in the presence and absence of 0.01 M Na-tartarate were compared to estimate the activity of lysosomal acid phosphatase, which was sensitive to tartarate [12, 13]. Under the conditions, the activity determined was found to be proportional to the amount of the enzyme. RESULTS AND DISCUSSION As illustrated in Fig. la, a greater part of the membrane-bounded yolk sphere of a blastula is surrounded by a thin YCL. The YCL is con- tinuous to YSL, or marginal and subblastodermal periblasts, in animal pole region. Oil drops Yolk Sphere of Fish Embryo 893 soot Fo AT Cm ae S oes Fic. 1. Isolation of intact yolk spheres from Oryzias latipes embryos. a, A diagrammatic illustration of a mid-blastula. B; blastoderm, O; oil drops, YS; yolk sphere, YCL; yolk cytoplasmic layer, YSL; yolk syncytial layer. A small square represents the locale of a section shown in Fig. 1c. b, Some specimens obtained in the process of isolation of yolk spheres. 1; dechorionated Day 1 embryos (2 mid-blastulae and 1 early gastrula), 2; a midblastula whose YCL is in the midst of removal, 3; a mid-blastula whose YCL is, together with YSL and a blastoderm, almost removed. Oil drops are adhering to YCL and YSL in this psecimen. 4; an isolated yolk sphere. Scale bar, 1 mm. c, An electron micrograph of a section in the locale shown in Fig. 1a as a small square. YS; york sphere, YSL; yolk syncytial layer. d, An electron micrograph showing a surface of an isolated yolk sphere. Arrows in c and d indicate a membrane enveloping the yolk sphere. Scale bars in c and d, 1 um. locating in YCL are stuck in the yolk sphere at the vegetal pole. Fig. 1b shows some specimens in the process of isolation of intact yolk spheres from mid-blastulae or early gastrulae. The dechorion- ated embryos are usually posed animal pole side down in water, as large oil drops had migrated and attached to the vegetal pole side. On incubation in the medium containing EDTA, the oil drops come outward slightly and a small space appears beneath the loosened membrane of YCL around the oil drops. YCL can be peeled off from this space toward animal pole with sharp tweezers under a binocular micropscope. The oil drops are released and float to the surface of the medium. Finally, all YCL can be removed together with YSL and blastoderm, and an intact yolk sphere is obtained. 894 M. Murakami, I. IucHI AND K. YAMAGAMI a | |) a: EF 3 —s 5 j We. 1 2s 4 Fic. 2. Disc electrophoretic patterns of soluble proteins and acid phosphatase of whole embryos (blastulae) and yolk spheres isolated from blastulae. The extract equivalent to one embryo or one yolk sphere was applied to each column. 1 and 2; whole blastula, 3 and 4; yolk sphere. 1 and 4; soluble proteins, 2 and 3; acid phosphatase. Phosvitins are not stained by coomassie brilliant blue [4] but are faintly visible in the columns for phosphatase stain- ing (x). As shown in Fig. 1c and 1d, the yolk spheres, both in situ and in isolation, are enveloped by a membrane, which we can identify more clearly in an isolated specimen. No YCL is found to be adhering to the isolated yolk spheres. This mem- brane has been documented for the embryos of this fish [14] and Fundulus heteroclitus [6, 15}. Yolk platelets in amphibian embrycs are also surrounded by a similar membrane [16]. Disc PAGE patterns of proteins and acid phos- phatase activity for whole embryo and the isolated yolk sphere are shown in Fig. 2. The pattern and density in a whole embryo are quite similar to those in an isolated yolk sphere, indicating that the acid phosphatase is represented by a single protein band and that most of the acid phosphatase as well as soluble proteins in a whole embryo are localized to the yolk sphere. In Oryzias latipes embryos, acid phosphatase is considered to be closely related to dephosphorylation of yolk phosphopro- teins during early embryonic development, as there is a high similarity between the autolytic dephosphorylation of yolk phosphoproteins and the acid phosphatase activity with respect to the pH-activity relationship and the effects of some =) N 0.1 = ro) WE Fic. 3. Acid phosphatase activity in whole embryos and isolated yolk spheres. The activities per two whole midblastulae (WE) and two isolated yolk spheres (YS) in the presence (white column) or absence (hatched column) of 0.01M Na-tartarate are ex- pressed as the average of 16 experiments. YS Acid phosphatase activity (A.os) ions on the activity and there is an active acid phosphatase but little alkaline phosphatase in the early embryos [2, 3]. Thus, it is highly probable that the acid phosphatase visualized on the disc PAGE is responsible for dephosphorylation of yolk phosphoproteins [2-4]. As shown in Fig. 3, the present results indicate that about 75% of the acid phosphatase in a whole Day 1 embryo is localized to the yolk sphere itself. About 76% of the activity in whole embryo and about 84% of that of the isolated yolk sphere are sensitive to tartarate. Therefore, a large part of the tartarate- sensitive (or lysosomal) acid phosphatase of whole embryo is located in the yolk sphere. The tartarate-sensitive phosphatase are grouped into ethanol-sensitive and -insensitive enzyme [12]. Most activity of the acid phosphatase in the yolk of Oryzias latipes is found to be insensitive to ethanol (Murakami ef al., unpublished). Although a detailed mechanism of deposition of fluid yolk in Oryzias latipes oocytes is obscure, it is highly probable that lysosomes participate in transforma- Yolk Sphere of Fish Embryo 895 tion of the endocytosed vitellogenin into deposited yolk proteins (cf. [17]). The present results directly confirm that a yolk sphere is closely related to lysosomes in terms of the localization and the nature of the acid phosphatase. Moreover, it seems highly probable that dephosphorylation of yolk phosphoproteins occurs inside the yolk sphere. ACKNOWLEDGMENTS The authors wish to thank Dr. T. S. Hamazaki for his invaluable advice on electron microscopy. REFERENCES 1 Yamagami, K. (1960) Sci. Pap. Coll. Gen. Educ., Univ. Tokyo, 10: 99-108. 2 Yamagami, K. (1961) Sci. Pap. Coll. Gen. Educ., Univ. Tokyo, 11: 153-161. 3 Yamagami, K. (1963) Sci. Pap. Coll. Gen. Educ., Univ. Tokyo, 13: 223-229. 4 Murakami, M., Iuchi, I. and Yamagami, K. (1990) Dev. Growth Diff., 32: 619-627. 5 Murakami, M., Iuchi, I. and Yamagami, K. (1991) Comp. Biochem. Physiol., 100: 587-593. 6 Betchaku, T. and Trinkaus, J. P. (1978) J. Exp. Zool., 206: 381-426. Heming, T. A. and Buddington, R. K. (1988) In “Fish Physiology”. Ed. by W. S. Hoar and D. J. Randall. Academic Press, San Diego, Vol. 11A, pp. 407-446. Yasumasu, S., Iuchi, I. and Yamagami, K. (1988) J. Biochem., 105: 204-211. Williams, D. E. and Reisfeld, R. A. (1964) Ann. N. Y. Acad. Sci., 121: 373-381. Gabriel, O. (1971) In “Methods in Enzymology”. Ed. by W. B. Jakoby, Academic Press, N. Y., Vol. 22, pp. 578-604. Bessey, O. A., Lowry, O. H. and Brock, M. J. (1946) J. Biol. Chem., 164: 321-329. Abul-Fadl, M. A. M. and King, E. J. (1949) Biochem. J., 45: 51-60. Dipietro, D. L. and Zengerle, F. S. (1967) J. Biol. Chem., 242: 3391-3396. Yamamoto, M. (1965) J. Fac. Sci., Univ. Tokyo, IV 10: 483-490. Lentz, T. L. and Trinkaus, J. P. (1967) J. Cell Biol., 32: 121-153. Richter, H. P. (1987) Roux’s Arch. Dev. Biol., 196: 367-371. Wallace, R. A. (1985) In “Developmental Biology”. Ed. by L. W. Browder. Plenum Press, N. Y., Vol. 1, pp. 127-177. ey i ‘A 17 Mi iy ditt cassia rh, i, hie? we Sanan it. rain shit , ‘Why j pany hal one Hi tas ye ‘Vee Peed, - a he ent iE Pe ET ee Gh 4 ae eer) Octabmels fp iy dryadind a is ir ; ni fot hat Te a ee a ih afl , 7 fed Ay j Peak inti , i . it ane fit oF As: it tof aL Ti ee mh hae WH] eee we | t ti Ae ibs } las \ } i f , ¢ A Aras ia ed ie cyt) tad) airy Ga Mew oA, ve dige Aa CZ ir dof eorpal Agee ~ JF noivahepont a iy , al’ m Vie Tby Bi in vais 1 / ‘Sasite A ay ur f +, re ely a gi Aint I / iu ‘2: el Dd. raed nat CHa «ioe eV tsgampall wale (ei (fT) 0 oa ha at kN at Alo PE baguia Py va yoda ativiry pyr titietiane HOM ZOOLOGICAL SCIENCE 9: 897-900 (1992) [COMMUNICATION] © 1992 Zoological Society of Japan First Electron Microscopical Study on the Sperm Morphology of the Sea Lily (Crinoidea, Echinodermata) SHONAN Amemiya! and Yoko NAKAJIMA~ ‘Misaki Marine Biological Station, University of Tokyo, Miura-shi, Kanagawa 238-02, and *Department of Biology, Keio University, Hiyoshi, Yokohama 223, Japan ABSTRACT—The sperm morphology of the sea lily (stalked crinoid) is described for an isocrinid species, Metacrinus rotundus. Scanning electron microscopy shows that the head of the sperm is spherical with an acorn-like shape. The acrosome appears as a nipple- shaped hemispherical protuberance on the apical tip of the sperm head. The middle piece occupies the basal one-third of the head, forming the “cap” of the acorn. The sperm head is 2.0 ~m long and 1.7 «m wide, and the acrosome area is 0.23 ~m long. The sperm flagellum is 0.15 4m thick and 43 ~m long. The sperm morphology of the sea lily is similar to that of the feather stars. INTRODUCTION Sea lilies (stalked crinoids) flourished in the sea during the Palaeozoic era, and have survived through to the present day, keeping their morphol- ogy essentially unchanged [1, 2]. These animals (class Crinoidea) are the only living Pelmatozoa among the six classes of Echinodermata, and are considered to occupy an extremely important position for understanding the phylogeny of the echinoderms [1, 3]. They once had a wide range of bathymetric distribution from shallow to deep water, but most extant species live in seas deeper than several hundred meters. Because of the difficulty in obtaining live specimens, sea lilies have been regarded for a long time as biologically mysterious organisms [4], and details of their gamete morphology, reproduction and develop- ment have remained largely unknown. Recently, Accepted June 5, 1992 Received May 6, 1992 we succeeded in obtaining sperms of an isocrinid sea lily, Metacrinus rotundus, and observed their morphology by scanning electron microscopy (SEM). This report is the first to give details of the outer features of sea lily gametes. MATERIALS AND METHODS Specimens of the isocrinid sea lily, Metacrinus rotundus Carpenter, were collected by dredging from 150m depth at Suruga bay, on the Pacific coast of central Japan. The sperms were obtained by dissecting the testis. Fixation of the sperms was performed essentially by the procedure reported by Holland and Kubota [5] with some modifica- tions. In brief, the sperms placed on filter paper were fixed in 2% glutaraldehyde in 70% artificial seawater (ASW, Jamarine U, Osaka), post-fixed in 2% osmium tetroxide in 2.5% NaHCO3, and dehydrated with an ethanol series. The specimens were then critical-point dried, sputter-coated with gold and examined with a Hitachi S-510 scanning electron microscope at 25 kV. RESULTS AND DISCUSSION The testes of Metacrinus rotundus were found in the genital pinnules and in the arms at the points of branching of the pinnules in males (Fig. 1). In females, the ovaries were located in the same place as the testes in males. Some fractions (if not all) of the live sperm obtained by dissection of the testes swam slowly, indicating that the sperm were S. AMEMIYA AND Y. NAKAJIMA 898 Fic. 1. A part of an arm bearing some pinnules of a male isocrinid sea lily, Metacrinus rotundus, observed by dissection microscopy. The testes (arrow-heads) are located in the genital pinnules (small arrows) and in the arm (large arrow) at the points of branching of the genital pinnules. Bar represents 5 mm. Fic. 2. Sperms of the isocrinid sea lily, Metacrinus rotundus, observed by scanning electron microscopy. The acrosome (A) is surrounded by a circular groove (arrow). M, middle piece; F, flagellum. Bar represents 2 ~m. Sea Lily Sperm 899 mature or almost mature. The head of each sperm was almost spherical with an acorn-like shape (Fig. 2). The acrosome formed a nipple-like hemispher- ical protuberance on the apical tip of the sperm head. The middle piece occupied the basal one-third of the head, forming the “cap” of the acorn. The head size estimated from the SEM photograph was about 2.0 vm in length with the acrosome region measuring 0.23 um, or one-ninth of the total head length. The width of the head represented by the size of a nucleus which was located between a mitochondrion and an acro- some, was about 1.7 zm. The head size (2.0 ~m long) was comparable to that of two other species of sea lily (Calamocrinus diomedae and Holopus rangii) reported by Holland [6] based on light microscopical observations. The flagellum of the sperm was 0.15 ~m thick and 43 um long. How- ever, the preparation procedure for SEM observa- tion might have caused underestimation of the size. An oblique view around the apical tip of the sperm head showed details of the acrosome and its neighborhood (Fig. 2). The hemispherical nipple of the acrosome was surrounded by a circular groove 0.15 “m wide. The sperm of the feather star, Florometra serratissima [7], showed remarkable similarity in the shape of sperm head to M. rotundus. The head length described for F. serratissima was 2.3 um, almost the same as that of M. rotundus. The shapes of the middle piece and acrosome were also very similar. The acrosome was somewhat larger in M. rotundus than in F. serratissima and in other echinoderm species with a spherical sperm shape [7]. In three subphyla (Crinozoa, Asterozoa and Echinozoa) of Echinodermata [1], two of three Asterozoa (ophiuroids and asteroids) species known so far have sperms with spherical heads. The sperm morphology of the newly discovered Asterozoa, concentricycloids, is V-shaped as in the phoronids, and has no similarity with any other echinoderm species [8]. Most species of holothu- rians, one of the Echinozoa, have sperms with spherical heads, but some species have the sperm of cylindrical heads [7]. All sperms of another member of the Echinozoa, the echinoids, have conical heads [7], although the sperm heads of some species are highly elongated [9, 10]. As to the Crinozoa, the sperm morphology has been examined electron microscopically for some spe- cies of feather stars (Antedon petasus, Comanthus japonicus and F. serratissima), and has been reported to be spherical [7]. In contrast, Holland [6] has reported that the sperm head of a feather star, Antedon bifida, is conical. Information on the sperms of sea lilies is extremely limited, and no electron microscopical study has yet been re- ported. Based on light microscopical studies, only four species of sea lily have been reported so far with regard to sperm morphology. These are Rhizocrinus lofotensis by Ludwig [11], Bathycrinus carpenteri by Danielsen [12], and Calamocrinus diomedae and Holopus rangii by Holland [6]. These authors reported that the sperm heads of the four species are spherical. The present electron microscopical study on the sperm morphology of M. rotundus confirmed that the sperms of this sea lily have spherical heads. Chia et al. [7] suggested that the spherical sperm head is the original form, and that the sperms of many species of Echinozoa have been modified to have conical or cylindrical heads, whereas the sperms of Crinozoa and Aster- ozoa have retained the original form. However, more recent information on the sperm morphology of concentricycloids [8] and some feather stars [6] suggests that further analysis is necessary to confirm the relationship between sperm morpholo- gy and echinoderm phylogeny. Further examination of the details of the sperm morphology of M. rotundus, based mainly on transmission electron microscopy, is now in prog- ress and the findings will be reported in the near future. ACKNOWLEDGMENTS We would like to thank Dr. T. Oji for his assistance to collect the materials. REFERENCES 1 Nichols, D. (1962) Echinoderms. Hutchinson and Co. Ltd., London. 2 Paul, C. R. and Smith, A. B. (1984) Biol. Rev., 59: 443-481. 3 Hyman, L. H. (1955) The Invertebrates: Echi- 900 nodermata. McGraw-Hill, New York. Amemiya, S. and Oji, T. (1992) Nature, 357: 546— 547. Holland, N. D. and Kubota, H. (1975) Annot. Zool. Jap., 48: 83-89. Holland, N. D. (1991) Echinoderms and Lophopho- rates. In “Reproduction of Marine Invertebrates”. Ed. by A. C. Giese, J. S. Pearse and V. B. Pearse, The Boxwood Press, Pacific Grove, California, Vol. VI, pp. 247-299. Chia, F-S., Atwood, D. and Crawford, W. (1975) American Zoologists 15: 553-565. 8 S. AMEMIYA AND Y. NAKAJIMA Rowe, F. W. E., Anderson, D. T. and Healy, J. M. (1991) Echinoderms and Lophophorates. In “Re- production of Marine Invertebrates”. Ed. by A. C. Giese, J. S. Pearse and V. B. Pearse, The Boxwood Press, Pacific Grove, California, Vol. VI, pp. 751- 760. Amemiya, S., Suyemitsu, T. and Uemura, I. (1980) Develop. Growth Differ., 22: 327-335. Raff, R. A., Herlands, L., Morris, V. B. and Healy, J. (1990) Develop. Growth Differ., 32: 283-291. Ludwig, H. (1877) Zeitschrwiss. Zool., 29: 47-76. Danielsen, D. C. (1892) Zoology, 5: 1-28. D evel O m en { Published Bimonthly by the Japanese Society of Pp Developmental Biologists Distributed by Business Center for Academic Growth & Differentiation Societies Japan, Academic Press, Inc. Papers in Vol. 34, No. 4. (August 1992) 40. H. Fang, T. Sakuma and K. Yanagisawa: Ammonia Determines the Alternative Pathways of Sexual or Asexual Development in the Cellular Slime Mold Dictyostelium discoideum 41. K. Mitsunaga-Nakatsubo, A. Fujiwara and I. Yasumasu: Change in the Activity of Na‘, K*-ATPase in Embryos of the Sea Urchin, Hemicentrotus pulcherrimus, during Early Development 42. H. Nakamura and N. Itasaki: Expression of en in the Prosencephalon Heterotopically Transplanted into the Mesencephalon 43. H. Dupont, P. Deparis, M. Tujague, D. Saboulard, C. Ardourel, C. Daguzan and J.-C. Beetschen: Differential Protein Distribution Related to Dorsoventral Polarity in Pleurodeles waltl Cleaving Egg 44. K. Hoshino, T. Shimizu, Y. Sendai, T. Harumi and N. Suzuki: Differential Effects of the Egg Jelly Molecules FSG and SAP-I on Elevation of Intracellular Ca** and pH in Sea Urchin Spermatozoa 45. C. Inoue, M. Kiyomoto and H. Shirai: Germ Cell Differentiation in Starfish: The Posterior Enterocoel as the Origin of Germ Cells in Asterina pectinifera 46. A. Shinagawa: Relative Timing of Stiffening with Various Combinations of Nucleate and Enucleated Egg Fragments of Xenopus laevis 47. K. Kawamura and N. Yamashiki: The Determination of Spindle Polarity in Early Mitotic Stages of the Dividing Grasshopper Neuroblasts 48. K. Hara, S. Fujiwara and K. Kawamura: Retinoic Acid can Induce a Secondary Axis in Developing Buds of a Colonial Ascidian, Polyandrocarpa misakiensis 49. R. Yanagimachi, G. N. Cherr, M. C. Pillai and J. D. Baldwin: Factors Controlling Sperm Entry into the Micropyles of Salmonid and Herring Eggs 50. S. Fujiwara and K. Kawamura: Ascidian Budding as a Transdifferentiation-Like System: Multipotent Epithelium is not Undifferentiated 51. S. Nagata, M. G. Rosenfeld and K. Inoue: Development of Prolactin and Growth Hormone Production in the Fetal Rat Pituitary: An Immunochemical Study 52. L.-N. Wei, C.-H. Lee, S.-L. Chang and Y.-S. Chu: Pathogenesis in Transgenic Mice Expressing Bovine Cellular Retinoic Acid-Binding Protein Development, Growth and Differentiation (ISSN 0012-1592) is published bimonthly by The Japanese Society of Developmental Biologists, Department of Developmental Biology, 1990: Volume 32. Annual subscription for Vol. 33, 1991: U. S.$ 162,00, U.S. and Canada: U.S. $ 178,00, all other countries except Japan. A\ll prices include postage, handling and air speed delivery except Japan. Second class postage paid at Jamaica, N.Y. 11431, U.S.A. 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The Ultimate Name in Micromanipulation Ease of operation, and the most advanced 1) Can be combined with New WR & MX series he Model MX-1 3-D Micromanipulator Model MxX-2 3-D Micromanipulator Model WR-87 One Dimensional Aqua Purificate Micromanipulator Model SR-6 Model PP -83 Stereotaxic Instrument for Rat Glass Microelectrode Puller * * & Send enquiries for request for MODIFICATIONS or IMPROVEMENTS * * * Physiological, Pharmacological, Zoological & Neurosciences Research Equipments (NARISHIGE) NARISHIGE SCIENTIFIC INSTRUMENT LAB. CUIRERING 9-28 KASUYA 4-CHOME SETAGAYA-KU, TOKYO 157, JAPAN PHONE (INT-L) 81-3-3308-8233, FAX (INT-L)81-3-3308-2005 CABLE: NARISHIGE LABO, TELEX, NARISHIGE J27781 (Contents continued from back cover) of hypothalamo-pituitary-ovarian axis in the TER, coi. RoE gOS RE eee nice en eae 791 Sawada, K. and T. Noumura: Differential effects of testosterone and 5a-dihydrotes- tosterone on growth in mouse submandibu- lar gland Morphology Shirai, S. and K. Nakaya: Functional mor- phology of feeding apparatus of the cookie- cutter shark, Jsistius brasiliensis (Elasmo- branchii, Dalatiinae) Ando, K. and S. Arai: Neuropeptide Y in- nervation of cerebral arteries in microchiro- PUCKAMMD ALS ye ne alae ech us nie ane a cee avem 823 Ecology Matsumoto, T.: Familial association, nym- phal development and population density in the Australian giant burrowing cockroach, (Blattaria: Macropanesthia __ rhinoceros Blaberidae) Taxonomy Nagatomi, A.: Notes on the phylogeny of various taxa of the orthorrhaphous Brachycera (Insecta: Diptera) ............ 843 Ohtsuka, S., R. Huys, G. A. Boxshall and T. It6: Misophriopsis okinawensis sp. nov. (Crustacea: Copepoda) from hyperbenthic waters off Okinawa, South Japan, with definitions of related genera Misophria boeck, 1864 and Stygomisophria gen. nov. Huys, R., S. Ohtsuka, G. A. Boxshall and T. It6: Itoitantulus misophricola gen. et sp. nov.: First record of Tantulocarida (Crus- tacea: Maxillopoda) in the North Pacific re- AKON. ac @oeteae cet rte ca Ween a 875 ZOOLOGICAL SCIENCE VOLUME 9 NUMBER 4 AUGUST 1992 CONTENTS REVIEWS Matsumoto, A.: Hormonally induced synap- tic plasticity in the adult neuroendocrine Bray “waccuitigasnaice sao eae ae eer 679 Bock, W. J.: The species concept in theory and Practice .i cc. cdeac eee oo uae 697 ORIGINAL PAPERS Physiology Naitoh, T. and R. J. Wassersug: The emetic response of urodele amphibians .......... 713 Miro, J. L., S. Araneda and B. Canguilhem: Origin of serotonergic innervation of olfac- tory bulbs in the Europian hamster, Cricetus cricetus: An autoradiographic study using [PH]S-HT retrograde labelling Yamashita, S.: mination of the brain on the phototactic Effect of monochromatic illu- behavior of orb weaving spiders, Argiope amoena and Nephila clavata (COMMUNI- CATION) Developmental Biology Endo, K., S. Ueno, M. Matsufuji and Y. Kakuo: Photoperiodic control of the deter- mination of two different seasonal diphen- isms of the Asian comma butterfly, Polygo- nia c-aureum L. Ukeshima, A.: Scanning electron microscopy of differentiating chick ovaries during embryonic period Murakami, M., I. Iuchi and K. Yamagami: Isolation of intact yolk spheres of fish embryos, which contain the majority of lyso- somal acid phosphatase responsible for yolk Nakamura, S., R. Kagotani, H. Fujisaki and M. K. Kojima: The acid-insoluble organic matrix of spicules in the sea urchin Hemicentrotus pulcherrimus .........-..--. 741 Ohya, Y., K. Watanabe, N. Shimamoto and M. Amano: Scleral fibroblasts of the chick embryo can proliferate without transferrin in protein-free culture Inoue, C. and Y. Kakinuma: Symbiosis be- tween Cytaeis sp. (Hydrozoa) and Niotha livescens (Gastropoda) starts during their larval stage Amemiya, S. and Y. Nakajima: First electron microscopical study on the sperm morpholo- gy of the sea lily (Crinoidea, Echinodermata) (COMMUNICATION) Endocrinology Kobayashi, M., M. Amano, Y. Hasegawa, K. Okuzawa and K. Aida: Effects of olfactory tract section on brain GnRH distribution, plasma gonadotropin levels, and gonadal stage in goldfish Madsen, S. S. and H. A. Bern: Antagonism of prolactin and growth hormone: Impact on seawater adaptation in two salmonids, Salmo trutta and Oncorhynchus mykiss Kai-ya, H., J. Okuyama, T. Ishijima, Y. Sasayama, H. Yoshizawa and C. Oguro: Effects of Ca concentrations in culture medium on the release of calcitonin from incubated ultimobranchial glands of the bull- frog, Rana catesbeiana Kobayashi, Y., S. Kawashima, S. Takahashi and K. Wakabayashi: Effects of chronic phosphoprotein metabolism (COM- treatment with chlorpromazine on the aging MUNICATION)? = 2seetaseeeeaeene ene 891 (Contents continued on inside back cover) INDEXED IN: Issued on August 15 Current Contents/LS and AB & ES, Science Citation Index, ISI Online Database, CABS Database, INFOBIB Printed by Daigaku Letterpress Co., Ltd., Hiroshima, Japan ees October 1992 : An International Journal PHYSIOLOGY CELL and MOLECULAR BIOLOGY GENETICS IMMUNOLOGY BIOCHEMISTRY DEVELOPMENTAL BIOLOGY REPRODUCTIVE BIOLOGY ENDOCRINOLOGY BEHAVIOR BIOLOGY ENVIRONMENTAL BIOLOGY and ECOLOGY SYSTEMATICS and TAXONOMY Business Center for Academic Societies Japan VSP, Zeist, The Netherlands \ published by Zoological Society of Japan i) distributed by | | ak 0289-0003 — 1 | ee | LOOLOGICAL SCIENCE The Official Journal of the Zoological Society of Japan Editors-in-Chief: The Zoological Society of Japan: Seiichiro Kawashima (Tokyo) Toshin-building, Hongo 2-27-2, Bunkyo-ku, Hideshi Kobayashi (Tokyo) Tokyo 113, Japan. 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Publication of Zoological Science has been supported in part by a Grant-in-Aid for Publication of Scientific Research Results from the Ministry of Education, Science and Culture, Japan. ZOOLOGICAL SCIENCE 9: 901-924 (1992) REVIEW Heterogeneity and Development of Somatotrophs and Mammotrophs in the Rat SuMIO TAKAHASHI en Department of Biology, Faculty of Science, Okayama University, Tsushima, Okayama 700, Japan ABSTRACT— Growth hormone (GH) and prolactin (PRL) are secreted from corresponding secretory cells, somatotrophs (GH cells) and mammotrophs (PRL cells) in the pituitary gland. A novel cell type, the mammosomatotroph (MS cell), which contains both GH and PRL in the same cell, has been found in many species including rats, mice and humans. In this article, we demonstrate the heterogeneity of GH cells and PRL cells at various levels, mainly in the rat, and discuss the developmental and functional significance of these heterogeneities. Age-related alteration in GH and PRL secretion is summarized, since it is one of the most important aspects of the developmental changes in pituitary glands. Heterogeneities of GH and PRL cells may be the outcome of various difference in the molecular variance of hormones, the intracellular age of hormones, the difference in receptors on the pituitary cells for hypothalamic regulatory hormones, the different mechanisms of intracellular signal transduction, and the location of pituitary cells in the gland, and may also reflect the maturation of pituitary cells. The possible relationship among GH cell, PRL cell and MS cells is © 1992 Zoological Society of Japan discussed. INTRODUCTION Growth hormone (GH) and prolactin (PRL) are protein hormones that are produced in corre- sponding specific pituitary secretory cells, somato- trophs (GH cells) and mammotrophs (PRL cells) [1]. A significant amount of evidence has indicated that each cell population of GH cells and PRL cells consists of morphologically and functionally heter- ogeneous cells [2, 3]. Furthermore, variants of hormone molecules have been found. Such heter- ogeneity observed may reflect the functional dif- ference or maturating process of pituitary cells and hormone molecules. A novel pituitary cell, a mammosomatotroph (MS cell) or a somatomammotroph, which con- tained GH and PRL in the same cell, was found in several species. In this paper, the term mammoso- matotroph is used to describe this secretory cell. The developmental and functional significance of Received July 10, 1992 MS cells is not yet fully understood. It is notewor- thy that GH and PRL genes are believed to be derived from a common ancestral gene [4, 5], and are regulated by a common transcription factor [6, 7]. Accordingly, MS cells are considered to be closely related to GH cells and PRL cells. Im- muno-electron-microscopical studies showed sub- types of GH cells and PRL cells in rat pituitaries. MS cells in the rat closely resembled one of the subtypes of GH cells and PRL cells. This finding suggests that MS cells may be involved in the genesis of GH cells and PRL cells. In this paper, the morphological and functional heterogeneity of GH cells and PRL cells, and the relationship among the three pituitary cells, GH, PRL and MS cells, will be discussed using examples mainly from the rat. As one of the most important develop- mental aspects in pituitary glands, age-related al- terations in GH and PRL secretion will be exten- sively studied in order to understand the signif- icance of the heterogeneities of GH and PRL cells. The molecular heterogeneity of both hormones 902 S. TAKAHASHI will also be briefly considered. I. SOMATOTROPHS 1. Identification of GH cells Size, shape and distribution Immunocytochem- ically identified GH cells in rat pituitaries ranged from ovoid to pyramidal in shape, and were usual- ly situated along sinusoids. They were evenly dispersed bilaterally and rostrocaudally, but un- evenly distributed dorsoventrally [8]. GH cells were not found near the intermediate lobe, nor were they found in the anteroventral portion of the gland [9, 10]. Differentiation of GH cells The ontogeny of GH cells has been immunocytochemically studied in several laboratories. GH cells were first observed at 18 or 19 days of gestation in the rat (the day on which the vaginal plug was detected is designated as day 0 of gestation) [11-13]. Using a combination of the immunocytochemistry and the in situ hybridization method [14], GH im- munoreactivity was detected from day 18 of gesta- tion, but GH-mRNA positive cells were detected on day 19 of gestation. GH cells had substantially increased in number by day 19 of gestation. Pitui- tary specific transcription factor GHF-1 (Pit-1), which was responsible for activating GH and PRL genes, was detected in the anterior pituitaries on day 16 of gestation [15], although GH-mRNA and GH immunoreactivity were first expressed on day 18-19 of gestation as stated above. In mouse pituitaries, temporal and spatial correlation be- tween the GHF-1 gene expression and GH gene expression was clearly observed [16]. GHF-1 transcript was first detected on day 13 of gestation, and had significantly increased by day 15 of gesta- tion. GH-mRNA was first detected on day 15 of gestation. GH synthesis increased sharply be- tween the 16th and 17th day of gestation [17]. GH cells were morphologically detected on day 16 of gestation [16]. 2. Morphological heterogeneity of GH cells Immuno-electron-microscopical studies have shown that there are three subtypes of GH cells in the rat [18]. GH cells were classified based on the size of secretory granules (Fig. 1). The Type I GH cell contained large secretory granules (diameter, 250-350 nm, Fig. 2). The Type II GH cell con- tained large and small secretory granules (di- ameter, 100-150 nm, Fig. 3). The Type II] GH cell contained small secretory granules (Fig. 4). Type I and II cells are polygonal or oval in shape, and medium in cell size. Type III cells are oval or sometimes irregular in shape, and small in size. The rough endoplasmic reticulum and the Golgi apparatus were moderately developed in Type I and II cells. Type I and II GH cells had been originally identified as somatotrophs. The relative proportion of each subtype of GH cells differed between male and female rats (Fig.5), and changed with several hormonal treatments and age [19]. This will be discussed later. 3. Development of GH cells Proliferation of pituitary cells The pituitary gland is an “expanding organ” (see Goss, [20]), and its development is characterized by the fact that cell differentiation is not incompatible with mitosis, and every pituitary cell probably has the capacity to divide during its life span [21]. Mitotic GH cells were immunocytochemically identified in several reports [22-25]. These observations clear- ly indicate that differentiated GH cells divide in a similar manner to other types of pituitary cells [26- 30]. Pituitary cells proliferate using a mode of self-duplication, and the differentiated pituitary cells divide into the same two differentiated cells. However, another mode of proliferation cannot be ruled out. For example, undifferentiated “stem” cells, may divide and then one of the divided cells may terminally differentiate into a specific secre- tory cell. Sexual difference in the percentage of GH cells A sexual difference in the number of GH cells was found in rats [19, 31] and in mice [32]. Our study showed the percentage of GH cells in male and female rats at various ages (Table). The percent- age of GH cells was higher in male rats than that in female rats, and decreased with age in both sexes. Similar findings were reported by a reverse hemolytic plaque assay [33]. On the contrary, Dada er al. [34] could not detect the sexual differ- ence in the percentage of GH cells, although the GH and PRL Cells in the Rat 903 Fic. 1. Secretory granules immunocytochemically stained with rat GH antiserum by the protein A gold colloid method. Large secretory granules with a diameter of 250-350 nm (arrows) and small secretory granules with a diameter of 100-150 nm (arrowheads) are seen. Bar: 200nm. (from Takahashi [19]). Fic. 2. Type I GH cell in a 6-month-old female rat. Large secretory granules are seen throughout the cytoplasm. Bar: 1.0 4m. (from Takahashi [19]). Fic. 3. Type Il GH cell in a 6-month-old female rat. Large and small secretory granules are seen throughout the cytoplasm. Bar: 1.0 zm. (from Takahashi [19]). Fic. 4. Type III GH cell in a 6-month-old female rat. Small secretory granules are seen throughout the cytoplasm. Bar: 1.0m. (from Takahashi [19]). reason for this discrepancy is not clear. proliferation of GH cells [35]. Thus, hypothalamic Hormonal effects on GH cells Growth hor- __ peptides affect the proliferation of pituitary cells mone-releasing hormone (GHRH) stimulates the _[30, 36]. GHF-1, a transcription factor of the GH 904 Fic. 5. S. TAKAHASHI CJ Type | Type Il Male 3 an ) ° fo} ° ox sy to} 20 Percentage of GH cell types Female Percentage of GHcell types o to} Age Age-related changes in the percentages of GH cell types in male and female rats. The number above the GB Type 1! (months) columns depicts the number of rats. Bars depict the standard errors of means. The percentage of Type I cell increased, whereas the percentages of Type II and III cells decreased at 2 and 6 months of age. At 12 and 18 months of ages, the percentages of Type II and III cells increased. (from Takahashi [19]). TABLE Percentage of GH cells and PRL cells in male and female rats Percentage of cells (%)” Sex Age(months) GH cell PRL cell Male 68.34+2.2)" 15.5+1.4 12 59.7+4.1° 22.1+1.6 18 40.5+4.2" 18.9+3.0 Female 40.6+2.0° 34.0+4.4% 12 34.8+4.1° 48.7+2.9" 18 PB NAA Spies (le ') Mean+S.E. >) In each age-group five rats were used for the determination of the percentages of GH cells and PRL cells. Statistical significance was tested by ANOVA. When significant, the differences among age-groups of each sex were determined by Duncan’s multiple range test. In each sex, there is a_ significant difference between the values for the age-groups with the same superscripts. a, P<0.05; b, c,d, P< 0.01. (from Takahashi er al. [31]). gene, may function in the proliferation of GH cells [37]. This suggests that stimulation of GH gene expression may stimulate the cell division of GH cells. Estrogen decreases the percentage of GH cells, but androgen increases it [38, 39]. Estrogen in- creased the percentages of Type II and III GH cells (Fig. 6), and androgen increased the percen- tage of Type I cells and decreased the percentage of Type II cells (Fig. 7). As estrogen is known to inhibit GH secretion, and androgen is known to stimulate [39], it is concluded that the inhibitory factors for GH secretion decrease the percentage of GH cells, and the relative proportion of Type I cells, and, on the contrary, the stimulatory factors for GH secretion increase the percentage of GH cells and the relative proportion of Type I GH cells. T3 is known to stimulate fetal somatotroph differentiation probably by a synergistic action with cortisol [40] and GH production [41]. T GH and PRL Cells in the Rat 905 » L_J]Type! KNtypel! Btype i 60 40 20 Percentage of GH cell types (0) Vehicle ES Fic. 6. Effects of injection of 50 ug estradiol-172 (E>) daily for 5 days on the percentage of GH cell types in male rats. In E>-treated rats, the percentage of Type I cells decreased and the percentage of Type II and III cells increased. The number above the column depicts the number of rats. Bars depict the standard errors of means. *P<0.05, **P<0.01 vs vehicle. (from Takahashi [19]). treatment (10 ~g/100 g BW daily twice for 5 days) significantly increased the percentage of Type III GH cells from the control level of 3.4+1.1% (n= 6) to 6.9+0.8% (n=6). This increase in Type III cells may indicate the formation of immature GH cells (Type III) from undifferentiated cells, pro- vided that the Type HII GH cell is an immature type of GH cell. Thus, the relative proportion of GH cell subtypes changed, together with changes in GH secretion, indicating that three morphologi- cally different GH cell types may have different secretory activities. Development of GH cells Perinatal develop- ment of GH cell subtypes was reported by Kuro- sumi and Tosaka [42], and postnatal changes (from immature ages through to aged ones) were studied by Takahashi [19]. The Type III GH cell was the predominant type of GH cell during the prenatal period, and the percentages of Type I and II cells 2 L ]typel KYtypen Bi typem Kk Vehicle Fic. 7. Effects of injection of 100 ug testosterone prop- ionate (TP) daily for 5 days on the percentages of GH cell types in male rats. In TP-treated rats, the percentage of Type I cells increased, but the per- centage of Type II cells decreased. *P<0.05 vs vehicle. (from Takahashi [19]). gradually increased by the term. After birth, the Type I GH cell became the predominant type. Such morphological changes in GH cell popula- tions have been extensively described [19]. Type I cells, containing large secretory granules (250-350 nm in diameter) predominated throughout the life (Fig. 5). The proportion of Type I cells was highest at 6 months of age. The proportion of Type Il and of Type III cells decreased from 1 month to 6 months of age, but increased there- after. This suggests that when GH secretion is more active (age-related changes in GH secretion will be discussed later), the proportion of Type I cell increases, and when GH secretion is less active, the proportion of Type II and III cells increases. GH cell-populations, morphologically classified, changed in accordance with GH secre- tory activity. Thus, it is highly probable that morphological heterogeneity of GH cells reflects 906 S. TAKAHASHI functional heterogeneity and/or the maturating process of GH cells. Therefore, as originally stated by Kurosumi er al. [18], the Type III GH cell may be an immature type of GH cell, the Type I cell the mature type, and the Type II cell may be an intermediate type, although no direct evidence for this hypothesis has been presented. The total volume of each GH cell type had been estimated [19] (Fig. 8), although the number of each type of GH cell was not examined. Type I cell populations peaked in volume at 6 months of age, and decreased thereafter. Type II and III cell populations increased in volume with age. The change in volume of GH cell populations may be partly due to the changes of the number of GH cells. Type I GH cells may increase with age until at least 6 months of age. Type II and III cells may gradually increase with age (Fig.8). These changes in percentages and estimated number of each GH cell type may be explained in several ways. One is that the increase in the percentage and number of GH cells is caused either by the proliferation of a specific type of GH cells, and/or the cell death of other specific types of GH cells. Another way is the conversion of one type of GH Male Female 3 Type I 2 2 =i oO 3c eS © 0 Type Il © 1 aot TT 3 © oO > 1 Type Ill ) 1 2 61218 1 2 6 1218 Age (months) Fic. 8. Estimated total volumes of each GH cell type. Volumes were expressed in arbitrary units. Age- related differences were detected in each GH cell type of both sexes (male, for each cell type: P< 0.01; female, Type I: P<0.01, Type II, Ill: P<0.05). (from Takahashi [19]). cell to another type of GH cell. It is also possible that GH cells are generated from stem cells or progenitor cells, although their presence has still not been proved. The interconversion of one type of GH cell to another type of GH cell is the most probable among the three possibilities. We have not direct evidence for it, but if large secretory granules are formed as a result of more intense stimulation from the hypothalamus (probably GHRH), the new formation of large secretory granules in Type III cells is to convert Type III cells to Type II cells. In Type II cells the formation of the small secre- tory granules may gradually slow or stop, and the proportion of the small granules becomes even- tually smaller. As the result, the mature Type I cells will finally appear. Alternatively, the small secretory granules may be fused to be a large secretory granule as previously reported in PRL cells [43]. For another example, estrogen in- creased the percentage and number of Type II GH cells (Fig. 6), and it had already been verified that the proliferation of GH cells is not stimulated by such estrogen treatment [44]. Therefore, estrogen appears to cause the conversion of Type I cells to Type II cells. The small secretory granules may be newly formed in the Golgi apparatus, or the large secretory granules may be disintegrated to the small granules under estrogen treatment. The transition from the Type I cell to the Type II cell is more probable than the proliferation or genesis of the Type II cell. 4. Functional heterogeneity of GH cells Heterogeneity in GH synthesis Uptake of [*H]- leucine into dissociated GH cells was studied using electron microscopic autoradiography. Only half of the GH cells were heavily labelled, indicating that GH synthetic ability differed among GH cell populations [45]. Furthermore, dissociated pitui- tary cells were separated into two subpopulations of GH cells by density gradient centrifugation. One of the two GH-cell subpopulations, which is less dense (the light fraction), produce more GH than the other [46]. Heterogeneity in GH release GH release from dissociated individual GH cells was analyzed by a reverse hemolytic plaque assay. Figure 9 shows GH and PRL Cells in the Rat 30 nN a — nN oO Frequency (% of total plaques) ° a on ee a \\7 \4 \. ee ee, eee 0 le} 1 2 345 10 20 40 80 160 Plaque area (um’x 10°) Fic. 9. The composite distribution pattern of plaque areas formed by GH cells from four hemolytic plaque assays. Each points are means of 4 separate assays with the standard errors. In each assay about three hundred plaques were measured. the bimodal frequency pattern of the plaque areas formed by GH cells of adult female rats. One subpopulation formed the larger hemolytic pla- ques than the other. As the plaque area is pro- portional to the amount of hormones secreted, this result indicates that one group of GH cells secretes more GH than the other. Similar result has been already reported by Frawley and Neill [47], although the bimodal distribution of the plaque area was detected only in GHRH-treated pituita- ties. The subpopulation of Type III GH cells was smaller than the other two subpopulations of Type I and II cells. Consequently, contribution of Type III GH cells in the reverse hemolytic plaque assay is quite small, and can be neglected. In young female pituitaries, the relative proportion of Type II cell-populations to Type I cell-populations was 41%. It is probable that the two subpopulation, morphologically divided, may correspond to the two subpopulation detected in the reverse hemoly- tic plaque assay. Further study is needed to clarify this correlation. 5. Possible mechanisms of functional heteroge- neity Preferential release of newly-synthesized hor- mones Chen et al. [48] found, using the reverse 907 hemolytic plaque assay, that in basal secretion of GH, one subpopulation of GH cells secreted a larger amount of GH than the other. The former subpopulation was likely to preferentially release newly-synthesized hormones for basal secretion, whereas the other population was likely to release stored hormones for basal secretion, even without stimulation. Therefore, this heterogeneity of GH cell population may be due to the difference in the intracellular content of the preferentially releas- able hormone component among GH cell popula- tions. Difference in GH cell-location A tissue-slicing method clarified another functional heterogeneity of GH cells [8]. The responsiveness of GH cells to GHRH on GH release is different, depending on the location of GH cells within the gland. GHRH- induced GH release was only detected in GH cells derived from the left dorsorostral, right ventro- caudal and right ventrorostral parts of the gland. This location-dependent functional heterogeneity of GH cells may be due to the differential blood supply, the different concentrations of hypothala- mic hormones in different portal vessels, or the effect of cell-to-cell communication (the paracrine effect). Differences in intracellular signal transduction Localization of protein kinase-C (PK-C) subtypes in the pituitary gland was studied immunocytoche- mically [49]. Not all of the pituitary cells contained PK-C. As for GH cells, only 9% of all GH cells contained PK-C. This finding suggests that GH cell populations can be divided into two sub- populations by the difference in the PK-C system. The function of PK-C in pituitary glands is not fully understood, although numerous physiological functions of PK-C are known [50]. As PK-C plays a key part in intracellular signal transduction, the GH cells which are deficient in PK-C may be controlled by other signal transduction mecha- nisms, Or may not have some functions (for exam- ple, as PK-C is known to be involved in cell proliferation in some types of cells, PK-C deficient cells may not have the ability to proliferate). 6. Molecular heterogeneity of GH Multiple molecular forms of GH are found in the rat pituitary gland [51-54]. Farrington and 908 S. TAKAHASHI Hymer [54], for example, demonstrated that 11 variants of rat GH exist, ranging in molecular weight from 11 kDa to 88 kDa (Fig. 10). Some of them were dimeric and glycosylated (24 K, Bollen- gier et al., [53]). These variants might be related to the functional heterogeneity of GH hormones, and the morphological heterogeneity of secretory gra- nules. Multiple forms of GH molecules were also well known in human, bovine and ovine pituitaries [S55, 56]. The most extensive studies were carried out on human GH variants. Human pituitary extracts contained at least half a dozen peptides. The major GH components in pituitary extract are 20 kDa, 22 kDa (the major component), and 45 kDa (dimer) [57, 58]. Using transgenic mice. 22 kDa, 20kDa and 5 kDa variants of human GH were evaluated [59]. Both 22 kDa and 20kDa forms stimulated linear body growth and liver hypertrophy. The linear growth mediated by the 22 kDa variant did not correlate with an increase in blood IGF-1 level. The 5 kDa variant did not Absorbance o o +++ | B at J ow i o+ x at é ar 5 OT —] 80-88K > 5 i o wn 60-66K ee E) Oar , 3 geen: Bs See 40-44K = ie a # N sagt) = a o 25k > | 2a = ; 22K ot 22K of 20K ‘ = 20K 14K > = 14K <= ! as s Fic. 10. Representative Western blots of rat pituitary GH contained in extracts electrophoresed under nonreducing (A) or reducing (B) conditions (see, Farrington and Hymer [54]). The reflectance optical density tracking of blot in A is shown in the middle. (from Farrington, M. and Hymer, W. C., Growth hormone aggregates in the rat adenohypophysis, Endocrinology, 126: 1630-1638, 1990; © The Endo- crine Society, with written permission). elicit any obvious activity. Thus, molecular variants of hormones may have different functions, although it has still not been established whether each molecular variant has a physiological function [S58]. Further studies on the mechanism of dif- ferential production of each variant and on cellular localization of each variant will probably give us the answer to this issue. 7. Age-related changes in GH secretion Physiological significance Multiple physiolo- gical roles of GH have been reported previously [60]. GH is one of the most important anabolic hormones. Dysfunction of the GH _ secretory mechanism may cause severe anomalies in various body functions. Sonntag ef al. [61] found a de- crease in protein synthesis, and GH administration reversed this. Takahashi and Meites [62] also reported the alterations in liver GH receptors with age, and GH administration in old rats reversed the age-related changes. The decreased GH secre- tion resulted in a low plasma somatomedin-C level [62]. Therefore, it is important to study the age-related changes in GH secretion. Changes in morphology of GH cells with age Morphological changes of GH cells have already been explained in the section Development of GH cells. DNA content of GH cell-populations, which indicate the number of GH cells, was estimated from pituitary DNA content data and the percen- tage of GH cells. The estimated DNA content constituting the GH-cell population did not change at 6, 12 and 18 months of age in male rats (16.1+ 2.4, 18.9+2.1 and 13.9+1.8 ug, respectively), but increased during this period in female rats (11.3+ 1.6, 18.4+2.1 and 18.1+0.4 ug, respectively). Changes in GH release with age GH is released in a pulsatile fashion. In male rats, the pulse interval is 3-4 hours, and in female rats it is about 70 minutes [63, 64]. The pulse amplitude is higher in male rats than in female rats. GH secretory patterns in male rats continued to remain un- changed throughout the day and night, but swiched to a rapid, highly pulsatile pattern at night in female rats [65]. There was no clear relationship between the GH secretory pattern and the phase of estrous cycle [65]. These sexual differences in GH secretion are clearly discussed in the review by GH and PRL Cells in the Rat 909 ny fo} oO Plasma GH (ng/ml) ~< ° Cc =} a 11 12 13 14 15 16 11 Fic. 11. Middle-aged 12 13 14 15 16 11 Clock time Mean plasma GH concentrations in young (5 months), middle-aged (11 months) and old (25-29 months) Old 12 ie 4 WS We female rats of SD strain. Each point represents the mean of GH levels in 17 young, 6 middle-aged and 13 old rats. Bars depict the standard errors of means. Plasma GH levels in young females were higher than in middle-aged and old female rats. Plasma GH levels in old rats tended to be somewhat lower than in middle-aged rats. (from Takahashi et al. [68]). Jansson et al. [66]. In the rat, GH secretion diminished in both sexes with age [67, 68] (Fig. 11). The pulse intervals did not change with age, but the pulse amplitudes were significantly lower in old rats than in young rats. GHRH responsiveness to GH release was reduced in old rats in vivo [69, 70], although Weherenberg et al. [71] reported the opposite result that there were no age-related changes in the responsiveness to GHRH. A recent study showed, using a hemolytic plaque assay, that GH release from individual GH cells was less in old female rats compared to young females (Taka- hashi, unpublished observation). The mean pla- que area produced by GH cells was significantly lower in 20-21 month-old females (7.7 +2.6 x 10° ym?) than that in 3-4 month old young females (14.9+2.4x 10° wm’). Responsiveness to GHRH was reduced in old female rats than in young rats. Accordingly, the diminished GH secretion in old rats is due partly to the reduced secretion of GH from individual GH cells. Changes in GH synthesis with age Pituitary GH content decreased with aging [19]. GH synthesis in young, middle-aged and old rats was studied at the GH-mRNA level [31]. GH concentration per single GH cell was significantly lower in old rats than in young rats (Fig. 12). Also, GH-mRNA concentration per single GH cell was significantly lower in old female rats (Fig. 13). Thus, GH synthesis diminished with age at the transcription level of the GH gene. These results were in good agreement with a recent in situ hybridization study [72]. This decrease may be due partly to the reduced release of hypothalamic GHRH in old rats [73-74], and the reduced binding sites of GHRH in old rats [75]. An uncoupling between the Male Female 10007 100 (2) GH content 800 8 = GH concentration 80 600 60 400 * 40 @ (WNC [199-H® 371/377) uonemuaou09 HO 200 tes Kk 20 GH_ content (wg/gland) a fo} 6 12 18 6 12 18 C) prt content 8 RK 200 GB PRL concentration KK 15 10 PRL content (wg/gland) (VN 1199-Tud 371/317) uoenusouos 7 yd (1) (o} 6 12 18 6 12 18 Age (month) Fic. 12. Pituitary GH and PRL content (per gland) and concentration (per ~g GH-cell DNA or PRL-cell DNA) in male and female rats at 6, 12 and 18 month of age. Female rats at estrus or persistent estrus (middle-aged and old) were used. *P<0.05, **P< 0.01 compared with 6-month-old rats. *P<0.05, **xP<0.01 compared with 12-month-old rats. (from Takahashi et al. [31]). 910 S. TAKAHASHI *lo Male Female 120 100 mo) & = 80 Z w~ 60 = = 5 40 20 7 7 to) 6 12 18 6 12 18 °lo 120 Z A 100 8 80 a0) Os * oD = s 40 fod — 20 <= = i i 7 6 12 18 6 12 18 Age (month) Fic. 13. Total pituitary GH mRNA content (per gland) and concentration (per ~g GH-cell DNA) in male and female rats at the age of 6, 12 and 18 months. Female rats at estrus or persistent estrus (12 and 18 months) were used. *P<0.05 compared with 6- month-old rats. (from Takahashi ef al. [31]). GHRH receptor and the G protein occurred in old male rats, resulting in a weaker response to GHRH in old pituitaries [76]. In male mice GH mRNA levels decreased with age [77]. Il. MAMMOTROPHS 1. Identification of PRL cells Size, shape and distribution PRL cells were found sparsely in the anterior-ventral portion of the gland, and found in the areas near the in- termediate lobe in the rat [9, 10, 78]. Regional distribution of PRL cells was reported by Sasaki and Iwama [79] in mice. The densities of PRL cells in the rostral and caudal pituitaries of mice were significantly greater than those of GH cells. The number and size of PRL cells differed significantly between male and female rats. PRL cells were polygonal, elongated and frequently cup-shaped and surrounded by large oval gonadotrophic cells [80]. Differentiation of PRL cells There are several reports about the first appearance of PRL cells in rat pituitaries, and these are somewhat contradic- tory. Sétal6 and Nakane [11] found PRL cells on day 16 of gestation. Chatelain ef al. [12] reported that PRL cells were detected on day 21 of gesta- tion, and Watanabe and Daikoku [13] reported that PRL cells were first detected postnatally. Nogami et al. [14] observed immunocytochemical- ly and by in situ hybridization that PRL was detected on day 18-19 of gestation, and PRL- mRNA was also detected on day 18-19 of gesta- tion. PRL gene expression during the neonatal period had been studied [81]. PRL genes were expressed by at least 3 days of age, but the translation of the PRL message was, interestingly, reported to be blocked by the lack of association of the PRL message with ribosomes. In the mouse pituitary, immunoreactive PRL cells were detected at birth, but PRL cells might possibly appear in fetal pituitary glands [82]. In mice pituitaries an in situ hybridization with a PRL riboprobe, actually showed a few PRL cells 15.5 days after conception, and the number of PRL cells decreased 16.5 and 17.5 days after conception, although GH cells remarkably increased in number during this period [16]. In another study, PRL synthesis in mice was first detected at 8 days of age by a two-dimensional Fic. 14. Type I PRL cell in an adult female rat. The cell is elongated and a round nucleus is located slightly eccentrically. The rough endoplasmic reticulum and the Golgi apparatus are well developed. Large round or irregularly shaped secretory granules are located in the peripheral cytoplasm. Bar=1.0 um. (from Takahashi and Miyatake [88]). Fic. 15. Type II PRL cell in an adult female rat. Type II cells contain round secretory granules with a diameter of 150-250 nm. The number of secretory granules is larger than that in Type I cells. Bar=1.0 ~m. (from Takahashi and Miyatake [88]). at sin the R and PRL Cell GH 912 S. TAKAHASHI electrophoresis [17]. This discrepancy is partly due to the difference in the sensitivity of assays used (in situ. hybridization, immunocytochemistry, and two-dimensional electrophoresis). 2. Morphological heterogeneity of PRL cells Sato [80] studied postnatal development of PRL cells in the rat, and suggested from an immunocy- tochemical study that the oval PRL cells were premature, the polygonal ones mature, and the cup-shaped ones particularly differentiated. The ultrastructure of PRL cells had been extensively studied and three types of PRL cells, mainly based on the size of the secretory granules, were found [83-85]. Smets er al. [78] subdivided rat PRL cells into two types, one containing large polymorphic granules, and the other small round granules. Harigaya et al. [86] also classified mouse PRL cells into three types by immuno-electron-microscopy. Electron microscopically, the PRL mRNA was localized in the rat pituitary, and two types of PRL-synthesizing cells were identified [87]. One type was characterized by large secretory granules, and the other by small secretory granules. Taka- hashi and Miyatake [88] observed three subtypes of PRL cells in the rat, and classified them based on Kurosumi’s classification (Kurosumi et al. [89]). Type I cells contained irregularly shaped large secretory granules with a diameter of 300-700 nm (Fig. 14). Type II cells contained spherical gra- nules with a diameter of 150-250 nm (Fig. 15). Type III cell contained small round granules with a diameter of 100 nm (Fig. 16). Type I PRL cells had been originally identified as mammotrophs. 3. Development of PRL cells Sex difference in PRL cells Sex differences in PRL cell number were immunocytochemically found in mice [32] and in rats [90]. On the contrary, Dada et al. [34] reported that sex differ- Fic. 16. Type III PRL cell in an adult female rat. The cell is characterized by the small amount of cytoplasm containing small secretory granules with a diameter of about 100 nm. Cell organelles are less developed. Bar= 1.0 um. (from Takahashi and Miyatake [88]). GH and PRL Cells in the Rat 913 ences were not detected in adult rats. Using the reverse hemolytic plaque assay, postnatal develop- ment of PRL cells was studied, and it was clearly shown that the percentage of PRL-secreting cells did not differ between male and female rats at immature ages, but significantly increased in adult female rats as previously reported [33, 91]. These sex differences in the percentage and the number of PRL cells were caused by the difference in estrogen level [90, 92-94]. Proliferation of PRL cells The proliferation of PRL cells is stimulated by estrogen [29], and is closely correlated with PRL secretion. Bromocrip- tine, a dopamine agonist, inhibited not only PRL % 1007 () Wee! Type Il BB ype ill 80 ” o roe = > 60 (6) —l ioe a i) oD & 40 Cc o = o ra 20 (o) Control Estradiol Fic. 17. Effects of injection of 50 4g estradiol-17 (E>) daily for 5 days on the percentage of PRL cell types in male rats. In E;-treated rats, the percentage of Type I cells increased and the percentage of Type II and III cells decreased. The number above the column depicts the number of rats. Bars depict the standard errors of means. Estrogen affected the relative proportion of each subtype of PRL cells (P <0.01). (from Takahashi and Miyatake [88]). secretion, but also the mitosis of PRL cells [44, 95]. The difference in the number of PRL cells may partly result from the difference in the mitotic activity of PRL cells. The sex difference in the number of PRL cells is explained by the difference in the mitotic activity of PRL cell. Actually, the mitotic activity of PRL cells in estrous female rats was significantly higher than that in male rats [29]. Development of PRL cells Estrogen increased the percentage of Type I PRL cells, and decreased the percentages of Type II and III cells (Fig. 17). On the contrary, ovariectomy and bromocriptine decreased the percentage of Type I PRL cells and increased the percentages of the other two types (Fig. 18). Thus, the relative proportion of PRL cells changed in accordance with the change in % eS L_] Type! NS Type I = Type Ill 80 ” o Q or 5 OO a ra ao i) o ro) S 40 c ® 2 o a 20 (0) Control Bromocryptine Fic. 18. Effects of bromocriptine treatment on the per- centages of PRL cell types in adult female rats. The number above the column depicts the number of rats. Bars depict the standard errors of means. Bromocriptine affected the relative proportion of each subtype of PRL cells (P<0.01). (from Taka- hashi and Miyatake [88]). 914 S. TAKAHASHI PRL secretion. The Type III PRL cell, containing small secretory granules, was predominantly pre- sent at immature ages (Fig. 19), and was small in size. Therefore, the Type III PRL cell is consid- ered to be an immature type of PRL cell. Type I PRL cell, containing large secretory granules, constitutes most of the PRL cell population in adult female rats and is large in size. Thus, the Type I PRL cell is considered to be a mature type of PRL cell. The Type II PRL cell is considered to be an intermediate cell between the Type I and III cell [88, 89]. The change of secretory granules in size and shape is explained by the fusion and lysosomal degradation of preexist- ing secretory granules, which had previously been shown by Farquhar et al. [43]. irregularly-shaped 100 (-] Type! Type | [EB Type tll a => 80 3 5 —| 60 a ro N a 40 N s N = N % 20 N 2 Ne oa N M F F M F 10 30 60 Age (days) Fic. 19. Postnatal development of the percentages of PRL cell types in male (M) and female (F) rats. The number above the columns depicts the number of rats. Bars depict the standard errors of means. There are significant differences (P<0.01) between these groups: 10-day-old males vs. 30-day-old males, 10-day-old females vs. 30-day-old females, 30-day- old females vs. 60-day-old females. (from Taka- hashi and Miyatake [88]). 4. Functional heterogeneity of PRL cells Heterogeneity in PRL synthesis and release Hymer et al. [96] separated PRL cell populations using the differences in unit gravity, that is, the difference in the cell shape and secretory granule content. This method revealed that the intracellu- lar content of PRL differed among the separated PRL cell fractions, and the amount of PRL re- leased during the culture period of 14 days was positively correlated with the initial intracellular PRL content [97]. Swearingen [98] first found the heterogeneity in turnover of PRL in in vivo and in vitro studies. Walker and Farquhar [99] further clarified heterogeneity in PRL cells with respect to the PRL synthetic rate, which was autoradio- graphically visualized using the difference in the uptake of [*H]-leucine in PRL cells. They also found a subpopulation of PRL cells which secreted preferentialy newly synthesized PRL. Velkeniers et al. [100] separated PRL cell populations into high density and low density populations using the discontinous Percoll gradient, and found that low density PRL cells have a high basal secretory activity and a higher PRL-mRNA content, and high density PRL cells have a low basal secretory activity and a lower PRL-mRNA content, but a higher responsiveness to vasoactive intestinal polypeptide. Functional heterogeneity of rat PRL cells was also shown by the reverse hemolytic plaque assay [38, 101, 102]. The bimodal distribution of plaque sizes indicated that the amount of hormones re- leased from dissociated individual cells differed among PRL cells [101]. PRL cells were heter- ogeneous with respect to basal hormone secretion and responsiveness to TRH. Thus, there apparently seemed to be at least two subpopula- tions of PRL cells. Other evidence for the functional heterogeneity of PRL cell populations was reported by Arita er al. {103, 104] using the sequential cell immunoblot assay. Their study reported that there is a heter- ogeneity in PRL cell populations with respect to dopamine and TRH. Heterogeneity in PRL-cell surface antigen Another morphological heterogeneity in anti-PRL cell-surface immunoreactivity was shown in the rat pituitary [105]. Only half of all PRL cells from female rat pituitaries contained a cell-surface PRL immunoreactivity. This finding implies the pre- sence of PRL receptors on the cell surface, or some of the released PRL is retained on the surface of these cells. From this finding, PRL cell populations may also be divided into at least two subpopulations. However, it is not easy to corre- late this heterogeneity of PRL cell populations to the PRL cell types stated above. GH and PRL Cells in the Rat 915 5. Possible mechanism of functional heterogeneity of PRL cells Difference in PRL cell-location dependent functional heterogeneity in PRL cells was shown by the reverse hemolytic plaque assay [106], similar to the findings that have already been described in GH cells. In this study, PRL cells from the peripheral rim (outer zone) responded greatly to TRH, but only moderately to dopamine. PRL cells from the central region (inner zone) were affected slightly by TRH, but were markedly inhibited by dopamine. These regional differences in pituitary cells may be derived from the regional differences in the portal blood levels of hypothala- mic releasing/inhibiting hormones. Another pos- sibility is the paracrine effect on pituitary cells from the neighboring cells. Difference in the molecular variants secreted Diethylstibestrol-induced prolactinomas consisted of three different subpopulations of PRL cells [102]. In their study by gravitational seidmenta- tion, PRL cells were divided into large-, intermedi- ate- and small-sized PRL cells, which differed in their content and release of PRL. Large- and intermediate-sized PRL cells contained typical pleiomorphic secretory granules, but small-sized PRL cells were sparsely granulated or agranular. Small-sized PRL cell-populations contained uni- que PRL variants, whose molecular weights were 10-14K Dalton. This study suggests that there may be a relationship between the molecular heterogeneity of PRL and the diversity of mor- phology and function of PRL cells. Molecular variants of PRL will be discussed later. Difference in electrophysiological properties of PRL cells and dopamine receptors on PRL cells PRL cell populations were electrophys- iologically divided into two subpopulations, which, in turn, correspond to two groups separated by a BSA density gradient separation, the light and heavy groups [107]. Most of PRL cells of the light fraction showed a type 1 response; dopamine induced a hyperpolarization of the membrane potential from the resting potential. The other PRL cells of the heavy fraction mostly do not respond to a dopamine (type 2 response), but when the membrane potential has been depola- A. location- rized, dopamine induces a repolarization. The expression of the two dopaminergic D2 receptors, D24,5 and D2444, was studied and was found to be different in these two PRL cell populations [108]. The ratio D24;5/D2444 was higher in the light fraction of PRL cells than in the heavy fraction. This result indicates that the two different re- sponses to dopamine in PRL cells could be associ- ated with the differnetial expression of two differ- ent D2 receptors. Such differences may eventually bring about a difference in PRL secretion, and/or even in the morphology of PRL cells. Differences in the intracellular age of PRL The intracellular age of PRL molecules in the pituitary cells may be another important factor for the functional heterogeneity of PRL cell populations [109]. Dopamine had a significantly lower inhibi- tory effect on mature PRL (4-8 hr after synthesis) than newly synthesized and older stored PRL. TRH had a greater stimulatory effect on mature PRL (4-8 hr after synthesis), indicating that ma- ture PRL molecules are more readily released than on newly synthesized and old stored PRL. Thus, functional heterogeneity in PRL cell populations may be accounted for by the difference in the intracellular age of PRL, which is determined by whether it is newly synthesized or old (stored). 6. Molecular heterogeneity of PRL Several laboratories described molecular variants of PRL [110]. Hymer and Motter [102] reported in diethylstilbestrol-induced prolactino- mas that several variants of PRL molecules ranged from 12 kDa to 64kDa. Bollengier et al. [53] also showed molecular heterogeneity of PRL. That is, 23K, doublet 25K-26K, 40K and 42K. A variant of 26 kDa is considered to be glycosylated PRL. High molecular weight variants occur as a product of disulfide linkage between monomeric units. Oetting and Walker [111] reported an interesting finding that three variants of PRL, whose molecular weights were the same (24K), were different in their net charge (isoform 1, least negatively charged isoform of PRL; isoform 2; isoform 3, most negatively charged isoform), and considered them to be synthesized in PRL cells. Isoform 2 was the predominant form inside the cell and isoform 1 was the predominant secreted form, 916 S. TAKAHASHI although all three isoforms were released. Physiological significance of molecular hetero- geneity Frawley et al. [112] indicated the possi- bility that each molecular variant of PRL differs in biological activity, and suggested that each molecular form may have specific target cells, and consequently, have specific physiological roles. A good example to demonstrate the possible phys- iological significance of molecular variants of PRL was recently reported in ram pituitaries. The study clearly showed that production of variant forms of PRL in ram pituitary glands varied seasonably [113]. In their study, the 23 K form is a primary hormone, and the 25K form is a glycosylated form. High molecular weight-forms (more than 25 K), which are aggregated by a disulfide linkage between monomers, are significant in winter, and may be for storage. During the season when PRL secretion is active, high molecular forms dis- appeared. An explanation for this could be that synthesized hormones may be rapidly released into circulation, and are not stored in the cell. On the contrary, during the season when PRL secretion is low or inhibited, synthesized hormones are more likely to aggregate and to become the stored type. Thus, it is probable that changes in molecular forms of a hormone may be parallel to changes in the secretory activities of hormones. 7. Age-related changes in PRL secretion Physiological significance A number of phys- iological actions of PRL have been reported [114]. The altered PRL secretion induces various dis- eases (eg. [115, 116]). Therefore, it is valuable to study age-related changes in PRL secretion. Changes in morphology of PRL cells with age Kawashima [117] reported, electron microscopi- cally, the morphological changes in pituitary cells, particularly hypertrophy and hyperplasia of PRL cells in female rats, although an immunocyto- chemical identification had not been done. Age- related changes in immunocytochemically iden- tified-PRL cells were reported by Takahashi and Kawashima [90]. The percentage of PRL cells significantly increased in female rats with age (Table). The total number of PRL cells had not been measured, but it had been estimated from the pituitary DNA content and the percentage of PRL cells as described in GH cells. Actually, the DNA contents constituting the PRL-cell population at 6, 12 and 18 months were as follows; in male rats, 3.7 +0.6ug (7), 7.0+0.8 (8) and 6.5+0.8 (5), and in female rats, 9.5+1.4 (8), 25.8+2.9 (8) and 40.3 + 2.4 (7). The DNA contents of PRL cells increased in both sexes with age, but more markedly in old female rats, suggesting the significant increase of PRL cells in number. This was confirmed by Chuknyiska ef al. [118]. The increase in the number of PRL cells with aging was caused by the Ovarian estrogen, the stimulatory factor for the proliferation of PRL cells [119]. Prepubertal ovar- iectomy prevented the increase of PRL cells in number in old female rats. Age-related changes in PRL cell mitosis in the rat were observed (Fig. 20). Even in 2-year-old female rats mitotic pituitary cells were encoun- tered. Immuno-electron-microscopical studies have been done in male rats [120]. The relative proportion of each type changes with age in male rats. One type of PRL cell, containing small round secretory granules (Type III cells in Kurosumi’s classification [89]), increased in percentage, and on the contrary, another type of PRL cell with large irregularly-shaped secretory granules (Type I cells in Kurosumi’s elassification) decreased in percen- tage in old male rats. Changes in PRL synthesis and secretion with age PRL secretion increased with age, and the enhanced secretion of PRL is partly due to the dysfunction of the hypothalamic dopaminergic mechanism [121-123]. A reverse hemolytic plaque assay revealed that the amount of PRL released per cell decreased in old rats [124]. Pituitary PRL content significantly increased with age in female rats, but PRL concentration per PRL cell de- creased [31] (Fig. 5). PRL mRNA levels per PRL cell decreased with age in both sexes (Fig. 21). PRL synthesis in each PRL cell decreased at the transcription level with age. However, since PRL cells significantly increased in number in old female pituitaries of the Wistar/Tw rats [90], the total amount of PRL significantly increased with age. Stewart ef al. [125] recently reported no significant change in PRL mRNA concentrations (per measured amount of pituitary DNA) with age in female rats, but did report a significant increase GH and PRL Cells in the Rat 917 69.5%. 8 80 - (8) 73.7°lo = () Male E wall [2] Female “ 25.2% positive cell 3 (8) 95.79, : (6) = 20) |e =< o Uv S nl 65.3% iS 201% NN 13.5% 15.5% a) 3 oe = 11.9% 30.4% 25.0% = (5) (6) (6) § (6 N (6) (7) (4) E D2 E D2 E D2 PE PD 20 30 60 90 150 - 180 ca360 ca.730 Age (days) Fic. 20. Age-related changes in the mitotic indices of pituitary cells and PRL cells in male and female rats. The colchicine-arrested mitotic cells and immunocytochemically-identified PRL cells (positive cells) were observed. For the detail of the method, see Takahashi er al. [29]. Female rats at estrus (E), 2nd day of diestrus (D3), persistent estrus (PE) and persistent diestrus (PD) were used. The percentage above the each column depicts the percentage of mitotic PRL cells in total mitotic pituitary cells. The number in parenthesis depicts the number of rats, and bars depict the standard errors of means. in serum PRL level. We did not find any signif- icant difference in PRL mRNA concentrations , Male Foner * (per yg of pituitary cell DNA) in female rats, 200 either (data not shown). Crew et al. [77] reported 2 an age-related decrease in PRL mRNA in male > 150 mice. < Zz = 5 100 Ill. MAMMOSOMATOTROPHS & eK 50 * 1. Identification of MS cells : | z Mammosomatotrophs (MS cells), which con- ) De BR AB tained both GH and PRL in the same cell, were ony! immuno-electron-microscopically described in in- lo tact adult rats [126-128]. MS cells were small in s 120 size and irregular in shape. Secretory granules, F100 50-150 nm in diameter, contained both hormones rs) caieners [126]. x Oo. 2 60 * nee = ok Fic. 21. Total pituitary PRL mRNA content (per s 40 ** gland) and concentration (per ng PRL-cell DNA) in iz 7K male and female rats at the age of 6, 12 and 18 = 20 fe months. Female rats at estrus or persistent estrus o 5 7 [al 7 (middle-aged and old) were used. *P<0.05, **P< 6 12 18 6 12 18 0.01 compared with 6-month-old rats. P<0.05 compared with 12-month-old rats. (from Takahashi Age (month) et al. [31]). 918 S. TAKAHASHI MS cells in various animals MS cells were quite rare in normal adult rats, but MS cells were usually encountered in lactating and pregnant females [126, 127]. Adenomatous rat pituitaries contained MS cells [128]. MS cells were also observed in mice [32, 129], musk shrews [127], bats [130], cows [131], sheep [132, 133], rhesus monkeys [134], and humans (fetal, [135-137]; normal adult, [138]; adenomatous adult, [139-141]). MS cells in rat pituitary tumor lines are well known [142-144]. However, Shirasawa et al. [145] could not detect any MS cells in the fetal and male adult bovine pituitary glands using three different immunohis- tochemical methods. The difference between the report of Fumagalli and Zanini [131] (nursing cows and virgin cows) and that of Shirasawa et al. [145] (fetal and adult bulls) is partly due to the differ- ence in the age and sex of animals used. In mice, MS cells were further divided into two subtypes, the small, round, solid secretory granular type and the vesicular secretory granular type [129]. Ishibashi and Shiino [127] found two types of colocalization of GH and PRL. One type was that GH and PRL are colocalized in the same secretory granules within a single cell, as described by Niki- tovitch-Winer et al. [126]. The other type is that GH-secretory granules and PRL-secretory gra- nules were intermixed within closely aggregated and interdigitated cell-clusters which consist of GH and PRL cells in pregnant rats and female musk shrews. This type is similar to the multinucleated mammosomatotrophs in cows reported by Fuma- galli and Zanini [131]. This finding suggest a possibility that the enhanced stimulation of hor- mone secretion iduce the fusion of the secretory cells. 2. Development of MS cells Using the reverse hemolytic plaque assay, MS cells were detected in neonatal and adult male and female rat pituitaries [146], in bovine pituitaries [147] and also in human pituitaries [135, 138]. Hoeffler et al. [33] reported that MS cells were 35.8% of all GH and/or PRL secreting cells of 5-day-old male rats. In adult male rats about one third of all GH and/or PRL secreting cells are MS cells [146]. Leong er al. [148] reported that about 5% of all pituitary cells were MS cells in adult male Pues Fig. 22. which was identified by the double immunocytoche- mical method using antisera to GH and PRL. GH was labelled with small gold particles and PRL was labelled with large gold particles. Bar=500 nm. rats. The data of the relative proportion of MS cells shown above could not be directly compared, because the mode of data description was different between the two reports. Chatelain et al. [12] immunocytochemically observed MS cells in rats at 21 days of fetal age. In neonatal rats, MS cells were found and these cells resembled the type III cells of GH or PRL cells (Fig. 22). The frequency of occurrence of MS cells during the neonatal period was not so high as reported by Hoeffler er al. [33]. In fetal mice at 15.5 days of gestation, a few pituitary cells colocal- ized GH- and PRL-mRNA, but the majority of cells containing PRL-mRNA did not express GH- mRNA [16]. 3. Developmental and physiological significance of MS cells Several possibilities were presented to explain the significance of MS cells. (i) One is that MS GH and PRL Cells in the Rat 919 cells are a transitional cell type for the conversion of GH cells to PRL cells, or PRL cells to GH cells. (ii) Another possibility is that MS cells are pro- genitor cells for GH and PRL cells [135]. (iti) The other possibility is that the MS cell is an independ- ent type of cell, and may be terminally differenti- ated. Analysis of the data which had been re- ported so far, and possible future data may clarify the genesis and physiological roles of MS cells. The following reports are favorble to the tran- sitional cell hypothesis. GH cells appeared earlier than PRL cells in rats and humans [14, 149]. In fetal mice, GH synthesis preceded PRL synthesis [17]. Stratmann and Ezrin [150] previously showed the possibility of the transition of GH cells to PRL cells by estrogen treatment using both electron micriscopy and autoradiographical detection of 3[H]-thymidine uptake. They stated that some of the previously existing GH cells proliferated and were converted into PRL cells. Frawley’s group had reported a large amount of evidence for the transition from GH cell to PRL cell, or PRL cell to GH cell using a hemolytic plaque assay [33, 38, 151, 152]. Using transgenic mice, Borrelli et al. [153] clearly showed that some stem-PRL cells were derived from part of the stem-GH cells, stating that PRL cells originated from the GH cell lineage. One of the transcription factors for GH and PRL genes was the same, GHF-1 or Pit-1 [6, 154-156]. GH and PRL molecules were consid- ered to be derived from a common ancestor mole- cule [157, 158]. Lira et al. [156] suggested that thyroid stimulating hormone-secreting cells (thy- rotroph) as well as GH and PRL cells are derived from a common lineage of pituitary cells. The analysis of factors of the transition of one type to the other type is required. Borreli et al. [153] stated that estrogen is essential for the gene- sis of stem-PRL cells from stem-GH cells in mice. Insulin inhibited GH synthesis and secretion [159], and also reduced the number of fetal GH cells in vitro [160]. On the contrary, insulin stimulated PRL synthesis through the activation of a PRL gene promoter [161]. Inoue et al. [162] recently induced the transition of GH secreting cells to PRL secreting cells by insulin or insulin-like growth factor (IGF-1) in their newly established pituitary clonal cell line [163]. Thus, insulin, and/ or IGF-1, is closely associated with the develop- ment of GH and PRL cells, and probably MS cells. If stimulation of PRL synthesis and secretion can induce the transition from GH cell to PRL cell, which may occur through the transitional cell of an MS cell, excessive stimulation of PRL secretion may enhance the occurrence of MS cells. Our preliminary study showed that estrogen treatment (50 ~g for 3 days) increased the number of MS cells (Fig. 22) about twice in the neonatal male rats. Similarly, estradiol increased the proportion of MS cells in a monolayer culture of male pitui- tary cells [164]. Chronic stimulation of GHRH using transgenic mice caused a hyperplasia of MS cells [165], although some of the MS cells in those transgenic mice were morphologically similar to those in adult mice, but others were morphologically different, and relatively close to those in adenomatous hu- man pituitaries. Provided that the MS cell is the common progenitor cell, and this progenitor cell of MS cells exists even in adult pituitaries, MS cells in adenomatous tissues may be derived from unreg- ulated proliferation of preexisting MS cells. The volume of data accumulated so far seems to strongly support the theory that the MS cell is a transitional cell from a GH cell to a PRL cell. However, it is probable that PRL cells transform to GH cells through MS cells as shown in Porter et al. [151, 152]. If such bidirectional conversion between GH cells and PRL cells occurs in rat pituitaries, MS cells may be the common progeni- tor cells. Currently, further study is still needed to determine which possibility stated above holds for the rat pituitary. IV. CONCLUSIONS A large amount of evidence indicated that the GH cell and PRL cell populations were morpholo- gically and functionally heterogeneous. A correla- tion between morphologically and functionally different subtypes remains to be studied. Analysis at a single cell level is needed for further clarifica- tion. Multiple molecular variants of GH and PRL have been reported. It is probable that such molecular variants of the two hormones may play different physiological roles. 920 S. TAKAHASHI Heterogeneity of pituitary GH and PRL cells at different levels (morphological, functional and molecular) may be the integrated outcome of various differences in the molecular variants of hormones, the intracellular age of hormones, the difference in receptors on the pituitary cells for hypothalamic regulatory hormones, the difference in mechanisms of intracellular signal transduction, and the location of pituitary cells in the gland. The maturating process of GH and PRL cells may be associated with these heterogeneities. Heter- ogeneity of GH and PRL cells in itself alters with age: the relative proportion of each subtype of GH and PRL cells changed with age. Age-related changes in GH and PRL secretion were studied, and the decrease in GH and PRL syntheses were clearly explained. MS cells may be the transitional cell between GH cells and PRL cells, or a common progenitor cell of GH and PRL cells. Further study is needed to clarify the significance of MS cells. From the Committed progenitor cell on 8; view of the developmental and maturating process of pituitary cells, the hypothetical schema for explaining the morphological heterogeneity of GH and RPL cells, and the relationship among GH, PRL and MS cells is described (Fig. 23). The overview of heterogeneities of GH cells and PRL cells gives the impression that such a wide spectra of heterogeneities, at various levels from the molecular to the pituitary level, could probably be highly helpful for endocrine functions. Such heterogeneity can give the pituitary gland enough flexibility to respond to any demands for hormone secretion. ACKNOWLEDGMENTS The author would like to express cordial thanks to Dr. K. Wakabayashi, Gunma University (Maebashi, Japan) for kindly supplying of the antisera against GH and PRL, and to Dr. J. A. Martial, University of Liege (Belgium) for the rat GH cDNA and rat PRL cDNA. The author is also grateful to Dr. S. Raiti, the National Hormone and oF BRU STATA Sr Obes (Estrogen) Typell GH cell Type Il bre Androgen (Mammosomato troph) Eee Pluripotent stem cell . PRL © Af Simple duplication\ y pe I type ll ot Simple duptication Estrogen ae “ y \ © LHIFSH & Simple duplication —— imple dupli © (GHRH) TSH cell ACTH cell Fic. 23. Typel GH cell Possible relationship among GH, PRL and MS cells. (Estrogen) Simple duplication (Eatrogen) PRL cell Pituitary glands consist of GH cells, PRL cells, gonadotrophs (LH/FSH cells), thyrotrophs (TSH cells), corticotrophs (ACTH cells) and folliculo-stellate cells (not shown in the figure). development of GH the mode of self-duplication. Pituitary specific transcription factor GHF-1 is known to be involved in the and PRL cells, and probably TSH cell. androgen, and that of PRL cells by estrogen. This conversion may be bidirectional. GHRH stimulates the mitosiss of GH cells, and estrogen that of PRL cell. Mammosomatotrophs (MS cells) may be the transitional cells between GH cells and PRL cells. reports, part of PRL cells may be derived from part of GH cells. The conversion of GH cells is stimulated by Pituitary cells proliferate by From several This transition from stem-GH cells into stem-PRL cells may probably be stimulated by estrogen. GH and PRL Cells in the Rat 921 Pituitary Program (University of Maryland School of Medicine, Baltimore, MD, USA) and the NIDDK, NIH (Bethesda, MD, USA) for the RIA kit. This study was supported in part by Grants-in-Aid for Scientific Re- search from the Ministry of Education, Science and Culture, Japan, and by the Itoh Science Foundation. REFERENCES 1 Kurosumi, K. (1986) J. Clin. 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(1989) Endocrinology, 125: 2710-2718. ZOOLOGICAL SCIENCE 9: 925-939 (1992) © 1992 Zoological Society of Japan REVIEW Chloride Pumps in Biological Membranes Georce A. GERENCSER! and BLANKA ZELEZNA Department of Physiology, College of Medicine, University of Florida, Box 100274, Gainesville, FL 32610-0274, U.S.A. INTRODUCTION The electrical activity of isolated biological epithelia has been a source of intense interest and much scientific study since the early reports of DuBois-Reymond [1] and Galeotti [2]. However, it was not until the brilliant and creative studies of Ussing [3] on isolated frog skin and, later, those of Leaf [4] and his co-workers on isolated toad urin- ary bladder that defined the nature of the bioelec- tric potential. The defined interrelationship be- tween bioelectric potential and active Na* trans- port ushered in the modern era of ion transport study in epithelia. Skou [5] molecularly defined the nature of Na* transport with his discovery of the (Nat +K*)-stimulated ATPase enzyme. For years thereafter active Na‘ transport across epithelia has occupied the collective focus of trans- port physiologists with Cl~ assuming a secondary role of passive counterion. However, within the past 20 years there has been an intensive interest in transmembrane Cl~ transport primarily because Cl” has been found to move actively in a very wide range of species [6, 7]. Within the last 20 years three general mecha- nisms of transepithelial Cl” transport have been reasonably well established. The first of these is a strictly passive means of Cl” transport coupled electrically and/or chemically to primary active Na®* transport and is exemplified by isolated frog skin [8] and toad urinary bladder [4]. The second well-accepted Cl” transport process is secondarily Received July 3, 1992 ' To whom all correspondence should be addressed. active and is thought to be effected through an electrically neutral Na*-coupled carrier mecha- nism which drives Cl” uphill into epithelial cells via the inward flow of Na* down a favorable electrochemical potential gradient. This NaCl symport process is located within the apical mem- brane if Cl~ is actively absorbed by the epithelium or is located within the basolateral membrane (BLM) if Cl~ is actively secreted. Extrusion of Na* from the cell, therefore maintenance of the favorable Na* electrochemical potential gradient, occurs by the ouabain-sensitive (Na*+K‘)- stimulated ATPase (i.e., primary active Na* transport) located within the BLM. Epithelia which exemplify NaCl symport absorption include prawn intestine [9], flounder intestine [10, 11], sculpin intestine [12], marine eel intestine [13], flounder urinary bladder [14], trout urinary blad- der [15], Necturus gallbladder [16, 17], Necturus proximal tubule [18], bullfrog small intestine [19, 20], frog skin [21, 22], bovine rumen [23], rat colon [24], rabbit gallbladder [25], rabbit ileum [26] and human intestine [27]. Those epithelia in which Na*-coupled Cl” secretion has been demons- trated include killifish operculum [28], pinfish gills [29], shark rectal gland [30], frog stomach [31], frog cornea [32, 33], rabbit ileum [34] and dog trachea [35]. In these systems Na * is thought to be actively recycled at the BLM by the Na* pump while Cl~ moves energetically downhill from cyto- sol to the mucosa via a cAMP-enhanced Cl~ conductance [36]. The third widely accepted epithelial Cl~ transport process is also secondarily active and involves Cl” /anion antiport and is found, for example, in anal papillae of mosquito 926 G. A. GERENCSER AND B. ZELEZNA larvae [37], fish gills [38-40], frog skin [21], urodele intestine [41], turtle bladder [42], rat intes- tine [43], rabbit colon [44] and human small intes- tine [27]. The energy source for this process is unknown, but it has been suggested that uphill Cl~ transport is energized by a favorable downhill electrochemical potential gradient for the counter anion [6]. However, a considerable amount of Cl” trans- port data has accumulated in the transport litera- ture that does not conform to any of the three well established models described above. For instance, Hanrahan and Phillips [45] have provided evidence for an electrogenic Cl~ accumulative mechanism located in the mucosal membrane of locust rectal epithelium. This mechanism is activated and stimulated directly by K* and is also independent of Na* and HCO; . Observations of plant cell membranes [46, 47], as well as bacterial mem- branes [48] have yielded Cl -ATPase activity and associated Cl~ accumulation which are inconsis- tent with the three models for Cl transport described previously (vide supra). Perhaps, the strongest and most compelling evidence for a primary active transport mechanism of Cl” (Cl~ pump) resides with the observations of Gerencser [49] and Shiroya et al. [50] who have characterized Cl~-ATPase activity and ATP-dependent Cl~ transport in the same plasma membrane system as well as reconstituting these activities in a liposome system [51]. Indeed, the speculation by Frizzell et al. [6], Schultz [52], and DePont and Bonting [53] that Cl~-stimulated ATPases are not involved in biological Cl~ transport may have been too pre- sumptuous and premature considering the recent ground-swell of possible evidence to the contrary. EXISTENCE AND GENERAL PROPERTIES Since the time Durbin and Kasbekar [54] first demonstrated anion-stimulated ATPase activity in a microsomal fraction of frog gastric mucosa, there has been little question as to the existence of, at least, the biochemical manifestation of the en- zyme. The distribution of anion-stimulated ATPase activity seems to be as widely distributed throughout biology as the number of different plants and animals studied [53, 55, 56]. Anion-stimulated ATPase activity, and there- fore possibly Cl” pump existence, has been de- monstrated in both microsomal and mitochondrial fractions of many tissues (Table 1) in which HCO; , Cl” or H™ transport occurs, suggesting a transport function for this enzyme. DeRenzis and Bornancin [57] demonstrated the existence of Cl” /HCO3 -stimulated ATPase in goldfish gill epithelia. It was not until this observation that HCO; -stimulated ATPase activity was linked with possible primary active Cl” transport, be- cause Cl” stimulation of this enzyme had not been previously demonstrated. As the name of the enzyme implies, it is directly stimulated by anions, especially HCO; and Cl. Bicarbonate stimulation of the enzyme has occu- pied the predominant focus of attention primarily because of cellular acid-base implications and also because of possible simultaneous proton secretion in gastric mucosal systems [53]. However, HCO3~ can be replaced by several other anions, especially Cl” and the oxy-anions such as arsenate, arsenite, borate, selenite, sulfate and sulfite [58-61]. As can be surmised, however, there are considerable dif- ferences in effectiveness of the various anions in different tissues [62]. As an extreme example, glucaronate stimulates ATPase activity in lizard gastric mucosa [63] while it inhibits, presumably, the same enzyme in frog gastric mucosa [64]. As emphasized by Schuurmans Stekhoven and Bont- ing [55], this species and tissue variability may very well be caused by affinity differences of the various anions for the enzyme. ATP is the preferred substrate for the anion- stimulated ATPase, with an optimal Mg?* /ATP ratio ranging from 0.5 to 2.0 [62, 65, 66]. GTP and ITP are less preferred substrates than ATP for the anion-stimulated ATPase, whereas UTP and CTP are slightly hydrolyzed or not hydrolyzed at all by the enzyme [58, 65]. The divalent cation Mg** is absolutely required for maximal anion-stimulated ATPase activity, but inhibits at high concentrations [64], as are also the case for the cation-stimulated enzymes: (Na* + K*)-ATPase and (Ca7+ +Mg**)-ATPase. Mn?* can substitute for Mg** in the gastric mucosal enzyme [67], but does so to a lesser extent in the pancreatic enzyme [65]. Generally Na* or K* has Chloride Pumps in Biology 927 TABLE |. Some biological tissues in which Cl -stimulated ATPase activity has been localized to cellular plasma membranes or microsomal fractions TISSUE SPECIES REFERENCE BACTERIA Cytoplasmic membrane Halobacteria [99] PLANTS Cell membrane Algae [110] Salt gland cell membrane Salt marsh [122] ANIMALS Gill Goldfish [57] Eel [84] Trout [85, 51] Fiddler Crab [89] Blue Crab [90] Kidney Rat [68] Rectum Larval Dragonfly [91] Locust [96] Intestine Rat [68] Aplysia [72] Mantle Oyster [78] Spinal Motoneurons Rat [108] Brain Rat [50] Embryo Sea Urchin [97] Lens Cow [98] little or no effect on the activity [64, 65], but K* was shown to have a stimulatory effect on the enzyme in rat salivary glands [61]. NH," appears to inhibit anion-stimulated ATPase activity [67]. LOCATION Presently, without question, the greatest argu- ment regarding Cl” - pump activity is its localiza- tion within the subarchitecture of cells. It seems that Cl -stimulated ATPase activity resides in both microsomal and mitochondrial fractions [53] of cell homogenates. However, DePont and Bont- ing [53] and Schuurmans Stekhoven and Bonting [55] have declared that microsomal or plasma membrane localization of this enzyme is entirely due to mitochondrial contamination, hence the dispute. If Cl~-stimulated ATPase activity is exclusively of mitochondrial origin, it is very dif- ficult to conceive a mechanism which is the ATPase that can drive net Cl” transport across plasma membranes. On the other hand, if the Cl -stimulated ATPase is located in the plasma membrane, it would not be difficult to envision primary active Cl~ transport by this enzyme analo- gous to the (Na* +K*)-stimulated ATPase and its role in the net transport of Na* and K* across the plasma membranes [5]. Without argument, the primary site of Cl7- stimulated ATPase activity within plant or animal cells appears to be in the mitochondria; that is, a property of the mitochondrial Ht-ATPase [68]. 928 G. A. GERENCSER AND B. ZELEZNA Obviously, the key question is: what is the origin of the Cl -stimulated ATPase activity of non mitochondrial organelles? Is it as Schuurmans Stekhoven and Bonting [55] have dogmatically stated, that all nonmitochondrial organelles which exhibit Cl” -stimulated ATPase activity have been contaminated with the mitochondrial-based en- zyme, or is there a true, separate and distinct Cl -stimulated ATPase that is localized within the cellular plasma membranes, and which therefore can possibly act as the prime mover of net Cl™ transport between the intracellular and extracellu- lar space? Van Amelsvoort et al. [62] provided extensive evidence via differential and density gradient cen- trifugation techniques on epithelia from trout gill, rabbit kidney and rabbit stomach that most, if not all, anion-stimulated ATPase activity is of mito- chondrial origin. Their speculative conclusions negated any plasma membrane anion-stimulated ATPase localization found in other studies [61, 69, 70] on the basis that the results from these studies possibly artefactual due to improper homogenization and density gradient centrifuga- tion techniques. They stated that excessive or “drastic” homogenization may inactivate the mitochondrial anion-stimulated ATPase by release of the endogenous mitochondrial inhibitory pro- tein [71], therefore this effect would amplify, in a were relative sense, mitochondrial contamination observed in non-mitochondrial organelles. However, they did not comment why the mitochondrial inhibitory protein also would not inactivate the mitochondrial contaminant, anion- stimulated ATPase found in non-mitochondrial organelles. Surprisingly, in the same study Van Amelsvoort et al. [62] observed low cytochrome oxidase activity in presumably mitochondrial-rich rabbit kidney and stated that cytochrome oxidase was either specifically inacti- vated, or that loss of the mitochondrial inhibitory protein led to an exaggerated anion-stimulated ATPase activity in these fractions. They did not present data nor did they speculate on how these fractions of mechanisms were actuated in light of the apparent contradiction based on the argument that they put forth for “drastic” homogenization effects. They also stated that “drastic” homogenization techni- ques may yield extremely small submitochondrial particles which may not reach their equilibrium position in normal empirically determined times of density gradient centrifugation, which could also account for erroneous plasma membrane localiza- tion of anion-stimulated ATPase activity. It had been the preceding studies that negated any inter- pretation, other than anion-ATPase being a prop- erty of mitochondrial H*-ATPase, that stultified progress in this most complex research area for a period extending from the mid-1970’s through the mid-1980’s. As suggested earlier (vide supra), there are numerous examples of those tissues that transport Cl” whose processes of transfer have been mod- eled mechanistically, but thermodynamically have not been rigorously defined or tested. Invoking a cellular active Cl” transport mechanism on energetic grounds justifies the search for such a process in the one cellular organelle that regulates the transfer of material and information (Cl) between the external world and intracellular con- tents, the plasma membrane. The hallmark study demonstrating, unequivo- cally, the existence of Cl --ATPase activity in a plasma membrane system free from any possible mitochondrial contaminant ATPase was that by Gerencser and Lee [72]. They presented evidence which indicated that the BLM of Aplysia foregut absorptive cells contein Cl -ATPase activity. Their finding that the BLM subcellular membrane fraction had a high specific activity in (Na* + K*)-ATPase, but had no perceptible cytochrome c oxidase activity and a significantly reduced succinic dehydrogenase activity, supported this conclusion (Table 2). The observation that there was very little NADPH-cytochrome c reductase activity in the membrane fraction (Table 2) suggested that the BLM in this fraction were also relatively free from endoplasmic reticulum and Golgi body mem- brane contamination [73]. The failure of oligomy- cin to inhibit Cl --ATPase activity in the BLM fraction was also consistent with the nonmi- tochondrial origin of the Cl" -ATPase. Supporting this contention was the corollary finding that oli- gomycin inhibited mitochondrial Cl -stimulated ATPase activity. The finding that efrapeptin, a direct inhibitor of mitochondrial F,;-ATPase activ- Chloride Pumps in Biology 929 TABLE 2. from Aplysia Distribution of marker enzymes and anion-stimulated ATPase during preparation of gut BLM S-II Enzyme Homogenate (Mitochondria) Nene Total protein (mg) 208.59 + 24.95 15.13 + 1.95 5.47 + 1.09 Na*-K*-ATPase 0.85+0.11 0.25 +0.23 BA oar B10) 5’-Nucleotidase 0.41 +0.17 0.37+0.11 0.89 +0.33 Cytochrome c oxidase 0.50+0.12 0.97 £0.23 ND Succinic dehydrogenase 22.30+4.90 83.40 + 27.60 4.17+2.40 NADPH-cytochrome c reductase 3.73 £0.28 7.44+1.25 1.32+0.18 Mg>*-ATPase 2.69+0.57 5.10+0.84 9.16+1.82 HCO; -ATPase 3.85 +0.83 7.26+0.91 14.12+2.02 Cl -ATPase 2.51+0.59 2.50+0.79 5.77 £2.26 Values are means+SE from 9-11 different preparations. Enzyme activity is expressed as ~mol-h '-mg protein.' for Na*-K*-ATPase and 5’-nucleotidase; Alog (ferrocytochrome c)-min '-mg protein! for cytochrome c oxidase; ymol-min '-mg protein ' for succinic dehydrogenase; nmol-min‘-mg protein! for NADPH-cytochrome c reductase; “mol-15 min~'-mg protein _' for Mg**-, HCO, , and Cl _-ATPase. P, pellets from 9,500-g centrifugation; S-III, 40-50% sucrose interface; ND=not detectable. was ~1.5g. Conditions for enzyme assays were as described in text. permission. ity [74], significantly inhibited Mg**-ATPase activity in the mitochondrial and not in the BLM fraction [72] unequivocally supported the notion that the plasma membrane fraction is of extrami- tochondrial origin. Additionally, Gerencser and Lee [75] showed that vanadate (an inhibitor of only “P-type ATPases”) inhibited Cl” -ATPase activity in the purified BLM fraction. Taken together, all of these observations strongly support the hypothesis that Cl -stimulated ATPase activ- ity exists in, at least, one subcellular locus other than mitochondria. It appears that in numerous biological cells, which transport Cl”, Cl -sti- mulated ATPase activity forms an integral part of the plasma membrane [68], [76-78], and Table 1. FUNCTION To impart a direct role of Cl~ transmembrane transport to an ATPase, the ATPase should be shown to be an integral component of the plasma membrane surrounding the cell periphery. The energy for active transport of Cl~ can therefore, in principle, be obtained from the hydrolysis of ATP. Both of these prerequisites have been amply sa- tisfied (Existence and General Properties section Starting gut mucosa Table from Gerencser and Lee [72] with and Table 4). Therefore, the next question that need be asked is: Is the anion-stimulated ATPase identical with a primary active transport mechan- ism (“pump”) for anions? Hopefully the following discussion can lend some insight into this most controversial question [6, 7, 52, 53, 79]. Countertransport of Cl” and HCO3~ has been reported in molluscan neurons [80, 81] and mouse soleus muscle [82] that is sensitve to 4-acetamido- 4’-isothiocyanostilbene-2,2’-disulfonic acid (SITS) and is not inhibited by thiocyanate in mouse soleus. It has also been reported in numerous epithelia [7] that this anion exchange process exists and is sensitive to the stilbene derivatives. The stilbene-sensitive countertransport or exchange mechanism does not seem to require ATP and, therefore, in all probability, is not an ATPase [83]. It was not until the following observations that HCO; -stimulated ATPase activity was linked with Cl” pumping because no Cl activation of this enzyme had been observed. DeRenzis & Bornancin [57] were the first to demonstrate the membrane presence of a (Cl +HCO; )- stimulated ATPase in goldfish gill epithelium and suggested that the enzyme could participate in the branchial Cl~/HCO3° exchange mechanism. 930, G. A. GERENCSER AND B. ZELEZNA Bornancin et al. [84] confirmed these results in freshwater eel gill epithelium as did Bornancin et al. [85] in freshwater trout gill epithelium. Kinetic studies in these three gill epithelial systems strong- ly suggested that a (Cl /HCO3_)-stimulated ATPase is involved in the Cl” /HCO3” exchange mechanism and therefore in the acid-base regula- tion of freshwater fish. These authors reported a parallelism between the affinities of the ATPase for Cl” and both the Cl” affinity for the gill transport mechanism and the Cl” influx rate. The affinity constants for the Cl” -stimulated ATPase were 1.0, 5.9 and 23.0 meq/L for the goldfish [57], freshwater trout [85], and freshwater eel [84] gill epithelium, respectively. The affinity of Cl” for the transport systems in vivo was 0.07, 0.25 and 1.3 meq/L for the goldfish [57], freshwater trout [85] and freshwater eel [84] gill epithelium, respective- ly, while the corresponding maximal Cl~ influxes were 55.0, 19.6, and 0.36 weq/hr/100 g. In addi- tion, the finding that Cl” activation of anion- stimulated ATPase activity was inhibited by thiocyanate [57] was consistent with transport stu- dies which showed that Cl~ influxes were inhibited by thiocyanate [86]. These studies on gill epithe- lium strongly support the hypothesis that the Cl” - stimulated ATPase is involved in gill anion ex- changes that are related to mineral and acid-base homeostasis in freshwater fish. The fiddler crab gill has been shown to actively absorb Cl” from low salinities [87] and actively extrude Cl” in high-salinity media [88]. In concert with these findings DePew & Towle [89] demon- strated the existence of an anion-stimulated ATPase in the gill cell plasma membrane of fiddler crab and suggested that this enzyme is so situated with its environment that it is highly accessible to Cl” and HCO; _, and thus many play a direct role in acitve Cl” /HCO3 exchange. Lee [90] used an additional approach to the question concerning correspondence between transport and anion-stimulated ATPase activity. After it was established that anion-stimulated ATPase activity existed in the plasma membrane of blue crab gill epithelium, the animals were adapted to low salinities. This thinking presumed that Cl” /HCO3” exchange should increase under these osmotic stressful conditions, therefore this transport activity should be reflected in an increase in the activity of anion-stimulated ATPase activity. This was indeed the case and Lee [90] suggested that anion-stimulated ATPase activity appears likely to play an important role in anion transport for osmoregulatory and/or acid-base homeostasis in marine organisms. Komnick et al. [91] reported the presence of (Cl” +HCO3 )-stimulated ATPase activity in plasma membranes of larval dragonfly rectum. The Cl -stimulated ATPase activity was inhibited by thiocyanate as was the Cl” influx into the rectal epithelia. These results suggested the possible existence of an ATPase-mediated, active Cl~ transport mechanism located in the plasma mem- brane of larval dragonfly rectal epithelial cells. In the eel (Anguilla japonica) intestine, elec- trophysiological experiments have shown that ac- tive transport of Cl” coupled with water transport markedly increases during seawater adaptation [92, 93]. The observed increase in Cl” absorption raised the question of an associated increase in activity of an enzyme contributing to the transport process. It was demonstrated by Morisawa and Utida [94] that anion-stimulated ATPase activity existed in an oligomycin-insensitive, thiocyanate- sensitive membrane fraction of eel intestinal en- terocytes that was also relatively deficient of cytochrome oxidase activity. Seawater adaptation increased the enzyme activity commensurate with changes in Cl” and water transport. From these considerations, these authors concluded that the anion-stimulated ATPase played a direct role in Cl” transport in the eel intestine. The hindgut of the desert locust possesses an unusual chloride transport system [95]. The iso- lated locust rectum absorbs chloride from the mucosal (lumen) to the serosal (haemolymph) side at a rate which is equal to the short-circuit current (I,.). Net chloride transport (J$%,) persists in nominally Na-free or HCO 3(CO3)-free saline, is insensitive to normal inhibitors of NaCl co- transport and anion exchange, and is independent of the net electrochemical gradient for sodium across the apical membrane. However, active chloride transport is strongly dependent on mucos- al potassium (K,=5.3mM-K). Chloride entry across the apical membrane is active, whereas the Chloride Pumps in Biology 931 net electrochemical gradient across the basal mem- brane favors passive Cl exit from the cell. Although mucosal potassium directly stimulates “uphill” chloride entry, there is no evidence for coupled KCl co-transport, nor would co-entry with potassium be advantageous energetically. This Cl~ absorption is electrogenic, not depen- dent on Na* or HCO; /COs, and insensitive to inhibitors of NaCl cotransport or HCO; /Cl~ exchange [96]. To determine if active Cl” trans- port across rectal epithelia might be due to an anion-stimulated ATPase, a microsomal fraction was obtained by differential centrifugation. Micro- somal ATPase activity was stimulated in the fol- lowing sequence: sulphite >bicarbonate >chlor-- ide. Maximal ATPase activity was obtained at 25 mM HCO3° or25mM Cl. Thiocyanate (10 mM) inhibited 90% of the anion-stimulated ATPase activity. The microsomal fraction was enriched in the plasma membrane markers, leucine ami- nopeptidase, alkaline phosphatase, 5’-nucleo tidase, and gamma-glutamyltranspeptidase, and had little contamination of the mitochondrial en- zymes, succinate cytochrome c reductase and cytochrome oxidase. Na, K-ATPase was enriched in the mitochondrial fraction. Microscopic ex- amination confirmed that basolateral membranes were associated with mitochondria following dif- ferential centrifugation, while the microsomal frac- tion contained little mitochondrial contamination. These results indicate the presence of an anion- stimulated ATPase activity that could be responsi- ble for active Cl~ transport across locust recta. In cultured cells derived from isolated micro- meres of sea urchin eggs, Cl’ /HCO3 -ATPase activity was found in the plasma membrane and the microsome fractions before and after the initia- tion of spicule formation [97]. After initiation, the skeletal vacuole fraction was obtained from sub- cellular structures containing specules. Cl 7/ HCO; -ATPase in the skeletal vacuole membrane probably mediates HCO; transport into the vacuoles to supply HCO; for spicule formation. An anion-stimulated ouabain-insensitive Mg? *- ATPase activity has been found in fresh homogen- ates prepared from capsules and epithelia of bovine lenses [98]. Approximately equal activity was observed in the presence of HCO; or of Cl. The stimulation of each anion obeys saturation kinetics, with an optimum at approximately 20 mM Cl” or HCO; _. In keeping with other tissues, the diuretic drugs furosemide and ethacrynic acid are inhibitory. ATP is the primary substrate for the enzyme, which also shows some activity on GTP, ITP, and even ADP. Little Na‘/K*- dependent ATPase activity was observed in the fresh homogenate, but it increased in lyophilized preparations. In contrast, the lyophilized prepara- tions showed no anion-dependent ATPase activity. It is postulated that active bicarbonate ion trans- port in the lens may be mediated by this anion- dependent ATPase. Halorhodopsin [99, 100], one of the retinal proteins in the cytoplasmic membrane of halobac- teria, is an inward-directed light-driven elec- trogenic pump for chloride ions that generates an inside-negative membrane potential similar to that of bacteriorhodopsin, which transports protons out of the cell interior. However, the physiological role of halorhodopsin might be not only to gener- ate a transient proton-motive force on illumina- tion, but also to maintain cell volume [101]. This is because in these organisms the high (several mo- lar) external NaCl concentration in the medium is balanced mostly by intracellular KCI, and although the replacement of Na* with K* can be accom- plished, as in many other systems, by a combina- tion of a K*/Na° antiporter [102, 103] and elec- trogenic K* uptake [104, 105], the net uptake of Cl” requires an active accumulation system. In- deed, as with protons in the case of bacterio- rhodopsin, a second transport pathway for active Cl transport exists in the dark [106], apparently driven independently, by proton-motive force. The elements of the foregoing hypothesis can be observed in vesicles prepared from Halobacterium halobium cell envelopes containing halorhodopsin [107]. Thus, in the absence of K* (e.g., in 3M NaCl), illumination causes the inward flow of Cl", which is detectable by direct determination of the accumulated Cl in vesicles equilibrated first with Na SO, or phosphate. When the illumination is started, there is an initial passive influx of protons, which slows as a concentration gradient for pro- tons (inside acid) develops. During this time, Na* takes over as the main counterion to the Cl” 932 G. A. GERENCSER AND B. ZELEZNA movement. Once the protonmotive force approaches zero, the net proton flux ceases, and light will drive the continued uptake of NaCl instead. Indeed, illumination is seen to cause swelling of the vesicles, particularly when gramici- din is added to increase the electrical potential- driven secondary Na‘ uptake. Halorhodopsin, as bacteriorhodopsin, requires no other component than the opsin, a small (MW above 26,000) integral membrane protein, and the retinal, for the light-driven transport. In both proteins the retinal is attached to a lysine via a protonated Schiff base. The intimate association of the retinal with various amino acid residues in halo-opsin is indicated by the fact that the wavelength maximum of the pigment is shifted from 440 nm, that of a protonated retinal Schiff base in solution, to 578 nm. Thus, halorhodopsin, as bacteriorhodopsin, is a purple protein. Absorp- tion of a photon causes the isomerization of the retinal from all-trans to 13-cis; this initiates a sequence of thermally driven reactions which lead back to the parent pigment in a few tens of milliseconds (the “photocycle”). This character- ization provides the first strong evidence for the existence of a Cl pump mechanism residing in bacteria. The following studies on rat brain motoneurons provided the strongest evidence in vertebrates for the existence of a Cl” pump. Shiroya eft al. [50] demonstrated that EDTA-treated microsomes prepared from rat brain mainly consisted of sealed membrane vesicles 200-500 nm in diameter and were rich in both Cl--ATPase and Na”, K’*- ATPase activities. Such Cl” -ATPase-rich mem- brane vesicles accumulated Cl~ in an ATP- dependent and osmotically reactive manner in the presence of 1 mM ouabain. The Cl” uptake was maximally stimulated by ATP with a K,, value of 1.5 mM; GTP, ITP, and UTP partially stimulated Cl” uptake, but CTP, beta, gamma-methylene ATP, ADP, and AMP did not. The ATP- dependent Cl” uptake was accelerated by an in- crease in the medium Cl~ concentration with a K,, value of 7.4mM. Such stimulation of Cl” uptake by ATP was dependent on the pH of the medium, with an optimal pH of 7.4, and also on the temperature of the medium, with an optimal range of 37-42°C. Ethacrynic acid dose-dependently inhibited the ATP-dependent Cl” uptake with a concentration for half-maximal inhibition at 57 uM. N-ethylmaleimide (0.1 mM) completely inhi- bited and sodium vanadate (1 mM) partially inhi- bited the ATP-dependent Cl” uptake. The mem- brane vesicles did not accumulate H~ in the Cl— uptake assay medium. The ATP-dependent Cl— uptake profile agreed with that of Cl--ATPase activity reported previously [108], and this strongly supports the idea that Cl” -ATPase in the brain actively transports Cl. Gradmann and his colleagues [109] have pro- vided electrophysiological data and ATP synthesis by the Cl” pump through reversal of Cl~ elec- trochemical gradients in Acetabularia which pro- vided strong evidence for the existence of a Cl~ pump in algae. Buttressing these conclusions were those of Ikeda and Oesterhelt [110], who showed a Mg?*-ATPase, isolated from Acetabularia, recon- stituted into liposomes and tested for a Cl - translocating activity. A significant increase in °°Cl~ efflux from the negative and neutral lipo- somes was observed by addition of ATP in the presence of valinomycin after incorporation of the enzyme by short-term dialysis. The ATP-driven °°CI~ efflux was inhibited by addition of azide, an inhibitor of the ATPase. When chloride was replaced by sulfate, no ATP-dependent sulfate efflux was detectable from the proteoliposomes. Proton-translocating activity of the enzyme was also tested, and no fluorescent quenching of 9- ACMA was observed. These observations strong- ly suggested the existence of a Cl” pump in Acetabularia. Graves and Gutknecht [111] have provided evi- dence for an electrogenic Cl” pump with similar properties in the membrane of Halicystis, another marine alga that is related to Acetabularia. For the question of the physiological significance of the electrogenic Cl” pump in Acetabularia, Gradmann [109] favors a “Mitchellian” answer. This primary, electrogenic ion pump would create an electroche- mical driving force to fuel secondary, elec- trophoretic (or electroneutral) transport proces- ses, such as uptake of sugars or amino acids. However, the most rigorous and definitive proof for a Cl” pump’s existence and its mode of opera- Chloride Pumps in Biology 933 tion rests with the following group of experiments by Gerencser and his colleagues [56, 72, 109]. Gerencser and Lee [68, 72] presented evidence which indicated that the BLM of Aplysia foregut absorptive cells contains Cl” -ATPase activity. Biochemical properties of the Aplysia foregut absorptive cells BLM-localized Cl -stimulated ATPase include the following: 1) pH optimum= 7.8; 2) ATP being the most effective nucleotide hydrolyzed; 3) also stimulated by HCO;—, SO3”, and S03”, but inhibited by NO; , and no effect elicited by NO3” and SO,~7, 4) apparent K,, for Cl” =10.3 mM while the apparent K,, for ATP= 2.6 mM; and 5) a requirement for Mg** which has an optimal concentration of 3mM [72]. Coin- cidentally, Cl~ has an intracellular activity approx- imating its apparent K,, for the Mg**-dependent ATPase (vide supra), which supports the notion for its physiological and biochemical activities. Additionally, the ATPase activity stimulated by Cl~ was strongly inhibited by thiocyanate, vana- date, and acetazolamide but not inhibited by oua- bain (Table3). These results with inhibitors strongly suggested a possible participation by the Cl -stimulated ATPase in net chloride absorption by the Aplysia gut [72]. The finding that anion- stimulated ATPase is inhibited by thiocyanate, but not by ouabain has also been demonstrated in many tissues known to perform active anion trans- port and to contain anion-stimulated ATPase activity [112]. This coincidental inverse parallel between ouabain insensitivity and thiocyanate sen- sitivity to Cl” -stimulated ATPase activity and net TABLE 3. active Cl” absorption in the Aplysia gut warranted conjecture that the active Cl” absorptive mechan- ism could be driven by a Cl -stimulated ATPase found in the BLM of the foregut absorptive cell. Additional support for this contention rested with the finding that Cl” -stimulated ATPase activity of the BLM is inhibited by vanadate (Table 3). Vanadate has been shown to inhibit ATPases, which form high-energy phosphorylated inter- mediates while having no effect on the mito- chondrial anion-sensitive ATPase [113]. These results strongly suggested that the Cl -stimulated ATPase is an ion-transporting ATPase of the “P” variety rather than the “Fo—F,” or “V” types. Acetazolamide inhibited Cl -stimulated ATPase activity in the Aplysia gut (Table 3). This finding has also been demonstrated in blue crab gill HCO; -ATPase [90]. Although acetazolamide has been shown to be a specific inhibitor of carbo- nic anhydrase [114], it has also been demonstrated to be a Cl” transport inhibitor [115]. Additionally, it has been shown by Gerencser [116] that carbonic anhydrase does not exist in the BLM of the Aplysia gut absorptive cell. Thus, the data further strengthen the notion that the Cl -stimulated ATPase, which is inhibited by acetazolamide, may be involved in net Cl~ transport across the mollus- can gut. Furthermore, Gerencser and Lee [75] demon- strated an ATP-dependent Cl” uptake in Aplysia inside-out gut absorptive cell BLM vesicles that was inhibitable by thiocyanate, vanadate, and also by acetazolamide. The ATP-driven Cl” uptake Effects of Inhibitors on Cl -ATPase Activity alsibition Concentration Specific Activity % (Gay Me?* +Cl--ATPase Cl--ATPase LQMDLOR Control 16.8+0.3 6.7 0 Thiocyanate 10.1 7.7+0.6 4.5 33 Acetazolamide 1.0 11.1+1.0 1.4 79 Acetazolamide 2.0 VW se 20) 0.4 94 Ouabain 1.0 16.5+0.5 7.0 0 Vanadate 0.5 14.0+1.4 4.4 34 Vanadate 1.0 10.5+1.1 2.5 63 Values are means+SE from 3-5 different experiments. Table from Gerencser and Lee [72] with permission. protein” '. Inhibitors were as described in text. Specific activity is expressed as ~mol-15 min” '-mg 934 G. A. GERENCSER AND B. ZELEZNA TABLE 4. Effect of ATP on Transport Parameters in Basolateral Membrane Vesicles Experimental Condition Cl~ Uptake (nmol/mg protein) Vesicular Membrane Potential Difference (mV) +ATP 102.7+7.9 —ATP A) 7] 3ES)9) +Nonhydrolyzable ATP analog 59.6+8.3 (5’-adenylyl-imidodiphosphate) NO; for Cl” (mole for mole) Values are means + SE; n=number of experiments. was obtained in the absence of Na’, K*, HCO; , or a pH gradient between the intra- and ex- travesicular space, which is strong suggestive evi- dence that the Na*-K*-ATPase enzyme, Na™ / Cl~ symport, K*/Cl~ symport, Na*/K*/Cl7 symport, Cl” /HCO3 ° or Cl /OH antiport and K*t/H* antiport were not mechanisms that are involved in the accumulation of Cl” within the vesicles. To further elucidate the electrogenic nature of the ATP-dependent Cl~ transport porcess, several experimental maneuvers were performed by Gerencser [49] as follows. First, an inwardly directed valinomycin-induced K* diffusion poten- tial, making the BLM inside-out vesicle interior electrically positive, enhanced ATP-driven Cl~ uptake compared with vesicles lacking the ionophore. Second, an inwardly directed FCCP- induced H* electrodiffusion potential, making the BLM inside-out vesicle interior less negative, in- creased ATP-dependent Cl~ uptake compared to control. Third, ATP increased intravesicular nega- tivity measured by lipophilic TPMP™~ distribution across the vesicular membrane (Table 4). Addi- tionally, both ATP and Cl” appeared to be neces- sary for generating the negative intravesicular membrane potential, because substituting a nonhydrolyzable ATP analog for ATP, in the presence of Cl” in the extravesicular medium, did not generate a potential above that of control ({56], and Table 4). Likewise, substituting NO3~ for Cl~ in the extra- and intravesicular media, in the presence of extravesicular ATP, caused no change in potential difference above that of con- trol (Table 4). These results also suggested that hydrolysis of ATP is necessary for the accumula- 3 = 34! Q2e 2.5 12 3 0.0+5.2 12 3 = 1.3)25(0).) 12 +3.0+4.6 3 Table is taken from Gerencser, et al. [109] with permission. tion of Cl” in the vesicles. Furthermore, vana- date, acetazolamide, and thiocyanate inhibited the (ATP+Cl~)-dependent intravesicular negativity [109]; and in addition, it had been demonstrated that the pH optimum of the Cl -stimulated ATPase [72] coincided exactly with the pH opti- mum of 7.8 of the ATP-dependent Cl” transport in the same fraction of Aplysia foregut absorptive cell BLM vesicles [49]. Therefore, both aspects of the Cl” pump (ATPase and ATP-dependent Cl— transport) have the same pH optimum, which suggests that these properties are part of the same molecular mechanism. SULFHYDRYL LIGANDS OF Cl” PUMP It appears that the catalytic, Cl -stimulated ATPase activity, and its corollary transport com- ponents, ATP-dependent Cl~ transport and ATP- detendent Ay in the BLM of Aplysia foregut absorptive cells are dependent on intact sulfhydryl ligands [117, 118]. P-chloromercuribenzensul- fonate (PCMBS) forms a mercaptide complex with sulfhydryl ligands of the Cl” pump which inhibit Cl" -stimulated ATPase activity [117], ATP- dependent Cl” accumulation and ATP-dependent 4y in BLM vesicles [118]. These catalytic and transport inhibitions of Cl” pump activity are totally reversed by dithiothreitol (DTT), which is a specific thiol reducing agent [119]. This result provides strong evidence that the ligands involved in both hydrolysis of ATP and accumulative Cl— transport are sulfhydryl and not carboxyl, phos- phoryl, tyrosyl or amino [119]. In addition, it appears that the sulfhydryl ligands of the Cl ~ pump that are responsible for its catalytic and Chloride Pumps in Biology 935 transport activities are located on the cytoplasmic surface of the BLM of Aplysia gut absorptive cells, for PCMBS has been shown to have a very low lipid solubility [119], and this restricts its action to surface and not intramembranous sulfhydryl ligands. RECONSTITUTION OF THE Cl” PUMP Reconstitution of a membrane protein into a liposome provides one of the few methods needed to rigorously demonstrate the existence of a sepa- rate and distinct biochemical and physiological molecular entity. This method also provides evi- dence that all components of the solubilized pro- tein haye been extracted intact. With this premise in mind, Gerencser [51] reconstituted both aspects of the Cl~ pump; that is, the catalytic (ATPase) and transport components from the BLM of Aply- TABLE 5. (A) Proteoliposome ATPase Activity sia gut absorptive cells, as shown in Table 5. Table 5 shows Cl -stimulated ATPase activity exists significantly (P<0.05) above Mg’*-stimulated ATPase activity found in the proteoliposome population extracted and generated with digitonin. Vanadate (0.1 mM) inhibited this Cl -stimulated ATPase activity by 99%. From this digitonin- generated proteoliposome population, it is also seen in Table 5 that there is a significant ATP- dependent Cl~ uptake into these proteoliposomes above that of control (P<0.05), and that this ATP-dependent Cl~ uptake is also inhibited by 0.1 mM vanadate. Not detected in the proteolipo- somes solubilized and formed by digitonin were Na‘ /K+t-ATPase, alkaline phosphatase, (Ca** + Mg?*)-ATPase, or cytochrome c oxidase activities and, coupled with a previous observation that FCCP had a stimulatory and not an inhibitory effect on ATP-driven Cl” accumulation in the Reconstitution of Cl” Catalytic and Transport Activities Extractive and Mg?t- (Moat Glp)s wanl(Mezn Gln) reconsitutive ATPase ATPase ATPase detergents + vanadate Cholate n.d. n.d. — Octyl glucoside n.d. n.d. — Lubrol PX n.d. n.d. _ Digitonin 2.8+0.4 11.2+2.0 2.9+0.4 (B) Cl Uptake Into Proteoliposomes Extractive and —ATP + ATP +ATP reconstitutive + vanadate detergents Cholate 87.5+5.6 82.7+6.9 80.6+8.3 Octyl glucoside 82.7+8.0 73.6+9.2 SO) SE(}.0) Lubrol PX 28.3+11.1 39.Q3E 13},9) 39.3+ 14.0 Digitonin 91.2+6.0 192.5+9.3 93.1+7.9 Values are means+SE from four individual determinations. Conditions for the enzyme assay are described in Materials Time period of assay for V; determined previously. Vanadate (0.1 mM) had no significant effect Either vanadate (0.1mM) was preincubated with the proteoliposomes in the mg protein for Mg**- and (Mg** +Cl_)-ATPase. and Methods. on Mg**-ATPase activity. Enzyme activity is expressed as ~mol/15 min per reaction mixture (50 1 containing 10 mM imidazole-HCl, 250 mM sucrose, 3 mM MgSO,, and 25 mM choline chloride) at pH 7.8 for 10 min at 25°C, or 5mM ATP was added to the reaction mixture to initiate the incubation for the transport experiments. The incubation for the uptake of *°Cl~ was measured for 10 min at 25°C. Time for steady-state values for both ATP-independent and ATP-dependent *°Cl~ uptakes was based on previous observations. *°Cl~ uptake is expressed as nmol/mg protein. reaction mixture; — represents its absence; n.d., not detectable. permission. + Represents a compound’s presence in the Table is taken from Gerencser [51] with 936 G. A. GERENCSER AND B. ZELEZNA BLM vesicles, it is suggested that none of these enzymes nor eukaryotic vacuolar H‘-ATPases could express Cl” pump activity. These data also suggested that these two major observations are manifestations of one molecular mechanism: the Cl~ pump. Support of this contention rested with the findings that vanadate (an inhibitor of P-type ATPases) inhibited both Cl” -stimulated ATPase activity and ATP-dependent Cl” transport in the digitonin-based proteoliposomes (Table 5). Even though Krogh [120] first coined the term “Cl— Pump,” it was not until the reconstitution of all of its components into an artificial liposomal system through the study mentioned above [51] that the existence of this mechanism (primary active trans- port mechanism) was rigorously proven. MOLECULAR WEIGHT OF THE Cl" PUMP Utilizing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) techniques to digitonin-generated proteoliposomes containing the Cl” pump components from Aplysia gut absorptive cells as shown previously [51], the approximate molecular weight of the Cl” pump was ascertained. Since both aspects of the Cl~ pump were inhibited by vanadate (Table 5), it was surmised that the approximate molecular weight of the Cl” pump of Aplysia gut BLM should be approximately 100 K daltons since vanadate only inhibited “P” type ATPases and not “FoF,” or “V” type ATPases [121]. The alpha-subunit of all “P” type ATPases approximates 100K daltons in molecular weight [121]. Two protein bands were eluted through SDS-PAGE, one of which was a major band at 116 K daltons and the other being a minor band at 97.5 K daltons. These two protein bands were obtained whether 5-mercaptoethanol was present or not in the buffer medium. This finding indicates that the Cl” pump of Aplysia is not dependent upon the integrity of subunit- linking sulfhydryl ligands. Also, these purified proteins of the Cl” pump have been subjected to phosphorylation within the proteoliposome and the reaction sequence and kinetics of the reaction sequence of the enzyme have been determined: Mg?* causing phosphorylation, Cl~ causing de- phosphorylation, and all in a time frame consistent with an acyl phosphate linkage. Hydroxylamine and high pH destablize this phosphorylation. Orthovanadate (10~’M) almost completely in- hibit the Mg**-driven phosphorylation reaction. CONCLUSIONS In summary, it is quite apparent that in the past few years there has been an increasing number of convincing studies in a variety of animal tissues that have provided indirect, correlative evidence that active Cl” transport is primary by nature. The active translocation of Cl” by an enzyme that directly utilizes the energy from ATP hydrolysis is not unlike that observed in plants [122, 123]. Indeed the evidence for primary active Cl” trans- port in these simple living things is almost as convincing as that presented for (Na* +K7~)- stimulated ATPase and (Ca** +Mg?*)-stimulated ATPase in their respective roles for actively trans- ferring Na*, K*, and Ca?* across animal plasma membranes. As alluded to by DePont and Bonting [53], future experimental steps in assuring that an animal Cl -stimulated ATPase is involved in pri- mary Cl transmembrane movement should approximate the following: 1) a specific inhibitor for the enzyme should be found or synthesized (e.g., an antibody), and this inhibtor should be shown to inhibit the transport process; and/or 2) the Cl -stimulated ATPase should be biochemi- cally isolated or purified and after its incorporation in liposomes should then be shown to support active Cl” transport. This demonstration of re- constitution has been shown in the present review which provided the first direct evidece for the existence of a new “P” type ATPase: the Cl~ pump. . ACKNOWLEDGMENTS The authors wish to acknowledge their gratitude to their technologists, students, and collaborators for their able contributions to the studies reviewed and performed herein. These studies were supported by NIAMD Train- ing Grant (1-TO1-AM-05697-02 and -03), NIH Grants (No. AM17361 and RCDA No. AM00367), D.S.R. Seed Award (NO. 229K15), Whitehall Foundation Grant (No. 78-156 ck-1), D.S.R. Award (No. 122101010), and The Eppley Foundation for Research, Inc. 10 11 20 21 22 2B 24 25 26 Chloride Pumps in Biology REFERENCES DuBois-Reymond, E. (1848) Berlin. Galeotti, G. (1904) Z. Physik. Chem., 49: 542- 562. Ussing, H. H. and Zerahn, K. (1951) Acta Physiol. Seand., 23: 110-127. Leaf, A. (1965) Ergeb. Physiol. Chem. Exp. Pharmakol., 56: 216-263. Skou, J. C. (1965) Physiol. Rev., 45: 596-617. Frizzell, R. A., Field, and Schultz, S. G. (1979) Am. J. Physiol., 236: F1-F8. Gerencser, G. A. (1983) Am. J. Physiol., 244: R127-R129. Ussing, H. H. (1960) Springer-Verlag, Berlin. Ahearn, G. A., Maginnis, L. A., Song, Y. K. and Tornguist, A. (1977) In “Water Relations in Mem- brane Transport in Plants and Animals.” Ed. by A. M. Jungreis, T. K. Hodges, A. Kleinzeller and S. 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Physiol., 259: Rh a? \ he 7 NH a | i ie At a ot a mae Re Salt vt ewuid PMP (aks ,Aae im » 2 ators hi es, en ¢ +P PA Nees Sar) — vw To 1 pte 7TH ee as de a Tn ti Apt A AT ea i sv) mt Avira if A CAT rau? - ‘pies 7 { ‘ns ! ye a { ; x ee ii. t 4 ; rt t ’ j doy sage pith: jena ey 0s) uC oi beg et Coe SPL, (OED (80 ck DS tue sv AA, Piawnl it taty easily ety ht Riba" cinerea ate tart Sree Ls Debi etetind LEY ie ; “Na ; i al Lae brtig, fel Vaal eee MOS areca A cee t Bitsy A ul wii Bae avhocktt (ote | ys ae iu Mi wy Li ‘ “spoknertot. 5H | a tiett a Qe a (Or send slept ROTI Oe ee he iy aj gv , hi! Cae ke . Meier r., (2) dhorteeneQ> foo M1 ee 0) ee a velenomehe dott | UOC okt j we." BRON wwe (OO) Mol Sere ‘ “ETeY ae F wotrehetes tr, CGA cae ae MAgwaer Lee BU EET a , 7 i 2) Dern butte Cae eR ALE | ote A) Og a ’ 1) Siam ; brrkotcatnes (iA ay Unit dp t ZOOLOGICAL SCIENCE 9: 941-946 (1992) © 1992 Zoological Society of Japan HPLC Analysis of Retinoids Extracted from the Planarian, Dugesia japonica Katsu Azuma!, Naouiko Iwasakt!, MASAMI AZUMA2, TAKAO SHINOZAWA°® and Tatsuo Suzuki‘ 'Department of Biology, Osaka Medical College, Takatsuki, Osaka 569, "Department of Health Science, Osaka Kyoiku University, Osaka 547, 3Department of Biologyical and Chemical Engineering, Faculty of Engineering, University of Gunma, Kiryu, Gunma 376, and “Department of Pharmacology., Hyogo College of Medicine, Nishinomiya, Hyogo 663, Japan ABSTRACT—Retinoids extracted from the planarian, Dugesia japonica were analyzed by high- pressure liquid chromatography (HPLC). All-trans retinal, all-trans retinol, and all-trans retinyl ester were detected in the extracts from the head and tail pieces of the worm, while 11-cis retinal was detected in the extracts from the head pieces. The amounts of all-trans retinal, 11-cis retinal and all-trans retinol including the retinyl ester were 0.1-1.1, 0.11-0.19, and 20-50 pmol/head, respectively. The planarian contained many oil-droplets which emitted the green-yellow fluorescence probably derived from retinol and retinyl ester. These results suggest that the planarian contains all-trans retinol and the retinyl ester in oil-droplets and 11-cis retinal as the chromophore of the visual pigment in the eye. INTRODUCTION The planarian is one of the lowest metazoans. The eye of the planarian consists of pigmented cells and photoreceptors of a microvillar type [1]. Extracellular microelectrode recordings from the eye of the planarian Dugesia tigrina suggested the presence of a rhodopsin-like photopigment whose absorption maximum was at about 508 nm [2]. Spectral phototaxis experiments showed the sensi- tivity maximum of the planarian eye (Planaria lugubris) at about 475nm [3] and 530nm for Dendrocoelum lacteum [3]. The differences in those maxima may have been caused by a con- tribution from the dermal photoreceptors [2], an effect of screening pigments [2] or, perhaps, simply a species difference. At present, the visual pig- ment of the planarian has hardly been investigated by spectrophotometric methods, because the worm contains so little visual pigment. In histo- Accepted June 8, 1992 Received April 6, 1992 chemical experiments, it has been reported that the photopigment of the planaria (Dugesia japoni- ca) is a chromoprotein which possesses retinal- dehyde as the chromophore [4]. Recently an immunochemical study suggested the presence of rhodopsin-like protein in the head of the planarian Dugesia japonica by use of anti-frog-rhodopsin rabbit IgG [5]. Although 11-cis-retinal is the most ubiquitous as the chromophore in the vertebrate and inverte- brate rhodopsin [6], a variation in the chromo- phore of visual pigment is found in other spe- cies: 11-cis 3-dehydroretinal is found in many fresh water vertebrates [7, 8] and invertebrates [9, 10]; 11-cis 3-hydroxyretinal is found in the insects [11]; and 11-cis 4-hydroxyretinal is in a bioluminescent squid [9]. In addition all-trans retinal and 13-cis retinal are seen in Halobacterium halobium [12]. It is unknown whether or not the chromophore of the visual pigment of the planarian is 11-cis retinal. The purpose of this study is to estimate the con- figuration of the chromophore of the planarian visual pigment by high-pressure liquid chroma- 942 K. Azuma, N. IwasAkI et al. tography (HPLC) analysis of retinoids extracted from the worms. Our results indicate that 11-cis retinal is one of the most plausible candidates for the chromophore. MATERIALS AND METHODS Materials The planarian worms, Dugesia japonica were collected from streams in the suburbs of Kyoto city (Kyoto prefecture, Japan) and Kiryu city (Gunma prefecture, Japan). Kyoto worms were maintained by feeding on fresh beef livers, and used for extractions of retinoids from their whole bodies or both the head pieces (anterior part containing the eyes) and the tail pieces (the tissues without the head). Head pieces of kiryu worms were stored as frozen materials and used for extraction of re- tinoids. Extractions of retinoids Usually, retinoids were extracted from the fresh or frozen head pieces by the oxime method which was developed to extract the retinal from biologic- al materials as retinaloximes (syn- and anti-forms) in the original isomeric configuration without ther- mal isomerization [13, 14]. The planarian samples were homogenized in a solution of 100mM NH,OH (pH 7.2) and methanol (final concentra- tion of methanol was 60-70%) using a homogeniz- er (Physcotron NS-50, Nichion Irikakikai Seisa- kusho Co. Ltd., Japan). The homogenate was mixed with dichloromethane and n-hexane (1:2, vol/vol), shaken vigorously and centrifuged at 2,500r.p.m. for 15min. The upper layer (dichloromethane/hexane layer) was collected. This extraction was repeated three times. The collected solution was stored as extracts of re- tinoids. In a few cases, the planarian samples were freeze-dried for hexane extraction of retinoids. The freeze-dried samples were shaken vigorously in hexane solvent and centrifuged at 2,500 r.p.m for 15min. The supernatant was collected and hexane extraction was repeated 3 times. The collected solution contained retinol and retinyl ester. Precipitates were gently aspirated to evapo- rate hexane solvent and served for the extraction of retinoids by the oxime method as described above. The obtained solution (dichloromethane / hexane extract) contained retinaloxime and retinyl ester. After evaporating solvents of the extracts mentioned above, the residues were dissolved in 50 ul of hexane/diethylether/ethanol (90/10/0.1, vol/vol) and analyzed by HPLC. All procedures were carried out under dim red light. Detection of retinoids in the samples Extracts of retinoids from the planarian samples were analyzed by the HPLC method as reported previously [15]. An HPLC system equipped with a 4.6250 mm column of YMC-Pack A-003-3 SIL (Yamamura Chemical Labo. Co. Ltd., Japan) and a pump (TRI ROTER, JASCO, Japan) was used. The eluent was a mixture of n-hexane, diethylether and ethanol (90:10:0.1, vol/vol) and was used at the flow rate of 1.3 ml/min for 50min. The absorbances of the fractions at 350 nm and at 280- 500 nm were measured with a detector UVDEC- 100-III (JASCO, Japan), and with a multi- wavelength detector MULTI-340 (JASCO, Japan), respectively. The measurement with MULTI-340 was carried out in order to obtain the absorption spectra of the fractions over the wide range of wavelengths, although the sensitivity of the detector was less than that of the UVIDEC- 100-III detector. Quantities of several retinoids were estimated from their abosrption coefficients and the peak areas of known amount of standard retinoids. The fractions of 2-8 min (retention time) under our HPLC conditions were used as the sample of retinylester. The solvent was evaporated from the fractions and the residue was incubated in 6% KOH-methanol solution at 25°C for 1 hr for sapo- nification. The amount of retinyl ester was calcu- lated from that of retinol produced by the sapo- nification. Observation of fluorescent images of oil-droplets in planarian tissues The planarian worm of 5 mm in length was put on non-fluorescent slide glass, covered with a thin coverslip and spread by the squash method. The fluorescent images of the oil-droplets in the spread HPLC Analysis of Planarian Retinoids 943 a b A350 gai @ Cc oOo Qa (= fo} uw OQ — <0) > 5 oO 1 2 a oO 10 min Fic. 1. 300 340 380 420 Wavelength (nm) (a) HPLC profile of retinoids extracted from 18 bodies of planarian worms. (b) Absorption spectra of peak fractions indicated by numbers 1 and 2. The worms were dark-adapted overnight in aged tap water at 20°C. Extractions were carried out by the oxime methods. specimen were observed using a flouorescence microscope (Olympus inverted-microscope, IMT- 2, equipped with Olympus incident-illumination type fluorescence apparatus, IMT2-RFL). The Specimen was excited by light (50 W halogen lamp) passing through an excitation-filter (UG-1) and observed through a filter (L420) and a dichroic mirror (DM 400). The photograph of fluorescent images was taken using the color film (Fujichrome DX 400D). RESULTS Retinoids detected in the planarian Figure la shows an HPLC profile of the extract of retinoids from the whole bodies of 18 planarian worms of about 10 mm in length. This figure was obtained by recording the absorbaces of the frac- tions at 350 nm with the UVIDEC-100-III detec- tor. Numbers 1 and 2 indicate peaks close to the retention time of standard syn all-trans retinalox- ime and all-trans-retinol, respectively. Two big peaks between 1 and 2 are not identified. The relative absorption spectra (300-420 nm) of the fractions corresponding to peaks 1 and 2 were obtained by the MULTI-340 detector, indicated as curves 1 and 2 in Fig. 1b, respectively. The absorption maxima of curves 1 and 2 clearly match those of the standard syn all-trans retinaloxime (Amax=358 nm) and all-trans retinol (Amax=325 Fic. 2. planarian body. The specimen was obtained by the squash methods. Bar=50 um. Fluorescent micrograph of oil-droplets in the nm), respectively. The fractions between 2 and 8 min, which contain retinyl esters, were collected and saponified as described in Materials and Methods. Then the material obtained after the saponification was analyzed by HPLC. From these analyses, the amounts of all-trans retinal, all-trans retinol and all-trans retinyl ester were calculated as 2.5, 14.7 and 99.4 pmol/body, respectively. Figure 2 shows fluorescent images of several 944 K. Azuma, N. Iwasaki et al. oil-droplets in the planarian specimens obtained by the suqash method. The light color of fluoresence was green-yellow suggesting the presence of re- tinol and/or retinyl ester. Probably, the planarian worms store the all-trans retinyl ester in oil- droplets, because the worms large amounts of the retinyl esters, as mentioned above (more than 85 mol% of total retinoids). Figure 3 shows HPLC profiles of extracts from the head (a) and the tail (b) pieces of 28 planar- ians. Peaks numbered 1 and 2 are corresponding to syn all-trans retinaloxime and all-trans retinol, respectively, as estimated from their retention times and abosorption spectra (data not shown). Thus all-trans retinal and all-trans retinol were found in the head and tail pieces of the planarian. contain D 2 A350 q 2-004 a con 10 min 10 min Fic. 3. HPLC profile of retinoids extracted from both the head (a) and tail (b) pieces of 28 planarian bodies. The worms were dark-adapted overnight in aged tap water at 20°C, separated into head and tail pieces under dim red light and were extracted by the oxime methods. Retinals in the planarian head pieces Figure 4a shows an HPLC profile of the extract of retinoids from head pieces. The head pieces were cut off from the 600 bodies of the planarian under room light, then dark-adapted overnight in aged tap water at about 20°C and stored at —20°C until use. The chromatogram was obtained by recording the absorbances of the fractions at 350 nm with the MULTI-340. Retention times of a 0 10 20 30 40 50 Time (min) Relative Absorbance 300 340 380 420 Wavelength (nm) Fic. 4. (a) HPLC chromatogram of retinoids extracted from head pieces of the planarian worms. (b) Absorption spectra of peak fractions indicated by numbers 1, 2 and 3. The planarian head pieces were separated from 600 bopdies of the planarian worms under room light, then dark-adapted overnight in aged tap water at 20°C and stored at —20°C until use. Extractions were carried out by the oxime methods. peaks 1, 2 and 3 are close to those of standard syn 11-cis retinaloxime, syn all-trans retinaloxime and all-trans retinol, respectively. The relative absorp- tion spectra of fractions corresponding to peaks 1, 2 and 3 are represented as curves 1, 2 and 3 in Figure 4b respectively. Curves 2 and 3 are due to the absorption spectra of syn all-trans retinaloxime and all-trans retinol, respectively, as indicated in Figure 1. Curve 1 seems to be corresponding to the absorption spectrum of syn 11-cis retinaloxime, because the shape of curve 1 is different from that of surve 2 due to syn all-trans retinaloxime. The amounts of all-trans retinal, 11-cis retinal and all- trans retinol including the retinyl ester in different preparations were ().1-1.1, 0.11-0.19 and 20-50 pmol/head, respectively. In order to elucidate whether or not 11-cis and HPLC Analysis of Planarian Retinoids 945 D 3 350 0.004 2 10 min Relative Absorbance oO 300 340 380 420 Wavelength (nm) Fic.5. (a) HPLC chromatogram of hexane extract from freeze-dried sample of the planarian heads prepared as mentioned in Fig. 4. (b) HPLC chro- matogram of retinoids extracted by the oxime method from the residues after the hexane extrac- tion. (c) Absorption spectra of peak fractions indicated by numbers 2 and 3 (solid lines) and standard syn 11-cis and syn all-trans retinaloximes (dotted lines). all-trans retinals were bound to any protein in the planarian tissues, we carried out an experiment as follows. The hexane extract from the freeze-dried samples of planarian heads was analyzed by HPLC. As shown in Figure 5a, the peak of all- trans retinol (peak 1) was quite large, while the peaks due to 11-cis and all-trans retinals were not found. Figure 5b is an HPLC profile of retinoids extracted from the residues by the oxime methods after the hexane extraction. The figure indicates substantial peaks, numbered 2 and 3, correspond- ing to syn 11-cis retinaloxime and syn alll-trans retinaloxime, respectively. Figure 5c indicates the relative absorption spectra of fractions corres- ponding to peaks 2 and 3 as curves 2 and 3. These spectra are very similar to those of standard syn 11-cis retinaloxime and syn all-trans retinaloxime (indicated by dotted lines), respectively. The early fractions (2-8 min) in Figure 5a have peaks which are much larger than those in Figure 5b, indicating that retinyl esters were mostly extracted by the hexane extraction. Thus hexane extracted almost all of the all-trans retinol along with retinyl esters in the planarian tissues leaving 11-cis and all-trans retinals. DISCUSSION As shown in Figure 1, retinoids extracted from the homogenates of 18 bodies of the planarian were composed of all-trans retinal (2.1 mol%), all-trans retinol (12.6 mol%) and all-trans retinyl ester (85.2 mol%). Retinyl ester is probably a main storage form of retinoids in the planarian body and seems to exist in the oil-droplets, which emit the green-yellow fluorescence as seen in Figure 2. It has long been known that vitamin A is stored mainly as retinyl ester in the livers of a numbers of vertebrate species and that these re- tinyl esters are present in oil-droplets of the liver fat-storing cells [16]. The planarian has oil- droplets in the fixed parenchymal cells [17]. Prob- ably the planarian is capable of storing retinol in ester form in the parenchymal cells. The hexane extract from the freeze-dried planar- ian heads contained almost all of the all-trans retinol and the retinyl ester in the tissues. How- ever, all-trans and 11-cis retinals could not be detected in the extract. Both of the retinals were extracted from the residues, after the hexane ex- traction, as the oximes. It is well known that retinals combined with amino group of a protein in tissues (e.g. vertebrate and invertebrate retinas) are not extracted by hexane. Therefore the retin- als in the planarian may be bound to an uniden- tified proteins in the tissues. All-trans retinal was detected in extracts of both head and tail of 28 planarians (see Fig. 3), while 11-cis retinal was detected in extracts derived from 600 planarian head pieces (see Fig. 4 and Fig. 5). It is reasonable to infer that the 11-cis retinal was derived from the chromophore of visual pigment of the planarian photoreceptor. The eye of the planarian used in this experiment is assumed to be a sphere of about 90 4m in diameter. The rhodop- sin concentration of invertebrate photoreceptors is 946 0.3-0.4 mM [18]. We can estimate the amount of the chromophore per planarian eye, if the eye is assumed to be filled with microvilli which contain rhodopsin in the concentrations of 0.3-0.4 mM. The calculated value of the amount of chromo- phore was 0.11-0.15 pmol per eye, which was close to the amount of 11-cis retinal indicated in this experiment i.e., 0.11-0.19 pmol per head. ACKNOWLEDGMENTS The authors thank professor Dr. T. P. Williams (Flor- ida State University) for critical reading of the manu- script and Messrs. H. Fujino, T. Tanaka, A. Yuda and M. Miyazaki for collections of the planarian worms. REFERENCES 1 Tamamaki, N. (1990) Evidence for the phagocytotic removal of photoreceptive membrane by pigment cells in the eye of the planarian, Dugesia japonica. Zool. Sci., 7: 385-393. 2 Brown, H. M. and Ogden, T. E. (1968) The electrical response of the planarian ocellus. J. gen. Physiol., 51: 237-253. 3 Menzel, R. (1979) Spectral sensitivity and colour vision in invertebrate. In “Handbook of sensory physiology”. Ed. by H. Autrum, Springer-Verlag, Berlin Heidelberg New York, VII/6A, pp. 503-580. 4 Ozaki, K., Hara, R. and Hara, T. (1983) Histoche- mical localization of retinochrome and rhodopsin studied by fluorescence microscopy. Cell Tissue Res., 233: 335-345. 5 Fujita, J., Sakurai, N. and Shinozawa, T. (1991) Presence of rhodopsin-like proteins in the planarian head. Hydrobiologia, 227: 93-94. 6 Knowles, A. and Dartnall, H. J. A. (1977) Habitat, habit and visual pigments. In “The Eye, 2B”. Ed. by H. Davson, Academic Press, New York, pp. 581- 648. 7 Wald, G. (1941) The visual system of euryhaline 10 11 12 15 16 K. Azuma, N. Iwasaki et al. fishes. J. gen. Physiol., 25: 235-245. Wald, G. (1957) The metamorphosis of visual systems in the sea lamprey. J. gen. Physiol., 40: 901-914. Matsui, S., Seidou, M., Uchiyama, I., Sekiya, N., Hiraki, K., Yoshihara, K. and Kito, Y. (1988) 4-Hydroxyretinal, a new visual pigment chro- mophore found in the bioluminescence squid, Watasenia scintillans. Biochim. Biophys. Acta, 966: 370-374. Suzuki, T. and Eguchi, E. (1987) A survey of 3-dehydroretinal as a visual pigment chromophore in various species of crayfish and other freshwater crustaceans. Experientia, 43: 1111-1113. Vogt, K. and Kirschfeld, K. (1984) Chemical identi- ty of the chromophores of fly visual pigment. Natur- wiss., 71: 211-213. Spudich, J. L. and Bogomolni, R. L. (1988) Sensory thodopsins of Halobacteria. Ann. Rev. Biophys. Chem., 17: 193-215. Groenendijk, G. W. T., De Grip, W. J. and Dae- men, F. J. M. (1980) Quantitative determination of retinals with complete retention of their geometric configuration. Biochim. Biophys. Acta, 617: 430- 438. Suzuki, T. and Makino-Tasaka, M. (1983) Analysis of retinal and 3-dehydroretinal in the retina by high-ressure liquid chromatography. Anal. Biochem., 129: 111-119. Azuma, M. and Azuma, K. (1988) Retinoid changes in the in vitro regeneration of frog visual pigments. J. exp. Biol., 135: 317-327. Goodman, De. S. and Williams, S. B. (1984) Biosynthesis, absorption, and hepatic metabolism of retinol. In “The retinoids”. Ed. by M. B. Sporn, A. B. Roberts and D. S. Goodman, Academic press, London, Vol. 2, pp. 2-34. Ishida, S. (1987) “Biology of planarians.” Ed. by W. Teshirogi, Kyouritu press, Tokyo, pp. 36-46. Liebman, P. A., (1972) Microspectrophotometry of photoreceptors. In “Handbook of Sensory physiolo- gy”. Ed. by H. J. A. Dartnall, Springer-Verlag, Berlin Heidelberg New York, VII/1, pp. 479-528. ZOOLOGICAL SCIENCE 9: 947-954 (1992) Effect of Disuse on Muscle Energy Metabolism Studied by in vivo 31-Phosphorus Magnetic Resonance Spectroscopy SHoy] FUKUTA, TAKAAKI IkATA and Iwao Miura! Department of Orthopaedic Surgery, School of Medicine, the University of Tokushima, 3-18-15, Kuramoto-cho, Tokushima city, Tokushima 770, Japan ABSTRACT— Chronic unloading induces disuse atrophy in rat hindlimb muscles. The purpose of this study was to examine the effect of hindlimb suspension on energy metabolism in hindlimb muscles during tetanic contraction using 31-phosphorus magnetic resonance spectroscopy (*1P-MRS). Muscular contraction was induced by electrical stimulation of the sciatic nerve at 40 Hz. Energy metabolism was evaluated by measuring the relative intensities of phosphocreatine (PCr), inorganic phosphate (Pi), B-ATP and phosphomonoester (PME). The intracellular pH was also estimated from the chemical shift of Pi. Two weeks of hindlimb suspension induced a greater weight loss in the soleus compared with the gastrocnemius muscle. The maximum tension at 40 Hz was significantly lower (P< 0.05) in the hindlimb suspended group than in the control group. However, fatigability did not differ significantly. The energy level at rest was similar in the two groups. During electrical stimulation, the Pi/(Pi+ PCr) ratio was higher in the hindlimb suspended group compared with the control group significantly (P<0.01), but the intracellular pH did not differ. These results suggest that the metabolic capacity of hindlimb muscle decreases after a prolonged period of disuse. © 1992 Zoological Society of Japan INTRODUCTION Disuse atrophy of skeletal muscle has been studied in several experimental models, including small cage restraint [1], spinal cord transection [2], denervation [3, 4], tenotomy [3, 5], cast immobi- lization [6, 7], joint fixation [8] or paralysis with tetrodotoxin [9]. In 1979, the hindlimb suspension model was developed by Morey to simulate the influence of weightlessness [10]. With the hind- limb suspension model, in contrast to previous models, the hindlimbs are made completely non- weight bearing and the animal is able to both contract and relax the hindlimb muscle freely. Numerous investigators have begun to use this model to examine the effect of unloading on skeletal muscle and study the mechanism by which disuse atrophy is undergone. The changes which occur in the suspended hind- Accepted July 22, 1992 Received March 19, 1992 ' Bioenergetics Res. Center, Tokushima Inst., Otsuka Pharmaceutical Co., Ltd. 463-10, Kagasuno, Kawau- chi-cho, Tokushima city, Tokushima 771-01, Japan limb have been studied histochemically. The per- centage of slow-twitch fibers decreases in the soleus after the hindlimb is suspended [11, 12], while no such changes was observed in the gastroc- nemius, tibialis anterior [13], and extensor digitor- um longus muscles [12]. Significant changes in contractile properties, therefore, may be due to changes in the fiber composition of an unloaded muscle. Histochemical and biochemical enzyme assays have shown that both anaerobic and aerobic capac- ity increase in slow-twitch muscle [11-13] while the aerobic capacity in fast-twitch muscles decreases during hindlimb suspension [13]. These studies were based on measurements made in vitro of isolated muscles. However, it is best to measure the enzymatic adaptation which affects muscle energy metabolism during contraction in vivo. 31-phosphorus magnetic resonance spectroscopy is being used to study the energy metabolism and mitochondrial function of skeletal muscle [14, 15]. This technique permits non-invasive and repetitive measurement of high energy phosphate, inorganic phosphate, and sugar phosphate level, as well as 948 S. Fuxata, T. IkATA AND I. Miura estimation of the intracellular pH. The purpose of this study was to examine the effect of prolonged muscle unloading on energy metabolism in vivo during muscular contraction. MATERIALS AND METHODS Animal care Male Wistar Kyoto rats (20-24 weeks) were used in this study. Temperature (25°C) and light-dark cycles (12:12 h) were main- tained throughout the experimental period. Ex- perimental rats were divided randomly into the control and hindlimb suspended (HS) groups. Hindlimb suspension procedure The suspen- sion method used in this study was a modification of Morey’s tail-suspension model [10]. After the rat was anesthetized with pentobarbital sodium (50 mg/kg body weight), 1.2 mm diameter holes were drilled carefully through the third and fourth tail bones without disturbing blood flow to the tail. A stainless steel wire was inserted and attached to the top of the cage by a ball-bearing wheel swivel which allows 360° rotation. The suspension height was adjusted to prevent the hindlimbs from con- tacting the floor or the sides of the cage while the forelimbs were allowed to support their weight and to obtain food and water ad libitum. Experimental preparation and muscle stimulation After 2 weeks of suspension, the rat was anesthetized with pentobarbital sodium (50 mg/kg body weight). The right sciatic nerve was exposed in the gluteal region and placed in contact with a small bipolar electrode. The right hindlimb was placed in a 2cm diameter solenoid coil and was fixed on a small platform at both the knee and ankle with full extended position. The distal tendon of the gastrocmenius-plantaris-soleus mus- cle group was exposed at the ankle, cut, and attached to a strain gauge (TB611, NIHON KOHDEN) with a noncompliant thread, the out- put of which was displayed on a Polygraphy system (RM-6000, NIHON KOHDEN). The rat was inserted in the magnet. The hindlimb muscles were stimulated electrically (SEN-3301, NIHON KOHDEN) through an electrode with a 0.5 ms square wave pulse at 40 Hz for 20 min. Stimula- tion was performed with intermittent trains of 1 sec in duration, repeated every 2 sec. Stimulation voltage was 4 V which was supramaximal in both the control and HS groups. 31-phosphorus magnetic resonance spectroscopy (“P-MRS) _*'P-MRS spectra were recorded with a BEM 250/80 spectrometer (OTSUKA ELECTRONICS U.S.A. INC.) operating at 32.3 MHz. The spectrometer was operated in the Fourier transform mode with a pulse duration of 15 ws (90°) and a pulse interval of 2sec. Each spectrum was averaged over 2 min, which was from 60 free induction decays (FIDs). The phos- phocreatine peak (PCr) was assigned as a chemical shift of zero. The levels of tissue PCr, inorganic phosphate (Pi), @-ATP and phosphomonoester (PME) were estimated from the areas under indi- vidual peaks. The cellular energy level was evalu- ated by the Pi/(Pi+PCr) ratio and the relative concentration of Pi, PCr, ATP, and PME were calculated as fractions of the total 7'P-MRS signal. Pulse interval of 2 sec reduced the PCr and Pi amplitudes to about 60% of their respective maxi- mum. These saturation factor were incorporated into the calculation of relative concentration. The intracellular pH was estimated from the chemical shift of Pi relative to the PCr peak (6) and calcu- lated as follows [16]: Intracellular pH=6.90— |(6—6.81)/(3.29—6)| Measurement of muscle weight At the end of the experiment the rat was killed with an overdose of pentobarbital sodium. The hindlimb muscles were removed, cleaned of connective tissue, and weighed. Weights were expressed in absolute (mg) or relative (mg/g body weight). Data analysis +S.E. Differences were tested for significance by Student’s f-test. Values were expressed as mean RESULTS The absolute and relative muscle weights from the control and the HS groups are shown in Table 1. They were significantly lower in the HS group than in the control group. The muscle mass loss *'P-MRS Study of Disuse Muscle Atrophy 949 TABLE 1. body weight ratios Effect of hindlimb suspension on body weight, muscle weight and muscle weight to Control hindlimb suspension Yo change (6) (6) BW(g) 378.0+3.1 349.0+5.0 = 57° MW(mg) soleus 170.8+4.2 104.4+3.6 = 38,9" plantaris 421.3+5.6 350.5 +4.0 =loi3"" gastrocnemius 1991.7+47.2 1557.6 +53.6 = ils." MW/BW(mg/g) soleus 0.448 + 0.009 0.297 +0.013 = 33.1" plantaris 1.111+0.014 1.006 + 0.021 = 95°" gastrocnemius 5.020 +0.136 4.462 +0.128 = iii Values are means+S.E. The number of rats in each group is given in parentheses. weight; MW, muscle weight; MW/BW, muscle weight to body weighg ratios. BW, body Significant differences between control and hindlimb suspension groups are indicated by *(P<0.05) and ** (P<0.01). TABLE 2. Effect of hindlimb suspension on contrac- tile properties in the soleus-plantaris-gastro- cnemius muscle group control hindlimb suspension Ychange (6) P.(g) 413.6+5.9 336.5+13.7 —18.6* P./BW 167.1+1.1 167.0+1.5 Values are means+S.E. The number of rats is given in parentheses. Abbreviations: P,, maxi- mum tension at 40Hz; BW, body weight. A significant difference between control and hindlimb Suspension is indicated by * (P<0.05). 100 80 % tension Time Fic. 1. 10 20 after the onset of stimulation Time course of changes in the % tension during electrical stimulation. HS, hindlimb suspension. was greater in the slow-twitch soleus than in the predominantly fast-twitch plantaris and gastrocne- mius muscles. The maximum tension at 40 Hz in the control and HS groups were 413.6+5.9 g and 336.6+13.7 g, respectively (Table 2). It was significantly lower in the HS group when compared with the control group (P<0.01). However the tension per gram of muscle did not differ between the control and HS groups. The percent decline in tension (% ten- sion) 2 min after the onset of electrical stimulation —c— Control (min) 950, S. Fuxata, T. IKATA AND I. Miura was 39.1+2.0% in the control group and 33.6+ 2.3% in the HS group. At the end of the stimula- tion % tensions were 21.3+0.2 and 21.1+0.2%, respectively. There was no significant difference in the % tension decline between the two groups (Fig. 1). The relative signal intensities of PCr, ATP, Pi, PME and the intracellular pH did not differ signifi- cantly at rest between the control and HS groups (Table 3). Typical *!P-MRS spectra taken at rest, during electrical stimulation, and during recovery are TABLE 3. Relative signal intensities at rest of phos- phomonoester (PME), inorganic phosphate (Pi), phosphocreatine (PCr), adenosine triphsphate (ATP) and intracellular pH in control and hind- limb suspension control hindlimb suspension (6) PME 4.25+0.28 3.43 +0.41 Pi 4.03 +0.28 4.28+0.22 PCr 71.93 £0.45 72.42+0.39 B-ATP 20.67 £0.72 19.87+0.76 intracellular pH 7.08 £0.03 7.08 +0.03 Values are means+S.E. The number of rats is given in parentheses. Control Fic. 2. shown in Figure 2. Decreases in the PCr peak and increases in the Pi peak were observed during electrical stimulation. A lower level of PCr peak and higher level of Pi peak were maintained in the HS group when compared with the control group. After 10 min of recovery, the Pi and PCr peaks in both groups returned to the resting values. The time course of changes in the Pi/(Pi+ PCr) ratio is shown in Figure 3(A). In the first 2 min of the stimulation, the Pi/(Pi+PCr) ratio in the control and HS groups increased to 0.71 + 0.03 and 0.84+0.02, respectively. After that, they returned gradually with time and were 0.55+0.04 and 0.74 +0.03 at the end of the stimulation. The Pi/(Pi+ PCr) ratio was significantly higher in the HS group than in the control group during the stimulation. During the first 8 min of recovery, the Pi/(Pi+ PCr) ratio in both groups decreased rapidly and returned to the resting values without significant difference. The time course of changes in the intracellular PH is shown in Figure 3(B). After the onset of the stimulation, the pH decreased and reached its lowest level within 2 min (6.36 +0.02 in the control group and 6.45 +0.03 in the HS group, respective- ly). From 2 min to the end of the stimulation, the intracellular pH increased gradually and returned A typical set of *'P-MRS spectra at rest, during stimulation, and during recovery. HS, hindlimb suspension; PME, phosphomomoester; Pi, inorganic phosphate; and PCr, phosphocreatine. IP_MRS Study of Disuse Muscle Atrophy 951 (A) : } Rest Stimulation Recovery Ale [-——=19} (Clojnilitpell| X=---k HS 6) & a OF a 40. (min) Time after the onset of stimulation (B) Rest Stimulation Recovery Ht t 1 e—5 CON X----x HS ae Q. iS 2 oO (3) ee ie 2 = 4 + + an 1 0 40 20 30 40 (mln) Time after the onset of stimulation Fic. 3. Time course of changes in the P1/(Pi+ PCr) ratio (A) and the intracellular pH (B) at rest, during stimulation, and during recovery. Values are means+S.E. The number of rats in 6 in each group. HS, hindlimb suspension; Pi, inorganic phosphate; and PCr, phosphocreatine. Significant differences between control and HS groups are represented by * (P<0.05) and ** (P<0.01). to the resting value within 10 min of recovery. There was no significant difference in the intra- UISOUSISIROS cellular pH between the two groups throughout Chronic unloading induces disuse atrophy in rat the experimental period. hindlimb muscles but the degree of muscle atrophy varies in which the slow-twitch muscle is more 952 S. FuKATA, T. IKATA AND I. Miura susceptible to unloading that fast-twitch muscle. In the present study, the decrease of wet weight was greater for the soleus muscle than for the gastrocnemius muscle. These results are consistent with those of previous studies [13, 17-19]. A singificant decrease in the maximum tension was demonstrated after 2 weeks of suspension. The control and HS groups were similar with respect to the tension developed per gram of muscle. These results indicate that muscle atrophy may occur without selective myofibrillar protein loss during hindlimb suspension. The percent decline in tension development did not differ sig- nificantly between the two groups. In contrast, Fell et al. [20] reported faster rates of fatigue in gastrocnemius muscle after 1 week of suspension. This discrepancy might be due to differences in the stimulation parameters. Fitts and Hollozsy [21] reported that the degree of fatigue measured dur- ing a period of stimulation varied with the stimula- tion parameters used for the measurements. The stimulation parametes that we used induced con- traction at a higher intensity than did those used by Fell et al. In suspended rat the gastrocnemius muscle, therefore, is thought to be more fatigable during stimulation at a lower inensity. Chronic unloading affects musclar energy state as well as mechanical properties in the hindlimb muscle. Biochemical study of high energy phos- phate concentrations in atrophied muscles re- ported that the concentrations of PCr and ATP were unchanged in the soleus while a significant reduction in ATP concentrations was noted in the extensor digitorum longus muscle [12]. In con- trast, Jaspers et al. [24] reported that the ATP concentration increases in the soleus muscle after 6 days of tail-cast suspension, although they did not examine that in fast-twitch muscles. However, the ATP concentration relative to total adenosine nucleotide was unchanged in both the soleus and the extensor digitorum longus muscle [12]. In this study, relative concentrations of high energy phos- phates were calculated for the estimation of energy state. At rest, high energy phosphates did not differ significantly between the control and HS groups. This result agrees with in vitro assay. There are some *'P-MRS studies of energy levels in skeletal muscle during disuse. Zochodne et al. [22] reported that the patients with denervated muscles had a lower PCr concentration and a higher intracellular pH than did healthy control resting muscles. In contrast, the patients who had cast immobilization did not exhibit these changes. This difference is likely due to the type of disuse. Denervation removes trophic neural influence, by which the metabolic properties in skeletal may be affected. High energy phosphates during electrical stimu- lation was significantly lower in the HS group than in the control group, whereas no difference was observed in the intracellular pH during electrical stimulation. Similar phenomena were observed in the patients with mitochondrial myopathy [23, 24]. In the patients with mitochondrial myopathy, the PCr concentration decreases to low values during exercise but without an accompaning severe in- tracellular acidosis in contrast to the observation in Since free ADP is believed to be the principal driving force of mitochondrial respiration in skeletal muscle, the increase Pi concentration and the decreased PCr concentration suggest that there exists the elevated ADP concentration due to poor coupled oxidative phosphorylation in sus- pended hindlimb muscle. These results suggest that the decrease in aerobic capacity after hindlimb suspension induced low energy level during elec- trical stimulation. The enzymatic adaptation of rat hindlimb muscle after hindlimb suspension has been re- ported [11, 13]. It is suggested that the capacity for glycolysis increases in slow-twitch and fast-twitch oxidative glycolytic fibers while fast-twitch glyco- lytic fibers are affected minimally. Aerobic capac- ity is reported to increase in slow-twitch fibers but decreases in fast twitch fibers. The result obtained by *'P-MRS are consistent with the enzymatic changes in fast-twitch fibers during hindlimb sus- pension. However, enzyme activities may not be the only factor to muscular energy metabolism in vivo. Oxygen delivery to the hindlimb muscle also affects the energy metabolism during musclar con- tractions. Since the ratio of capillaries per fiber in the soleus decreases 46% [12], oxygen delivery may also decrease. Sogabe et al. [25] reported that controls. low inspired oxygen caused low energy level dur- ‘IP-MRS Study of Disuse Muscle Atrophy ing muscular contraction without intracellular acidosis. Both changes in enzyme activites and oxygen delivery may induce lower energy state in suspended hindlimb muscle during contraction. While further study is required to assess the mechanism of metabolic adaptation during unload- ing of the hindlimb by the suspension, the present study demonstrates that *'P-MRS may be used to assess the biochemical changes which occur in skeletal muscle under a variety of pathological conditions. 10 11 REFERENCES Branski, S., Kwarechi, K., Szmikielski, S. Rozynski, J. (1971) Histochmistry of skeletal mus- cle fibers in rats undergoing long term experimental hypokinesia. Folia. Histochem. Cytochem., 9: 381- 386. Roy, R. R., Sacks, R. D., Baldwin, K. M., Short, M. and Edgerton, V. R. (1984) Inter-relationships of contraction tome, V max and Myosin ATPase after spinal transection. J. 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Neurosci., 5: 2177-2188. Morey, E. R. (1979) Spaceflight and bone turnover: Correlation with a new rat model for weightlessness. Bioscience, 29: 168-172. Haushka, E. O., Roy, R. R. and Edgerton, V. R. (1987) Size and metabolic properties of single muscle fibers in rat soleus after hindlimb suspension. J. Appl. Physiol., 65: 2338-2347. and 12 13 14 16 7 18 19 20 21 DD, 23 953 Desplanches, D., Meyer, M. H., Sempore, B. and Flandrois, R. (1987) Structural and functional re- sponses to prolionged hindlimb suspension in rat muscle. J. Appl. Physiol., 63: 558-563. Roy, R. R., Bello, M. A., Bouissou, P. and Edger- ton, V. R. (1987) Size and metabolic properties of fibers in rat fast-twitch muscle after hindlimb sus- pension. J. Appl. Physiol., 62: 2348-2357. Chance, B., Leigh, Jr. J. S., Clark, B. J., Maris, J., Kent, J., Noika, S. and Smith, D. (1985) Control of oxidative metabolism and oxygen delivery in human skeletal muscle: a steady-state analysis of work/ energy cost transfer function. 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(1984) Influence of suspension hypokynesia on rat soleus muscle. J. Appl. Physiol., 56: 278-286. Fell, R. D., Gladden, L. B., Steffen, J. M. and Musacchia, X. J. (1985) Fatigue and contraction of slow and fast muscles in hypokinetic/hypodynamic rats. J. Appl. Physiol., 58: 65-69. Fitts, R. G. and Hollszy, J. O. (1977) Contractile properties of rat soleus muscle: effects of training and fatigue. Am. J. Phsiol., 233 (Cell Physiol. 2) C86-C91. Zochodne, D. W., Tompson, R. T., Driedger, A. A., Strong, M. J., Gravelle, D. and Bolton, C. F. (1988) Metabolic changes in human muscle de- nervation: Topical 31P NMR spectroscopy studies. Magn. Reson. Med., 7: 373-383. Arnold, D. L., Taylor, D. J. and Radda, G. K. (1985) Investigation of human mitochondrial myopathies by phosphorus magnetic resonance spectroscopy. Ann. Neurol., 18: 189-196. Argov, Z., Bank, W. J., Maria, J., Peterson, P. and Chance, B. (1987) Bioenergetic heterogeneity of 954 S. Fukata, T. IKATA AND I. Miura human mitochondrial myopathies: phosphorus of inspired oxygen on energy state of contracting magnetic resonance spectroscopy study. Neurology, skeletal muscle in rats. Igaku no Ayumi, 148: 119- 37: 257-262. 120. 25 Sogabe, T., Tsubo, M. and Miura, I. (1989) Effect ZOOLOGICAL SCIENCE 9: 955-969 (1992) © 1992 Zoological Society of Japan Initiation of Spermatophore Protrusion Behavior in the Male Cricket Gryllus bimaculatus DeGeer TAKAHIRO OotsuBo and MASAKI SAKAI’ Department of Biology, Faculty of Science, Okayama University, Tsushima-Naka-3-1-1, Okayama 700, Japan ABSTRACT—Spermatophore protrusion is the reproductive act of the male cricket ejecting the immature spermatophore to the external genitalia to solidify. Forty-five seconds prior to this act spermatophore materials are secreted into the ejaculatory duct. Initiation of spermatophore protrusion was facilitated by contact with the female: the interval between the end of copulation and sperma- tophore protrusion was considerably shorter in the male with the female (6 min) compared to the male with no female (>60 min) after copulation. It was also accelerated to a similar extent through increased sensitivity to the female when males were deprived of females for at least 12-24 hr prior to testing. On the other hand, the occurrence of spermatophore protrusion was delayed or prevented when the male was restrained in an abnormal posture. Spermatophore protrusion still occurred, though more slowly, in males with the connectives transected between the thorax and the abdomen. INTRODUCTION During copulation, the male insect gives the female a spermatophore, a proteinous capsule containing sperm. In the cricket Gryllus bimacula- tus, the male starts tc prepare a new sperma- tophore for the next mating about 6 min after copulation [1]: the white milky mass is ejected from the genital cavity into a space between the ventral lobes of the external genitalia. This act is called spermatophore protrusion. Once it occurs the male begins to sing a calling song at a fixed interval [2-5]. Thus, spermatophore protrusion is taken as the first step in the sequence of the male cycle. Although the structure and formation of sper- matophores of insects have been studied extensive- ly [6-8], the initiation mechanism of sperma- tophore protrusion is not yet known. Behavioral study indicates that the female does not play a role in stimulating the initiation of spermatophore protrusion [3]. Our previous study showed that the interval (CPPT) between the end of copulation Accepted July 23, 1992 Received May 23, 1992 J ' To whom reprint requests should be addressed. and spermatophore protrusion varied among males [5]. CPPT was extremely long in some males [5], and considerably prolonged by adverse stim- ulation of the genitalia [1, 4]. These suggest that the process of spermatophore protrusion may be affected by some external and internal factors which is different from the process of the calling song starting with a constant latency after sperma- tophore protrusion [2-5]. On the other hand, physiological study indicates that communication between the brain and the terminal abdominal ganglion was indispensable for spermatophore protrusion [9]. In particular, the pars intercerebra- lis of the brain seemed to be essential because its coagulation abolished this behavior [10]. There is, however, a conflicting result that elimination of the corpora allata or the corpus cardiacum, which is the terminal regions of the projection neurons in the pars intercerebralis [11], failed to abolish sper- matophore protrusion [2]. Thus, we investigated external and internal fac- tors that may affect the initiation of sperma- tophore protrusion and estimated nervous com- munication between the brain and the terminal abdominal ganglion. 956 T. OorsuBo AND M. SAKAI MATERIALS AND METHODS Animals Crickets Gryllus bimaculatus DeGeer reared at a 12L:12D photoperiod at 27+2°C were used. We previously reported CPPT varied considerably among individuals reared alone and gradually de- clined as males matured [5]. In the present study, we used males reared in a group because a large number of insects were used for various experi- ments. The daily change in CPPT (Fig. 1A) was checked in males (n=30) reared in a group with females (n=60) in a container (45 x 30X25 cm). CPPT was much longer and more variable, as compared with that in males reared individually isolated (Fig. 1B) and those (n=30) reared in a group with males (Fig. 1C) for the first 6 days after 60 aN (e) = nN {e) {e) (e) Median of CPPT (min) ine) [e) molt and then reached a constant level after day 8. Thus, crickets aged 8-10 days after molt were used. Males reared in the two latter conditions (B and C) were not used here except in only one experiment. Behavior observation and data analysis The male and female were paired in a 200 ml beaker. Behavioral events were checked by eye, but antennal and abdominal movements were re- corded on video tape and later analyzed by slow speed play back [1]. After they copulated once, the time of reproductive events, i.e. the pairing of male and female, beginning of the calling song, spermatophore extrusion during copulation, the opening of the subgenital plate and spermatophore protrusion was recorded with a stop watch. In the present experiments, the end of copulation was W Ee E kK o OS tS aida MC 8 10 ° Ae z [| 8 10 a rr ois 4 5 6 8 10 Days after imaginal molt Fic. 1. Developmental changes in CPPT of spermatophore protrusion in males reared under 3 different conditions after molt. A: males reared in a group with females. B: males reared alone. C: males reared in a group with males. Each experiment started with 30 males but the number reduced to 29 in A and C, and 24 in B on day 10th owing to death. Each point represents the median value of CPPT and each bar, the 95% confidence interval (C.I.). The dotted line in A indicates the | hr observation time limit. Some of the same conventions are adopted in the subsequent figures. Spermatophore Protrusion in Crickets 957 defined as the moment of genitalia separation (GS in Fig. 2) which regularly occurred 10sec after spermatophore extrusion [1], i.e., extrusion of the attachment plate of the spermatophore from the dorsal pouch [1]. Spermatophore protrusion was defined as the moment of ejection of an immature spermatophore from the genital cavity (SPPT in Fig. 2). Thus, CPPT is represented as the interval between GS and SPPT. CPPT was measured in minutes rounding off fractions to one decimal place. Data were expressed as medians with 95% confidence intervals (C.I.). Statistical significance on CPPT was set at P<0.05 by the Mann-Whitney U test unless otherwise stated. Separation from the female To examine the effect of the female on CPPT, two different procedures were used. During _ testing: When copulation accomplished, the female was removed from the beaker (“male” condition) and CPPT was timed. For comparison, the female was replaced with a male (“male and male” condition). Control was the “male and female” condition in which the male and female were simply left after copulation. Was Prior to testing : ly removed from the container before experiments and males experienced female-deprivation for 4, 8, 12, 24 or 72 hr prior to pairing. According to Fig. 1C, CPPT was expected to be shortened by these treatment. They were tested in the “male” condi- tion. The reason for this will be described later. Modifications of these procedures will be de- scribed in the places concerned. All females were temporari- Restraint of males In order to examine whether spermatophore protrusion could occur in abnormal conditions, males were restrained in four different conditions immediately after copulation (Table 2). 1) Posture normal, legs contacted; the male was glued by the dorsal pronotum to a metal bar in a normal posture and placed on a movable styrol ball. 2) Posture normal, legs restrained; each tarsus of the 6 legs was fixed to the substrate with a piece of Scotch tape. 3) Posture reversed, legs contacted; the male was glued by the dorsal pronotum to a wooden mound with the ventral side up. A wooden plate was set over the male to contact with the legs on it. 4) Posture reversed, legs suspended; the same as in 3) except for no plate. All the males were given a periodic brushing of their antennae with female’s antennae for 2—3sec every 40sec as female sti- mulation. If the male did not show spermatophore protrusion within a | hr observation period, it was checked again after 24 hr during which they were left without female stimulation. Inspection of the spermatophore in the ejaculatory duct The ejaculatory duct was examined for an imma- ture spermatophore in males at 4 different phases of the male cycle (Table 1); after spermatophore extrusion in copulation, after subgenital plate opening, after spermatophore protrusion, and an arbitrary time after the start of courtship before copulation. Dissection was made soon after freez- ing the males in liquid nitrogen (—192°C). Ablation of sensory organs and reproductive organs To block visual input, the compound eyes and oscelli were covered by black lacquer. To reduce chemical input, the antennae, palpi and tarsi were cut off with scissors. For reproductive organs, the accessory glands or the testes were removed with a pair of forceps after opening the cuticles of the respective abdominal segments. This was done on day 3 after molt for the males to recover sufficient- ly before testing. Transection of the nervous system After the male copulated, it was anesthesized by cooling at 4°C, fixed on a cork board, and the cuticles over the intended level of the connectives or brain were cut (Fig. 9 inset). The connectives were cut at different levels; between the brain and the suboesophageal ganglion (Fig. 9e), between the metathoracic ganglion and the 1st abdominal ganglion (Fig. 9c), and between the 4th abdominal ganglion and the 5th abdominal ganglion (Fig. 9b). The connectives between the suboesophageal ganglion and the prothoracic ganglion was sepa- rated by cutting off the head at the neck (decapita- tion Fig. 9d). The brain was cut 3 ways with a 958 T. OorsuBo AND M. SAKAI razor blade; cut horizontally to separate the anter- ior region (Fig. 9f), cut vertically at the midline to separate it into the left and right (Fig. 9j), or cut diagonally to separate the antero-lateral regions (Fig. 9h) or the antero-median region including the pars intercerebralis (Fig. 9g). The corpora allata and the corpus cardiacum were removed together with forceps (Fig. 91). Control males were only anesthetized for the same duration as transected males. All tests started on the day of surgery. Spermatophore checking in nervous system tran- sected males To examine the operated male’s ability to pro- duce the spermatophore, the interior of the sub- genital plate was checked every 6 hr for 3 succes- sive days during which each male was kept isolated in a 200 ml beaker. A spermatophore was marked by India ink to discriminate from a new one. The number of males that produced spermatophore(s) during the 3 days, and the number of sperma- tophore(s) produced per male per day were counted. RESULTS Process to spermatophore protrusion Behavior Main reproductive events are shown in abbre- viations in the middle of Fig. 2. The male copu- lates with the female some time after pairing (MFP). When the genitalia separated after sper- matophore extrusion (GS in Fig. 2), all bodily movements stopped. In 1 sec (C.I. 1-2, n=21), a half (51%, n=41) of the males, then, exhibited post-copulatory body thrust [5] similar to copula- tory movement for 18sec (C.I. 14-30, n=21). These movements occasionally continued for up to a few minutes interposed by short rests. It finally ceased when the male lowered its abdomen itself (71%, n=21) but, in the remaining 29%, the body thrust was terminated by the female’s movements on the male’s back. Then, the male crept forward from under the female with his body rocking in a typically aggressive expression (MEFS in Fig. 2). A distribution of the time interval between genitalia separation (GS) and male-female separation (MES) is shown in Fig. 2A. The GS-MSF interval distributed from 5 to 80 sec with a median value of 18sec (C.J. 8-23, n=29). The interval (30sec (C.I. 6-43, n=21)) of males exhibiting body thrust was significantly longer than that (8 sec (C.I. 4-12, n=20)) of those not exhibiting body thrust. This post-copulatory body thrust developmentally emerged on day 4th after molt [5] and may play a role in keeping the female quiet longer to secure the settlement of the spermatophore in the genital chamber of the female. After male-female separation, the male exhi- bited watching behavior [12] or guarding behavior [3]. Occasionally two kinds of characteristic move- ments were observed; one, abdominal contraction similar to those seen in evacuation of feces in a strained posture and the other, rapid antennal vibration at 10 Hz in the antero-postero direction [13]. One example of the occurrences of these movements in the male is shown by bars on the two lines in the middle of the Figure 2. The frequency of the abdominal contraction reached a maximum around spermatophore protrusion. It may reflect the activity of the reproductive organs to prepare a new spermatophore or warm-up for its ejection. Shortly before the spermatophore was ejected, the male often repeated 2-3 small openings of the subgenital plate. Then, the plate was widely pulled down (Fig. 2B inset) referred to here as subgenital plate opening (SGPO). The interval between male-female separation (MFS) and subgenital plate opening (SGPO) was 4 min (C.I. 3-9, n=29, Fig. 2B). Then, the abdominal contraction with the back- ward pushing of the genitalia complex became frequent and the immature spermatophore was ejected through the genital opening to the space between the ventral lobes, that is, the moment of spermatophore protrusion (Fig. 2C inset). The interval between subgenital plate opening (SGPO) and spermatophore protrusion (SPPT) was 45 sec (C.I. 43-49, n=28), very constant for its shortness with a small C.I. (Fig. 2C). Timing of spermatophore materials secretion into the ejaculatory duct Prior to spermatophore protrusion, the mate- Spermatophore Protrusion in Crickets B mc x M=4min Mes © 40 ey SE @o {= 5 2 20 (Ss) iS 0 Jen Oo Lo ao Gogo Interval (min) Ant. movement Abd. movement A AA A MFP GS MFS SGPO SPPT = A 6Of MN Occurrence (%) Interval (sec) 959 60 | as M=45sec : 5 an O 20r Occurrence (%) 0 2 40 GO Interval (sec) Fic. 2. Reproductive events and time intervals between them. In the center, the occurrences of antennal (upper) and abdominal (lower) movements shown by vertical bars. The male was paired with a female at MFP. Scale bar, 10 sec. A-C: histograms of the time intervals. Time bin, 5 sec in A and C, and5 minin B. A: distribution of the time interval between genitalia separation (GS) and male-female separation (MFS). Solid line, males (n=20) not exhibiting post-copulatory body thrust. Dotted line, males (n=21) exhibiting post-copulatory body thrust. B: distribution of the time interval (n=29) between MFS and subgenital plate opening (SGPO). C: distribution of the time interval (n=28) between SGPO and spermatophore protrusion (SPPT). The histogram representing >60 min in B indicates males which failed to exhibit SGPO within the time limit. Some of these conventions are adopted in Fig. 4. M, median value; S, spermatophore; SGP, subgenital plate; VL, ventral lobes. rials of the spermatophore are secreted from the testes and accessory glands into the ejaculatory duct. To determine this timing, the inside of the ejaculatory duct was examined using males quickly frozen at different phases in the male cycle. The spermatophore was not found in any phase except the very short period between the subgenital plate opening and spermatophore protrusion (Table 1). This means that spermatophore formation is initi- ated at the moment of subgenital plate opening. Some critical observations are shown in Fig. 3. The ejeculatory duct was vacant in males in the period between the end of copulation and sub- genital plate opening (Fig. 3A). Ten seconds after subgenital plate opening, a white mass containing spermatozoa and the pressure body are seen (Fig. 3B). Thirty seconds after subgenital plate open- ing, the spermatophore moves toward the genital opening (Fig. 3C). We observed that a white mass emerged from the accessory glands into the vacant ejaculatory duct in the male in phases other than the one between subgenital plate opening and sperma- tophore protrusion when the dissected male was left to thaw at room temperature. This curious phenomenon seems to be an artifact caused by a 960 T. OorsuBo AND M. SAKAI TABLE 1. Presence or absence of the spermatophore in the ejaculatory duct and ventral lobes Time after reproductive events Ejaculatory duct Ventral lobes n Spermatophore 0 x x 10 extrusion 10 (min) x x 5 30 x x 5 Subgenital plate 0 @©5 Xx x 10 opening 10 (sec) O x 10 30 © x 5 Spermatophore 0 x O 10 protrusion 10 (min) x O 5 30 x O 5 Calling song >0 (min) x O 10 x absence of spermatophore. © presence of spermatophore. ©, x presence of spermatophore in only 4 males. n, number of males used. A pre-SGPO B post-—SGPOt0 c post—SGPO30 Fic. 3. Timing of the emergence of the spermatophore in the ejaculatory duct. Insects were frozen at 3 different phases in the male cycle. A: between the end of copulation and subgenital plate opening (SGPO). B: 10sec after SGPO. C: 30sec after SGPO. AG, accessory glands; DP, dorsal pouch; ED, ejaculatory duct; GO genital opening; PB, pressure body; Sz, spermatozoa; VL, ventral lobes. Scale bar, 1 mm. loosening of the accessory glands due to tissue role in intiation of spermatophore protrusion, destruction. CPPT was measured in 3 different conditions (see ' methods); “male and female” (Fig. 4A), “male Role of the female in spermatophore protrusion and male” (Fig. 4B) and “male” (Fig. 4C). In A as a control, 82% (n=32) of the males showed sper- Removal of the female during testing matophore protrusion within 10 min. The median To examine whether the female plays a major — of CPPT was 6 min (C.I. 5—8) with 3 min being the Spermatophore Protrusion in Crickets 961 LA +2 .B G+S .C & M=6min M=>60min M=>6O0min 60r #l 60r r L % w 40} 4o} a oO ‘S 60 CPPT (min) CPPT (min) CPPT (min) Fic. 4. Effect of the female presence after copulation on CPPT. A: control (“male and female”, n=32). B: the male paired with a male after copulation (“male and male”, n=26). C: the male separated from the female after copulation (“male”, n=36). Time bin, 5 min. shortest. In contrast, the male paired with a male after copulation made spermatophore protrusion in only 31% (n=26) within 1 hr (B). They were often fighting at the beginning. No males showed a CPPT shorter than 10 min. In C, 42% of males (n =36), which were kept quiet with no female after copulation, made spermatophore protrusion with a CPPT of >60 min (C.J. 12->60). Twenty five percent of them showed a CPPT shorter than 10 min. There was a significant difference between B and C in the number of males exhibiting sperma- tophore protrusion within 1 hr. In B and C, males tend to split into two groups: those exhibiting spermatophore protrusion within half an hour and those not exhibiting it within 1 hr. In sum, these results reveal that spermatophore protrusion is considerably facilitated by the presence of the female. Then, the effect of the female’s presnece on CPPT was examined by changing the duration of pairing after copulation. Figure 5 shows the re- sults. CPPT did not significantly decrease until the duration of the female presence was prolonged to 5 Median of CPPT (min) Fic. Duration of female presence after copulation (min) 5. Time dependence of female presence on CPPT. The abscissa represents the duration (min) of the presence of the female after copulation in the beak- er. Asterisk, the male paired with a female again for only 1 min between 4 and 5 min after copulation. Each point represents the median for 30 males. 962 T. OorsuBo AND M. SAKAI min (C.I. 5-19, n=30). However, this may be due to the change in male’s internal state starting several min after copulation. Yo examine this possibility, the male was paired with a female for only 1 min between 4 and 5 min after copulation (asterisk). This short presence of the female was not sufficient to facilitate the initiation of sperma- tophore protrusion as seen in the CPPT of 41 min (C.I. 18->60, n=30). Block of sensory input To study the nature of female stimulation, visual or chemical input was eliminated. CPPT in blind males (E in Fig. 6A) was 5.5 min (C.I. 3-7, n=20) which was not significantly different from that of intact males (Int in Fig. 6A). However, CPPT in A 60 eee c cece cece cccccccccccccscccccccs 60 e S 40 Fk ae OW O == (@) = 2 2O n@) ® = Int E Fic. 6. =20) whose compound eyes and oscelli were covered with black lacquer. A,P&T Analysis of female factors. A: the effect of ablation of sensory organs. Int, intact (n=32). E, blind males (n males with antennae, palpi and tarsi removed (A, P & T in Fig. 6A) was 12 min (C.I. 7-23, n=25) which was much shorter than that of males tested without a female (Fig. 4C) but significantly longer than that of the intact males. This means the shortening of CPPT still occurred via male’s chemoreceptors on the body surface as well as those on the antennae, palpi and tarsi. To deter- mine whether receptors were olfactory or taste, the female was suspended by a string 8 cm over the male without contact (non-C in Fig. 6B). CPPT was >60 min (C.I. 18->60, n=28). For control, a female was suspended 3cm above the male allowing contact with her body. CPPT was 10.5 min (C.J. 5-18, n=20) which was much shorter than that of the non-C group and not significantly B fe) nonc C A, P & T, males (n=25) whose antenna, palpi and tarsi were removed. Note CPPT in A, P & T is much shorter than that of non-C, but still significantly longer than that of the Int. B: the effect of contact with the female. non-C, males (n=28) which could not contact with the female. C: males (n=20) which could contact with the female. A female was suspended 8 cm (non-C) and 3 cm (C) above the male, respectively. Spermatophore Protrusion in Crickets 963 60 Cece cc ccccccccccceccccccesccs pS oO pe) O Median of CPPT (min) Pee ecrescceseseseseseseseesesssessoesseoseceoes Lendl o" Cone lame: aE Sapo aT 172 Duration of deprivation (hr) Fic. 7. Effect of female-deprivation prior to testing on CPPT. The abscissa represents the time (hr) during which males (n=30) had been separated from females prior to testing. Note the long latency at 0, 4, and 8 hr is characteristic of males tested in the “male” condition as in Fig. 4C. longer than that of the intact group (Fig. 6A), This result reveals that female stimulation is not olfac- tory, but presumably contact-mediated. Female-deprivation prior to testing Males were temporarily separated from the females for a fixed time prior to pairing since CPPT was shorter in female-deprived males during the sexual maturation process after molt (Fig. 1B, C). During testing, CPPT was measured in the “male” condition so that the results would be much clearer than in the “male and female” condition because CPPT was expected to be shortened by deprivation (compare Fig. 4A with Fig. 4C). As shown in Fig. 7, the control (Ohr deprivation group) had a median value of >60 min (C.I. 12-> 60, n=36) similar to that of Fig. 4C. The CPPTs for the 4 hr and 8 hr deprivation groups were >60 min (C.I. 40->60, n=27) and 17 min (C.I. 8-57, n=31) respectively which were not significantly different from that of the control partly because of the large C.I.s. However, CPPT for 12 hr was 17 min (C.I. 4-43, n=27) which was significantly shorter than the control. At 24 hr, CPPT reduced markedly, as small as 4 min (C.I. 3-8, n=29). The interval, however, did not further decrease in the additional 48 hr of deprivation since CPPT was 4.5 min (C.J. 3-8, n=30) at 72 hr. The shortening of CPPT may be due to a reduction of spermatophore production as a result To test this possibility, males with accessory glands removed were used. The accessory gland ablated males could not make spermatophores but exhibit a spermatophore protrusion-like behavior [1] some- time after a vaccum copulation with the female [2— 5]. Thus, CPPT was defined here as the time between genitalia separation and spermatophore protrusion-like behavior. Fig. 8 shows the results of the female-deprivation test in the “male” condi- tion using accessory gland ablated males. CPPT of the control (AG (0)) was 47 min (C.I. 10->60, n of no copulation in the container. 964 T. OotsuBo AND M. SAKAI Bas oO Median of CPPT (min) ine) oO AG AG (QO) (24) Fic. 8. Effect of female-deprivation in males with accessory glands removed on CPPT. AG(0), males (n=30) with no deprivation as control. AG(24), males (n=25) with 24 hr deprivation. =30) while that of the 24 hr deprived group (AG (24)) was 11 min (C.I. 7-25, n=25). It is obvious that the shortening of CPPT occurred even in the males lacking the ability of spermatophore produc- tion. In addition, males whose testes were re- moved showed a similar result with a CPPT of 7 min (C.I. 5-8, n=36). These results indicate that the effect of female-deprivation can not be corre- lated with the lowered activity of spermatophore production. The shortening of CPPT may be due to the lack of genitalia coupling [1] and subsequent sperma- tophore extrusion. Males were then placed with females for 24hr whose subgenital plates were closed with wax to prevent males from hanging the epiphallus onto the female subgenital plate but not to prevent other mating acts. They were restricted in only genitalia coupling and spermatophore ex- trusion. The result indicated that the shortening of CPPT did not occur (CPPT>60 min (C.J. 28-> 60, n=20)). This revealed that the shortening of CPPT was not correlated with the inability of the genitalia to couple and to extrude the sperma- tophore. It is worth mentioning that males reared with males (they were not used so far in the experi- ments) occasionally showed an extremely short CPPT when they were tested in the “male and female” condition. Although it was not apparent in the males in Fig. 1C, in one case using a different batch, CPPT was as short as 3 min (C.I. 3-4, n=28). The shortest of these was 1 min. Such a short CPPT was seldom observed in males reared with females (Fig. 1A). This reveals that the effects of the female-chemical substances and female-deprivation could be additive. Effect of restraint in spermatophore protrusion Table 2 shows the effects of the forced posturing on spermatophore protrusion. Sixty-five per cent of the “posture normal, leg contacted” males ex- hibited spermatophore protrusion with a relatively long latency (40 min (C.I. 19->60)), while only 10% of the “posture normal, leg restrained” males did so. In contrast, no males of the “posture reversed” groups ejected the spermatophores within a | hr and the following 24 hr. These results indicate that the male must be kept in a normal posture to protrude the spermatophore with the legs freely moving in contact with the substrate. In an additional experiment, the male was im- mediately paired with a female in a beaker at the end of stress testing (“posture reversed, legs sus- pended”). They showed spermatophore protru- TABLE 2. Spermatophore protrusion under re- strained conditions Conditions Spermatophore : Posture Legs protrusion (%) CPET. (ain) Normal Contacted 65 [30] 40(C.1. 19->60) Restrained 10 [35] 18, 55 Reversed Contacted 0 [0] — Suspended 0 [0] —_ Number in each group, 20. Number in square brackets, males produced spermatophore(s) within 24hr after the end of a 1 hr observation. Spermatophore Protrusion in Crickets 965 sion with a CPPT of 9 min after pairing (C.I. 8-17, n=20) which was not significantly different from the control (8 min (C.1. 8-10, n=33)) tested simp- ly under the “male and female” condition using the same group. This indicates that spermatophore protrusion was inhibited during heavy stress but there was no aftereffect. Transition from SGPO to SPPT Disturbance As shown in Fig. 2-C, the fact that the interval between subgenital plate opening and sperma- tophore protrusion was very constant suggested that both actions were linked together. Thus, whether spermatophore protrusion can be pre- vented by heavily distracting the male immediately after subgenital plate opening was examined. The male was quickly picked up at subgenital plate opening and kept gripped in the palm. In spite of such a harsh treatment, all the males showed spermatophore protrusion in the normal time in- terval of 45 sec (C.I. 42-51, n=25). Transection of the connnectives To estimate the involvement of the anterior nervous system in initiation and execution of sper- matophore protrusion, the connectives were cut at the neck by decapitation or at the junction be- tween the thorax and the abdomen immediately after subgenital plate opening. Both the groups showed spermatophore protrusion with the normal time interval of 45 sec (C.I. 41-48, n=20) and 45 sec (C.I. 42-51, n=20), respectively. These re- sults revealed that spermatophore protrusion is an unstoppable process once the subgenital plate Opens and is accomplished under the control of only the abdominal ganglia. In contrast, as will be described below, when the connective transection was made before subgenital plate opening, sper- matophore protrusion within the normal time range was prevented. Effects of nerve and brain transection When the male was kept with a female in a beaker, spermatophore protrusion occurred reg- ularly after every copulation. The number of spermatophores reached an average of 16 per day in males on day 10 after molt [5]. On the other hand, when the male was kept alone in the beaker, it showed spontaneous cycle renewal [1] in which the male exhibited spermatophore protrusion without any mating actions. In the present experi- ments, 96% of the control males (Fig. 9, a) pro- duced one or two spermatphores per day with an average of 1.6+0.7. No males (Fig. 9, b) with the bilateral connectives cut between the 4th and the Sth abdominal ganglion produced sperma- tophores. Only males in this group showed feces discharge all the time indicating that they had problems in the control of intestinal and abdomin- al movements. In contrast, males with a unilateral connective cut at the same level produced sperma- tophores similarly to the control. In males (Fig. 9, c) with bilateral connectives cut between the tho- rax and abdomen, the average number of sperma- tophores was about half the control. Unilateral transection again altered neither the number of the males producing spermatophores nor the number of spermatophores produced. Decapitated males (Fig. 9, d) and decerebrated males (Fig. 9, e) also showed a significant decrease in the numbers of males producing sperma- tophores (71% and 50%) and the number of spermatophores produced (1.0+1.4 and 0.4+0.5) compared with the control. The brain was cut in different ways to separate the particular regions. Horizontal transection (Fig. 9, f) caused a significant decrease in the numbers of males producing spermatophores (65%) and spermatophores produced (0.6+0.8). Males with the antero-median region eliminated (Fig. 9, g) had a significant decrease in both the numbers of males (68%) and spermatophores (0.5 +(0.6). On the other hand, males with the antero- lateral regions eliminated (Fig. 9, h) showed only a significant derease in the number of sperma- tophores (0.8 +0.6) with a similar number of males producing spermatophores (79%) as the control. The median transection (Fig. 9, j; n=22) caused a significant decrease in the number of sperma- tophores produced but not in the number of males producing them (88%). Finally, the removal of the corpora allata and the corps cardiacum (Fig. 9, i; n =12) did not alter the number of males (91%) or spermatophores (1.3+0.8). Only the disconnection of the bilateral nerve 966 T. OotsuBo AND M. SAKAI Males with spermatophores (%) Mean of spermatophores / day O b © a e f el, MY, wv oO neon 5 A pecs case Fic. 9. Effects of transection of the connectives, separation of the brain, and removal of the corpora allata and corpus cardiacum. A: percentage of males producing mature spermatophores at least once in 3 successive days following surgery. B: the average number of mature spermatophores produced per male per day. Small letters (b-j) represent the types of surgery corresponding to those in inset. Each group consisted of the following number of males; 24(a), 13(b), 20(c), 14(d), 12(e), 20(f), 22(g), 19(h), 12(i), and 22(j). Asterisks indicate significant differences (A: 2? test, P<0.05; B: t-test, P<0.05) between (a=control) and (b-j). cords between the 4th and the 5th abdominal ganglion abolished spermatophore protrusion. These results indicate that spermatophore protru- sion can be initiated and executed without any influence from the head or throacic ganglia. DISCUSSION The present study shows the starting event of spermatophore protrusion, and external and inter- nal factors that influence the latency of sperma- tophore protrusion in the male cricket Gry/lus Spermatophore Protrusion in Crickets 967 bimaculatus. Initiation of spermatophore protru- sion is considerably facilitated both by contact with the female and by an increase in sensitivity to the female. On the other hand, spermatophore protrusion was delayed or prevented under stress- ful conditions such as persistent restraint in an abnormal posture. Spermatophore protrusion could occur, though more slowly, in males with the connectives cut, except connectives between the 4th and the 5th abdominal ganglion. Our results provide the basis for future neurophysiological study to elucidate the initiation mechanism of spermatophore protrusion. Start of spermatophore production Spermatophore protrusion occurs with a more variable CPPT compared with that (about 1 hr) of the calling song [1]. In extreme cases, CPPT was more than 1 hr (see Fig. 2B) while only 1 min at the shortest. These findings suggest that the pro- cess of spermatophore protrusion is influenced by some external and internal factors in contrast to the process of the calling song which is dependent on an internal timer [4]. Spermatophore protrusion occurs 45 sec after subgenital plate opening. Transection of the con- nectives revealed that these two actions were a continuous process. Inspection of the sperma- tophore in the ejaculatory duct indicated that the secretion of sperm from the testes and proteinous substances from the accessory glands occurred at the moment of subgenital plate opening. These facts indicate that spermatophore protrusion, which we defined a priori as the moment of immature spermatophore ejection through the genital cavity to the exterior, actually starts at the moment of accessory gland contraction to secrete the spermatophore materials into the ejaculatory duct [14] possibly mediated by dorsal unpaired median (DUM) neurons [15]: i.e. the male cycle starts at the time of subgenital plate opening. Previously, one paper [4] reported that a new spermatophore was already present in the ejacula- tory duct soon after spermatophore protrusion and stayed there until the next spermatophore protru- sion, while the other paper [14] did not confirm this and rather agreed with our present result. We suspect the observations by the former authors might be an artifact due to freezing and thawing. Facilitation of spermatophore protrusion CPPT was much shorter in the male kept con- tinuously with the female after copulation. This suggests that some contact-mediated chemical sub- stances are facilitating spermatophore protrusion. Chemo-receptors mediating spermatophore protrusion may be distributed over the body sur- face because the shortening of CPPT was still apparent in males without the antennae, palpi and tarsi. Previously in Teleogryllus, the female had no effect on spermatophore protrusion [3] though its contact chemoreceptors certainly facilitated mat- ing actions [16]. The discrepancy may be due to the difference in species or the difference in the size of the space where animals were tested. The compartment they used was a little wider than our 200 ml beaker, which might have lowered chances of contact with each other. It is interesting that males split into those with CPPT less than 30 min and those with more than 1 hr when tested in no female conditions (Fig. 4B, C). Probably an indi- vidual difference in male reproductive activity was exaggerated through some accommodation which increased the threshold of spermatophore protru- sion under less stimulative conditions. Spermatophore protrusion was also facilitated after males were temporarily kept isolated from females. This was already seen from day 3 after molt, and matched our empirical observations that males reared without females were sexually more excitable than those with females [1]. Our sys- tematic study of female-deprivation found that the shortening of CPPT became pronounced after a 12-24 hr separation. Our results indicated that this effect was neither due to a reduction of spermatophore production nor due to the lack of genital coupling and spermatophore extrusion dur- ing isolation. It is probably caused by an increase in sensitivity to the chemical substances of the female. Inhibition of spermatophore protrusion Stress, such as forced posturing, significantly delayed or even prevented spermatophore protru- sion. In particular, males fixed in the upside-down position were unable to push out the sperma- 968 T. OotsuBso AND M. Sakal tophores, indicating that normal posture with the legs kept freely on the substrate is a prerequisite. A similar result was previously obtained by fixing the body to a board with paraffin [4]. These results give cautions against neurophysiological study where animal restraint would be inevitable. Simi- lar but not large delay in CPPT was also found under a different type of stress, 1.e., fighting with a male (Fig. 4B). It should be worth comparing the resistivity to stress between the two interval states: genitalia separation to spermatophore protrusion vs. sper- matophore protrusion to calling song. As long as the male is under heavy stress, it is natural that the male cannot initiate either spermatophore protru- sion or calling song. However, the male sang the calling song with the normal latency (about 1 hr) from spermatophore protrusion when stress was terminated in less than 1 hr after spermatophore protrusion [4]. Stress did not disturb the running timer. On the other hand, the male exhibited spermatophore protrusion with a latency of 1h plus the normal CPPT when stress was terminated 1 hr after copulation and the male was paired with a female. Stress inhibited the processing necessary for spermatophore protrusion. Role of the brain in spermatophore protrusion It is known that nervous communication be- tween the brain and the terminal abdominal gan- glion is necessary for the male cricket to perform mating behavior normally [9]. Here, we state more concretely that male crickets with both con- nectives cut at the thoracic level can perform spermatophore protrusion but not produce as many spermatophores as intact males. This result means that the motor program for spermatophore production resides totally within the abdominal ganglia and can proceed without command of the anterior ganglia. Normally, however, sperma- tophore protrusion is certainly triggered by a com- mand in the brain because decerebrated males failed in spermatophore protrusion within the nor- mal time range after copulation. It would be interesting to record the descending neural activity conveying signals for sperma- tophore protrusion which should arrive at the abdominal ganglia before subgenital plate open- ing. The fact that spermatophore protrusion re- quired 45 sec from subgenital plate opening and that the minimum CPPT (genitalia separation to spermatophore protrusion) was only | min suggest that information on mating termination generated in association with the dorsal pouch contraction to extrude the spermatophore [1] can be processed immediately in the brain and in turn can trigger the pattern generator in the abdominal ganglia. At the same time, some neural events associated with DUM neuron activity for secretion of the sperma- tophore materials into the ejaculatory duct may set the timer for the calling song. The resultant accessory gland contraction itself or the ensuing spermatophore ejection to the external genitalia is not concerned with the timer setting since males without accessory glands [2] and those with sper- matophores but incapable of spermatophore protrusion [1] still retain the normal timer func- tion. Regarding the effects of partial elimination of the brain, determination of brain regions critical for spermatophore protrusion was virtually im- possible because the activity of spermatophore production in intact males was very low (1 or 2 per day) and that in the experimentals was not zero but 0-1 per day: the differences were too small to compare. The total inability of males with the connectives transected between the 4th and the Sth abdominal ganglion to perform spermatophore protrusion is probably due to the paralysis of abdominal segments as suggested by continuous discharge of feces. The results of the corpora allata and corpus cardiacum removal was in agree- ment with the previous report that elimination of either organ failed to abolish mating behavior [2]. ACKNOWLEDGMENTS The study was supported by Grant-in-Aid (No. 2640557) for Scientific Research from the Japanese Ministry of Education, Science and Culture. REFERENCES 1 Sakai, M., Taoda, Y., Mori, K., Fujino, M. and Ohta, C. (1991) Copulation sequence and mating termination in the male cricket Gryllus bimacuatus DeGeer. J. Insect Physiol., 37: 599-615. Spermatophore Protrusion in Crickets 969 Beck, R. (1974) The neural and endocrine control of mating behaviour in the male house cricket, Acheta domesticus L.. University of Nottingham, doctoral thesis. Loher, W. and Rence, B. (1978) The mating behavior of Teleogryllus commodus (Walker) and its central and peripheral control. Z. Tierpsychol., 46: 225-259. Nagao, T. and Shimozawa, T. (1987) A fixed time-interval between two behavioural elements in the mating behaviour of male cricket, Gryllus bima- culatus. Anim. Behav., 35: 122-130. Sakai, M., Katayama, T. and Toada Y. (1990) Postembryonic development of mating behavior in the male cricket Gryllus bimaculatus DeGeer. J. Comp. Physiol., 166: 775-784. Khalifa, A. (1949) The mechanism of insemination and the mode of action of the spermatophore in Gryllus domesticus. Q. J. Microsc. Sci., 90: 281- PDD. Gregory, G. E. (1965) The formation and fate of the spermatophore in the African migratory locust, Locusta migratoria migratorioides Reiche and Fair- maire. Trans. R. ent. Soc. Lond., 117: 33-66. Mann, T. (1984) Spermatophores: Development, structure, Biochemical Attributes and Role in the Transfer of Spermatozoa. Springer-Verlag, Berlin Heidelberg New York Tokyo, pp. 107-115. Huber, F. (1955) Sitz und Bedeutung nerovoser Zentren flr Instinkthandlungen beim Mannchen 10 11 12 13 14 von Gryllus comperstris L.. Z. Vierpsychol., 12: 12- 48. Loher, W. (1974) Circadian control of sperma- tophore formation in the cricket Teleogryllus com- mondus Walker. J. Insect Physiol., 20: 1155-1172. Moor, D. and Loher, W. (1988) Axonal projection within the brain-retrocerebral complex of the crick- et, Teleogryllus commodus. Cell Tissue Res., 252: 501-514. Khalifa, A. (1950) Sexual behaviour in Gryllus domesticus L.. Behav., 2: 264-274. Alexander, R. D. (1961) Aggressiveness, territori- ality, and sexual behavior in field crickets (Ortho- ptera: Gryllidae). Behaviour, 17: 130-223. Kimura, T., Yasuyama, K. and Yamaguchi, T. (1989) Proctolinergic innervation of the accessory gland in male crickets (Gryllus bimaculatus): detec- tion of proctolin and some pharmacological prop- erties of myogenically and neurogenically evoked contractions. J. Insect Physiol., 35-3: 251-264. Yasuyama, K., Kimura, T. and Yamaguchi, T. (1988) Masculature and innervation of the internal reproductive organs in the male cricket, with special reference to the projection of unpaired median neurons of the terminal abdominal ganglion. Zool. Sci., 5: 767-780. Rence, B. and Loher, W. (1977) Contact chemoreceptive sex recognition in the male cricket, Teleogryllus commodus. Physiol. Entomol., 2: 225- 236. ee. \ a ; : i 4s bri os i =) ; dhe AP iG’ Peel@ehinneyy.. , a . , pate Ay ‘ bh aN, vy P Ac . : Wino! : iSefetiey i ho teatibanlt © uy ie ac) Lani “ett 4 deel epteerts thy pi “i ian. Tu awa: —ciieelwany Mode, ar UPaLy ikat ‘ eh Pal eat alt gi po of vere Te) gitar aby E ¢! cytidine irs inv a ny rai ol 2 iy 2 . ; feed ire} " fu EE Cie: 1 oetiithaed “LAR bin a weet nh ry Att k P| b ‘ ON Hp ER A i at ; atts Fone | nba tl a Ge ti Ai } acti : f a pit a sp aaenetldY CRO At 7 1 wll ve reese di | Pe ET huede dit CU hal bmi Vinda” 43 yet sheds se deanna : : ‘ : i ae i ~§ ya beriag ger et 7 OE oi ga a 1 bobs ‘ J é pe . tars hs Oe yeti PSE ASP a 5 e- “ky Ara re ro My whi cadi\ vi tevin Leahy : Tht ; j if 5 Rica rakes by alias a aii tide hk) ae : bes AY ty Gi ATALE TEROEE & F ; ay eh tee? v rit \ LJuat j t ’ r re eu t 7 MASE yH 1a wert ib ooh wisi E |! ; ‘ ay eh tie i ii ' : cn 4 Lin rts to a ae i ; : 5 p bins yhdtee oA. bated oo | ‘ecpabie’ st. 7 b lal PRS TLS A, CUS iD ee 4 ‘ ey oak i . ie { oft od of, ba salu” | foe : rst ie ee 1 : iy icy fr esr Tega ik i ¥ ' d i abdekt ( Fh sak O VAP eT ee en”: endl : ( le gefeaendblay prekoad je rmwcantoriaeld 4 ZOOLOGICAL SCIENCE 9: 971-981 (1992) © 1992 Zoological Society of Japan Ultrastructure of Spermatozoa and Spermatogenesis in Nepomorpha (Insecta: Heteroptera) with Special Reference to Phylogeny YouNG Hwan Lee! and CHANG Eon LEE* 'Department of Biology Education, College of Education, Taegu University, Kyungsan 713-714, and *Department of Biology, College of Natural Sciences, Kyungpook National University, Taegu 702-701, Korea ABSTRACT — Ultrastructural study of spermatozoa was made for five species and four genera in two families of Nepomorpha, Belostomatidae and Nepidae (Insecta: Heteroptera). The following five synapomorphic relationships were recognized for the fine structures of spermatozoa: 1) Nepomorpha have acrosome displaced to a lateral position with respect to the nucleus; 2) the centriole adjunct is well developed in mature spermatozoa; 3) each of the two mitochondrial derivatives contains two or three crystalline bodies; 4) the derivatives are linked to the axoneme by two bridges at the level of doublets 1 and 5; and 5) accessory bodies are absent in this group. A brief comments on the phylogenetic relationships among three families (Belostomatidae, Nepidae and Notonectidae) of Nepomorpha is given. INTRODUCTION The aquatic heteropteran group Nepomorpha (=Cryptocerata or MHydrocorisa; Insecta) re- corded in Korea comprise 32 species, belonging to 16 genera of 8 families. Lee [1] attempted the reconstruction of Nepomorpha phylogeny based on adult characters, in particular fine structures of genitalia. Though comparative spermatology seems to provide useful information for heteropteran sys- tematics, only few papers have been published on Nepomorpha. Itaya er al. [2] described fine struc- tures of the later stages of spermiogenesis in Leptocoris trivittatus Provancher (Corixidae), but did not refer to mature spermatozoon. Descrip- tions of the sperm in Nepa rubra L. (Nepidae) were given by Werner [3]. In Nepomorpha as a whole, a few works on mature spermatozoa of the following species have been made: Diplonychus esakii Miyamoto et Lee [4] (Belostomatidae), Ranatra unicolor Scott [5] and Laccotrephes japonensis Scott [6] (Nepidae) and Notonecta glauca L. [3, 7-9] (Notonectidae). In Notonectidae no information on the centriole Accepted May 30, 1992 Received October 26, 1991 adjunct is available. Recently Lee and Lee [5] reported the centriole adjunct of mature spermato- zoa of Ranatra unicolor Scott. In this paper we report and compare some ultrastructures of the spermatozoa and sperma- togenesis in Nepomorpha. MATERIALS AND METHODS The following five species of Nepomorpha were examined in this study: Belostomatidae— Muljarus japonicus, Diplonychus esakii; Nepidae —Ranatra unicolor, R. chinensis, Laccotrephes Japonensis. They were all collected near Taegu, Korea. For transmission electron microscopy, testes and seminal vesicles of the adult males were fixed in 2.5% glutaraldehyde in a 0.1 M cacodylate buffer, rinsed in 0.1M cacodylate buffer, pH 7.2, post- fixed in 1% osmium tetroxide for 40 minutes, dehydrated in ethanol, and embedded in Epon 812. Ultrathin sections were stained with uranyl acetate and lead citrate and examined in a JEOL 100 S and a Hitachi H-600. For light microscopy, a drop of suspension of spermatozoa obtained by squeezing the seminal vesicle in cacodylate buffer was smeared on a slide, covered with a slide cover and photographed with 972 Olympus multipurpose microscope. RESULTS AND DISCUSSIONS Morphology of the spermatozoa and sperma- togenesis in Nepomorpha Spermatozoa and the structural changes that Y. H. LEE anp C. E. LEE occur during spermatogenesis In the five species of Nepomorpha have been examined. The spermato- zoa of the Nepomorpha vary in length in different species, from 210 4m or less to 16.5 mm or more. The head is from 20 4m to 1.16 mm long (Table 1). 1.1 Belostomatidae Muljarus japonicus has spermatozoa measuring B: Belostomatidae; Ne: Nepidae; N: Notonectidae. TABLE 1. Summary of the morphology of the sperms and spermatogenic cells in 6 species of Nepomorpha B Ne N M D R R L N Character state Character j e u c j g = = 200-220 um or less = — 221-240 um = 241-260 ~m '— Total length of the sperm = 350-370 vm — 15mm or more H =z = | 30-40 ~m or less — _ SL SU att ‘— Head length = 61-70 em = 1mm or more = oe ee eee ee |e Lateral disposition o- —_— | 0.1-10 pm? ~— '— Acrosome — | 11-50 zm? = 51-100 zm? i— volume + a | 10,000 pm or more — = oe eee | eee Compact _— Texture of acrosome a Not compact = Absent a ett Cane ce Microtubules within acrosome Present aan mee eee eee ee oe Tubular type Subacrosomal lumen oe ee fee ees |S Circular type — : ; Nuclear section — | Kidney type a om ee eee ee |CND Long = ’ Centriole adjunct =e ee Abundant = | oe eee eee eee see | Axoneme (9+ 9+2) aa em ee oe Bridges between A and MD -— Tail oe eee eee ee ee MD crystallization ~ Equal — Eh a ee i MD symmetry wee | Not equal = (2 ee ee ee ||! Mj: Muljarus japonicus; De: Diplonychus esakti; Ru: Ranatra unicolor; Re: Ranatra chinensis, Lj: Loccotrephes japonensis, Ng: Notonecta glauca, ND: no data. Spermatogenesis of Nepomorpha 973 about 260 “m in total length (Fig. 1), 0.4 ~m in diameter at the head and 0.6 um at the tail, the head being 30-35 um long. D. esakii has sperma- tozoa measuring 210 “m in total length, the head being 65 um long (Fig. 10). Spermatozoa of D. esakii are longer and the heads are shorter than those of M. japonicus. The acrosome is displaced to a lateral position with respect to the nucleus in the family (Fig. 2) as well as in the other examined groups of Nepomor- pha (Figs. 11, 16, 26, 30). The family has an acrosome of very small size, approximately 1.4 yam®* in volume in the early spermatid of Muljarus japonicus (Fig. 6) and 0.9 um in Diplonychus esakiit. The acrosome of M. japonicus is composed of an electron-dense material during the spermatid differentiation (Fig. 6), whereas that of D. esakii is very low in electron density (Fig. 11). The nucleus of the sperm shows kidney-shape in its cross section during the differentiation of the spermatid (Figs. 3, 13). The morphology of the nucleus is distinctly different when compared with those of other families of Nepomorpha (Table 1). The nuclear membrane holds in its posterior con- cavity the centriole adjunct in early spermatid and they form the nucleus-centriole adjunct complex (Fig. 5). The spermatid tail elongates tangentially at the proximal portion of the nucleus (Fig. 4). The centriole adjunct is abundant and displaced to a lateral nucleus in mature spermatozoa (Figs. 8, 12). The sperm of this family has a “9+9-+2” axonemal pattern (Figs. 9, 14). A sperm bundle contains 1024 spermatozoa (Fig. 7), the mitosis occurring eight times at the spermatogonia. 1.2 Nepidae Ranatra unicolor spematozoa are about 225 um long (Fig. 15); the head about 20 ~m long and 0.5 ym wide at the base and 0.4 wm wide at the tip. The mature acrosome is approximately 5.83 ~m long and 0.33 um thick (Fig. 16). The acrosome contains 220-250 A wide longitudinal tubules through its length (Fig. 17) [5]. The acrosome of the early spermatid is approximately 30 ~m* in volume (Fig. 18). R. chinensis spermatozoa are about 220 4m long (Fig. 24); the head about 20 um _long and 0.8 ~m wide at the base, 0.5 ~m wide at the tip. The early acrosome of approximately 20 7m? in volume is smaller than that of R. unicolor. The filiform spermatozoa of japonensis are about 370 um long (Fig. 29); the head about 45 ~m long and 0.33 ~m wide at the tip and 0.5 ~m wide at the base. A sperm bundle of Laccotrephes L. japonensis contains 512 spermatozoa (Fig. 35). The acrosome forms a spear with the hooked tip. The posterior surface of the acrosome invaginates to the one side of the nucleus (Fig. 30). The acrosome of the early spermatid is approximately 2.5 wm in diameter and 65.4 ~m* in volume (Fig. 36), which is the largest one of the Nepidae. The acrosome of Nepidae is much longer and larger than that of Belostomatidae and also forms an apical cap, extending on one side along the anterior nucleus (Figs. 16, 30). The family has a prominent acrosome which contains tightly packed tubules (Fig. 17). An acrosome with tubules has been described from Gerromorpha but not from acrosomes of other animals [10, 11]. Dallai and Afzelius [12] have proposed that this type of acrosome represents an adaptation to life in fresh water. The subacrosomal space forms an elon- gated tube extending to its anterior end through the acrosome. The nucleus is a cylindrical rod with highly condensed chromatin, but apically is narrowed and surrounds the acrosome which is in cross section embedded in an opened thick ring of the nucleus (Figs. 19, 26, 31). The anterior and posterior nuclear cross-sections contain the acrosome and the centriole adjunct of crescent-shape respective- ly (Figs. 21, 26, 27, 34). The family has well-developed centriole adjunct of very elongated and electron dense structure in mature sperm (Figs. 20, 21, 25, 27, 33, 34). The centriole adjunct of R. unicolor lengthens to at least 10 ~m more in nearly mature spermatozoa (Fig. 20). It extends anteriorly lateral to the basal nucleus and surrounds posteriorly the outside of the mitochondrial derivatives and flagellum. The lateral position of the centriole adjunct is very characteristic in Nepidae as well as Belosto- matidae. In particular two families have abundant centriole adjunct displaced to a lateral nucleus in mature spermatozoa (Figs. 5, 8, 12, 20, 25, 33). Y. H. LEE AND C. E. LEE 974 Spermatogenesis of Nepomorpha 975 Fics. 1-9. Muljarus japonicus (Belostomatidae). Abbreviations in these and subsequent figures: A=acrosome; Ax=axoneme; N=nucleus; CA=centriole adjunct; MD=nmitochondrial derivatives x 30,000. Fic. 1. Mature spermatozoa. Scale bar; 25 um. Fic. 2. Acrosome displaced to a lateral nucleus. 6,000. Fic. 3. Cross section of nucleus in spermatid. 18,000. Fic. 4. Tail elongating tangentially at the proximal portion of the nucleus. Fic. 5. Longitudinal section of head-tail junction. x 15,000. Fic. 6. Acrosome of the early spermatid. x 10,000. Fic. 7. Cross section of a sperm bundle. 2,300. Fic. 8. Cross section of nucleus-centriole adjunct complex. 20,000. Fic. 9. Cross section of tail. 30,000. The nucleus-centriole adjunct complex follows two mitochondrial derivatives which surround the axoneme. The microtubules are ordered in hex- agonal and clover arrangement around the nuc- leus-centriole adjunct complex during the sperma- tid differentiation (Figs. 23, 37). The tail appears symmetrical. It is made up of an ordinary axoneme and two mitochondrial de- Tivatives of equal size. These components are arranged throughout the length of the tail. The mitochondrial derivatives are the bilateral sym- metry of the axoneme (Fig. 22). They surround most of the axoneme in Nepidae (Figs. 22, 28, 32). The horizontally oriented cristae exhibit a very regular periodicity of approximately 55—-S7 nm in R. unicolor (Fig. 20), 47 nm in R. chinensis (Fig. 25) and 41 nm in Laccotrephes (Fig. 33). The axoneme belongs to the basic 9+-9+-2 pat- tern, having evident radial links with prominent heads, evident dynein arms on the doublets, and a 976 Y. H. Lee AND C. E. LEE a Be Fics. 10-14. Diplonychus esakii (Belostomatidae). Acrosome showing very low electron density in spermatid. 42,000. x 69,000. Fic. 10. Mature spermatozoa. Scale bar; 25 um. Fic. 11. Fic. 12. Cross section of nucleus-centriole adjunct complex. Fic. 13. Cross section of nucleus in spermatid. X 12,000. Fic. 14. Cross section of tail. 49,000. layer of 9 accessory tubules. The diameter of the crystallization of the mitochondrial derivatives may measures more or less half of the section in the all examined group (Figs. 9, 22, 28, 32). The axoneme is flanked by two mitochondrial deriva- tives but lacks accessory bodies, such as are found in spermatozoa from Homoptera and many related groups of insects [13]. Three further features are also characteristic in Nepomorpha as follows: (1) the presence of two bridges that join the mitochondrial derivatives to the axoneme at the level of the microtubular doublets nos. | and 5; (2) the occurrence of two or three crystalline bodies in the mitochondrial de- rivatives, rather than a single one as is common in pterygotes; (3) the abundant centriole adjunct in mature sperm. The third feature seems to be unique to spermatozoa of Nepomorpha, such as is found in spermatid of Pentatomidae but has not been observed in mature sperm [14]. 1.3 Notonectidae 7 Estimations of the sperm length have ranged between 14mm for Notonecta maculata [15] to 16.5 mm for N. glauca [16]. They are the longest one among the animal kindgom. N. glauca de- scribed previously [3, 7-9] are reanalysed in the light of phylogeny of Nepomorpha. Two special structures appear in connection with the nucleus during spermatogenesis in Notonecta species. These are pseudochromosomes in sper- matocytes and nuclear calottes in mid spermatids. Spermatogenesis of Nepomorpha Fics. 15-23. Ranatra unicolor (Nepidae). Fic. Fic. Fic. Fic. Fic. Fic. Fic. Fic. Fic. 1), 16. 7. 18. 19. 20. 21. 22. 23. Mature spermatozoon. Scale bar; 50 ~m. Longitudinal section of acrosome. X 12,000. Cross section of acrosome containing tightly packed tubules. 30,000. Acrosome of the early spermatid. 12,000. Cross section of the head. 24,000. Longitudinal section of centriole adjunct. 8,000. Cross section of nucleus-centriole adjunct complex. 24,000. Cross section of-tail. 60,000. Cross section of microtubules between the nucleus and a membranous sleeve. x 30,000. Y. H. Lee ann C. E. LEE Longitudinal section of sperms showing nucleus-centriole adjunct complex and tail. 30,000. Fics. 24-28. Ranatra chinensis (Nepidae). Fic. 24. Mature spermatozoon. Scale bar; 25 um. Fic. 25. Fic. 26. Cross section of head. 32,000. Fic. 27. Cross section of nucleus-centriole adjunct complex. 32,000. Fic. 28. Cross section of tail. 32,000. In spermatocytes the membrane system consists of a shell of two or three parallel cisterns enclosing the whole nucleus at a certain distance from its envelope. In young spermatids remnants of the former shell change to spherical calottes and be- come attached to the nucleus. After separation from the nucleus it ultimately disintegrates to whirls of cisterns still visible in late spermatids [8]. The acrosome grows to a sphere measuring about 20 um in diameter and 33493.33 ~m®* in volume. During elongation the acrosome attains spindle shape, the lateral process gradually becom- ing its anterior part [7]. The acrosome of the backswimmer resembles somewhat that of two other hemipterans, Nepa rubra L. [17] and Gerris remigis [10]. At its posterior end the acrosome has a lateral groove. It appears empty at some levels but contains the nucleus and the centriole at a more posterior level. The axoneme is of a common type among insects with a 9+9-+ 2 pattern, and it is unusual only by its great length. The larger mitochondrion occupies Spermatogenesis of Nepomorpha Fics. 29-34. Laccotrephes japonensis (Nepidae). Fic. 29. Fic. 30. Fic. 31. Fic. 32. Fic. 33. Fic. 34. Mature spermatozoon. Scale bar; 50 um. Longitudinal section of head. 16,000. Cross section of head. 9,000. Cross section of tail. 30,000. 979 Longitudinal section of sperms showing nucleus-centriole adjunct complex and tail. 12,000. Cross section of nucleus-centriole adjunct complex. x 30,000. 980 Y. H. Lee AND C. E. LEE Fic. 35. Cross section of a sperm bundle. 4,000. Fic. 36. Acrosome of the early spermatid. 10,000. Fic. 37. x 18,000. 70-80% of the tail cross-section, the smaller one 10-15%. The two mitochondria are completely embracing the axoneme. The mitochondria have regularly arranged cristae. The three paracrystal- line bodies can be recognized in both the small and the large mitochondrion. The shapes of the bodies differ in the two mitochondria. The paracrystalline bodies occupy most of the mitochondrial volume [7]. 2. Phylogenetic relationships among three fami- lies in Nepomorpha based on the spermatozoal ultrastructures In an investigation of spermatozoal ultrastruc- ture from six species of Nepomorpha, the position of the acrosome and centriole adjunct appears to be specific for Nepomorpha and the centriole adjunct is well developed in mature spermatozoa. Diplonychus esakti and Muljarus japonicus are Cross section of microtubules between the nucleus-contriole adjunct complex and a membranous sleeve. easily distinguished by the following characteris- tics: (1) the total length of the sperm of M. Japonicus is longer than that of D. esakii, on the other hand the head length is shorter than that in D. esakii; and (2) the acrosome has low electron density in the spermatid of D. esakii, while M. japonicus has a dense electron acrosome through the sperm differentiation. The differences be- tween the two species are evident and they are not closely related, although they are grouped to the same genus, until Lee [1] established a new genus Muljarus for the species of japonicus groups. Two species of Ranatra are closely related by the characteristics showed in Table 1. Laccotrephes has many resemblances to Ranatra in nucleus, acrosome and centriole adjunct and two genera should be related to each other closely grouped. Both Belostomatidae and Nepidae are closely related based on the ultrastructures of acrosome, Spermatogenesis of Nepomorpha 981 subacrosomal lumen, centriole adjunct and mito- chondrial derivatives, though they are different in the morphology of the nucleus during the dif- ferentiation and in the microtubules within the acrosome. On the contrary, Notonecta are char- acterized by the ultrastructure of mitochondrial derivatives with two different sizes and the giant sperm and the acrosome. Miyamoto [18] considered that the Nepidae and Belostomatidae are of the same evolutional direc- tions in the structures of the alimentary organs. Cobben [19] lined Nepidae with Belostomatidae based on the distribution of the larval abdominal glands and the structures of stylets. Lee [1] also grouped the Belostomatidae with Nepidae based on the genital structures and subdivided Nepomor- pha into four family groups, Belostomatidae and Nepidae; Notonectidae, Pleidae and Helotrepi- dae; Aphelocheiridae, Naucoridae, Gelastocor- idae and Ochteridae; and Corixidae. Moreover he suggested that they do not show any phylogenic trend in genital structures of the four groups and the different characters might have been derived from the polyphyletic terrestrial origins. ACKNOWLEDGMENTS This study was supported in part by the Basic Science Research Institute Program, Ministry of Education, Korea, 1991. REFERENCES 1 Lee, C. E. (1991) Morphological and phylogenetic studies on the true water bugs (Hemiptera, Heterop- tera). Nature & Life (J. Nat. Hist. Soc. Korea), 21: 1-183. 2 Itaya, P. W., Thompson, S. A. and Heidger, P. M. (1980) Fine structure of late stages of sper- miogenesis in Leptocoris trivittatus Say (Hemiptera, Corizidae). Int. J. Insect Morphol. & Embryol., 9: 135-145. 3 Werner, G. (1986) Change in the Golgi apparatus during spermiogenesis in the backswimmer, Notonecta glauca. Biol. Cell, 57: 169-180. 4 Lee, H. S. and Lee, Y. H. (1988) Ultrastructure of spermatogenesis in Diplonychus esakii (Heterop- tera; Belostomatidae). Korean J. Entomol., 18: 49- 63. 5) 10 11 12 13 14 15 16 17 18 19 Lee, Y. H. and Lee, C. E. (1991) Ultrastructural study of spermiogenesis and mature spermatozoa of Ranatra unicolor with special reference to centriole adjunct (Heteroptera, Nepidae). Japanese J. En- tomol. 59: 71-82. Lee, Y. H. and Lee, C. E. (1987) Ultrastructural studies of spermiogenesis in Laccotrephes Japonensis. Korean J. Entomol., 17: 199-214. Afzelius, B. A., Baccetti, B. and Dallai, R. (1976) The giant spermatozoon of Notonecta. J. Submic- rosc. Cytol., 8: 149-161. Werner, G. (1985) So-called pseudochromosomes and nuclear calottes and their changes during sper- matogenesis in the backswimmier, Notonecta glauca. Biol. Cell, 53: 127-140. Werner, G., Afzelius, B. A. and Mosler, B. (1988) Acrosome formation during spermiogenesis in Notonecta glauca L. (Heteroptera). J. Submicrosc. Cytol. Pathol., 20(1): 123-135. Tandler, B. and Moriber, L. G. (1966) Microtubu- lar structures associated with the acrosome during spermiogenesis in the water-strider Gerris remigis (Say). J. Ultrastruct. Res., 14: 391-404. Lee, Y. H. (1985) Spermatogenesis of the water strider, Gerris paludum (Heteroptera, Gerridae). J. Ultrastruct. Res., 90: 235-250. Dallai, R. and Afzelius, B. A. (1980) Characteris- tics of the sperm structure in Heteroptera (Hemip- tera, Insecta). J. Morph., 164: 301-309. Phillips, D. M. (1970) Ultrastructure of spermato- zoa of the woolly opossum, Caluromys philander. J. Ultrastruct. Res., 33: 381-397. Trandaburu, V. (1973) The acrosome and the centriole adjunct in Eurydema ventralis Kol. (Heter- optera, Pentatomidae). Trav. Mus. Hist. Nat. “Gri- gore Antipa.”, 13: 137-145. Poisson, R. (1927) Recherches sur quelques proces- sus spermatogénétiques observés dans les éléments sexuels jeunes. Arch. zool. exptl. Gen., 66: 23-70. Larsen, O. (1938) Untersuchungen tber den Ges- chlechts-Apparat der Aquatilen Wanzen. Opusc. Entomol., Suppl. 1: 1-388. Werner, G. (1966) Untersuchungen tiber die Sper- miogenese bei einem Laufkafer, Carabus catenula- tus Scop., und der Skorpion-Wasserwanze, Nepa rubra L. Z. Zellforsch., 73: 576-599. Miyamoto, S. (1961) Comparative morphology of alimentary organs of Heteroptera, with the phy- logenetic consideration. Sieboldia, Fukuoka, 2: 197-259, Pls. 20-49. Cobben, R. H. (1978) Evolutionary trends in Heter- optera, Part II. Mouthpart-structures and feeding strategies. Meded. Landbouwhogeschool Wage- ningen, Nederland, 78-5: 27-38, 187-254. q sla perv arpa cas at opto ae: es fe es Toe eb es e me : : vr Pa i taal | wagutite ow om _ VR aig ; role Pus ay A te Mi ; aR oe nee Ope ain Oe rs fi, “el \Saeohie seal m get Ate ree A i: Pa ue fi ) f a by i Ty Tole 4 del > 8 ate 5b i } \ i iL ‘ , ‘ iy : fe Rm | 7 | ZOOLOGICAL SCIENCE 9: 983-988 (1992) © 1992 Zoological Society of Japan Self-Nonself Recognition in the Colonial Protochordate Botryllus schlosseri from Mutsu Bay, Japan‘ Barucu RINKEVICH~ and YASUNORI SAITO” National Institute of Oceanography, Israel Oceanographic & Limnological Research, Tel-Shikmona, P.O.B. 8030, Haifa 31080, Israel and 3Shimoda Marine Research Center, University of Tsukuba 5-10-1 Shimoda-shi, Shizuoka 415, Japan ABSTRACT— Wild Botryllus schlosseri collected from a 5X5 m area in Mutsu Bay (Aomori Prefecture, Japan) were tested for alloresponses in intrapopulation colony allorecognition assays (CAAs). Results indicate that rejection patterns are similar to those recorded previously in the populations from Monterey and Santa Barbara, California, from the Mediterranean coast of Israel, and from the Venetian lagoon, Italy. The only difference was the marked accumulation of bright-yellow blood cells in the tips of interacting ampullae. Pairwise CAAs which were performed on all combinations (n=91) from 14 colonies resulted in 12.1% fusions, which gives a populational estimation of 32 alleles on the fusibility locus. A fusibility chart for these 14 genotypes revealed 18-19 different, not equally frequent, allorecognition alleles, of which 12 were assigned to only one genotype, each; 5 occurred twice, and each one of 2 alleles was present in three different genotypes. It is concluded that the sampled area was too small to represent the probable higher number of fusibility alleles residing in this population. INTRODUCTION It is now almost 90 years that studies on the colonial tunicate Botryllus schlosseri (Pallas) have shown its capacity for colony specificity [1], a histocompatibility system which resembles the ma- jor histocompatibility complex (MHC) of the vertebrates in many aspects [2]. The genetic basis for the colony specificity resides in a single, highly polymorphic haplotype (called the fusibility /histo- compatibility locus, Fu/HC [3]), which possesses multiple codominantly expressed alleles. Interact- ing colonies which do not share any allele on this locus reject each other, while colonies which share in common at least one allele on the Fu/HC locus may undergo a natural transplantation (fusion) by forming vascular anastemoses between their peripheral ampullae [2, 4-6]. B. schlosseri is a cosmopolitan inhabitant of shallow water, hard bottom communities [7, 8]. Accepted July 1, 1992 Received April 16, 1992 ' Contribution number 541 from the Shimoda Marine Research Center. * To whom all correspondence should be addressed. This species has been studied for intrapopulation allorecognition responses in 4 remote localities: the population from the Venetian lagoon, Italy [6, 9], from the Mediterranean coast of Israel [10], from Woods Hole, Massachusetts, Atlantic Ocean [2, 11, 12], and from the Monterey and Santa Barbara areas, California, Pacific Ocean [11-15]. Results indicated that allogenic interactions be- tween ampullae of noncompatible Botryllus con- specifics exhibited not only the species-specific characteritics of rejection processes, but also population-specific rejection types which were constantly expressed even in interpopulation en- counters [11, 12]. When two incompatible colonies of the Woods Hole population came into tunic- tunic contact, a limited fusion of the cortical layers of both colonies prevailed, which resulted in a continuous tunic matrix between both partners. This permitted a reciprocal ampullae penetration which was followed by ampullae amputation and/ or haemorrhages formation, the development of dark-brown necrotic areas, points of rejection (PORs). In the other 3 studied B. schlosseri populations, on the other hand, the outer layers of the tunics did not fuse during allogeneic encoun- 984 B. RINKEVICH AND Y. Saito ters. As a result, the cortical layers were clearly observed as demarcating lines between colonies. PORs, therefore, were developed without recip- rocal ampullae penetration. The above studies [12, 13] have suggested that an extended comparison of intrapopulation allogeneic interactions would be of great benefit for a better understanding of self/nonself histo- compatibility alloresponses of this cosmopolitan species. Here we study allorecognition of a B. schlosseri population from Mutsu Bay, Japan, and try to evaluate the polymorphism pattern of the Fu/HC locus by calculating frequencies of fusion within a population sampled from a small area. MATERIALS AND METHODS Wild B. schlosseri colonies were collected (November 1991) in Mutsu Bay, Aomori (Aomori Prefecture), where they were grown in shallow water on floating Pecten culturing cages. All colonies were collected from a5 x5 m area. Large healthy colonies were removed from substrates by razor blades, tied with thin cotton fibers onto 2.5 x 7.5cm glass slides, and shipped to Shimoda Marine Research Center, where they were kept vertically in slots within a wooden culture box, submerged in Nabeta Bay, Shimoda. For the colony allorecognition assays (CAAs), we used small groups of zooids at the growing edges of colonies, carefully isolated from colonies. These subclones were attached in pairs on 5X7.5 cm glass slides, as described previously [11-15]. Dur- ing the experiments, the colonies were kept in 17-liter standing seawater tanks, aerated by air- stones and maintained at 17-18°C by aquarium heaters. CAAs were observed daily and cleaned during the observations by soft small brush. Food was supplied daily (artificial diet; Liquifry Marine, England). RESULTS Intrapopulation alloresponses Contacts between extended ampullae of encoun- ter colonies were established within 24 hr after forming the CAA. Tips of marginal ampullae were reciprocally positioned in tip-tip orientation where the cortical layers of the allogeneic partners were clearly observed as demarcating lines along contact areas. When compatible pairs were assayed, the tunic matrices eventually fused in limited or in broader areas. Ampullae of both partners, or of only one partner in a pair, penetrated into the tunic matrix of the other colony through the fusion areas and were positioned in tip-ampulla base (proximal part) orientations. This resulted, within less than 24 hr, in allogeneic anastomosis of blood vessels, the formation of chimeras. Chimeras were followed up for a period of one month. During this period, chimerism in several assays already ended in the resorption of all zooids of one partner each. When nonfusible partners come into direct con- tact, tunics did not fuse together, so ampullae engaged each other reciprocally but indirectly, through both cortical layers (Fig. la-i). During this process, the tips of interacting ampullae, and sometimes also the tips of all peripheral ampullae (even those of the other sides of the colony), of one or both partners in a pair, became very distinctive in bright fluorescent yellow color. This resulted from aggregations of bright yellow blood cells which accumulated in the ampullar tips (Fig. la, b). This phenomenon was also observed in interacting colonies of the Monterey population [11, 12] and the Mediterranean colonies [10]. By employing histological examinations, we (Terta- kover and Rinkevich, unpublished) characterized the accumulated cells as morula cells. However, the Mediterranean and Monterey populations did not exhibit the deeper intensity in color and the high frequency of cases as it is recorded in the Japanese population. In most of the yellow col- ored tips, the intense yellow color gradually abated and disappeared within the next 24—72 hr, while in some of the tips the color became dark-brown, blood cells infiltrated out of the ampullar tips, and the formation of PORs through haemorrhages was documented (Fig. la-i). In a few cases, ampullae were amputated from the peripheral blood vessels and gradually disintegrated. Only a few (1-8) out of many (up to tens) of interacting ampullae produced cytotoxic lesions (Fig. la, e). Following the acute phase of allogeneic response, where all PORs were developed within a short period of a Allorecognition in Japanese Botryllus 985 Fic. 1. Allogeneic interactions between B. schlosseri colonies: genotype combination 3 (left) vs 2 (a-d), and genotype combination 10 (left) vs 3 (e-1). a-48 hr after CAA. Six small reciprocal PORs along the contact area (arrowheads) out of 12 vs 17 interacting ampullae. Tips of ampullae (genotype 3) still possessed aggregations of bright yellow cells in the contact area and in other peripheral ampullae (confined between two white arrowheads). _b-Two days thereafter. No more PORs were added. Ampullae were cleared of yellow cell aggregations, and only 9 vs 7 were still interacting. Genotype 2 started retreat growing [14]. c-Two days later. Only a few colony 2 ampullae were still in contact with those of colony 3. Colony 2 zooids retreated 1-2 mm. d-7 days later. Zooids of colony 2 retreated more than 3 mm from the initial location, leaving an unvascularized tunic matrix with rudiments of blood vessels/ampullae and masses of degenerated infiltrating blood cells (small black arrowheads). e-48 hr after doing CAA. 17 vs 14 interacting ampullae, respectively. 7 PORs (arrowheads) were all produced by colony 10, but some were not yeat well developed. In genotype 10, the upper peripheral ampullae still had the yellow color. f-Two days thereafter. All former PORs were fully developed and no more were added. Ampullae were cleared of yellow color. g-h-4 days later. Some of the large necrotic lesions were diffused and dispersed (arrowheads), forming a black line along the contact area between the two genotypes. Interacting ampullae of both colonies (predominantly of genotype 10) retreated, leaving behind degenerated ampullae and masses of dying cells (small black arrowheads). 1-5 days thereafter. Ampullae of both genotypes reciprocally retreated, leaving degenerating areas. Scale of bars: a-g, i=l mm. h=0.25 mm. few days (usually within 48 hr), other ampullae enhanced by the “retreat growth phenomenon” continued to interact for longer periods, but with- _[14], where fewer buds than zooids per generation out the formation of any more PORs (Fig. 1d, g-i). were developed in the contact area, resulting in a It was also evident in many cases that during POR directional colony growth form, away from in- development and thereafter, ampullae of one or _ teracting zones (Fig. 1b-d). both colonies in a pair withdrew from the contact areas, leaving a bare tunic which gradually deterio- Pairwise allorecognition assays rated (Fig. 1d, h, i). This process was sometimes We examined 14 Japanese B. schlosseri colonies 986 B. RINKEVICH AND Y. SAITO in a pairwise allorecognition panel of all 91 com- binations. Fusions were recorded in 11 (12.1%) allogeneic assays (Fig. 2a, striped squares) as well as in all the controls, the isogeneic combinations (Fig. 2a, striped triangles). An Fu/HC chart for the studied 14 genotypes (Fig. 2b) revealed 18 to 19 different fusibility alleles. Genotypes 11 and 2 are clearly distinguished from each other since they possess different color morphs. However, according to the predictions of the fusibility model (legend to Fig. 2b), they share in common either one or both Fu/HC alleles. Twelve out of the maximum 19 Fu/HC alleles (63.2%) are assigned to only one colony each, while 5 alleles (F, I, J, P, R) (26.3%) occurred twice, and 2 alleles (M, N) (10.5%) occurred three times. N | {| {| Colony Allelic combination Colony Allelic combination | | | | | ABCDEFGHIJK]| "° LMNOPQRS me Ng y Fic. 2. A pairwise mer panel between 14 Japanese B. schlosseri colonies (A) and their Fu/HC allelic combination chart (B). A-Each rejecting CAA set is marked by a blank square, fusible pair by a striped square. All isografts resulted in fusions (striped triangles). B-The chart depicting predictions for a single Mendelian locus model of partial genetic matching for histocompatibility [2, 4-6], an exclu- sive heterozygosity of the Fu/HC locus in wild genotypes [2, 12], and a codominant expression by Fu/HC alleles [2, 4-6]. Colony 11 is marked also by allele E in brackets to note that it can share with colony 2 one or both alleles on the Fu/HC ha- plotype. DISCUSSION Intrapopulation allorejection processes in B. schlosseri from Mutsu Bay, Japan, are of like pattern as in the populations from the Venetian lagoon, from the Mediterranean coast of Israel and from the Monterey-Santa Barbara areas [6, 9, 10— 15]. The only difference which we could detect which characterized the Japanese population from all the above is the marked accumulation of yellow colored cell aggregations in the tips of interacting ampullae. Therefore, out of the 5 studied B. schlosseri populations up to date, only the Woods Hole population differs significantly from all the others when comparing between the effector mechanisms which are expressed during intra- or interpopulation interactions [11, 12, 16]. Since B. schlosseri is found in many additional localities around the world [7, 8, 11], further study is required to find whether the Woods Hole popula- tion possesses a unique variation of alloresponses within this taxon. Different populations of B. schlosseri are prob- ably characterized by a highly polymorphic pattern of alloreactivity. In the Mutsu Bay population, sampling from a very small area of 5 by 5m revealed only 12.1% of fusions. In the Woods Hole population, two different studies have found 6.2% of fusions out of 1,262 contiguous borders of colonies [17] and 4.2% fusions out of 500 pairs of colonies collected randomly along a 20 m transect [18]. Similar low percentage numbers of fusions were obtained from the Israeli B. population (Porat and Rinkevich, unpublished). These studies and others [19] reflect the existence of approximately 100 histocompatibility alleles in each studied B. schlosseri population. In Botryllus primigenus, pairwise fusibility tests revealed a similar low percentage of fusions [5, 20, 21]. Botrylloides fuscus, on the other hand, panels of pairwise combinations between colonies which were collected from 3 localities, 1-3 km apart, resulted in 64.0-74.0% of fusions. This indicates a much lower polymorphism on the Fu/HC locus of schlosseri this species [22]. The 14 genotypes from Mutsu Bay which were collected randomly from a very small site possess 18 to 19 alleles on their fusibility locus, of which at least 2 are commonly shared with the Monterey, CA, population (colonies 5, 9, 11 fused with Monterey colonies [16]). Based on the results of 12.1% fusions, we may estimate [23] the numbers Allorecognition in Japanese Botryllus of alleles on the Fu/HC haplotype in the Mutsu Bay population as approximately 32. However, it is clear that this estimation is minimal and that our sample does not represent the probable higher number of alleles residing in the Fu/HC locus of this population, since colonies were collected from a very limited (5x5 m) area. Moreover, calcula- tions are based on the prediction that all the alleles on the fusibility locus are equally frequent [23], which is not the case of the present study (Fig. 2b). This skews the figure for the number of histocom- patibility alleles and also marks the difficulty of using CAAs as the only implementation for asses- sing polymorphism on the Fu/HC haplotype. Some of these colonies responded to intrapopula- tion allogeneic encounters by the “retreat growth phenomenon” [14]. In assays resulting in fusion, one of the partners in the chimera was usually resorbed [3, 9, 13], a result which was recorded before in other B. schlosseri populations as well. These outcomes suggest that botryllid ascidians have highly complex systems of effector mechan- isms, all controlled by a variety of histocompatibil- ity genes. ACKNOWLEDGEMENTS Thanks are due to Dr. Numakunai for helping in colony collection and to T. Ishii and A. Dasai for general assistance. The staff of the Shimoda Marine Research Center is acknowledged for their hospitality and assist- ance. This study was supported by a grant from S. Price, North American Friends of IOLR, by a JSPS Fellowship for Research in Japan (B.R.), and by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, nos. 03455008 and 01304007 (Y.S.). REFERENCES 1 Bancroft, F. W. (1903) Variation and fusion of colonies in compound ascidians. Proc. Calif. Acad. Sci., 3: 137-186. 2 Scofield, V. L., Schlumpberger, J. M., West, L. A. and Weissman, I. L. (1982) Protochordate allorecognition is controlled by an MHC-like gene system. Nature, 295: 499-502. 3 Weissman, I. L., Saito, Y. and Rinkevich, B. (1990) Allorecognition histocompatibility in a protochor- date species: Is the relationship to MHC semantic or structural? Immun. Rev., 113: 227-241. 4 10 16 Ww) 18 19 987 Oka, H. and Watanabe, H. (1957) Colony specific- ity in compound ascidians as tested by fusion experi- ments. Proc. Japan Acad., 33: 657-659. Oka, H. and Watanabe, H. (1960) Problems of colony specificity in compound ascidians. Bull. Mar. Biol. Stn. Asamushi, 10: 153-155. Sabbadin, A. (1962) La basi genetiche della capaci- ta di fusione fra colonie in Botryllus schlosseri (Ascidiacea). Rend. Accad. Lincei, 32: 1031-1035. Berrill, N. J. (1950) The Tunicata. Ray Society, London. Tokioka, T. (1953) Ascidians of Sagami Bay. Iwa- nami-Shoten, Tokyo. Sabbadin, A. and Astorri, C. 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Grosberg, D. Hedgecock and K. Nelson. Plenum, New York, pp. 93-109. Rinkevich, B. and Weissman, I. L. (1992) Incidents of rejection and indifference in Fu/HC incompatible protochordate colonies. J. Exp. Zool., 263: 105- ii. Rinkevich, B., Shapira, M., Weissman, I. L. and Saito, Y. (1992) Allogenic responses between three remote populations of the cosmopolitan ascidian Botryllus schlosseri. Zool. Sci., 9: 989-994. Karakashian, S. and Milkman, R. (1967) Colony fusion compatibility types in Botryllus schlosseri. Biol. Bull., 133: 473. Grosberg, R. K. and Quinn, J. F. (1986) The genetic control and consequences of kin recognition by the larvae of a colonial marine invertebrate. Nature, 322: 456-459. Scofield, V. L. and Nagashima, L. S. (1983) Mor- phology and genetics of rejection reactions between oozooids from the tunicate Botryllus schlosseri. Biol. Bull., 165: 733-744. 988 20 Tanaka, K. and Watanabe, H. (1973) Allogeneic 21 inhibition in a compound ascidian, Botryllus pri- migenus Oka. I. Processes and features of “nonfu- sion” reaction. Cell. Immun., 7: 410-426. Mukai, H. and Watanabe, H. (1975) Distribution of fusion incompatibility types in natural populations of the compound ascidian, Botryllus primigenus. Proc. Japan Acad., 51: 44-47. B. RINKEVICH AND ye Y. SAITO Hirose, E. (1990) Colony specificity and possible allorecognition sites in Botrylloides (Ascidiae, Com- positae). Ph. D. Dissertation, Univ. Tsukuba, Japan. Grosberg, R. K. (1988) The evolution of allorecognition specificity in colonial invertebrates. Quart. Rev. Biol., 63: 377-412. ZOOLOGICAL SCIENCE 9: 989-994 (1992) © 1992 Zoological Society of Japan Allogeneic Responses between Three Remote Populations of the Cosmopolitan Ascidian Botryllus schlosseri’ BarucuH RINKEVICH~, MICHAL SHAPIRA~, IRVING L. WEISSMAN” and YASUNORI SaITo* National Institute of Oceanography, Tel Shikmona P.O. Box 8030, Haifa 31080, Israel, Howard Hughes Medical Institute, Stanford University Medical Center, Stanford, CA 94305, USA, +Shimoda Marine Research Center, University of Tsukuba, 5-10-1 Shimoda-shi, Shizuoka 415, Japan ABSTRACT— Colony allorecognition assays (CAAs) were performed between colonies of the world- wide distributed tunicate Botryllus schlosseri, sampled from the Mediterranean coast of Israel (Is), from Monterey, California (Mon) and from Mutsu Bay, Japan (Ja). While all 48 Is vs Ja CAAs resulted in nonfusion responses, unexpectedly, 4.4% of the 45 Is vs Mon pairs and 12.0% of the 25 Ja vs Mon assays ended in colony fusions. Allogeneic effector mechanisms in all 3 populations were similar, except for the Ja population which was characterized additionally by the appearance of masses of bright yellow blood cells gathered in the tips of interacting ampullae. A total of 201 multiple CAAs on 24 Is vs Mon, 22 Is vs Ja and 21 Ja vs Mon rejecting pairs did not show an allospecific memory in the rejection phenomenon. Results are discussed in view of the accumulated data on allogeneic responses in 5 remote populations of B. schlosseri. INTRODUCTION Botryllus schlosseri, a world-wide distributed colonial ascidian, is a common member of shallow water hard bottom communities. This species is found in European waters, Japan, the eastern and western coasts of North America, Australia, Hong Kong, and in many other localities [1-4]. This species, most likely of Mediterranean Sea origin, became a cosmopolitan species probably by ances- tral colonies which attached to ship bottoms and were introduced into new localities. Like other botryllid ascidians [5, 6], B. schlosseri colonies show colony specificity resulting either in vascular anastomosis (fusion) between separate parts of the same colony or between two compati- ble colonies, or in rejection between non- compatible colonies [7-10]. This histocompatibil- Accepted July 1, 1992 Received April 16, 1992 ' Contribution number 542 from the Shimoda Marine Research Center. * To whom all correspondence should be addressed. ity discrimination is controlled by a single gene haplotype (termed the tunicate’s fusibility- histocompatibility locus, Fu/HC [11]) with multi- ple codominantly expressed alleles [6-8]. Two colonies sharing no alleles at the Fu/HC locus will reject each other, whereas colonies sharing in common at least one allele on this locus will fuse upon direct contact [5-8]. When studying allogeneic reactions between B. schlosseri colonies collected from Monterey, Cali- fornia (Pacific Ocean) and from Woods Hole, Massachusetts (Atlantic Ocean), Boyd et al. [3] pointed to an interesting result that rejections were usually confined within the tunic and the peripher- al ampullae (sausage-like termini of blood vessels) of only Woods Hole (WH) colonies. Four diffe- rent types of rejections were developed by only WH ampullae which included blood cell infiltra- tion, haemorrhage formation, retraction and ampullae amputation [4]. More intriguing is the result that rejection patterns are somehow diffe- rent in each one of the two tested North American B. schlosseri populations [3, 4]. 990) B. RINKEVICH Here we further analyze allorecognition re- sponses between B. schlosseri colonies from 3 extremely separated populations: an eastern Mediterranean population (from along the coast of Israel), an eastern Pacific population (from Mon- terey, CA) and a western Pacific/Japan Sea population (from Mutsu Bay, Japan). Previous intrapopulational allogeneic assays revealed that rejection patterns are similar in these 3 popula- tions [3, 4, 10, 12, 13]; in short, immediately after the first tunic-tunic contact was established be- tween interacting colonies, tips of the marginal ampullae actively extended in a tip-tip orientation. Rejections were documented without ampullae penetration into the tunic matrix of the opposite colony, and without true matrix fusion. MATERIALS AND METHODS We used laboratory reared B. schlosseri colonies from Israel (Is) and Monterey, CA (Mon), and wild colonies from Mutsu Bay, Japan (Ja). Origi- nally wild Mon colonies were collected from Mon- terey marina and were shipped to the National Institute of Oceanography, Haifa, Israel, in cool condition, where they were assayed against col- onies of the Israeli population. Wild Mediterra- nean colonies were collected from 3 locations along the Israeli coast (Tel-Shikmona, Caesarea, Michmoret). Is and Mon colonies were kept in the laboratory [3, 12, 14], and offspring were collected and reared as described [3, 14, 15]. Assays were performed mainly on reared offspring. Ja colonies were collected in Mutsu Bay (Aomori, Aomori Prefecture) and shipped to Shimoda Marine Re- search Center where they were reared on glass slides in a wooden culture box immersed in Nabeta Bay, Shimoda. Is and Mon cultures were shipped to Shimoda where they were maintained in 17-liter standing seawater tanks until assayed against Ja colonies. Colony allorecognition assays (CAAs) and observations were performed as described [4, 10], and secondary and tertiary tests of CAAs were performed as previously [4, 16]. , M. SHAPIRA ef al. RESULTS Interpopulational allogeneic reactions We performed 45 CAA pairs of Is vs Mon colonies, 48 CAAs of Is vs Ja pairs, and 25 CAAs of Ja vs Mon. While all allorecognition assays of Is vs Ja resulted in nonfusion reactions (either in rejections or in indifference [16]), unexpectedly we observed 2 cases of vascular fusions in Is vs Mon pairs (4.4%) and 3 fusible assays in interacting Ja vs Mon pairs (12.0%). Necrotic lesions (POR=points of rejection) were developed in assays of the three interpopula- tional combinations (Fig. la-i) in a similar way as they were established in _ intrapopulational allogeneic assays [3, 4, 10, 12, 13, 16]. The only significant difference in the allorecognition mor- phology between colonies of the 3 tested popula- tions was the accumulation of bright yellow blood cells (morula cells; Tertakover and Rinkevich, unpublished) which usually gathered in masses within the tips of interacting ampullae of Ja col- onies (this was recorded as well in Ja vs Ja com- binations [13]). These cells disappeared after the formation of a full set of PORs. PORs were produced by colonies belonging to the 3 popula- tions; however, in Ja vs Mon and Ja vs Is combina- tions, the peripheral ampullae of Ja genotypes were extended more actively towards those of their confrere colonies, and more cases were recorded where PORs were developed by Ja ampullae alone (in 50.5% of nonfusible Ja vs Mon cases; Fig. 1b-d, and in 60.0% in Is vs Ja pairs; Fig. la). Cases where PORs developed by only Mon or Is when confronted with Ja colonies were much lower (18.2% and 20.8%, respectively). We observed up to 11 PORs per assay although up to several tens of ampullae interacted reciprocally. Three out of the 4 different characteristic types of PORs [4], including haemorrhages formation (Fig. la-f), ampullae amputation (Fig. 1d, f-h) and formation of an “ampulla POR” were recorded. Withdrawal of interacting ampullae from contact areas was another characteristic outcome of inter- populational allogeneic interactions (Fig. 1b-d). Ampullae regression appeared in colonies of the 3 tested populations, and usually started within 24 hr CAAs between Botryllus Populations 991 “ah a: 5 days after Ja (left) Fic. 1. ampullae produced 2 large and diffused PORs, which resulted through ampullar bleeding (arrowheads). Both previous bleeding ampullae remained intact near PORs (small arrowheads). b: 48 hr after first ampulla-ampulla contacts. One of the Ja (left) ampullae formed an “ampullar POR” [4] and was amputated and formed a large POR (arrowhead). Another ampulla formed a typical POR through bleeding (small arrowhead). Zooids of both partners retreated up to 2mm from the previously interacting site. c-d: 24 and 48 hr, respectively, after first ampulla-ampulla contact was established. Initial Ja (right) ampullar bleeding, which started with 4 PORs (c, arrowheads), progressed by the formation of the Sth POR (d, arrowheads) and by ampullae amputation (small arrowheads). Zooids of both colonies and some of the Mon ampullae retreated from the contact zone. e-i: A case where Is (left) ampullae were amputated after developing PORs. e: 24 hr after CAA, 3 PORs (arrowheads). f: 24 hr later, 6 PORs (arrowheads), amputation of Is blood vessels leaving only 5 connecting vessels (small arrowheads) to peripheral ampullae. g: 24 hr thereafter, no more PORs but an additional blood vessel was amputated (small arrowhead). Ja ampullae started overgrowing the contact area. h: 4 days later, all connecting vessels were amputated, Ja colony progressed, overgrowing Is ampullae and tunic matrix. 1: 6 days later, Ja colony was removed to show 6 old PORs (arrowheads) and degenerated tunic of the Is colony. Scale bars: a, c, d =0.25 mm; b, e-i=0.5 mm. after the first POR was developed. After ampullar retreat, the remaining bare tunic gradually de- generated, forming an empty space between the interacting colonies. Multiple colony allogeneic interactions A total of 201 CAAs were carried out in prim- ary, secondary and tertiary interaction with 24 Is vs Mon, 22 Is vs Ja and 21 Ja vs Mon rejecting pairs (Table 1). Secondary and tertiary interpopulation- al allogeneic interactions resulted in similar out- comes as primary interaction, that is, all colonies continued to express their former mode of allorecognition responses where again, Ja col- onies, when confronting either Is or Mon colonies, developed the yellow color in the tips of interact- ing ampullae, and were more active in producing PORs than the colonies of the other populations. 992 B. RINKEVICH, M. SHAPIRA et al. TaBLE 1. Morphological analyses of colony allorecognition assays carried out in primary, secondary and tertiary Botryllus schlosseri repeated interpopulational allogeneic reactions Repeated Is vs Mon CAAs, Repeated Is vs Ja CAAs, Repeated Ja vs Mon CAAs, in test no.* in test no.* in test no.* 2 P, mi 3 P, Mon 3P,Ja 10d,in | 5P,Ja SI 4P,rec 6P, rec 2P,Ja 15d,in | 8d,in | 72, rec 5 P, Is 10d, in Seal eee 2P,Is 6P, mi 2P, Is 3P,Ja |3P,Mon SP, Is 2 P, Is 7P,Ja 1P,Ja | 4P,Ja 8d,in 1 P, Mon 4P, rec 3P,Ja | SP,Ja 5 P, rec 15 D,in 10d, in 15 d,in |2P, Mon ——! 4P,Ja 10d,in | 4P,Ja 4P,Is 4P, Mon 10 P, mi 1 P, Mon 4P,Ja 4P,Ja | 1P,Ja &; fy] ee} HR] wl! B]_w] nt] & =) 2 P, Is 2P, mi = 2P, Mon 4P, Mon 10d,in| I | 7P,Ja | SP, Ja 1 tm 1P,Ja 2P,Ja |1P,Mon | 1 P, Is 7P, rec 2P,Ja 3P,Ja | 8P,Ja 2 P, Is 2P, rec 10d, in 2P,Ja | 3P,Ja 2P, Is 6P, Mon 3 P, rec 10d,in |; 2P,Ja 2P,Is 4P, Mon 3P,Ja 2P,Ja | 4P,Ja Wl Ww] Wi Mm] Ww] ff 4P, Mon 2 P, Mon 3P,Ja 10d,in | 4P,Ja 4P, Mon 5 P, Mon — 10d, in 1P,Ja | 6P,Ja 1P, Is 3 P, Mon 2P, Is 2P,rec | 8d.in '1 P, Mon 4P, Mon 4P,Ja 1P,Ja | 4P,mi | I 3 P, mi 5 P, Is 3 | 10d,in 3 P, rec it 2 P, Mon 5 | 2P, rec 3 PIs 1P,Ja | 17d, in 5) | SP. Us LPs Var || 10d, in 7 10 P, Is 2P, Is 4P, Mon 5 | 8P, rec * Is, Mon and Ja refer to the Israeli, Monterey and Japanese colonies, respectively. ** Results in the first to third tests: P=number of PORs; area where PORs were observed: Is, Mon, Ja, rec (reciprocal), mi (middle, on the borderlines between the interacting colonies), in (indifference, no POR was recorded during the period in days [d] specified). " Int=time interval (in days) from the day on which a full set of PORs was completed until the next assay was performed. I=acase where a preceding CAA was done immediately after the specified period of indifference or immediately after the last POR of the former CAA was observed. CAAs between Botryllus Populations 993 Ja interactions with Is or Mon colonies resulted also in up to 2-3 times more cases of “indiffer- ence” within secondary and tertiary assays when compared to the primary assay of a specific set of colonies (Table 1). The number of PORs in each combination of colonies are developed irrespective of the number of the repeated assay, and their locations (reciprocally, in the middle between both colonies or within the tunic of only one partner in the CAA) were not confined to either one of the interacting colonies of each specific set of geno- types (Table 1). The results presented in Table 1 further indicate that secondary and tertiary inter- populational allogeneic assays done on the same pairs of colonies (immediately to 17 days after the last POR of the former CAA was observed; Table 1) do not reveal an allospecific memory in rejec- tion, for example in an accelerated formation of PORs and/or augmentation in POR numbers. These results confirm previous conclusions on assays done with Mon vs Mon [16] and Mon vs WH [4] interactions, and further point to the high variability in directionality of responses/number of PORs/repertoire of allorecognition responses characteristic of this species. DISCUSSION Recently, 5 B. schlosseri populations were stu- died for intrapopulation morphology or the gene- tics of allorecognition: the population from the Venetian Lagoon, Italy [7, 17], from the Mediterranean coast of Israel [12], from Woods Hole, MA, Atlantic Ocean [3, 4, 8], from Mon- terey and Santa Barbara, CA, eastern Pacific Ocean [3, 4, 10, 16, 18], and from Mutsu Bay, western Pacific Ocean/Japan Sea [13]. The results of these studies elucidate the capacity for distinc- tion of colony which is manifested by either vascu- lar fusion or the formation of necrotic lesions when two different genotypes meet each other through their peripheral ampullae. In 4 of the above studied B. schlosseri populations (except the WH population [3, 4]), tips of marginal ampullae of paired noncompatible encounters actively ex- tended towards each other in a tip-tip orientation, without forming true tunic-matrix fusion. This continued in the production of PORs without penetration of ampullae into the tunic of the confrere genotype. In WH noncompatible pairs, however, the cortical layers of both tunics usually become enmeshed and are dissolved in limited areas near the ampullar tips. This results in reciprocal penetration of ampullae into the tunic of the facing colony. On the other hand, all other B. schlosseri allospecific phenomena, including the morphology of PORs, are similar to all 5 studied populations, except for the intensive aggregations of yellow blood cells in tips of interacting ampul- lae, which is also characteristic of Ja intrapopula- tional interactions [13]. Four interpopulation combinations were studied until now, including the WH vs Mon [3, 4] and Ja vs Mon, Is vs Ja, Is vs Mon interactions (this study). We [3, 4 and unpubl.] established more than 150 WH vs Mon CAAs, which resulted in zero fusions, similarly to the 48 Is vs Ja CAAs studied here. It is therefore very surprising that fusions were obtained in high proportions in Is vs Mon interactions (4.4%) and Ja vs Mon pairs (12.0%). In the 25 CAAs of Ja vs Mon, we used 25 Mon and only 11 Ja genotypes, which revealed 3 fusions (done with Ja colonies Nos. 5, 9, 11 [13]). The allelic pattern of the Fu/HC locus of these 3 fusible Ja genotypes [13] reveals that at least 2-3 alleles on the fusibility haplotype are common to both Ja and Mon populations. Therefore, it is not only that these three extremely separated popula- tions (eastern Mediterranean vs eastern Pacific Ocean and eastern Pacific Ocean vs Japan Sea, respectively) belong to the same cosmopolitan species, namely B. schlosseri (as was concluded when comparing WH vs Mon populations [3]), but also these populations are found to possess the same histocompatibility alleles in each population- al genetic pool (from a pool of a size of at least 100 allorecognition alleles [9, 19]). This is probably the reason for the high percentage of interpopula- tional fusions recorded here. The results of the present study and of earlier studies on B. schlosseri interpopulation specific responses [3, 4] indicate that there are some differ- ences in allogeneic responses (ampullae penetra- tions into confronting tunic matrices and the gathering of yellow pigment cells in tips of interact- ing ampullae). These responses appear to be 994 characteristic of distinct populations. Additional studies on other B. schlosseri populations may elucidate the repertoire of alloresponses character- istic of this cosmopolitan species. ACKNOWLEDGMENTS We thank the staff of the Hopkins Marine Station, Pacific Grove, and Shimoda Marine Research Center for the warm hospitality and assistance in conducting this study. The Is vs Mon part was supported by a Tobias Landau Fellowship in Marine Biology (B.R.) and by a grant from the U.S.-Israel Binational Science Founda- tion (B.R. and I.L.W.). The Ja vs Mon and the Is vs Ja studies were supported by a JSPS Fellowship for Re- search in Japan (B.R.) and by a Grant in Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, nos. 01304007 and 03455008 (Y.S.). REFERENCES 1 Berrill, N. J. (1950) The Tunicata. Ray Society, London. 2 Tokioka, T. (1953) Ascidians of Sagami Bay. Iwa- nami-shoten, Tokyo. 3 Boyd, H. C., Weissman, I. L. and Saito, Y. (1990) Morphologic and genetic verification that Monterey Botryllus and Woods Hole Botryllus are the same species. Biol. Bull., 178: 239-250. 4 Rinkevich, B. and Weissman, I. L. (1991) Interpo- pulational allogeneic reactions in the colonial pro- tochordate Botryllus schlosseri. Int. Immun., 3: 1265-1272. 5 Taneda, Y., Saito, Y. and Watanabe, H. (1985) Self or nonself discrimination in ascidians. Zool. Sci., 2: 433-442. 6 Oka, H. and Watanabe, H. (1960) Problems of colony specificity in compound ascidians. Bull. Mar. Biol. Stn. Asamushi, 10: 153-155. 7 Sabbadin, A. (1962) La basi genetiche della capaci- ta di fusione fra colonie in Botryllus schlosseri (Ascidiancea). Rend. Accad. Lincei, 32: 1031-1035. 8 Scofield, V. L., Schlumpberger, J. M., West, L. A. and Weissman, I. L. (1982) Protochordate 11 12 B. RINKEvicH, M. SHAPIRA ef al. allorecognition is controlled by an MHC-like gene system. Nature, 295: 499-502. Scofield, V. L. and Nagashima, L. S. (1983) Mor- phology and genetics of rejection reactions between oozooids from the tunicate Borryllus schlosseri. Biol. Bull., 165: 733-744. Rinkevich, B. and Weissman, I. L. (1988) Retreat growth in the ascidian Botryllus schlosseri: a con- sequence of nonself recognition. In “Invertebrate Historecognition”. Ed. by R. K. Grosberg, D. Hedgecock and K. Nelson. Plenum, New York, pp. 93-109. Weissman, I. L., Saito, Y. and Rinkevich, B. (1990) Allorecognition histocompatibility in a protochor- date species: is the relationship to MHC semantic or structural? Immun. Rev., 113: 227-241. Lilker-Levav, T. (1992) Allogeneic responses in Botryllus schlosseri and Botrylloides leachi from the Mediterranean coast of Israel. M. Sc. Dissertation, Tel-Aviv University (with English summary). Rinkevich, B. and Saito, Y. (1992) Self-nonself recognition in the colonial protochordate Botryllus schlosseri from Mutsu Bay, Japan. Zool. Sci., 9: 983-988. Boyd, H. C., Brown, S. K., Harp, J. A. and Weissman, I. L. (1986) Growth and sexual matura- tion of laboratory cultured Montery Botryllus schlosseri. Biol. Bull., 170: 91-109. Rinkevich, B. and Weissman, I. L. (1987) The fate of Botryllus (Ascidiacea) larvae cosettled with parental colonies: beneficial or deleterious consequ- ences? Biol. Bull., 173: 474-488. Rinkevich, B. and Weissman, I. L. (1992) Incidents of rejection and indifference in Fu/HC incompatible protochordate colonies. J. Exp. Zool., 263: 105- 111. Sabbadin, A. and Astorri, C. (1988) Chimeras and histocompatibility in the colonial ascidian Botryllus schlosseri. Dev. Comp. Immun., 12: 737-747. Rinkevich, B. and Weissman, I. L. (1992) Chimeras vs genetically homogeneous individuals: potential fitness costs and benefits. Oikos, 63: 119-124. Grosberg, R. K. and Quinn, J. F. (1986) The genetic control and consequences of kin recognition by the larvae of a colonial marine invertebrate. Nature, 322: 456-459. ZOOLOGICAL SCIENCE 9: 995-1000 (1992) Antibody Production in the Goat: Immunokinetics and Epitope Specificity Using a Glycoprotein Immunogen HEATHER Fasry and Jerry L. Hepricx! Department of Biochemistry and Biophysics, University of California, Davis, California 95616, USA ABSTRACT—We have developed methods for the efficient production of antibodies in goats using a glycoprotein purified from the eggs of the frog Xenopus laevis. When emulsified with complete Freund’s adjuvant, the amount of immunogen required to elicit high titer antiserum was much less than commonly used (50 ug as opposed to mg quantities). A single immunizing injection produced a maximum antibody response and secondary immunizations were not beneficial. The IgG was directed toward the carbohydrate moiety when the glycoprotein was used as immunogen, although the protein moiety was immunogenic as shown by the use of the deglycosylated polypeptide. Goats immunized with glycosylated immunogen all produced maximum titer antisera from 35 to 45 days postimmunization. In contrast, maximum titer in goats injected with deglycosylated polypeptide was reached at 26 days postimmunization. We anticipate that the immunization methods reported here will improve the usefulness of the goat for preparing polyclonal antibodies. © 1992 Zoological Society of Japan INTRODUCTION Methods for producing polyclonal antibodies in laboratory rodents, such as mice and rabbits, are experimentally well defined and commonly used. Although goats are particularly useful for the production of antibodies because large amounts of antiserum can easily be collected, experimentally determined methods for immunization have not been published. Immunizing procedures for goats and sheep are usually extrapolated from those designed for laboratory rodents. For example, it has been stated that the amount of antigen neces- sary to stimulate production of antibodies is appro- ximately proportional to body weight. That is, if 100 4g of protein is optimal for a rabbit, 5-10 mg is required for a goat [1]. Secondary immunization (boosting) is also thought to be necessary to pro- duce maximum titer antisera. Although these extrapolated methods are currently used [2-4], there is an acknowledged need for a systematic study of the immune response in the goat [1, 5]. Accepted July 13, 1992 Received June 4, 1992 ' To whom correspondence should be addressed. The purpose of the research reported here was to define methods for the most efficient production of polyclonal antibodies in goats for our purposes i.e. the minimal amount of immunogen required to produce a maximal antibody response in a mini- mum amount of time using a single immunizing injection. We used as our immunogen a presum- ably antigenically foreign purified glycoprotein, the Xenopus laevis cortical granule lectin. The time required to produce maximum IgG and IgM titers as a function of immunization dose and effects of secondary immunization on titer were determined. We also characterized the epitope specificity of the antibodies produced to the gly- cosylated and deglycosylated forms of the lectin and their macromolecular and tissue specificity. MATERIALS AND METHODS Antigen preparation Xenopus laevis cortical granule lectin (CGL) was purified from eggs as described [6]. Trifluoromethane sulfonic acid was used to degly- cosylate the CGL [7]. Xenopus laevis egg en- velopes were prepared as previously described [8]. 996 H. Fasry AND J. L. HEprRIcKk Protein concentrations were determined using the bicinchoninic acid method with bovine serum albu- min as a standard [9]. Immunization procedure The dairy goats used were does or wethers of the French Alpine and Nubian breeds or cross breds. Goats’ ages were variable, ranging from less than one year to 10. No tissue necrosis was observed in any of the animals at the sites used for primary or secondary immunizations. After dissolving the antigen in 1 ml Tris buffered saline (10 mM Tris-Cl, 150 mM NaCl, pH 7.5), an emulsion was made with an equal volume of complete Freund’s adjuvant (Difco Laboratories, Detroit MI). Double hubbed needles attached to glass syringes were used to make the emulsion as described by Herbert [10]. One intramuscular injection was made between the clavicle and the base of the neck. The first boost immunization, if made, was prepared similarly except that incom- plete Freund’s adjuvant was used. These boost injections were generally given in the opposite side of the neck. Immunogen for the second boost injection was dissolved in 10 mM phosphate buf- fered saline, pH 7.4 and injected subcutaneously. Blood collection and titer determination Blood was collected by jugular venipuncture into evacuated untreated tubes (Vacutainer, Rutherford NJ) and stored at room temperature for approximately 6 hr to allow clot formation. After refrigeration overnight at 4°C, the clotted blood was centrifuged at 10,000 x g for 30 min, the serum decanted and frozen at —20°C until use. For large scale collection of blood, we routinely collected 250 ml and typically recovered 65% of the volume as serum. A kinetic ELISA (enzyme-linked immunosor- bant assay) was used to determine the titer of serum samples. Disposable ELISA plates (‘Easy Wash’, Corning Glass Works, Corning NY) were coated overnight at 4°C with 15 ng of antigen in 50 wl of buffer (100mM NasCO;3, pH9.5). After washing with TBST (10 mM Tris, 150 mM NaCl, 0.1% Tween 20), 100 «1 of serial 1:3 dilutions of serum (with TBST) were applied to the wells. For IgM titer determination, IgG was removed from serum samples using a protein G kit (Quik-Sep IgM, Isolab Inc., Akron OH). After incubation at room temperature for 1 hr, plates were washed and 100 wl of a horseradish peroxidase conjugated secondary antibody was added for lhr. Either rabbit anti-goat IgG (Fc) or rabbit anti-sheep IgM (uw chain) (both from Organon Teknika Corpora- tion, West Chester PA) was used as the secondary antibody. Plates were developed by the addition of substrate [50 mM citric acid, pH 4.0, 0.60 mM 2,2’-azino bis(3-ethylbenzthiazoline-6-sulfonic acid), 0.08% v/v H2O;] and the absorbance at 405 nm was measured every 8 sec for 2 min using a kinetic microplate reader (Molecular Devices, Menlo Park CA). The velocity, 4A49;/minute, was plotted against serum dilution. We defined the antibody titer of antiserum as the inverse of the serum dilution corresponding to half maximal velocity. To confirm that the reported titers represent immunologically specific binding, a number of controls were tested: 1) In the absence of antigen, insignificant amounts of primary antiserum bound to the ELISA wells. 2) The secondary antisera was tested for nonspecific binding both to the antigen and to the ELISA plate. 3) Finally, coincubation of purified soluble CGL with anti-CGL serum abolished serum binding to the CGL immobilized on the ELISA plate. Preparation of tissue extracts and analysis of serum specificity Aqueous extracts were made from fresh Xeno- pus laevis or mouse tissue by crushing diced organs between two frosted glass microscope slides in distilled water. Cellular debris was removed by low speed centrifugtion. The proteins in the supernatant solution were separated by reduced SDS-PAGE [11] and electroblotted onto ni- trocellulose [12]. Membranes were probed with antiserum essentially as described for an ELISA, except that the developing solution contained 50 ml 10mM Tris, 150mM NaCl pH 7.4, 30 mg 4- Chloro-1-Napthol/10 ml methanol and 50 «l 50% (w/w) H3Q>. Antibody Production in the Goat 997 RESULTS Immunokinetics of IgG production as a function of immunogen dose To investigate the minimum amount of im- munogen necessary to stimulate maximum IgG production, goats were immunized with 10 to 1000 pug of CGL. Blood samples were collected twice weekly and the IgG titer of the serum determined. Immunization with 50, 100 or 1000 ug of CGL gave virtually identical immune responses. IgG production began 3 to 8 days postimmunization and continued to increase until the maximum titer was reached 35 to 45 days postimmunization. Figure 1 illustrates the immune response of the goat immunized with 50 ~g CGL. Maximum titer values ranged from 30,000 to 60,000 titer units. In contrast, immunization with 10 “7g of CGL led toa protracted immune response which peaked at a lower titer. For example, on day 42 postimmuniza- tion, the IgG titer from the three goats immunized with at least 50 wg CGL was at the maximum, whereas the titer of the goat immunized with 10 ug LO@ iver 0 10 20 30 40 60 £460 Days postimmunization Fic. 1. IgG and IgM response after injection of 50 ug CGL. ; was around 20% of maximum and reached a maximum of 7000 titer units on day 65. Immunization with 100 or 1000 ug of deglycosy- lated CGL elicited an immune response of slightly different kinetics compared to CGL. These two goats’ immune responses were essentially identi- cal, producing maximum titer antiserum 26 days postimmunization (approximately 14 days earlier than goats immunized with 504g or more of CGL). In addition, maximum titer values were lower (4,500—6,000 titer units) than those from goats immunized with CGL. Effect of secondary immunization on IgG titer The two goats initially immunized with 100 or 1000 ng CGL were given boost injections in an attempt to elicit production of higher titer antisera than was produced after primary immunization. At the time of boosting (273 days postimmuniza- tion), the IgG titer had declined to 3% or 7% (respectively) of the maximum from primary im- munization. These animals were boosted on two occasions with a total of 80 ~g CGL. The first injection was given intramuscularly in an emulsion of incomplete Freund’s adjuvant and the second was injected subcutaneously as a solution. After each boost injection, the serum titer increased and reached a plateau, but did not exceed the titer reached after primary immunization (Fig. 2). Immunokinetics of IgM production Given the differences in time to reach maximum IgG titer upon injection of glycosylated and degly- cosylated immunogen, we looked for similar dif- ferences in the kinetics of IgM production. IgG depleted samples from the goat immunized with 50 ug, CGL were analyzed for IgM content using an IgM specific secondary antibody. Serum samples from goats immunized with 100 or 1000 ug of deglycosylated CGL were similarly analyzed for IgM content, but without removing IgG. The IgM titer of the goat immunized with 50 pg CGL was detectable 8 days postimmunization and peaked between 19 and 22 days postimmunization. After day 22, the IgM titer slowly decreased reaching basal levels about 42 days postimmuniza- tion (Fig. 1). The IgM response of both goats immunized with 998 Log titer Boost with | 50g Boost with 80g 290 300 310 H. Fasry AND J. L. Heprick 320 330 340 350 360 370 Days postimmunization Fic. 2. IgG response after secondary immunization. Goat initially immunized with 100 ~g CGL. Prior to boost, titer was 3% of maximum produced following primary immunization. deglycosylated CGL was initially similar to the goat injected with SO yg CGL in that IgM appeared 8 days postimmunization. The IgM response of the goat immunized with 1000 ug of deglycosylated CGL, however, seemed acceler- ated because the IgM titer peaked between 12 and 15 days postimmunization and returned to basal level 22 days after injection. In contrast, the goat injected with 100 ug of deglycosylated CGL exhi- bited kinetics similar to those of the goat immu- nized with 50 ug of CGL in that maximum IgM antiserum was produced on day 19 postimmuniza- tion and the IgM serum levels declined gradually. Epitope specificity of antibodies generated to CGL and deglycosylated CGL Upon testing the macromolecular and tissue specificity of antisera, we observed marked cross- reactivity of CGL antiserum with Xenopus laevis egg envelope glycoproteins. However, the CGL antiserum was tissue specific as it did not react with aqueous extracts made from Xenopus laevis skele- tal muscle, liver, gall bladder, spleen, lung, nerve, and activated egg cytosol. In addition, it did not react with extracts from mouse lung, kidney, liver, heart, or skeletal muscle (data not shown). This cross reactivity with egg envelope glycoproteins prompted an investigation of whether carbohy- drate or protein domains were recognized by the CGL antibody. When CGL was used as an im- munogen, approximately 96% of the antiserum reactivity was directed against the carbohydrate moiety (Fig. 3). In addition virtually all crossreac- 160 Antigen CGL dgCGL VE* dgVE* 120 S| E SS < E 0 Py a oO e) 3S o > 40 10} 10° io? 107 10" 10° oom Serum dilution Fic. 3. ELISA reactivity of CGL antiserum. Reactivity of anti-CGL serum with given antigens. w CGL; VE*%, vitelline egg envelopes derived from activated, dejellied eggs; @ dgVE*, deglycosylated vitelline egg envelopes; () Preimmune serum; dgCGL, degly- cosylated CGL. Antibody Production in the Goat 999 tivity with egg envelope glycoproteins was due to shared carbohydrate residues, as deglycosylation rendered envelope glycoproteins unreactive to the CGL antiserum (Fig. 3). The deglycosylated CGL antiserum showed no significant crossreactivity with egg envelope glycoproteins, thereby indicat- ing that the CGL and envelope protein moieties were non-homologous (as expected from the li- mited amount of amino acid sequence data avail- able on these glycoproteins [13, 14, H. Fabry, M. Oda, and J. Hedrick, unpublished observations]). DISCUSSION Although goats are commonly used to produce commercial secondary antisera, rabbits and mice are used more often for production of primary antisera both in the research laboratory as well as for commerce. Part of this preference likely stems from the belief that milligram quantities of antigen are required for immunization of larger animals. From our observations, only small amounts of immunogen are required for the goat. We observed no differences in the kinetics or magni- tude of the IgG response when goats were immu- nized with as little as 50 ~g or as much as 1000 ng of CGL. These goats all produced maximum titer antisera (30,000 to 60,000 titer units) 35 to 45 days postimmunization. Although the IgG response after immunization with 10 ~g CGL was markedly slower, useful antisera was eventually produced (7000 titer units, 65 days postimmunization). Most immunization schedules call for boost in- jections at intervals after the primary immuniza- tion to increase antiserum titer. In addition, it is often recommended to delay boosting until the titer in response to primary immunization has declined to a small percentage of maximum. Our observations were contrary to this common prac- tice. We boosted goats initially injected with 100 vg and 1000 ~g CGL on two occasions with a total of 80 wg of CGL. After each boost, serum titers increased and reached a plateau, but at no time was the post boost titer significantly higher than that produced after primary immunization. Thus, when adequate amounts of immunogen are used for the primary immunization using complete Freund’s adjuvant in the goat, boosting is not required for generation of maximum titer anti- serum. However, if relatively small amounts of immunogen are used (<50 xg), boost injections may aid in increasing antibody production. When goats were immunized with CGL, the dominant immune response was directed against the carbohydrate or oligosaccharide moiety of the glycoprotein. Characterization of the sugar moieties recognized by the CGL antiserum is currently being investigated and preliminary re- sults suggest that a modified sialic acid may be the immunodominant monosaccharide present on the oligosaccharide sidechains of CGL (H. Fabry, N. Wardrip, and J. Hedrick, unpublished observa- tions). Since the presence of highly immunogenic car- bohydrate structures on the lectin might lead to an atypical immune response, we immunized addi- tional goats with 100 yg and 1000 ug of deglycosy- lated CGL. The kinetics and magnitude of IgG production by these goats was not dose dependent, similar to what was observed when glycosylated CGL was used. However, the immune response was accelerated and of lower magnitude than the goats immunized with the glycosylated lectin. These differences may reflect genetic variance between animals or result from the relative im- munogenecity of the carbohydrate versus the pro- tein moieties of the CGL. We have used more than 15 goats in our studies and all have produced strong immunological re- sponses to small quantities of Xenopus laevis and Sous scrofa (pig) protein as well as glycoprotein immunogens. We feel, therefore, the methods described in this paper can be successfully applied to a variety of immunogens, and will improve the usefulness of the goat for production of primary antisera. ACKNOWLEDGMENTS We thank Dr. Randy Harris for his assistance in collecting antiserum and Dr. Dan Hardy for helping with the immunizations. This study was supported in part by a United States Public Health research grant HD-04906 to JLH. The care and use of animals followed institutional and USDA guidelines. 1000 REFERENCES Dresser, D. W. (1986) Immunization of ex- perimental animals. 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Weir, 3rd edition, Blackwell Scientific Publications, Palo Alto, pp. A3.1. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteri- ophage T4. Nature 227: 680-685. Towbin, H. T., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacryl- amide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76: 4350-4354. Bakos, M., Kurosky, A. and Hedrick, J. L. (1990) Physicochemical characterization of progressive changes in the Xenopus laevis egg envelope follow- ing oviductal transport and fertilization. Biochem- istry 29: 609-615. Chang, B., Wardrip, N. J. and Hedrick, J. L. (1990) The cloning of the cortical granule lectin cDNA. J. Cell Biol. 111: 487a. ZOOLOGICAL SCIENCE 9: 1001-1007 (1992) Appearance of a Nuclear Histone H1 Kinase at the Start of DNA Synthesis of Regenerating Rat Liver KouicH! ASAMI 'Division of Biology, National Institute of Radiological Sciences, Chiba-shi 263, Japan ABSTRACT—Phosphorylation of histone H1 and elevation of nuclear protein kinase activity occur at the start of DNA synthesis during regeneration of rat liver. X ray irradiation prior to partial hepatectomy depressed both elevation of the nuclear protein kinase activity and DNA synthesis. Thus, the protein kinase seemed to play a role in the start of DNA synthesis, but the property of the enzyme was not well characterized. The nuclear protein kinase activity was measured with casein or histone H1 as substrate. A protein kinase activity specific for histone H1 was found in the extract from the regenerating liver nuclei, but not that from the irradiated and partially hepatectomized liver nuclei. On the other hand, casein kinase activity was found in the extract from the regenerating liver irrespoctive of prior X ray irradiation. Gel filtration chromatography of the nuclear extract revealed the presence of the histone H1 kinase and two casein kinases in the regenerating liver. Histone H1 kinase was observed in the regenerating liver, but not in the non-hepatectomized nor the irradiated and hepatectomized liver. Two casein kinases were found in the non-hepatectomized liver and partial hepatectomy with or without prior X irradiation induced a little increase in the activites. The present results indicate that appearance of the nuclear histone H1 kinase activity is required for phosphorylation of histone H1 and the start of DNA synthesis in the regenerating liver. © 1992 Zoological Society of Japan INTRODUCTION Hepatocyte in the adult liver does not prolifer- ate in a normal physiological state. But when a part of the liver is removed, the rest of the liver starts proliferating and when the liver mass has gained the former size the cells return to the quiescent state. Proliferation of hepatocytes are stimulated by several growth factors, such as hepatocyte growth factor or hepatopoietin A [1]. Before the onset of DNA synthesis the stimulated cells show many responses such as increase in Na*- H™ exchange, polyamine metabolism, and amino acid transport [2]. Phosphorylation of his- tones and HMG proteins which belong to nuclear non-histone proteins, and synthesis of histones and HMG (high mobility group) proteins also occur before and during DNA synthesis, and the en- zymes of nucleotide motabolism are synthesized at Accepted July 15, 1992 Received May 8, 1992 ' Present address: Department of Biology, Sapporo Medical College. Sapporo 060, Japan. the same period [2]. However, causal re- latiopnships among these events are not clear. A dose of X rays enough to inhibit the onset of DNA synthesis inhibited phosphorylation and synthesis of histone H1, while it failed to inhibit phosphory- lation and synthesis of HMG proteins [2, 3]. Thus, phosphorylation of histone H1 seemed to be close- ly related to the onset of DNA synthesis. Changes in nuclear protein kinase activity were measured, therefore, during the prereplicative phase of re- generation in the liver of partially irradiated rat [4]. The protein kinase activity increased at the onset of DNA synthesis and the rise of the activity was inhibited by X ray irradiation delivered just before partial hepatectomy [4]. Consequently this protein kinase seemed to be responsible for phos- phorylation of histone H1, although its nature was unknown. Rat liver nuclei contain several kinds of protein kinases such as histone H1 kinase [5, 6], casein kinases [7, 8], protein kinase C [9], Ca/calmodu- lin-dependent protein kinase [10] and cAMP- dependent protein kinase [4, 11]. Our previous 1002 results suggested that growth-associated histone kinase of Langan, a protein kinase specific for histone H1 found in growing cells [5], was re- sponsible for the rise of the protein kinase activity in the nuclei, although participation of other kinase could not be excluded completely [4]. Therefore, we measured histone H1 kinase and casein kinase activities in parallel. The present results indicate that nuclear kinase specific for histone H1 is responsible for the rise of protein kinase activity at the onset of DNA synthesis. MATERIALS AND METHODS Animals and materials Male Wistar strain rats, supplied from our breeding colony, were used between 8 and 10 week old of age. [y-*’P]ATP was purchased from Amer- sham, Japan. PMSF (phenylmethylsulfony! fluoride), a-casein (Sigma), and Combithek (pro- tein standard set: Boeringer) were used. Histone H1 was extracted from rat liver according to the procedures of Sanders and Johns [12], and the acid soluble fraction was passed through a column of BioRex 70 (Bio-Rad Lab., USA) [13]. The frac- tion containing histone H1 was collected, freeze- dried, and dissolved in water. Fractions containing mainly histone Hlg were not used. Histone H1 solution, which showed a single band with polyac- rylamide gel electrophoresis, was divided in small aliquots and stored at —20°C until use. X ray irradiation and partial hepatectomy X ray irradiation and partial hepatectomy of rats were carried out as described previously [3, 4]. During anesthesia with pentobarbital, rats were irradiated to their upper abdomen including the liver with 4.8 Gy of X rays. The condition of irradiation was 200 kVp and 20mA and X rays were filtered through 0.5 mm Cu and 0.5 mm Al. Following irradiation, the median and the left lateral lobe of the liver were removed. The non-irradiated hepatectomized under anesthesia with pentobarbital. The right lateral and caudate lobes were used in the experi- rats were also ments of non-hepatectomized rats, since these lobes were used in the case of hepatectomized rats. K. ASAMI The hepatectomized rats were killed at 24h after partial hepatectomy, when DNA synthesis started [3] and the protein kinase activity increased [4]. Preparation of nuclear extract Isolation of nuclei, essentially according to the procedure of Blobel and Potter, and preparation of the nuclear extract were described previously [4]. Briefly, the nuclei were extracted with 0.4 M NaCl in buffer A, which consisted of 20mM TrisHCl pH 7.5, 1mM EDTA pH7.5, 0.1mM PMSF, SmM_ benzamidine, 2mM_ glycerol-2- phosphate and 10mM 2-mercaptothanol. After treatment with BioRex 70, the extract was mixed with potassium phosphate pH 7.5 and cAMP to give 0.1M and 1M respectively in order to separate cAMP-dependent protein kinase [5, 6]. The protein precipitated with ammonium sulfate between 17.5% and 35% saturation was collected. The precipitate from one rat (ca. 5g liver) was dissolved in 0.5 ml buffer A containing 0.4M NaCl. The extracts thus obtained were dialyzed overnight against about 50 volume of buffer A containing 10% glycerol. For resolution with chromatography the nuclei from 3 rats were com- bined before extraction and the extracts were used for gel filtration chromatography immediately af- ter extraction. Although 0.4M NaCl did not extract all the kinase activity, the portion of the extracted activity was not changed much during regeneration [4]. The protein kinase activities were hence compared on the nuclear extracts with 0.4 M NaCl in the present experiments. Column chromatography of the extract FPLC system (Pharmacia) was used. When Mono Q column was used to separate the extract, dialysis of the extract before chromatography caused aggregation of the proteins and almost all proteins remained on the top of the Mono Q column. Therefore, the extract was applied to the column of Superose 12 immediately after extrac- tion. The column was equilibrated in 0.4 M NaCl containing buffer A, and ca. 2 mg of the nuclear extracts were applied. Fractions of 1 ml were collected, and the enzyme activity and protein concentration were determined. Protein was de- termined according to the procedure of Bensadoun H1 Kinase in Regenerating Liver Nuclei and Weinstein [14]. Assay of protein kinase activity A similar procedure with the previous experi- ments was used with rat liver histone H1 and a-casein as substrate [4]. Briefly, the reaction mixture contained in 0.05 ml; 50 mM TrisHCl pH 7.5, 10mM MgCh, histone H1 (0.5 mg/ml) or a-casein (0.5 mg/ml), 0.5 mM ATP (containing 1 uCi [y-°P]ATP), 1mM EDTA pH7.5, 1mM dithiothreitol, and 20mM _ glycerol-2-phosphate. The reaction was started with addition of the enzyme. After incubation for 10 min at 37°C, the reaction was stopped with addition of 0.25 ml 20% trichloroacetic acid. The precipitate was collected on a Millipore filter and washed with 10% trichlor- oacetic acid. After drying in air, radioactivity of the filter was measured with a liquid scintillation counter. Since incubation without substante caused substantial uptake of radioactivity, net up- take of radioactivity was calculated by subtracting the values obtained without substrate. time value was subtracted. The zero RESULTS Normal liver nuclei contain a protein kinase or kinases and partial hepatectomy causes partial increase in kinase activity at the time of DNA synthesis [4]. More than one protein kinase could be measured simultaneously in the previous ex- periments, however, since histone mixture used as the substrate contained histone mixture as well as non-histone proteins (data not shown). In the present experiments, therefore, protein kinase activity was measured with more defined substrate; rat liver histone H1 and casein. Phosphorylation due to endogenous substrate was subtracted from the values with the substrates, and also shown in the results separately (Table 1). The protein kinase activity phosphorylating histone H1 was observed in the extract from regenerating liver nuclei, while in was not observed in the extract from the nuclei of the X ray-irradiated and partial- ly hepatectomized rat (Table 1). On the other hand, the protein kinase activity phosphorylating casein was observed in both the hepatectomized and the irradiated and hepatectomized nuclear 1003 TABLE 1. Protein Kinases in the Nuclear Extract of Regenerating Liver Activity (nmol/10 min/mg protein) Substrates Non-irradiated X-irradiated 0.63+0.57 P<0.05 histone H1 3.66 £0.93 casein 4.75 + 1.60 3.65 +0.80 NS (endogenous) 7.14+1.66 Sil se WS NS The nuclear extract was prepared from the rat at 24 h after partial hepatectomy. Mean of 3 indepen- dent experiments with SEM. extract. Phosphorylation of endogenous substrate was also observed on all the groups measured (Table 1). changes in the activity phosphorylating casein or endogenous substrate. These results indicated presence of histone H1 kinase distinct from casein kinase | and 2 and responsible for phosphorylation of histone H1 at the onset of DNA synthesis in the regenerating liver nuclei. To confirm the presence of a protein kinase more specific to histone H1 than casein in the nuclei, the nuclear extracts were fractionated with gel filtration chromatography using Superose 12 column. The nuclear extract dissolved in buffer A containing 0.4 M NaCl was immediately applied to Superose 12 column equilibrated in the same medium. The protein kinase activities of each fraction was measured in the presence of histone H1 or casein or without substrate addition (Fig. 1). Elution profile of proteins did not change much among the non-hepatectomized, the hepatecto- mized and the X-irradiated and hepatectomized liver. The activity phosphorylating endogenous substrate(s) was found in the fraction eluted at the void volume and decreased in the later fractions. Endogenous substrate(s) were probably associated with its protein kinase in the nuclear extract and pass through the column in the associated form. The activity phosphorylating casein was eluted in Irradiation caused no _ substantial two peaks, i.e. fraction 10 and 15 as maximal respectively, while that phosphorylating histone H1 was maximal in fraction 12. The liver nuclei have been reported to contain two casein kinases [7, 8]. The two peaks probably correspond to the nuclear casein kinase | (fraction 15) and 2 (fraction ENZYME ACTIVITY (pmol/10 min ) 1004 100 10) or NI and NII [7, 8]. The casein kinase activity after subtraction of the activity without substrate addition were divided into two parts, i.e. the fractions 13-17 and the fractions 7-12, and they were designated here as casein kinase 1 and 2 respectively. The integrated activities are shown in Table 2. Activities of casein kinases of hepatecto- mized liver are a little higher than those of non- hepatectomized liver, but X ray irradiation hardly affected the activities. An activity phosphorylating histone H1 was eluted in the fractions of regenerating liver be- tween casein kinase 2 and 1. This indicates that casein kinases and histone H1 kinase are different enzymes. The nuclear extract from the non- hepatectomized liver or from the X-irradiated and hepatectomized liver contained casein kinase 1 and 2, but no histone H1 kinase. The activity phophorylating endogenous substrate was sub- tracted from that in the presence of histone H1 for each fraction. Phosphorylating activities of frac- tions 8 to 9 or to 11 were lower in the presence of histone H1 than in its absence, indicating that histone H1 inhibited the phosphorylation of the endogenous substrate. The fractions, where the activity was higher in the preence of histone H1 Fic. 1. Gel filtration chromatography of the nuclear extracts from rat liver. Superose 12 attached to FPLC system was used. (A) Partially hepatecto- mized (2.54 mg protein), (B) partially irradiated and hepatectomized (2.58 mg protein), and (C) non- hepatectomized (2.58 mg protein). Histone H1 (.), 6 A 10 12 % IG j@ 20 casein (A) and none (@) were used as substrate. FRACTION NO. Activity in 25 “1 of the eluate was shown in the figure. ...., Protein contents. TABLE 2. Protein Kinases in the Nuclear Extract of Rat Liver 7 xX Now Activities (nmol/10 min/mg protein) at ae : Ss cays EXD. HK CKI CKI Non-HPX = 3 0.58 +0.09 10.87 +£0.78 3.88 + 0.54 HPX — 2 2.31 +0.46" 17.52+4.78 4.74 + 1.42 HPX + 3 0.62 + 0.36 15.30+ 1.38" 4.16+1.33 The nuclear extract from the non-hepatectomized (non-HPX) or the hepatectomized (HPX) rat was used for FPLC. Protein kinase activities and protein centents of each fraction were measured and summed up. HK; histone H1 kinase, CKI and CKII; casein kinase | and 2 respectively. The asterisks indicate a significant difference from the non-hepatectomized (P<0.05). H1 Kinase in Regenerating Liver Nuclei than in its absence, were regarded to contain histone H1 kinase and their differences are sum- med up (Table 2). Nuclear histone H1 kinase activity of the hepatectomized liver was higher than either the non-hepatectomized or the X ray- irradiated and hepatectomized liver. Partial in- activation of the histone H1 kinase however was noticed on the fractions after chromatography, since the specific activity after chromatography was similar to that before chromatography (com- pare Table 2 with Table 1). The reason of inactiva- tion is unknown, but instability of growth associ- ated histone kinase after chromatography was re- ported [6, 15]. Apparent molecular weight of three protein kinases were calculated from chromatography of the standard proteins; ferritin, catalase, aldolase, bovine serum albumin, egg albumin, chymotrypsi- nogen A and cytochrome c. Some of the standard proteins, especially ferritin, showed peakds with smaller molecular weight after chromatography. This would be due to depolymerization of the polymerized form of the proteins in the elution buffer containing 2-mercaptoethanol. These peaks with smaller molecular weight were also used for calculation. The obtained regression line was log(molecular weight)=5.32—0.137 x (retention time in min); r=—0.9646. The calculated sizes of the protein kinases were 381 kDa (casein kinase 2), 92 kDa (histone H1 kinase), and 12 kDa (ca- sein kinase 1), respectively (data not shown). The results shown in Figure 1 indicated that histone H1 inhibited phosphorylation of endoge- nous substrate by the kinase eluted simultaneous- ly. Phosphorylation of endogenous substrate and casein was then measured in the presence of different concentrations of histone H1, since ca- sein kinases were the protein kinase found in the nuclei in the present condition except for histone H1 kinase (Table 3). Histone H1 inhibited phos- porylation of casein by casein kinase 1 (fraction 15) or 2 (fraction 10). Phosphorylating activity of fraction 8 which contained endogenous substrate was activated with casein and the activity was inhibited by histone H1 irrespective of addition of casein. 1005 TABLE 3. Inhibition by histone H1 of the phos- phorylating activity of the nuclear extract with or without casein histone H1 (mg/ml reaction mixture) Enzyme Casein 0 0.5 1.0 nmol/10 min/mg protein Fr. 8 + 5.50 6.56 3,32 = 2.54 1.62 1.18 Fr. 10 ar 10.98 9.41 5.04 = 1.63 1.91 1.33 Fr. 15 3 37.17 17.32 16.73 - 0.80 1.33 1.98 The values with casein are not corrected for *°P uptake without casein. Fraction (Fr.) 10 and Fr. 15 contain casein kinase 2 and casein kinase | respec- tively. DISCUSSION Phosphorylation of histone H1 is closely related to the initiation of DNA synthesis of regenerating liver [2, 3]. However, it is still not known which enzyme is responsible for the phosphorylation. Although cAMP-dependent protein kinase was found in the nuclei [11], the enzyme was not related with phosphorylation of histone H1 at the time of DNA synthesis [4] and was removed from the nuclear extract in the present experiments through treatment with cAMP and ammonium sulfate precipitation [5, 6]. Protein kinase C and Ca** /calmodulin-dependent protein kinase II are also reported in rat liver nuclei [9, 10], but the present assay condition was not favorable for the Ca-dependent kinases, since the assay medium contained no Ca** but EDTA. The inhibitors of the protein kinase C did not inhibit the nuclear kinase [4]. These protein kinases, therefore, will not be considered further in this article. The present results show that the nuclear extract of the regenerating liver contained two kinds of casein kinases and one histone H1 kinase. The extract seemed to contain another protein kinase which was associated with endogenous substrate. This will be a casein kinase as discussed later. Two kinds of casein 1 (NI) and 2 (NII), although the molecular weight of casein kinases in the present results are not consistent with the reported 1006 values (e.g., 50 and 133 kDa [8] or 35 and 200 kDa [15] respectively). In the present paper however they are called casein kinase 1 and 2. Activities of casein kinases increased a little in the regenerating liver and X rays enough to inhibit phosphorylation of histone H1 scarcely affected the increase. A nuclear non-histone protein, HMG 14 is phos- phorylated with casein kinase 2 [8, 16] and X rays do not affect phosphorylation of HMG 14 [3]. These are in consistent with the results that X rays scarcely affected the activity of casein kinase. Both casein kinase 1 and 2 are inhibited by histone H1. An endogenous inhibitor protein of casein kinase was reported previously, which was a 25kDa protein found in high mobility group (HMG) protein fraction of rat liver, but it was not characterized further [17]. However, histone H1 was not removed from their HMG fraction, which usually contained histone H1, before separation of the inhibitor [17]. The present results suggest that this endogenous inhibitor protein of casein kinase is histone H1. Inhibition by histone H1 of nuclear casein kinase may have physiological significance for regulating phosphorylation of nuclear proteins, although more experiments should be carried out before concluding histone H1 as a natural inhibitor of nuclear casein kinase. A protein kinase is associated with endogenous substrate and that is the cause of high phosphoryla- tion rate without added substrate (Table 1). The enzyme was able to phosphorylate casein (or it was activated with casein) and it was inhibited by histone H1. It was reported that casein kinase 2 in the nuclear extract was associated firmly with high molecular weight protein such as nucleolin or progesterone receptor [18, 19]. Hence, the en- zyme phosphorylating endogenous substrate may be casein kinase 2. Histone H1 kinase was found in the regenerating nuclei, while it was not observed in the non- hepatectomized nuclei. X irradiation inhibited the appearence of the histone H1 kinase activity in the regnerating nuclei. Casein kinases had a very low affinity for histone H1 as substrate. Therefore, the nuclear histone H1 kinase will be responsible for phosphorylation of histone H1 at the time of DNA synthesis. K. ASAMI The present results coincide with the previous results that the enzyme responsible for phosphory- lation of histone H1 will be a growth associated histone kinase of Langan [4]. Apparent molecular mass of the kinase was 92 kDa. Molecular weight of growth associated histone kinase is not re- ported, but that of catalytic subunit is 67,000 [6]. Cyclin-p34°“* protein kinase is specific for histone H1 and is thought to be the same as growth associated histone kinase [20, 21]. Association of p34°4? with cyclin, 62 kDa, will constitute a com- plex of 96kDa, a similar value to the present results, although apparent molecular mass of cyc- lin-p34°"? complex is evaluated as 220 kDa from chromatography with Superose 12 [21]. Nuclear localization of the kinase has been shown [22, 23]. Although the cyclin-p34~ play a role in the G2 to M transition of the cell cycle, the same enzyme or its homologue is claimed to participate for the G1 to S transition [24-26]. Thus the histone H1 kinase found in the nuclei will be growth associated histone kinase or a kinase related to it, which will play a role in phosphorylation of histone H1 at the onset of DNA synthesis. The mechanism how X rays inhibit appearance of the histone H1 kinase in the nuclei is not known. It is not known whether X rays affect directly the appearance of histone H1 kinase in the nuclei. Instability of growth-associated histone H1 kinase has been reported [6, 15, 27]. Activity of p34 protein kinase can be controlled by binding of cyclin which is usually synthesized in each cell cycle, and the activity can be changed depending on the phosphorylation state of these two proteins [28, 29]. On the other hand, synthesis of p34‘? during the G1 phase of stimulated lymphocytes was Observed [25]. Hence X rays may suppress appearance of the histone H1 kinase in the nuclei by changing the cellular metabolism resulting in inactivation of the kinase, or by inhibiting de novo synthesis of protein(s) constituting the kinase. REFERENCES | Michalopoulos, G. K. (1990) Liver regeneration: molecular mechanism of growth control. FASEB J. 4: 176-187. Ord, M. G. and Stocken, L. (1984) Cell and Tissue Regeneration. Wiley, New York. to 10 11 12 13 14 15 16 H1 Kinase in Regenerating Liver Nuclei Asami, K. (1987) Synthesis and phosphorylation of histone H1 and high mobility group proteins in the regenerating rat liver after X irradiation. Radiat. Res. 109: 216-226. Asami, K., Kobayashi, H., Fujiwara, A. and Yasu- masu, I. (1989) Changes in the nuclear protein kinase activities in the regenerating liver of partially irradiated rat. Radiat. Res. 119: 500-510. Langan, T. (1978) Isolation of histone kinases. In “Methods in Cell Biology”. Ed. by G. Stein, J. Stein, and L. J. Kleinsmith, Academic Press, New York, Vol. XIX, pp. 143-152. Woodford, T. A. and Pardee, A. B. (1986) Histone H1 kinase in exponential and synchronous popula- tions of chinese hamster fibroblasts. J. Biol. Chem. 261: 4669-4676. Desjardins, P. R., Lue, R. F., Liew, C. C. and Gornall, A. G. (1972) Purification and properties of rat liver nuclear protein kinases. Canad. J. Biochem. 50: 1249-1259. Delpech, M., Levy-Favatier, F., Moisand, F. and Kruh, J. (1986) Rat liver nuclear protein kinases NI and NII. Eur. J. Biochem. 160: 333-341. Masmoudi, A., Labourdette, G., Mersel, M., Huang, F. L. Huang, K-P., Vincendon, G. and Malviya, A. H. 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EMBO J. 9: 675-683. it ial TD | bao ty paw me ee ty ho ’ f fi ey 9 WW 3 . ’ i j 4 it bY rR re “i «Ke ae i mat | he | ere reap ue warkd tins vortigath nt a ta ae 1a red ast a eee stint: TWAT Tis Ueda wil S ort Tita vas a 4 3 fie ¢'s * nr “ani ’ OK ~ « i oe. 7 ? ot Tewtaics i ry ih gh H j or 3 , 2) ’ t yuh é lad ee | 7 % free oe Ky hi todap ht Wart) lieved mh fh 44 hag nike art ine Tilt chen nr vied ew Sid vty vehi dire aeimanuncodyale mal resed, 1 RNR ER Kae bag hae ie ¥ hy fe Le ail ih dts. cee HHA! aa : daleri(ey cee DE ye tins Wha Hook a io , TALES aha Cee ee veel. ; Jone i1TvG att Sas : Ea lineewl A * eetnepedy " \ "i, @ LP Lapaniyh rae £1 ON yr nen soir a ae ae oo wer) ILA d OI 2 ig Aer ary Sey. Ps see! wih bi ds fi et om wie. ! Y : ens cas | om bee i Art onside ly, phen Ag LAL ee eo Lis ing AU jority Lay a a CP t2t oot) bed ode : } : Meee yf seas’ di ee wie deat 4e i ey Lb ate Thee 4 ij MJ ot. enltelel \ ole sete te if r 1 ee 1 oink Tae 4 men LO PAGE Ls ie OW Ad! are wT sichesniee Pale + Wath Wh Banka oa ae mod bod 2h a ut> A (a th | Depletion riP ttt ZOOLOGICAL SCIENCE 9: 1009-1015 (1992) Immunohistochemical Demonstration of Metallothionein in the Rat Epididymis and Spermatic Cord TAKANORI SUZUKI, HIDETOSHI YAMANAKA, Kew Suzukt', Katsuyuki NAKAJIMA~, KAzUSHI KANATANI, Masami Kimura® and Noriko Oraki‘ Department of Urology, Gunma University, School of Medicine, Maebashi, 'Department of Pathology, Medical Care and Technology, Gunma University, Maebashi, "Japan Immunoresearch Laboratories Co., LTD, Takasaki, *Department of Molecular Biology, Keio University School of Medicine, Tokyo, and *Central Institute of Experimental Animals, National Institute of Industrial Health, Kawasaki, Japan ABSTRACT—We studied the immunohistochemical localization of metallothionein (MT) in the epididymis and spermatic cord of male Wistar rats. In the head of the epididymis, no MT immunoreaction of epithelial cells in the ductuli efferentes or ductus epididymidis was observed, but basal cells of the ductus epididymidis showed a positive immunoreaction for MT. In the body of the epididymis, a few epithelial cells in the ductus epididymidis had a positive immunoreaction for MT, but no positive immunoreaction was observed in the basal cells. In the tail portion near the body, basal cells in the ductus epididymidis showed a positive immunoreaction for MT while the epithelial cells had a negative one. In the portion near the spermatic cord, both the epithelial and basal cells in the ductus epididymidis had a positive immunoreaction for MT. In the spermatic cord, basal cells and some epithelial cells had a positive immunoreaction for MT. The localization of MT was observed mainly in the cytoplasm of the cells, with some nuclei of the cells having a positive immunoreaction. Different immunoreactions for MT were demonstrated in the epithelial and basal cells of the epididymis and spermatic cord, which suggested that epithelial cells function differently in various portions of the epididymis. © 1992 Zoological Society of Japan INTRODUCTION Metallothionein (MT) is a known metal-binding protein with a low molecular weight, approximate- ly 6,000 MW, and an ability to bind closs II-B metals, such as zinc and cadmium [1]. It has been identified in a variety of organs, including the liver, kidneys, prostate, and testes [1-4]. MT is induced in response to various physiological stimuli and endogenous factors [5]. Although the exact func- tions of MT are not clearly known, it has been considered to play a role in the detoxication, Accepted July 2, 1992 Received May 6, 1992 storage, and metabolism of heavy metals [6]. The epithelial cells of the human and rat pros- tate secrete MT [3, 7, 8] and zinc, an action which correlates with infertility [9]. Recently, MT was reported to be localized in the spermatogenic cells [10, 11], but few studies on MT in the epididymis have thus far been undertaken. The purpose of the present study was to demonstrate the localization of MT in the epididymis and spermatic cord as part of an investigation of the function of MT as it relates to sperm. MATERIALS AND METHODS Male Wistar rats (10 weeks old) were purchased 1010 from Imai Experimental Animals (Saitama, Japan) and were anesthetized by ether. The tissues ex- amined by immunohistochemical staining were obtained from the epididymis (head, body, and tail) and spermatic cord. Preparation of tissues for immunohistochemical examination All tissues were fixed in 10% buffered formalin solution for 48 hours and embedded in paraffin. Specimens were cut at a thickness of 3 ~m and mounted on glass slides. Staining was performed using an avidin-biotin peroxidase method. Sections were deparaffinized and incu- bated in 0.1% trypsin solution for 30 minutes at 37°C. They were then immersed in 0.5% (5 mM) periodic acid solution for 10 minutes in order to inhibit endogeneous peroxidase, and incubated with normal goat serum for 20 minutes to block the non-specific binding sites. Primary antibody was mixed with a 200-fold concentration of anti-MT-1 and ascaris antigen to inhibit non-specific reaction to ascaris, because anti-MT-1 antibody was obtained from rabbit antiserum boosted by MT-1 emulsion coupled to ascaris antigen with glutaral- dehyde [7]. The slides were layered with primary antibody for 2 hours at room temperature and complex T. Suzuki, H. YAMANAKA ef al. washed with 0.01M PBS buffer (pH7.2). The secondary antibody was applied for 1 hour and sections were again washed with PBS. They were treated with ABC complex for 30 minutes and washed with PBS. Sections were submerged in 0.05% diaminobenzidine tetrahydrochloride in 0.05M Tris buffer (pH7.6) to which H>O> (0.01%) has been added just before use. These reagents were prepared using a VECTASTAIN ABC KIT (VECTOR Laboratories, Burlingame, USA). Subsequent counterstaining was performed with Mayer’s hematoxylin. The specificity of the staining reaction was determined in a prior absorp- tion of anti-MT antibody with pure liver MT antigen (MT-1 and/or MT-2) (Sigma Chem. Co., USA) and omission of primary antibody from the procedure. RESULTS Immunohistochemical control As shown in Figure 1, the use of primary anti- body that had been absorbed with pure rabbit liver MT-1 and MT-2 did not result in any specific immunoreaction in the epididymal head or tail or in the spermatic cord. Use of non-immune serum Fic. 1. Control section of the ductus epididymidis in the tail portion treated with preabsorbed primary antibody. Immunoreaction for MT was negative in the epithelial and basal cells (x 220). MT in Rat Epididymis and Spermatic Cord 1011 and PBS did not result in any immunoreaction in _ tained two ducts, the ductuli efferentes and ductus any sections. epididymidis. No immunoreaction for MT was observed in the epithelial cells of the ductuli efferentes (Fig. 2). In the ductus epididymidis, basal cells showed positive immunoreaction for Immunohistochemical findings The head of the epididymis. This portion con- Fic. 2. Photomicrograph of the ductuli efferentes.. Immunoreaction for MT was negative in the epithelial and basal cells and in connective tissues (Xx 560). Fic. 3. Photomicrograph of the ductus epididymidis in the epididymal head. Immunoreaction for MT was found in the basal cells, but not in the epithelial cells or connective tissues (560). 1012 T. Suzuki, H. YAMANAKA et al. MT, but epithelial cells and connective tissues The body of the epididymis. A few epithelial showed a negative one. MT immunoreaction was cells of the ductus epididymidis, about one or two localized mainly in the cytoplasm and partly inthe cells per one section of the ductus epididymidis, nuclei of epithelial cells (Fig. 3). showed a positive immunoreaction for MT, but , b as P : 5 Es a e <. 4 *. s 3 Fic. 4. Photomicrograph of the ductus epididymidis in the epididymal body. Immunoreaction for MT was found positive in one epithelial cells, and was localized mainly in the cytoplasm. The basal cells, sperm and fluid in the lumen, and connective tissues were negative (350). pein ee “IS SET SSS ETE. ED ere - ——SS GN A Ze = - So a ~ ~~ = a a Be OR OE a a SG SS ease “a : 2 = - Fic. 5. Photomicrograph of the ductus epididymidis in the epididymal tail portion near the body. Basal cells had a positive immunoreaction for MT, but epithelial cells and connective tissues and sperm in the lumen were negative (x 560). MT in Rat Epididymis and Spermatic Cord 1013 most epithelial cells were nagative. The basal plasm, which appeared as fine granular immunos- cells, sperm and fluids in the lumen, and connec- taining (Fig. 4). tive tissues had no immunoreaction. MT im- The tail of the epididymis. In the portion near munoreaction was localized mainly in the cyto- the body, basal cells of the ductus epididymidis y, ] ite SS Se Fic. 6. Photomicrograph of the ductus epididymidis in the epididymal portion near the spermatic cord. Im- munoreaction for MT was found strongly positive in the epithelial and basal cells, and was localized mainly in the cytoplasm, especially the apical area, and partly in the nucleus. Connective tissues and sperm in the lumen were negative (x 560). Fic. 7. Photomicrograph of the ductus deferens. Immunoreaction for MT was found positive in the basal cells and some tall epithelial cells. Connective tissues and sperm in the lumen were negative (x 350). 1014 exhibited a positive immunoreaction for MT, but epithelial cells showed a negative one. The sperm and fluids in the lumen and the connective tissues exhibited a negative immunoreaction (Fig. 5). In the portion near the spermatic cord, the epithelial and basal cells of the ductus epididymidis showed a strong positive immunoreaction for MT, which was localized mainly in the cytoplasm, especially the apical area, and partly in the nuclei. The staining in the cytoplasm had a fine granular character. The sperm and fluids in the lumen and the connec- tive tissues were negative for immunoreaction (Fig. 6). Spermatic cord. Some tall epithelial cells of the ductus deferens showed a positive MT im- munoreaction as did many basal cells. The loca- lization of MT immunoreaction was seen mainly in the cytoplasm and in some nuclei (Fig. 7). Epithe- lial cells of the portion near the urethra were mostly negative. The connective tissues and the sperm and fluids in the lumen were negative. The basal cells in the ejaculatory duct also showed a positive immunoreaction for MT. Connective tis- sues surrounding the ejaculatory ducts exhibited a negative immunoreaction. DISCUSSION The presence of MT in rat testes was reported by Nolan and Shaikh [12], and immunohistochemical- ly demonstrated by Danielson [4] and Nishimura er al. [10]. MT immunoreaction was observed in the spermatogenic cells in the seminiferous tubules, but not in mature sperm in the lumen. Recently, De et al. [11] reported that MT mRNA which accumulated after the initial differentiation of primary spermatocytes was maintained in sperma- tids, and was shown to be present at low levels in interstitial, spermatogonial, and matured sperm cells by Nothern blooting and in situ hybridization. Therefore, they suggested there being a role for MT in the process of spermatogenesis. In this study, matured sperm in the lumen of the ductuli efferentes, ductus epididymidis, and ductus de- ferens had a negative immunoreaction for MT, suggesting that they did not synthesize MT. MT was identified in the prostatic cells of rats and humans, and was shown to secrete into the T. Suzuki, H. YAMANAKA et al. prostatic fluids [3, 7, 8]. The seminal fluids mainly consisted of those derived from the testis and epididymis, the prostate, and the seminal vesicle. MT is known to bind to zinc, an action which is reported to correlate with infertility [9, 13]. However, few studies on MT in the epididymis have thus far been undertaken, and the physiolo- gical function of MT in the epididymis is unclear. Nishimura et al. [10] reported that MT im- munoreaction was observed in a limited number of epithelial cells and in some basal cells in the ductus epididymidis of the rat. The present study demon- strated the differences of immunoreactions for MT in the various portions of the ductus epididymidis and spermatic cord. The basal cells had a positive immunoreaction for MT in the head and tail portions of the ductus epididymidis, but not in the body. The epithelial cells had a positive im- munoreaction in the body and tail, especially in many of the epithelial cells in the tail portion near the spermatic cord. The spermatozoa first to leave the testis are incapable of fertilization and are described as being immature. The sperm in the tail of the epididymis are mature and capable of ferti- lization [14]. The charactristic feature of matura- tion is a change in the surface of the sperm, as well as the presence of glycoprotein secreted from the epididymal epithelial cells which has been reported to bind to the sperm surface [15]. The principal cells has numerous cisternae of rough endoplasmic reticulum and well-developed Golgi apparatus associated with secretory function, and the MT localized in the epithelial cells of the tail portion was suggested to be secreted into the epididymal fluids [16, 17]. In this study, however, the secre- tion of MT could not be clearly demonstrated under light microscopy, and an electron immuno- histochemical study was though to be necessary to demonstrate the correlation between the sperm and MT in the epididymis. In this study, the localization of MT was observed mainly in the cytoplasm and partly in the nuclei. Histologically, MT localization in the nuc- leus has been reported by Nishimura et al. [10] to be present in the spermatogonial cells, and in the prostatic cells of the rat by many investigators [3, 7, 10, 18]. The positive staining of epithelial and basal cell nuclei might conceivably result from MT in Rat Epididymis and Spermatic Cord contamination of nuclear proteins by the MT transposed during tissue preparation. However, no nuclei had ever shown a positive immunoreac- tion in the epididymis, reducing the likelihood of this explanation. Recently, large molecules con- taining the nuclear location signal sequence have been reported capable of being transported into the nucleus crossing the nuclear envelope of mammalian cells [19, 20]. This suggests that MT may also be transported into the nucleus and act as an enzyme activator by donation of zinc bound to it. In summary, the localization of MT and differ- ences of immunoreaction for MT in the various portions of the ductus epididymidis were demons- trated. Many epithelial cells in the tail had a positive immunoreaction for MT, and MT was suggested to function in the maturation of sperm. ACKNOWLEDGMENTS This work was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture (02454367), Japan. REFERENCES 1 Margoshes, M. and Vallee, B. L. (1957) A cadmium protein from equine kidney cortex. J. Am. Chem. Soc., 799: 4813-4814. 2 Danielson, K. G., Ohi, S. and Huang, P. C. (1982) Immunochemical localization of metallothionein in rat liver and kidney. J. Histochem. Cytochem., 30: 1033-1039. 3 Umeyama, T., Saruki, K., Imai, K., Yamanaka, H.., Suzuki, K., Ikei, N., Kodaira, T., Nakajima, K., Saito, H. and Kimura, M., (1987) Immunohistoche- mical demonstration of metallothionein in the rat prostate. Prostate, 10: 257-264. 4 Danielson, K. G., Ohi, S. and Huang, P. C. (1982) Immunochemical detection of metallothionein in specific epithelial cells of rat organs. Pre. Natl. Acad. Sci., 79: 2301-2304. 5 Bremner, I. and Davies, N. T. (1975) The induction of metallo-thionein in rat liver by zinc injection and restriction of food intake. Biochem. J., 149: 733- 738. 6 Kagi, J. H.R. and Vallee, B. L. (1960) Metallothio- nein: a cadmium- and zinc-containing protein from equine renal cortex. J. Biol. Chem., 235: 3460- 3465. 7 Suzuki, T., Umeyama, T., Ohma, C., Yamanaka, 14 15 16 17 18 19 20 1015 H., Suzuki, K., Nakajima, K. and Masami, K. (1991) Immunohistochemical study of metallothio- nein in normal and benign prostatic hyperplasia of human prostate. Prostate, 19: 35—42. Suzuki, T., Yamanaka, H., Tamura, Y., Nakajima, K., Kanatani, K., Masami, K. and Otaki, N. (1992) Metallothionein of prostatic tissues and fluids in rat and human. Tohoku J. Exp. Med., 166: 251-257. Saito, S., Bush, I. M. and Whitmore, W. F. Jr. (1967) Effects of certain metals and chelating agents on rat and dog epididymal spermatozoan motility. Fertil. Steril., 18: 517-529. Nishimura, H., Nishimura, N. and Tohyama, C. (1990) Localization of metallothionein in the genital organs of the male rat. J. Histochem. Cytochem., 38: 927-933. De, S. W., Enders, G. C. and Andrews, G. K. (1991) High levels of metallothionein messenger RNAs in male germ cells of the adult mouse. Mol. Endo., 5: 628-636. Nolan, C. V. and Shaikh, Z. A. (1986) An evalua- tion of tissue metallothionein and genetic resistance to cadmium toxicity in mice. Toxicol. Appl. Phar- macol., 85: 135-144. Morisawa, M. and Mohri, H. (1972) Heavy metals and spermatozoan motility. I. Distribution of iron, zinc and copper in Sea urchin spermatozoa. Exp. Cell Res., 70: 311-316. Glover, T. D. (1976) The epididymis. In “Scientific Foundations of Urology” Ed. by Williams, D. I. and Chisholm, G. D., William Heinemann Medical Books Ltd., London, pp. 176-186. Eddy, E. M. (1988) The Physiology of Reproduc- tion. Ed. by Knobil, E. et al., Raven Press, New York, pp. 27-68. Hoffer, A. P., Hamilton, D. W. and Fawcett, D. W. (1973) The ultrastructure of the principal cells and intraepithelial leucocytes in the initial segment of the rat epididymis. Anat. Rec., 175: 169-201. Moore, H. D. M. and Bedford, J. M. (1979) The differential absorptive activity of epithelial cells of the rat epididymis before and after castration. Anat. Rec., 193: 313-327. Bataineh, Z. M., Heidger, P. M. Jr., Thompson, S. A. and Timms, B. G. (1986) Immunocytochemical localization of metallothionein in the rat prostate gland. Prostate, 9: 397-410. Reynolds, C. R. and Tedeschi, H. (1984) Per- meability properties of mammlaian cells nuclei in living cells and in vitro. J. Cell Sci., 70: 197-207. Yoneda, Y., Arioka, T., Imamoto-Sonobe, N., Sugawa, H., Shimonishi, Y. and Uchida, T. (1987) Synthetic peptides containing a region of SV 40 large T-antigen involved in nuclear localization direct the transport of proteins into the nucleus. Exp. Cell Res., 170: 439-452. ZOOLOGICAL SCIENCE 9: 1017-1023 (1992) Analysis of Sex Steroids in Feces of Giant Pandas Kaoru Kusoxawa!”, Susumu IsHu’, HipeEo Tasmma® Kazuo Saitou® and Kouk! TANABE> '? Department of Biology, Waseda University, Shinjuku, Tokyo 169-50, and *Ueno Zoological Gardens, Ueno-Kouen, Taitoh-ku, Tokyo 110, Japan ABSTRACT—Concentrations of testosterone (T) and estradiol-172 (E) in feces were measured in giant pandas (Ailuropoda melenoleuca). Feces were collected almost monthly from October 1987 to August 1988 and from June 1989 to July 1990, from captured pandas (a male and a female adult pandas and their two children) at Ueno Zoo, Tokyo. The elder child born in 1986 was identified later as the female and the younger one born in 1988 as the male. The concentrations of T in feces of the adult male was always higher than that in the female when compared in the same month. The concentration of E> in feces of the adults did not show clear difference between sexes. In the adult female, a high E, peak was observed in March of 1988, when it showed estrus and was artificially inseminated. No E> peak was detected in the spring of 1990, when it did not show clear estrus. Concentrations of T and E> in the children, especially in the younger child, were relatively low and variable. They showed no consistent seasonal changes. These results suggest that the sexing of adult pandas is possible by comparing the concentration of T in feces. Peaks of the fecal steroid content seem to show roughly reproductive condition of an animal, suggesting a possibility that ovulation can be detected in the female panda by the fecal E> analysis. © 1992 Zoological Society of Japan INTRODUCTION As well-known, the giant panda (Ailuropoda melenoleuca) is an endangered mammalian species distributed only in limited areas in China. Although its ecology has been thoroughly studied (see Schaller et al. [1]), much remains to be clarified. Especially, it is important to know reproductive physiology in wild individuals of this species. Analysis of sex steroid hormones in blood plasma is a popular and indispensable method for studying reproductive physiology of vertebrates. However, it is impossible or extremely difficult to collect blood samples from wild individuals of this species in the field or even from captive individuals in many cases. In captive individuals, analysis of urinary steroid can be an alternative, but it is practically difficult to collect urinary samples espe- Accepted July 23, 1992 Received June 12, 1992 ' Present address: Minami-dai, Nakano 164, Japan. Laboratory of Molecular Biology, Ocean Research Institute, University of Tokyo, 1-15-1, cially from infant pandas. Attempts have been made to estimate gonadal endocrine activity of mammals and birds by ana- lyzing sex steroids in feces [2-8]. This non- invasive method was recommended by Risler et al. [2] as a useful tool in ecophysiological studies and diagnosis of pregnancy in wild mammals or mam- mals from which collection of blood or urinary sample is difficult. Czekala and Lasley [5], and Stavy et al. [6] employed this method for sexing of monomorphoic bird species. Motivated by the study of Risler et al. [2], the authors intended to study the relation between feal sex steroids and sex or reproductive condition in adult and infant giant pandas by measuring sex steroids in feces which were collected in different months from two infant giant pandas and their parents. 1018 K. Kusoxkawa, S. Isuti et al. MATERIALS AND METHODS Animals Two infant giant pandas and their parents reared in Ueno Zoo, Tokyo were used. The father (named Fei Fei) was born in 1967 in Sichuan (China), caputured in 1976 and brought to Ueno Zoo in November 1982. The mother (Huan Huan) was born in 1972 in Sichuan (China), captured in 1975 and brought to Ueno Zoo in January 1980. The infants (Tong Tong and You You) were born in June 1986 and June 1988, respectively, and have been reared in Ueno Zoo. It was unable to sex these infants at the time of their birth morphologi- cally. Three to four years later, the elder and younger infants were identified as the female and the male, respectively, from their behavior and the difference in the length between the anus and urogenital opening. Collection of feces Fecal samples were collected from the floor of cages in which animals were kept once a month on 20th day of each month at around 5 am during the following two periods: the first from October 1987 to August 1988 and the second from June 1989 to August 1990. The feces were considered to be excreted between 9 pm and 5 am, since the floor of the cages used to be cleaned at 9 pm every day. One of fecal droppings or masses found in the floor was randomly selected for each animal at each collection time and frozen in a freezer at —20°C soon after collection and stored for a few months until steroid analysis. The mean weight + standard deviation (SD) of the selected fecal dropping was 56.46 + 20.22 g in the father, 52.44+ 15.52 g in the mother, 54.39+19.16g in the elder infant and 49.54+22.24 g in the younger infant. Extraction We followed the method of Risler er al. [2] with slight modification. Feces were partially thawed at the room temperature, and thick threads of bam- boo contained were removed with forceps. Then, 30 to 100g of the feces were homogenized in a grinder with 10 volumes of a mixture of ethanol and acetone (8:2). After adding about 1,000 cpm of tritiated steroids for determining the recovery rate, the homogenate was centrifuged at 3,000 rpm for 10 min at 4°C. The supernatant was filtered through a cellulose membrane filter (0.2 ~m pore size, FR-20, Fuji Film Co. Ltd, Tokyo). After concentrating the volume to about 5 ml by centri- fuging evaporation in vacuo, the volume was ad- justed to 20 ml by adding ethanol and water (8:2). This solution was placed in a freezer at —20°C for 12 to 18 hr to precipitate fat. After centrifuging at 2,000 rpm at —10°C for 20 min, fat was removed. To the ethanolic supernatant, 5 volumes of pet- roleum ether was added. After vortexing, the petroleum ether layer was removed and discarded. The bottom layer was evaporated to 1 to 2 ml, and extracted two times with 5 volumes of ethyl ether. The combined ether phase was washed two times with 1 ml of 8% sodium bicarbonate solutions with pH 10 and 8, respectively, and then the ether phase was evaporated to dryness under nitrogen. The residue was dissolved in 0.5 ml of methanol and used as the sample for chromatography. Chromatography Estradiol-172 (E>) was separated from testoster- one and progestrone by the DEAE A-25 column chromatography according to the method of Risler et al. [2]. The DEAE A-25 sephadex was changed from the chloride form to the hydroxide form just before the column chromatography, and sus- pended in 100% methanol. Columns were made of Pasteur pipettes, and packed with 1.5 ml of the gel. After applying the sample in 0.5 ml methanol, the neutral steroids were eluted with Sml of methanol, and then the phenolic steroids with 5 ml of 0.1 M acetic acid in methanol. Each eluate was evaporated to dryness and dissolved in 0.5 ml of methanol. The mean recovery+SD through the whole course of the extraction and separation procedures was 73.6+21.3% in E> and 65.9+17.0% in testos- terone. Radioimmunoassay Testosterone and E> in the chromatographic fractions were determined by radioimmunoassay using testosterone-3-(O-carboxymethyl) oximino- (2-[!"*I]iodo-histamine) of Amersham, England, Fecal Steroids in Giant Panda 1019 respectively, as radioligands, and the rabbit anti- testosterone serum (HAC-AA61-02-RBP81) and the rabbit anti-estradiol serum (HAC-AA63-01- RBP7), respectively, provided by Prof. Katsumi Wakabayashi of Gunma University. The separa- tion of free and bound steroids was performed with the second antibody method using a goat anti- rabbit gamma G serum provided by Prof. Katsumi Wakabayashi. The standard (12.2 pg to 6.25 ng of testosterone or E>/0.05 ml) or an appropriately diluted unknown sample (0.05 ml) was preincu- bated with the corresponding first antiserum (0.05 ml) and buffer (0.1 ml) at 4°C for 24 hr. Then, the mixture of the sample and antiserum was reacted with corresponding radioligand (10,000 cpm/0.05 ml) at 4°C for 48hr. The in- cubation with the second antiserum (0.1 ml of the 200 times diluted serum) contained with 4% polyethylene glycol was performed at room temperature for 3 hr. The mean intra- and inter- assay coefficients of variation were 1.5 and 4.3%, respectively, in the testosterone assay. They were 2.9 and 7.87%, respectively, in the E> assay. Testosterone (ng/g) 1011 1234 87 198 678 8 RESULTS Testosterone and estradiol concentration in mature pandas The testosterone concentration in feces of the parents varied widely among months, but the range of the variation was smaller, between 8 and 880 pg/g, in the mother than in the father, be- tween 105 and 8,910 pg/g (Fig. 1). Especially, the fecal testosterone level in the mother was signi- ficantly lower than that in the father when com- pared in the same month (P<0.01 by the analysis of variance with two-way layout). In addition, seasonal changes in the testosterone level were similar between the two observation periods (one from June 1987 to August 1988 and the other from June of 1989 to July of 1990) in both the father and mother, although data of August and September in 1989 and April and May in 1990 were lacked accidentally in both individuals. In the father, there were two peaks of testoster- one, one in the autumn (October or earlier in 1987 and October in 1989) and the other in the spring or late winter (April in 1988 and February in 1990). Corresponding exactly to these spring peaks in the OQ, 0 5 e) : 9 Fo) So :! 3 * (2) 0 12) ore, A 6 891011121234 67 1989 1990 Fic. 1. Monthly changes of immunoreactive testosterone levels in feces of mature male panda (Fei Fei) (open circles and dotted line) and mature female panda (Huan Huan) (closed circles and solid line) from June of 1987 to August of 1988, and from June of 1989 to Jyly of 1990. Samples were collected in 20th of each month. Each point indicates the mean of duplicate determinations for each fecal dropping. 1020 father, there were peaks of testosterone around the same time also in the mother. Supporting this, Kendall’s rank correlation analysis revealed that there was a statistically significant positive correla- tion (t=0.75, P<0.05, n=9) in the fecal testo- sterone concentration between the father and the mother in the earlier half of the year (from January to June) but not significant correlation or even negative correlation (t= —0.60, P>0.05, n=10) in the variable in the later half of the year (from July to December). The concentration of estradiol-178 (E2) in feces as well as the concentration of testosterone varied widely among months in both the mother (near 0 to 700 pg/g) and the father (near 0 to 1,064 pg/g) (Fig. 2). However, unlike the testosterone con- centration, the estradiol concentration did not differ clearly between the father and the mother (P >0.1 by the analysis of variance). In the father, the E> level elevated conspicuous- ly in January and February in both observation periods, showing the highest peaks in February (Fig. 2). Peaks of E> were also found in June or 1000 800 ) fe) . (pg/g o fo} fo} Estradiol 400 200 67 10 11 1987 Fic. 2. K. Kusoxkawa, S. Isuui ef al. Jyly in both observation periods in the father. To confirm the parallelism of the seasonal changes in fecal E, between these two periods, the correlation analysis was applied. There was found a statistical- ly significant positive correlation (t=0.763, P< 0.05, n=7) in the estradiol concentration in the father between the two observation periods. In the mother, the concentration of E> in feces had the highest peak of the year in November in both observation periods. High estradiol levels were also observed in June and July in both observation periods. Only in the first observation period, there was an additional high E> peak in March. The correlation of the estradiol concentra- tion in the mother between the two observation periods was not statistically significant (c=0.112, P>0.05, n=7). Testosterone and estradiol-178 concentrations in children In the elder child, the concentration of testoster- one in feces was low in the first observation period, having three peaks in October 1987, January 1988 23 7 1990 8 91011 1 Monthly changes of immunoreactive estradiol levels in feces of mature male panda (Fei Fei) (open circles and dotted line) and mature female panda (Huan Huan) (closed circles and solid line) from June of 1987 to August of 1988, and from June of 1989 to Jyly of 1990. Samples were collected in 20th of each month. Each point indicates the mean of duplicate determinations for each fecal dropping. Fecal Steroids in Giant Panda 1021 ss Testosterone (ng/g) 7 1011 1234 678 6 891011121 23 67 1987 1988 1989 1990 Fic. 3. Monthly changes of immunoreactive testosterone levels in feces of infant elder panda (Tong Tong) (closed triangles and solid line) and younger panda (You You) (open triangles and dotted line) from June of 1987 to August of 1988, and from June of 1989 to July of 1990. Samples were collected in 20th of each month. Each point indicates the mean of duplicate determinations for each fecal dropping. =e " x 800 Estradiol (pg/g) o ° ro) 400 7 1011 1234 678 6 7 891011 123 7 1987 1988 1989 1990 Fic. 4. Monthly changes of immunoreactive estradiol levels in feces of infant elder panda (Tong Tong) (closed triangles and solid line) and younger panda (You You) (open triangles and dotted line) from June of 1987 to August of 1988, and from June of 1989 to July of 1990. Samples were collected in 20th of each month. Each point indicates the mean of duplicate determinations for each fecal dropping. 1022 and June 1988, respectively (Fig. 3). However, the range of the fluctuation was small, and the highest peak was about 1,000 pg/g. In the second observa- tion period, the testosterone levels were still low except the two peaks, one higher than 2,000 pg/g in December 1989 and the other higher than 4,000 pg/g in February 1990 (Fig. 3). These basal and peak testosterone levels in the elder child were clearly lower than the respective levels in the father. The two peaks in the second observation period in the elder child were higher than the highest testosterone peak in the mother, but the levels in the other months in the elder child were similar to the level in the mother. The concentration of E> in feces of the elder child had three peaks in each of the two observa- tion periods (Fig. 4). Their locations (October, January or February and June) coincided approx- imately between the two observation periods, although their heights were different. In the younger child, the concentration of testos- terone in feces was extremely low, always less than 400 pg/g (Fig.3). The concentrations of E> in feces of the younger child were low and had three peaks in November, February and presumably June (Fig. 4). DISCUSSION Validity of the method of fecal sex steroid analysis must be discussed first. Positive proof of validity of this method for estrogens including E> and progesterone was demonstrated first by Adler- creuz and Jarvenpaa [7] with human feces. They identified the steroids in the feces by gas chroma- tography and mass spectrometry. They also found that 85 to 90% of excreted estrogen occurs in unconjugated form and also that fecal estrogen and progesterone well reflect reproductive condi- tions of men and women. Most or a part of their results have been confirmed in Macaca by Risler et al. (2). In addition, analysis of fecal E> and testosterone was successfully applied for sexing in three species of birds by Stavy er al. [6] and for estimation of gonadal endocrine activity in the Japanese quail [8], although feces of birds contain urine. Thus, there will be little doubt about the validity of fecal K. Kusoxkawa, S. Isuti et al. sex steroid analysis for assessing gonadal endoc- rine activity in mammals and birds. It is obvious that feces of the father contain higher concentration of testosterone than feces of the mother. This difference can be due to either sexual difference or a coincident individual varia- tion. The former possibility is supported by the following facts revealed in the present study: 1) the fecal testosterone showed a peak in the breeding period of this species, 2) similar seasonal changes were observed in the two observation periods, 3) the testosterone levels in the father were higher than those in the children, and 4) peaks of the testosterone level appeared simultaneously in the breeding season also in the mother, although the peak levels were lower in the mother. It is also noteworthy that there were additional increases in fecal testosterone in the autumn in the father. This fact reminds us the report that the giant panda breeds some times in the autumn [1]. Furth- ermore, in Ueno Zoo, artificial collection of sper- matozoa became possible from October in the father, and they were available until May or June. Thus, the possibility of the difference in the testos- terone concentration in feces between the mother and the father is obviously high, and hence the possibility of a coincident individual variation is low. We have no proper explanation for the fact that the father excreted as large amounts of E> in feces as the mother. However, this is not so surprising, since Adlercreuz and Jarvenpaa [7] reported that men and postmenopausal women excreted similar mounts of estrogen in feces. The presence of the estradiol peaks in November in both of the two observation periods in the mother is favorable to the theory of the presence of the additional breed- ing period in the autumn. There was a high peak of estradiol in March of 1988, when the mother was artificially inseminated and pregnancy was induced. She gave birth of You You in June of the same year. However, there was no conspicuous estradiol peak in March in 1990, when artificial insemination was attempted again but no pregnan- cy was induced this time. Fecal testosterone excreted by the elder child in the first observation period was as small in amount as that excreted by the mother. However, in the Fecal Steroids in Giant Panda second observation period, there were found two high peaks of fecal testosterone in December and February which were higher than the highest peak in the mother but lower than that in the father. However, unlike the father, the elder child did not show high autumnal increase in the fecal testoster- one. Amounts of excreted testosterone in feces by the younger child were too low to discuss the seasonal excretion pattern. The elder child showed a high fecal estradiol level in February 1988. This level is as high as the peak levels observed in the parents. In 1990, there was found an extremely high estratiol excretion in feces in the elder child in June. This level was higher than any of the highest levels observed in the parents. Interpretation of this estradiol level in June is difficult. The younger child excreted small amounts of estradiol in feces in both 1989 and 1990. These amounts are similar to those excreted in months of 1987 by the elder child who is 2 years older. In conclusion, this study suggested with a high possibility that we can sex adult giant pandas by analysis of fecal sex steroids, especially testoster- one, although more examples are needed to con- clude. It may be also possible to estimate gonadal endocrine activity of giant pandas indirectly by analyzing fecal sex steroids successively from the same individual. However, sexing of infant giant pandas by analysis of fecal sex steroids may be difficult. ACKNOWLEDGMENTS The authors thank to Mr. T. Hahn, University of Washington for his reviewing the manuscript and Prof. 1023 K. Wakabayashi for providing the antisera. This work was supported by grants of the Ueno Zoological graden, Waseda University and Ministry of Education, Science and Culture. REFERENCES 1 Schaller, G. B., Hu, J., Pan, W. and Zhu, J. (1985) The Giant Pandas of Wolong. The University of Chicago Press, Chicago. 2 Risler, L., Wasser, S. K. and Sackett, G. P. (1987) Measurement of excreted steroids in Macaca nemes- trina. Am. J. Primatol., 12: 91-100. 3 Shille, V. M., Wing, A. E., Lasley, B. L. and Banks, J. A. (1980) Excretion of radiolabeled estradiol in the cat (Felis catus, L.): A preliminary report. Zool. Biol., 3: 201-209. 4 Perez, L. E., Czekala, N. M., Weisenseel, K. A. and Lasley, B. (1988) Excretion of radiolabeled estradiol metabolites in the slow loris (Nycticebus coucang). Am. J. Primatol., 16: 321-330. 5 Czekala, N. M. and Lasley, B. (1977) A technical not of sex determination in monomorphic birds using fecal steroid analysis. International Zoo Year book, 17: 209-211. 6 Stavy, M., Gilbert, D. and Martin, R. D. (1979) Routine determination of sex in monomorphic bird species using fecal steroid analysis. International Zool Year book, 19: 209-214. 7 Adilercreuz, H. and Jarvenpaa, P. (1982) Assay of estrogens in human feces. J. Steroid Biochem., 17: 639-645. 8 Ishii, S. (1985) Estimation of gonadal activity by means of fecal sex steroid analysis in the Japanese quail. Report of Japanese Ibis Breeding Project in 1985, Nugata Prefecture, pp. 27-30 (in Japanese). clon WOT Miah ka sear. all ell ee yD nia vel ‘ Pag Fhe diapers qn vbiettnnds fie beta tae PR ae Ueki Wit F wos Mak? revs ‘i a bt MAL eS L } ; wa | ; i : dent iy { vat a / dey ae citi nt weuaniat Bp Viegas a pe ele BSR cid, G f s 4 ia EP WIE TY tlideavh wnat” re sven pe aihetityal tied | inet sols Bevan) vane anvanlt hve bie cae sya aolceorea ed yale wel ror redo ag : ace Wikeshaliet ‘ily dt janet _ ieee aat Motelvear tt pea » ether Drain eg onli Healey eel hake fos te Tis eRe utes t tis ria int! ys fre See te SW ahr havi oa b eet brie letter tad igh ond ry bie eesti ab cts hetene 4 othe getico gl aed cele ee eV tlhe eh Nonny iy ih in ob al Mi Vinken Lucena no FO re RS 0 A oe i at a dat nec cipend bet ecsinetitd 4 hehe “EY ~ tle 1 whale a Ge iemaeygi i esi aay ete stint andl “Tyan a ae lhe ory ok Cte heel canny gett deel Pep 00h elite & bi Jini oye nage cnerrt Wiawieowave Logan Limi freq ae (ory 4 vin abies tacit be nara B ZOOLOGICAL SCIENCE 9: 1025-1035 (1992) Oviduct 17£-Estradiol Receptor in the Female Lizard, Podarcis s. sicula, during the Sexual Cycle: Relation to Plasma 17/- Estradiol Concentration and Its Binding Proteins M. Paotuccr’, M. M. Di Fiore and G. Crarcia? Dipartimento di Zoologia, via Mezzocannone, 8. Universita di Napoli, 80134 Napoli, and *Dipartimento di Biologia Evolutiva e Comparata, via Mezzocannone, 8. Universita di Napoli, 80134 Napoli, Italy ABSTRACT—In the oviparous lizard, Podarcis s. sicula, 17f-estradiol binding molecules were characterized in the oviduct and plasma. In addition, their concentration was evaluated throughout the annual cycle. In the oviduct, a 17f-estradio] receptor (ER) is present. It shows high affinity for ligand (4.9-7.0 x 10~'°M). It is decreased by ovariectomy, and induced by 17/-estradiol treatment. 17(-estradiol causes also a ER shift from the cytosol into the nuclei. On sucrose gradient ER behaviour is consistent with the properties of ER obtained from other 17f-estradiol target organs. At isoelectrofocusing the labeled © 1992 Zoological Society of Japan 17-estradiol binding molecules fall into three pH ranges: 5.2-5.6, 7.0-7.7, 8,0-8.7. Nuclear filled ER significantly increases as oviduct grows. stimulation by 17/-estradiol. This supports an ER role in oviduct In the plasma, 17-estradiol binding molecules show many physico-chemical and behavioural properties of steroid-binding proteins (SBPs). INTRODUCTION In the lizard, Podarcis s. sicula, like in most Oviparous reptiles living in temperate zones, the female genital apparatus is active during spring and keeps quiescent for the rest of the year [1]. In early spring, as animals leave their winter shelters, several ovarian follicles undergo vitellogenesis, becoming yolky and ripe in a few weeks. Ovarian resumption is accompanied by parallel progressive growth of the oviduct, which rapidly reaches maturity. Reproductive females usually lay one to three egg clutches (three to six eggs per clutch at 20-day intervals), starting from the first half of May. In late spring, the breeding season ceases, the ovary becomes quiescent, and the oviduct regresses assuming a typical thread-shaped aspect (25 Ble The mature oviduct shows complex gross mor- Accepted June 6, 1992 Received February 24, 1992 ' To whom all correspondence should be addressed. phology and includes four regions, cranio- caudally: infundibulum, tuba, uterus and vagina. Histologically, it is characterized by considerable hypertrophy of the epithelium and associated glands. Moreover, oviduct development and activ- ity give rise to several biochemical events involving RNA and protein synthesis [4, 5], as well as some hydrolytic enzyme activity [4]. Lizard eggs are supplied with little albumen and a coriaceous shell, which are secreted by tubal and uterine glands, respectively [6, 2]. Seasonal oviduct growth depends on ovarian sex hormones, since, in prereproductive females, it can be easily prevented by ovariectomy and res- tored by 17f-estradiol or testosterone injections into spayed females [7]. Moreover the presence of sex hormone-binding molecules in oviduct cytosol and nuclei, has been also reported [8]. This work was undertaken with the aim of defining the properties of oviduct 17/-estradiol binding molecules in lizards, and ascertaining their putative receptor nature. Moreover, the concen- tration of these molecules has been evaluated 1026 throughout the breeding period and in 17f- estradiol and/or progesterone treated and spayed females, in relation to the plasma titres of steroid binding proteins (SBPs) and sex hormones. MATERIALS AND METHODS Animals Adult females of Podarcis s. sicula were caught in the outskirts of Naples during October 1990-July 1991. Soon after capture, the animals were anaesthetized with ethyl ether and bled through a heparinized glass capillary inserted in the heart. After brief centrifugation, the plasma was stored in liquid nitrogen until use. At autopsy, the oviducts were rapidly removed, rinsed in 0.7% saline solution in order to eliminate any blood trace, weighed and plunged in liquid nitrogen. Macroscopical oviduct development and ovarian follicle diameters were assessed by direct inspec- tion, and the tissues were divided into four groups, according to the stage of oviduct development: 1. Quiescent stage (October—February). Ovi- ducts showed a thread-like aspect (about 19 mg weight each). The ovary contained only previtel- logenetic follicles. 2. Recovery stage (March-April). Oviducts were growing and each of them weighed about 30 mg. In the ovary, several follicles were engaged in vitellogenetic processes. 3. Full growth stage (April-June). Oviducts appeared full grown, and each of them weighed about 80mg. In each ovary 1 to 3 ripe follicles were ready to ovulate. 4. Secretive stage (June-July). Eggs were con- tained in the oviduct which weighed about 60 mg. For experimental studies lizards, caught both in October and June, were reared in terraria at a temperature of 28°C, with a photoperiod L:D= 16:8, and fed on meal worms and vegetables ad libitum. October lizards were ovariectomized; four weeks later they were divided into four groups, each of 20 animals, and treated for two weeks as follows. Group 1: injected every two days intraperi- toneally with 0.1 «g of 17P-estradiol (Sigma) dis- solved in 0.1 ml of 0.7% saline. Group 2: injected with 0.1 ug of progesterone (Sigma) dissolved as before. M. PaoLucci, M. M. Di Fiore AND G. CrarcIA Group 3: injected with both 0.1 ug of 17f- estradiol and 0.1 ug of progesterone. Group 4: injected with 0.1 ml of solvent. Twenty-four hours after the last injection, the lizards were killed and processed as reported be- fore. June intact lizards with secretive oviducts, were divided into two groups, each of 10 animals. Group 1 animals were injected intraperitoneally with 0.1 ug of 17@-estradiol (Sigma) dissolved in 0.1 ml of 0.7% saline. Group 2 animals received the solvent alone. The lizards were killed six and twenty-four hours later, and their oviducts were utilized as reported above. Preparation of oviduct subcellular fractions All procedures were carried out at 0-4°C. Analytical grade chemicals were used. Oviducts were weighed, minced and homogenized in 3.5 vol (w/ v) of TEMG (10 mM Tris-HCl, 1 mM EDTA, 1 mM 2-mercaptoethanol, 10% glycerol, pH 7.8), containing 0.05 M NaCl (homogenization buffer). The suspension was centrifuged at 800g for 10 min. The supernatant was centrifuged at 105,000 xg for 1 hour in order to obtain the cytosol. The 800g pellet was suspended in 10 vol of buffer Tris-HCl 10mM containing 3mM MgCh, 2mM monothioglycerol, 0.25 M sucrose, pH 7.5 (rinsing buffer), pelletted and rinsed twice with the same buffer volume. Final pellet was suspended in 3.5 vol. (w/v) of TEMG containing 0.7 M KCI (ex- traction buffer). This suspension was frozen, thawed, and left 1 hour in ice bath with occasional stirring. The suspension was thereafter centri- fuged at 105,000Xg for 1 hour. The supernatant constituted the nuclear extract. Measurement of *H-17{3-estradiol binding in cytosol and nuclear extract (2,4,6,7-°H)-17-estradiol (90/110 Ci/mmole) was purchased from Amer- sham Radiochemical Centre (Amersham, Bucks, U.K.); unlabeled steroids were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Endogenous steroids were previously stripped from samples by adding an equal volume of TEMG containing 0.05% (w/v) dextran (Dextran T-70, Pharmacia Fine Chemicals, Piscataway, NJ USA) and 0.5% (w/v) active charcoal (Norit A Estrogen Receptor in Lizard Oviduct charcoal-Sigma, St. Louis, MO USA). The mix- ture was vortexed and incubated for 10 min at 4°C. The charcoal was removed by centrifugation at 3000 rpm for 10min. The steroid-free super- natants were utilized for all subsequent analyses. Aliquots (0.2 ml) of cytosol and nuclear extract were incubated with 5nM labeled 17/-estradiol, with or without a 100-fold excess of diethylstylbe- strol (DES) (DES does not bind to steroid binding proteins in plasma, therefore it is used to discri- minate between 17/-estradiol binding sites in plas- ma and in tissues). After 16 hours incubation at 4°C, 0.6ml of active charcoal suspension was added to the incubation medium. The mixture was vortexed and kept for 5 min in ice bath; thereafter it was centrifuged at 800 x g for 10 min at 4°C. The supernatant was decanted in counting vials and added with Maxifluor scintillation fluid (Maxifluor, Packard, Milan. Italy). Radioactivity was mea- sured in a Liquid Scintillation Spectrometer (Pack- ard 1600-CA) at 45% counting efficiency. For Ky determinations, 0.2 ml aliquots of cyto- sol and nuclear extracts were added to tubes containing increasing amounts (0.3 to 5nM) of labeled 17-estradiol, with or without a 100-fold excess of DES. Incubations were carried out for 16 hours at 4C. Bound and unbound steroids were separated by adding 0.6 ml of charcoal-dextran suspension as reported before. Specific binding data were analyzed according to Scatchard’s graphic method [9]. Labeled 17f-estradiol unfilled (RU) binding sites were determined by incubation 0.2 ml of samples with 5nM labeled 17-estradiol with or without a 100-fold excess of DES for 30 min at 0°C. Total binding sites (RT) were determined by incubating similar mixtures for 30 min at 20°C. The filled binding sites (RF) were calculated by the formula: RT—RU=RF [10]. For binding specificity evaluation, 0.2 ml sam- ples were added to 5 nM of labeled 17/-estradiol with or without increasing amount (10° °-10-° M) of various unlabeled steroids. Incubation and separation of bound and unbound steroids were performed as reported before. Measurement of *H-17{-estradiol binding in the plasma Endogenous steroids were removed as 1027 reported above. For Kg determination, plasma aliquots (0.2 ml), diluted with TEMG to a protein concentration of 2mg/ml, were used. Proteins were determined by Lowry et al.’s (1951) method [11], using BSA as a standard. Samples were added to tubes containing increasing amounts (0.3 to 20 nM) of labeled 17/-estradiol, with or without a 100-fold excess of unlabeled 17/-estradiol. Tubes were incubated at 0°C for 1 hour and there- after added with 0.6 ml of dextran-charcoal sus- pension. The mixtures were briefly vortexed and incubated 1 min at 0°C. After centrifugation, supernatants were decanted in counting vials and added with 5 ml Maxifluor scintillation fluid to evaluate radioactivity as reported before. Binding specificity was determined by incubating samples (0.2 ml) with 20 nM of labeled 17-estradiol with increasing amounts (10° ’-10-° M) of various un- labeled steroids. Incubation procedures and radioactivity evaluation were as reported before. Isoelectrofocusing The method reported by Matsumada and Goldman was used [12]. Cytosolic and nuclear extract samples (0.2 ml) were preincu- bated with 5 nM labeled 17/-estradiol for 1 hour at 4°C; plasma samples were subjected to the same incubation procedure although with 20 nM labeled 17f-estradiol. A glass column (310.5 cm) was filled with a mixture of 12.5% sucrose in water containing 0.01% Triton X-100 and 3% Ampho- line (Pharmacia, Sweden) pH3.5-10. After a prerun of 1 hour at 4°C, 200 V, the samples were layered on top of the gradient. Electrofocusing was carried out for 16 hours at 4°C, 200 V. At the end of the run, 0.4 ml aliquots were removed from the bottom of the column and used for the evalua- tion of the pH gradient and 17/-estradiol binding as described above. Sucrose density gradient Aliquots (0.2 ml) of cytosol, nuclear extract and plasma were preincu- bated with labeled 17-estradiol (5 nM for tissue extracts and 20 nM for plasma) for 1 hour at 4°C. Afterwards they were layered on the top of 4.4 ml of 5-20% sucrose linear gradient in TEMG con- taining 0.7 M KCI, and centrifuged at 189,000 xg at 4°C for 16 hours. Ovalbumin (3.7 S), albumin (4.6 S) and catalase (10.5 S) (Sigma) were run as 1028 markers in parallel gradients. After centrifuga- tion, the gradient were fractionated by collecting 0.2 ml aliquots from the punctured bottom of the tubes. Samples were reincubated with 5 nM labeled 17-estradiol with or without a 100-fold excess of DES or 17f-estradiol, in order to evalu- ate the specific binding. Measurement of plasma 17{-estradiol and progeste- rone A radioimmunoassay (RIA) method adapted to Podarcis s. sicula plasma was employed [13]. Sensitivity was 3 pg for both 17-estradiol (intraassay variability 7%; interassay variability 13%) and progesterone (intraassay variability 6%; interassay variability 9%). Statistical analysis Numerical data were analy- zed by the ANOVA method, followed by the Duncan multirange test. RESULTS Labeled 17/-estradiol binding molecules were present both in the cytosol and the nuclei of the lizard oviduct, and showed a high ligand affinity (Kg=7.0X 107 !°M for cytosol and Kyg=4.9~x 10°-'°M_ for extract) (Fig. 1A, B). Labeled 17f-estradiol binding molecules were found also in the plasma, though showing lesser ligand affinity (Kg=0.8< 10° M) (Fig. 1C). The affinity values did not undergo significant modifica- tions in the various phases of the breeding cycle (Table 1). Fig. 2 reports the specificity of labeled 17/- estradiol molecules in cytosol, nuclear extract and plasma samples. Both 17/-estradiol and DES competed very well in the nuclear extract, though specificity curves suggest that labeled 17/-estradiol molecules bind 17/-estradiol thighter than DES, while corticosterone, progesterone and testoster- one competed poorly. 17/-estradiol competed very well in the cytosol, followed by progesterone, testosterone, DES and corticosterone. In the plasma, progesterone, testosterone and corticos- terone competed with labeled 17/-estradiol mole- cules, whereas DES was inefficacious. Table 2 reports the levels of oviduct unfilled (RU) and filled (RF) labeled 17/-estradiol binding nuclear M. PaAotucci, M. M. Di FiorE AND G. CIARCIA -=10 A Kd=7.0x10 M 20 2.5 5.0 C [3H]-Estradiol [dpm x10°] Kd:0.8x10° M 25 10 20 [SH]-Estradiol [nM] Fic. 1. ‘Saturation curve and Scatchard analysis of 17- estradiol binding molecules in the oviduct cytosol (A) and nuclear extract (B) and in the plasma (C) of lizard, Podarcis s. sicula during different phases of the reproductive period. (A) cytosol of quiescent oviduct; (B) nuclear extract of secretive oviduct; (C) plasma (February). T=Tolal; S=Specific NS= Not specific. sites during the breeding cycle. In the cytosol and nuclear extract both RU and RF binding sites were always present; however, RU sites constantly pre- vailed. RU reached the highest level in quiescent Estrogen Receptor in Lizard Oviduct 1029 TABLE 1. Kg of the *H-17/-estradiol binding activity in the lizard Podarcis s. sicula oviduct and plasma during the reproductive cycle Oviduct stage (n)* Cytosol Nuclear extract Plasma Quiescent 15 7.0+0.9x 107 '°M 12.1+3.4x10~'°M 0.8+0.3x10-*M Full-grown 8 n.d.** 1.3+0.2x107'°M 1.5+0.4x10°°M Secretive 8 8.9+1.1x10-'°M 4.7+0.5x 107 '°M 0.1+0.1x10~-*M ed Each value is the mean+SE of three different determinations. * number of animals for each determination. ** not calculated because abnormal scatchard curue profiles. 100 A Br Cc % Specific IO O ONO IOC MOF Mito! 10") io 0? Me’ Pia. Competitor Concentration (M) Fic. 2. Labeled 17-estradiol binding specificity in the oviduct cytosol (A) and nuclear extract (B), and in the plasma (C). Samples refers to Podarcis s. sicula having full grown oviducts. Incubation were carried out with labeled 17f-estradiol and increasing amounts of competitors (10° "-10~° M). Competition is expressed as a percentage of specific labeled 17f-estradiol binding. (A=Corticosterone; O=Diethylstylbestrol; B@=Testosterone; A= Progesterone; ®=17/-estradiol). TABLE 2. Changes in the level of oviduct estradiol binding activity, plasma estradiol binding activity and plasma estradiol and progesterone in the lizard, Podarcis s. sicula during the reproductive cycle Oviduct estradiol binding activity (fmol/g tissue) Plasma concentration Ovduct Plasma estradiol Mines (n)* Cytosol Nuclear extract binding activity Progesterone Estradiol 8 Receptor Receptor Receptor Receptor (fmol/mg protein) (ng/ml) (ng/ml) unfilled * filled* unfilled * filled* Quiescent 6 13108+1543 991+141 47114568 371+35 86.4+ 9.3 ASaelail il, Ase} Recovery 5 3577+ 365 405+ 49. 32664319 643+65 n.d.** ml," n.d.** Full-growth 2 220+ 35 145+ 11 2408+223 452+31 145.7+15.1 60.0+9.9 1.4+0.4 Secretive 3 1156+ 131 164+ 13 717+ 68 173+15 71.8+ 6.7 45+1.1 0.2+0.06 Each value is the mean+SE of four different determinations. * number of animals per each determination. ** not dosed. # Receptor filled=17/-estradiol binding sites occupied by the endogenous hormone at the time of the assay. Receptor unfilled=17/-estradiol binding sites not occupied by the endogenous hormone at the time of the assay. 1030 M. Pao.tucci, M. M. Di FiorE AND G. CIARCIA pH pH pH B G 8 8 8 6 (3H]- Estradiol [dpm «107 ] = (e%) on al ae 1 C (3H]-Estradiol [dpm «10° ] ak @ oO £ (o>) (3H ]-Estradiol [dpm ~ 10°) —_ Ww B b oO 10 1 5 10 1 5 10 Fractions Fractions Fractions _ a Fic. 3. Isoelectrofocusing profiles of labeled 17f-estradiol binding molecules in the oviduct cytosol (A) and nuclear extract (B), and in the plasma (C) of Podarcis s. sicula having full grown oviducts. Specific bound is shown. Oblique line represents the pH gradient. Each drawing is representative of three different experiments. ' \ 2 1 ‘ 1 5. 10). 15) a20) 525 1 5 10 15 20 25 Fractions Fractions Fic. 4. Sucrose gradient profile of labeled 17/-estradiol molecules in the nuclear extract (A) and in the plasma (B) of Podarcis s. sicula having full grown oviducts. Only the specific binding is shown. Each drawing is representative of three different experiments. The arrow indicates the Albumin (4.6 S) position along the sucrose gradient. wo (8H]-Estradiol [dpm «102] (7H]-Estradiol [dpm « 103] Estrogen Receptor in Lizard Oviduct oviducts and decreased as oviduct growth occured during the breeding period. RF sites showed a similar behaviour in the cytosol, but they were significantly higher in nuclear extracts of recover- ing and full grown oviducts. The labeled 17/-estradiol binding activity in the plasma increased during ovarian vitellogenesis; the same happened for plasma levels of progesterone and 17-estradiol (Table 2). At electrofocusing, the labeled 17/-estradiol binding molecules from both oviduct cytosol and nuclear extract fell into three pH ranges: 5.2-5.6, 7.0-7.7, and 8.0-8.7 (Fig.3A, B). pH 8.0-8.7 molecules were always present in the oviduct cyto- sol and nuclear extract, although their level was lower in recovering and full grown oviducts. pH 7.0-7.7 molecules were found in fairly good amount in quiescent oviducts, and in the nuclear extract of secretive oviducts. pH5.2-5.6 mole- cules were not detected in quiescent oviducts but were present in the other phases of the oviduct cycle, except in the cytosol of secretive oviducts. At electrofocusing, labeled 17/-estradiol bind- ing proteins in the plasma fell into three pH ranges: 5.7-5.9, 7.4-7.8, and 8.2-8.7 (Fig. 3C). These molecules were always detectable, although pH 5.7-5.9 molecules were the most abundant, and significantly increased during oviduct growth. On sucrose gradient, labeled 17/-estradiol bind- ing molecules of the oviduct cytosol sedimented at about 4.6S, regardless of the oviduct stage (not shown). Nuclear extract binding molecules, however, resolved into two peaks; one peak was always present and sedimented at about 4.6 S, the other sedimented at 5.5S and was detectable in grown and secretive oviducts (Fig. 4A). Plasma labeled 17f-estradiol binding molecules always sedimented at about 4.0 S (Fig. 4B). Figure 5 shows the effects of 17-estradiol injec- tion on the distribution of labeled 17/-estradiol binding molecules six hours after hormone admi- nistration. Estrogen induced decrease of RU and RF in the cytosol, and of RU in nuclean extract (P <0.01). Instead, RF in nucleen extract inereased slightly. 17-estradiol effect was similar twenty- four hours after the injection (not shown). Figure 6 reports the effects of ovariectomy and sex hormone chronic administration on labeled 1031 0.5 — ER fmol/mg tissue Oi a b c d Cytosol Nuclear Extract Fic. 5. Unfilled (GQ) and filled (™) binding sites (ER) concentration (fmol/mg tissue) in the cytosol and nuclear extract of Podarcis s. sicula secretive ovi- duct. a & c=animals injected with solvent; b & d= animals injected with 17-estradiol. Bars indicate the S.E. 17-estradiol binding molecules. Ovariectomy was followed by the RU decrease both in the oviduct cytosol and nuclear extract (P<0.01), as well as by the RF increase in the cytosol (P<0.01). In the oviduct of spayed females injected with 17/- estradiol or 17f-estradiol plus progesterone, a further RU and RF decrease occured in the cytosol (P<0.01), whereas both RU and RF increased in the nuclear extract (P<0.01). Progesterone admi- nistration induced a small RU decrease and a RF increase in the nuclear extract. DISCUSSION In the oviparous lizard, Podarcis s. sicula, sea- sonal oviduct growth and activity depend on ova- tian steroids, namely 17f-estradiol and testoster- one [7]. Proteins binding to these hormones have been found in the oviduct cytosol, and, during the breeding period, in the nuclei [8]. When injected in vivo into spayed females, however, 17/- estradiol is retained in the oviducal tissues, where- as testosterone is not; therefore the estrogen is supposed to play a leading role in physiological oviduct regulation [8]. Our data corroborate this assumption since we identified an 17/-estradiol receptor (ER) in the lizard oviduct which behaves according to the oviduct annual cycle. 178-estradiol binding molecules bind the ligand with high affinity (Kg=7.010~'° M for the cyto- 1032 38 A 34 e = (o) Estradiol Receptor fmol/mg tissu oO 1 a M. Pao.ucct, M. M. Di FiorE AND G. CIARCIA B 15 10 5 b c d e a b c d e Fic. 6. Effects of ovariectomy and 17f-estradiol and/or progesterone treatment on unfilled (G) and filled (m) 17f-estradiol binding sites concentration in the oviduct cytosol (A) and nuclear extract (B) of Podarcis s. sicula captured in October. a=intact lizards; b=ovariectomized lizards treated with solvent (saline); c=ovariecto- mized lizards treated with 17f-estradiol; d=ovariectomized lizards treated with 17-estradiol plus progesterone; e=ovariectomized lizards treated with progesterone. Bars indicate the S.E. sol and 4.9x10~-'°M for the nuclear extract). Ligand affinity does not significantly change during the cycle. These parameters are consistent with the properties of the oviduct 17@-estradiol receptor (ER) of several lower vertebrate species: elasmob- ranchs [14]; reptiles [15]; birds [16-19], and of the mammalian oviduct and uterus [20]. However, in the cytosol of full-grown oviduct, the specific 17-estradiol binding resolves in an abnormal profile of the Scatchard plot (not shown), which does not allow Kg calculation. The meaning of this result is obscure. The hormone-binding sites are present in the cytosol and nuclei of oviduct tissues. Binding activity is decreased by ovariectomy and is res- tored in spayed females after 17(-estradiol treat- ment. .In spayed 17f-estradiol treated females, moreover, cytosolic unfilled and filled binding sites decrease whereas nuclear filled sites increase. In oviducts of female treated with 17-estradiol for a short time, a displacement of the binding sites from the cytosol to the nucleus occurs, although the total amount appears to be decreased, as reported in several other studies [20, 21]. In our opinion this behaviour rules out an arti- factual origin of cytosol binding sites as shown in several vertebrate systems [22, 23]. Quite a similar model has been reported for liver ER of the Estrogen Receptor in Lizard Oviduct salmon [24] and Rana esculenta [21]. The level of 17f-estradiol binding sites changes during the oviduct cycle. In the cytosol, it is higher in quiescent oviducts, but decreases as the organ grows. During growth a significant increase of nuclear filled sites occurs, which is coupled with an increase in plasma 17/-estradiol and progesterone titres as well as plasma levels of steroid binding proteins (SBPs). In Podarcis s. sicula oviducts, the increase in 17f-estradiol-induced binding sites is not counter- acted by progesterone administration, as reported for avian and mammalian systems [25-27]. This result, however, is in line with that obtained in the oviduct of the turtle Trachemys scripta [28], and proposes a difference in 17f-estradiol receptor regulation between higher vertebrates and lizards. On sucrose gradient 17-estradiol binding mole- cules resolve into two discrete peaks with sedi- mentation coefficients of 4.6S and5.5S. The 4.6 S peak is always present, whereas the 5.5 S peak has been found only in the nuclear extract of growing or secretive oviducts, and, therefore, might represent an activated form of the 17/- estradiol receptor. Changes in the ER sedimenta- tion coefficient following activation have been reported [29-31]. The sedimentation coefficient values of lizard oviduct putative ER are consistent with those found for ER from several vertebrate systems, sedimented at high ionic strength [32]. At electrofocusing, the oviduct 17/-estradiol binding molecules fall into three discrete pH ranges, id. 5.2-5.6, 7.0-7.7, and 8.0-8.7. It is difficult to interpret these results, although the absence of 5.2-5.6 molecules in quiescent oviducts and their appearance in the nuclear compartment of growing and secretive oviducts suggest that they might represent the activated receptor. A change in receptor pI following activation has been found in the androgen receptor of the rat prostate [33]. In Podarcis s. sicula plasma, 17-estradiol bind- ing proteins (SBPs) behave as those found in the plasma of other lower vertebrates [34-36], although in Podarcis s. sicula, binding is displaced also by corticosterone, a property found only in plasma SBP of Nerodia sipedon [37]. The sedi- mentation coefficient of lizard SBP is 4.0 S, a value similar to that reported for SBP of Alligator missis- 1033 sipiensis [35], but lower than that found in SBP of other vertebrates [38-41]. At electrofocusing, lizard SBP fall into three pH ranges, i.d. 5.7-5.9, 7.4-7.8 and 8.2-8.7, although the first form is the most abundant. The plasma level of pH 5.7—5.9 molecules changes during the oviduct cycle; it is higher when vitellogenesis prog- resses in the ovary, and its level is related to 17f-estradiol and progesterone plasma titres. This behaviour confirms its role as plasma sex-hormone carrier [42-44]. SBP changes related to the sexual cycle phase have been reported in the females of Taricha granulosa [45], Alligator mississipiensis [35] and in the male of Podarcis s. sicula [36]. ACKNOWLEDGMENTS This work was supported by grants of the Ministero della Pubblica Istruzione, Italy (40% and 60%). REFERENCES 1 Angelini, F. and Ghiara, G. F. (1984) Reproductive modes and strategies in vertebrate evolution. Boll. Zool., 51: 121-203. 2 Botte, V. (1973a) Morphology and histochemistry of the oviduct in the lizard, Lacerta sicula, the annual cycle. Boll. Zool., 40: 305-314. 3 Filosa, S. (1973) Biological and cytological aspects of the ovarian cycle in Lacerta s. sicula Raf. Monit. Zool. It. 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ZOOLOGICAL SCIENCE 9: 1037-1045 (1992) © 1992 Zoological Society of Japan Endogenous Testosterone Controls Humoral Immunity in the Lizard, Chalcides ocellatus ABDEL HAKIM SAAD, MOHAMED HESHAM MANSOUR, MANSOUR EL YAZJI and NASsrRi BADIR Zoology Department, Faculty of Science, Cairo University, Cairo 12613, Egypt ABSTRACT— Study of the correlation between endogenous blood levels of testosterone (TS) and the humoral immune response of male lizards, Chalcides ocellatus led to three major findings: 1) Long-term testosterone propionate (TP) treatment in “summer” induced an elevation of serum TS and complete abrogation of primary anti-rat erythrocytes (RRBC) antibody response similar to normal intact lizards during “spring”; 2) Bilateral orchidectomy of male lizards during “spring” showed increased response to RRBC and suggested that orchidectomy potentiated immunity and more particularly definite stimula- tion of humoral immunity; 3) Long-term TP treatment of orchidectomized lizards failed to suppress immune response to RRBC. Indeed, orchidectomized lizards given TP had response to RRBC similar to male intact controls. The results are discussed from the perspective of the role played by TS in mediating the seasonal rhythms that affect reptilian immunity. INTRODUCTION Many decades ago scientists and clinicans alike observed that there were striking differences be- tween the immune responsiveness of males and that of females [1]. In general, female had super- ior humoral and cell-mediated immunity [2, 3]. It is very likely that this difference in the immune response between the sexes is mediated by the action of sex steroids hormones on the im- munoreactive cell populations [4]. Since the major source of these sex steroids is the gonads, it follows that removal of these organs by gonadectomy might be expected to alter the immune response. Although gonadectomy has been shown to alter the cell-mediated immune response, there is a considerable controversy concerning the effects of gonadectomy on antibody production [5-9]. In contrast to the wealth of information obtained from study of gonadectomy and immunocompe- tence of mammalian vertebrates, data available about fish [10] and amphibians [11] are inadequate and as yet limited. At the reptilian level, no Accepted June 18, 1992 Received February 14, 1992. attempts were made to study the influence of gonadectomy on different immunological para- meters. The present study is mainly based on previous observations from our laboratory showing that the immune system of the lizard, Chalcides ocellatus was moderately active during April throughout June, reaching its maximum development during July/August [12, 13]. This slowly vernal building of immune response was ascribed to be due to high levels of endogenous testosterone (TS) [12]. Therefore, the present study was initiated to ex- amine the effect of adult orchidectomy and testos- terone treatment on the immune response of C. ocellatus. his study was conducted in view of contributing further information to the endoge- nous hormonal factors that mediate seasonal changes in immunity of reptiles. MATERIALS AND METHODS Lizards Sexually mature male lizards, Chalcides ocella- tus (Scincidae, non-hibernator, viviparous), 30—40 g in weight were collected from gradens, fields and 1038 arid regions in the vicinity of Cairo. Lizards were maintained as described previously [12] and given wood lice and tap water ad libitum. The study was performed from March through September (temperature ranges of 25-38°C). Preparation of cell suspensions Lymphoid cells were separated from lizard spleen exactly as described in detail earlier [13]. After three washes in cold RPMI 1640 medium (Sigma Chemicals Co., St. Louis, MO., USA), lymphocytes were counted using the trypan blue dye exclusion test. Blood testosterone (TS) determination Serum testosterone was measured following essentially the method described earlier [12] using a commercial radio-immunoassay kit (Coat-A- Count, Diagnostic Products Corporation, CA, USA). |*I-labelled TS was supplied as the radioactive tracer. Tetosterone showed less than 1.5% cross reactivity with corticosterone, a major serum corticosteroids in C. ocellatus [13]. The mean intra- and interassay coefficient of variation of assays fell within the range of 4.0-7.0% and 8.0-15.0%, respectively. In these assays, the minimum detectable dose on the standard curve was 10 pg. Recovery of labelled and unlabelled steroids standard was 90-93%. Values shown in the “Results” are corrected for recovery. Primary immunization with RRBC Animals were allowed to acclimate to ambient environmental conditions for few days before im- munization. The lizards received intraperitoneal (i.p.) immunizing injections of 0.5 ml of 10% rat erythrocytes (RRBC) suspension in phosphate- buffered saline (PBS), pH=7.2. Unimmunized (control) lizards were injected i.p. with 0.5 ml PBS, pH=7.2 and included in every such experi- ments. After sacrifice by decapitation, blood was collected and allowed to clot at room temperature. Serum was then heat-inactivated at 56°C for 30 min. and stored for use in the haemagglutination test. Rosette-forming cell (RFC) assay Groups of animals were sacrificed at a specified A. H. Saap, M. H. Mansour et al. intervals after immunization. A single cell suspen- sion from the spleen was prepared as described and the viability was determined by trypan blue exclusion method. The rosette-forming cell (RFC) assay was followed to assess the number of anti- gen-binding cells as described earlier [14]. On the basis of actual number of cells for each sample, the RFC per 10° spleen cells was computed for each spleen. Plaque-forming cell (PFC) assay The technique of Cunningham and Szenberg [15] as modified for lizards by Kanakambika and Muthukkaruppan [16] was followed to assess the number of haemolytic antibody-producing cells. Plaques were counted under low power with the aid of a dark field phase contrast microscope. The number of PFC/10° viable spleen cells was calcu- lated. Determination of antibody (Ab) Haemagglutination (HA) titrations of the sera were performed in microtiter plates using two-fold serial dilution as described in detail previously [12]. Titers were expressed as the logs of the highest dilution showing microscopic agglutina- tion. Bilateral orchidectomy Adult male lizards were allowed to acclimate to laboratory conditions for at least one week before operation. Surgical operations were performed under ether anaethesia. Operation sites were washed with distilled water followed by 70% ethyl alcohol. Skin was firstly cut by a sharp cissors, while the abdominal muscles cut by means of a small scalpel. The left then the right testes were separated from their blood supply by means of a cautery then cut off their respective vasa deferen- tia. Sulpha and neomycin was sprayed on the wound then sewed by means of a small curved surgical needle. Operated animals were placed in a clean glass terrarium for one day to avoid sepsis. In corresponding sham-operated controls, the testes were delivered to the wound surface, re- turned to the abdominal cavity and the wound closed. No death occurred in either operated or sham-operated groups during the course of the Testosterone and Immunity in Lizards experiment. Both were kept at ambient tempera- ture under normal conditions in the laboratory and given live wood lice and water ad libitum. Testosterone propinate (TP) treatment Intact, orchidectomized lizards were allowed to acclimate to ambient en- vironemtnal conditions a few days before treat- ment. All lizards received at 5 day-intervals a total of 5 i.p. injections of 50 #g testosterone propion- ate (Sigma)/g body weight/injection. Another group of lizards was injected at the same intervals with 0.3 ml of PBS, pH =7.2 and used as control. No death occurred in either TP-treated or control groups during the course of the experiment. Both were kept at ambient temperature under normal conditions in the laboratory and given wood lice and water ad libitum. sham-operated or Statistical analysis Student’s f-test was used to determine levels of significance between control and experimental groups. Differences were considered to be signi- ficant at P values <0.05. RESULTS Effect of long-term TP. treatment on intact lizards: Circulating TS levels: As illustrated in Figure 1, TS levels of intact males injected with PBS exhibited minor changes within the normal range of about 9.5-26.3 ng/ml. However, TS levels after multiple injections of TP were markedly elevated to the range of 18-140 ng/ ml, and stabilized at this level until the end of the experiment. Ten days after cessation of TP, a decline to basal values proceeded sharply. Primary anti-RRBC response: In mid-summer, male lizards were given five 1.p. injections of TP (50 “g/g body weight/injection; 5 days apart). Another group of male lizards re- ceived PBS and served as controls. Two days later, all TP-treated and PBS-injected lizards received an i.p. injection of RRBC suspension. RFC and PFC assays were performed on individual animals using spleen cell suspensions on different days after 1039 Testosterone level (ng/ml) 0 5 10 15 20 30 40 = a & = = Days after TP treatment Fic. 1. The levels of serum TS in intact lizards, C. ocellatus, administered with multiple injections of 50 pg, TP/g body weight/injection. During mid- summer, lizards received 5 injections, 5 days-apart, of TP suspension. Each point depicts the level of TS of serum sample pooled each from two animals. Day O represents TS levels of intact lizards injected with PBS. immunization. Titrations of serum Ab titers were carried out simultaneously. As depicted in Figure2A, TP-treated male lizards elicited a low number of RFC comparable to that enumerated in PBS-injected lizards. However, significant (P<0.01) differences could be observed at the peak day (Fig. 2A). As shown in Fig. 2B, although the number of PFC higher in PBS-injected males than in TP-treated lizards, no significant differences could be observed at the peak day. Indeed, the shape of the curves was otherwise quite similar. As depicted in Figure 2C, in PBS-injected lizards, low levels of Ab titer was detected below background level on day 7. There- fore, the titer rose quickly exhibiting a peak at day 21 and remained elevated until day 28. However, in TP-treated lizards, Ab titer increased steadily reaching its peak value at day 21, then gradually declined. Effect of bilateral orchidectomy on intact lizards: Circulating TS level: Adult male lizards, C. ocellatus were collected 1040 A. H. Saap, M. H. Mansour et al. during early spring and some of these lizards were orchidectomized during mid-March. Another group was sham-operated and used as controls. Figure 3 illustrates the serum TS levels in male o i—} aT leen cells ~ o agg Nes 2” aa sham- operated and orchidectomized lizards dur- is ing March through July. Sham- operated lizards sea exhibited high levels of TS being in the range of i 110-140 ng/ml. However, bilateral orchidectomy 0 in C. ocellatus lowered the level of TS to about 2— 18 ng/ml. These abrogated levels were sustained 2 and lasted long, so that the mean values for all 2 operated lizards, at any given point were signi- ficantly (P<0.001) different from sham-operated controls. 3 7 14 21 28 Days post immunization a o a o 6 Number of PFC /10 spleen cells > o w o Testosterone level (ng/ml) iy o 10 sham- 15 day 1 2 3 month operated 0 iL Time after orchidectomy | fa : P HH The levels of serum TS obtained from adult C DAYS POS! imewametiion Fic. 3. e levels of serum obtained from adult C. ocellatus after bilateral orchidectomy. Each point on the curve represents the level of TS obtained from an individual sample pooled from two animals. Day O represents TS levels of sham-operated ai @9) lizards. Fic. 2. Kinetics of RFC response (A), PFC response (B) and mean serum haemagglutinin titers (C) of normal lizards, C. ocellatus, administered with mul- tiple injections of 50 ng TP/g body weight/injec- tion. During mid-summer, lizards received 5 injec- tions, 5 days-apart, of TP suspension. Two days later, lizards were received 1.p. injection of 0.5 ml of 10% RRBC suspension. Each point represent the mean response of 4-6 separate animals and the Reciprocal log, HA titer vertical bars indicate standard error of the mean. @—e Normal intact lizards, @---@ Normal intact lizards injected with TP. *=0.050.01) increase in the number of PFC which was peaked on day 14 and then gradually decreased (Fig. 4B). When sham- operated lizards received RRBC suspension, low levels of Ab titer was detectable below background level on day 7 (Fig. 4C). Thereafter, the titer rose slowly exhibited a peak at day 21, then gradually declined at day 28. However, in both groups of orchidectomized lizards, a sharp increase in cricu- lating Ab titer occurred in day 7. Thereafter, a gradual increase occurred in the Ab titer (Fig. 4C). Fic. 4. Kinetics of RFC response (A), PFC response (B) and mean haemagglutinin titers (C) of C. ocella- tus after bilateral orchidectomy. Animals were immumized i.p. on day 0 with 0.5 ml of 10% RRBC suspension. Each point represents the mean re- sponse of 4-6 separate animals and the vertical bars indicate standard error of the mean. *=0.05< P< 0.01 and NS=not significant. @—@ sham-operated lizards; A—A orchidectomized lizards, one month after operation; ©—Q© orchidectomized lizards, three months after operation. *=0.05 w NR — A. H. SAAD, M. H. Mansour et al. 28 Days post immunization a 3 7 14 21 28 Days post immunization 7 14 21 28 > Days post immunization Testosterone and Immunity in Lizards bited minor changes within the normal range of 1.1-5.7 ng/ml. In comparison, orchidectomized lizards receiving five injections of TP showed a sharp increase in serum TS levels to about 45-120 ng/ml which maintained until day 20. Primary anti-RRBC response: TP was administered to orchidectomized lizards to determine whether increasing TS levels would result in modulation of the immune response. Lizards were orchidectomized during May. One month later, one group of orchidectomized lizards received i.p. five fractionated dose of TP (50 ug/g body weight/injection; 5 days-apart). Control orchidectomized lizards received i.p. five injec- tions of PBS. Two days after the last injection, lizards received i.p. injection of RRBC suspen- sion. As shown in Figure6A, in TP-treated orchidectomized lizards, the kinetics of RFC was similar to that demonstrated in orchidectomized control lizards. Moreover, no significant differ- ences were essentially observed between the two animal groups at the peak day. As shown in Figure 6B, in both PBS-injected and TP-treated orchidectomized lizards, the shape of the curves was Otherwise quite similar. Although both groups of lizards showed a high response, no significant differences were observed at the peak day. As depicted in Figure 6C, despite the fact that in TP-treated orchidectomized lizards, the peak of primary Ab response to RRBC was somewhat delayed and diminished in comparison with PBS- injected orchidectomized lizards, statistical differ- ences in humoral response at some time point were essentially not significant. DISCUSSION In the present study experimental administra- tion of exogenous TP in lizards produced changes in serum TS levels which closely mimicked those Occurring as part of response of lizards to the seasonal environment [12]. As expected, long- term TP-treatment of intact summer lizards led to a complete and an irreversible abrogation of their proliferative response to RRBC. The results indi- cated that the cell(s) responsible for these func- 1043 tional alterations in lizards were particularly TP- sensitive; the target cell(s), however, remained undefined. It is obvious that several doses of exogenous TP induced a high and more long standing elevation of TS resulting in a depressed immune reactivity, which simulates the natural situation lizards experienced annually in spring [12]. In view of the above, the authors hypothesized that lowering an animal’s blood TS levels by “orchidectomy” did alter the immune response and that alterations might be opposite to those induced by the administration of exogenous TP. Thus, bilateral orchidectomy was performed to adult male C. ocellatus in early spring in order to suppress the high TS levels at that time of the year [12]. lizards based on actually reproducing endocrine The immune reconstitution of “spring” environment is theoretically possible but ex- perimentally difficult maneuver. Yet, the outcome has been fruitful. Although bilateral orchidectomy strongly reduced serum TS levels in C. ocellatus, it did not result in its complete disappearance. However, these concentrations were similar to the low values previously recorded throughout sum- mer months [12]. Moreover, orchidectomized lizards showed increased response to RRBC and the rate of lymphoid tissue hyperplasia correlated with this increased reactivity (data not shown). These data, therefore, suggested that orchidectomy potentiated immunity in general and more particularly humoral one. Whether this effect is due to the differentiation of lymphoid precursor cells to immunocompetent cells and/or the proliferation of pre-existing competent cells, which might be present in low level in the other lymphoid organs is difficult to decide. Although TS deprivation showed to cause im- munological potentiation, the central point re- maining is whether the effects are due directly to withdrawal of TS or they result from interference with the hormonal milieu. Therefore, TP was administered to orchidectomized lizards to deter- mine whether increasing the concentration of blood TS would result in modulation of the im- mune reactivity. One month after orchidectomy, orchidectomized lizards received five fractionated doses of TP. These orchidectomized lizards exhi- 1044 bited a spring-like sustained rise of serum TS levels until the end of the experiment. However, phys- iologic doses of TP used in the current study failed to suppress immune responses to RRBC in orchidectomized lizards. In fact, orchidectomized lizards given TP had response to RRBC which were similar to male These findings suggested that the presence of TS caused immune function to be “normal” in orchidecto- mized lizards. To our knowledge, no such study has been available in reptiles. However, the scientific litera- ture dealing with sex hormones effect on verte- brate immune responses appeared to be confused and scant. While in marine teleost, Sebasticus marmaratus, the humoral reactivity of mature females to RRBC was lower than males or imma- ture females in the spawning season, yet exoge- nous administration of sex hormones or orchidectomy did not display influence on Ab production during the mating season [10]. The thymus and spleen were morphometrically analy- zed in orchidectomized frogs, Rana perezi [11]. However, the authors failed to interpret their data, since such important physiological processes were undobtly the direct effect of TS deprivation [11]. In addition, recent studies indicated that neonatal gonadectomy in chicken exerted two effects on the humoral immune response: suppressive in young birds [17] and stimulatory in older chicken [18]. Moreover, orchidectomy resulted in immuno- intact controls. potentiation in many mammalian systems. For example, orchidectomy against viral or fungal [5-7] and bacterial infec- tions [8] and parasitic infections [9]. Moreover, orchidectomized animals reject allografts rapidly [19] and accelerated graft-versus-host reactions [20]. lymphoblasts transformation in culture [21] and potentiated humoral immune response to several However, in some ex- perimental situations, orchidectomy had no effect on the immune system [8]. In conclusion, all these results confirm the im- increased protection Furthermore, gonadectomy augmented T- heteroantigens [22, 23]. portance of sex hormones as causative agents of the seasonal variations of reptilian immune system and emphasized the lymphocyte destructtion as the main effect of sex hormones on the immune system A. H. Saap, M. H. MAnsour et al. of lower vertebrates. Mechanisms of action of sex hormones, target cells for sex hormones in the lymphoid organs and the way of interaction be- tween sex hormones and the immune axis in lower vertebrates deserve further research. REFERENCES 1 Ansar, Ahmed, S., Penhale, W. J. and Talal, N. (1985) Sex hormones, immune response and auto- immune diseases. Mechanisms of sex hormones ac- tion. Am. J. Pathol., 121: 531-551. Grossman, C. J. (1984) Regulation of the immune system by sex steroids. Endocrine Rev., 5: 438-455. 3 Stimson, W. H. (1987) Sex steroids, steroid recep- tors and immunity. In “Hormones and Immunity”. Ed. by I. BERCZI and K. KOVACS, MTP Press. Lancaster. i) 4 Berczi, I. (1989) Immunoregulation by neuroendoc- rine factors. Dev. Comp. Immunol., 13: 329-341. Berkoulch, S. and Ressel, M. (1967) Effect of sex on susceptibility of adult mice to coxackie B virus infections. Arch. Gesmate Virus Forsch, 22: 246- Pile 6 Rifkind, D. (1972) Influence of gonadectomy on Candida albicans urinary tract infection in CFW mice. Infect. Immunol., 5: 363-369. 7 Streng, C. B. and Nathan, P. (1973) The immune response in steroid deficient mice. Immunology, 24: 559-563. 8 Cohen, D. A. (1979) Sensitivity to androgen. A possible factor in sex differences in the immune response. Clin. Exp. Immunol., 38: 218-225. 9 Kittas, C. and Henry, L. (1979) Effect of sex hormones on the immune system of guinea pigs and on the development of toxoplasmic lesions in non- lymphoid organs. Clin. Exp. Immunol., 36; 16-20. 10 Nakanishi, T. (1986) Seasonal changes in the humoral immune response and the lymphoid tissues of the marine teleost, Sebastiscus marmoratus. Vet. Immunol. Immunopathol., 12: 336-342. 11 Zapata, A., Garrido, E. Gomariz, R. P. and Leceta, J. (1983) Relationship between neuroendocrine and immune systems in amphibians and reptiles. Dev. Comp. Immunol., 7: 771-774. 12. Saad, A.-H., Abdel Khalik, N. and El, Ridi, R. (1990) Blood testosterone level: a dependent factor regulating immune reactivity in lizards. Immunobiol., 180: 184-190. 13. Saad, A.-H. and EL, Ridi, R. (1989) Endogenous corticosteroids mediate seasonal cycle changes in immunity of lizard. Immunobiol., 177: 390-403. I4. Saad, A.-H. and Shoukrey, N. (1988) Sexual dimorphism on the immune responses of the snake, Psammophis sibilans. Immunobiol., 177: 404-419. Nn season- 16 17 18 19 Testosterone and Immunity in Lizards Cunningham, A. and Szenberg, A. (1968) A further improvements in the plaque-technique for detecting single antibody forming cells. Immunology, 14: 599 —600. Kanakambika, P. and Muthukkaruppan, V. R. (1972) The immune response to sheep erythrocytes in the lizard, Calotes versicolor. J. Immunol. 109: 415-419. Vujic, D., Milicevic, Z., Micic, M., Isakovic, K. and Jankovic, B. D. (1983) Atrophy of the thymus and bursa of Fabricius caused by neonatal gonadectomy. Period biol., 85: 107-109. Milicevic, Z., Micic, M. and Isakovic, K. (1986) The role of gonads in immunomodulation and aging of the immune system. Period. biol., 88: 13-14. Graff, R. J., Lappe, M. A. and Snell, G. D. (1969) 20 21 1045 The influence of the gonads and the adrenal glands on the immune response to skin grafts. Transplant.., 7: 105-111. Castro, J. E. (1974) The hormonal mechanism of immuno-modulation in mice after orchidectomy. J. Endocrinol., 62: 311-318. Grossman, G. J., Sholiton, L. G. and Roselle, G. (1982) Estradiol regulation of thymic lymphocyte function in the rat: mediation by serum thymic factors. J. Steroid Biochem., 16: 683-688. Eidinger, D. and Garret, T. J. (1972) Studies of the regulatory effects of the sex hormones on antibody formation and stem cell differentiation. J. Exp. Med., 136: 1098-1116. Castro, J. E. (1975) Immunological effects of orchidectomy. Br. J. Urol., 47: 89-96. | prt oD Tate fain )) pr EP a aa aT Wid e Ap ecanaamttgern x otelginnagt Ged coals spires ou shits ohh LRM, Mout bh al Perera rir = tea " ] my Lawes Ve hi 1 @rtego. Ut } = a! bestis: igi chi ie hed AU oa ft teal ina SRI: “ i Fy ee fie | r Th ‘Ls bee, th 16 error "y =} ; ; vanities ' ae M nf , Me Aan Aas se. eer pneuneet + wikeneyt ’ WE iiprndtly oll rehearse BP ae ; & dri ths Se edinu bal etieed biti A slvcdeal ott Ds aly bid ihre diets L rreorieh » bie putoead) coche shew giteqey ty aa cya oe anna iwit “ff pels erties hi ae ae bat! It ‘ah $8 ile ve TAA Se a es dees, Wt, ? SR PR Py an ea neat Maat iy ui bales Dani Wi ae a ae hbo et lia ted eo hae they bo5 Ua Ay peat (Oo rail. onk vine Gyipp atty ee r bees 1s “Vie i " y wh a | Hol Nee. ; te € teow t 1 uo 1 La ate il f * ‘ zi ’ ‘ i j - errs = t - * r 4 - j e 7 ' ‘| - . ares i re i 1 i a ay his 3 ‘ Wyner ie aye Neh aw : ZOOLOGICAL SCIENCE 9: 1047-1053 (1992) Effects of Photoperiod, Pinealectomy and Ophthalmectomy on Circulating Melatonin Rhythms in the Goldfish, Carassius auratus Hiroaki Kezuka!, MasayukI I1Go*, KtvosHt FURUKAWA, Katsumi Arpa® and Isao HANyu Department of Fisheries, Faculty of Agriculture, The University of Tokyo, Bunkyo, Tokyo 113, Japan ABSTRACT—Effects of photoperiod, pinealectomy and ophthalmectomy on circulating melatonin rhythms were studied in the goldfish, Carassius auratus. Under light-dark (LD) 16:8 or LD 8:16 photoperiod, plasma melatonin levels exhibited diurnal rhythms with high titers during the scotophase and low titers during the photophase. When the fish were transferred from LD 12:12 to continuous dark conditions, plasma melatonin levels exhibited circadian changes during the first 3 days under continuous dark-ness (DD). The rhythms, however, became indistrinct during days 7-8 and 14-15. When the fish were transferred to continuous light conditions, plasma melatonin remained at low levels. Pinealectomy abolished high melatonin levels in the plasma at mid-dark, but ophthalmectomy did not. These results clearly indicate that circulating melatonin levels are photoperiod-dependent and shows circadian rhythms under DD conditions, and that the plasma melatonin rhythm is mainly generated by © 1992 Zoological Society of Japan the pineal gland. INTRODUCTION The pineal gland of vertebrates synthesizes and secretes its indole hormone, melatonin (N-acetyl- 5-methoxytryptamine) into the blood and cere- brospinal flud. Melatonin has been considered as the time-keeping hormone because of its cyclic appearance: melatonin levels in the pineal gland, blood, and cerebrospinal fluid fluctuate in a rhyth- mic fashion that is coincident with a given photo- period. Under light-dark (LD) cycles, high meato- nin titers were observed during the scotophase while low values were seen during the photophase in all vertebrate classes including fishes [1-13]. Accepted June 25, 1992 Received January 16, 1992 Present address: Advanced Research Laboratory, Hitachi, Ltd., Hatoyama-machi, Saitama 350-03, Japan. > Present address: Department of Anatomy, St. Marianna University School of Medicine, Miyamae- ku, Kawasaki 216, Japan. To whom reprint requests should be addressed. The pineal gland of fishes is considered to be a circadian oscillator, since behavioral analysis re- vealed that the pineal gland is implicated in the control of circadian organization and rhythmicity in locomotor activities [14, 15]. In addition, en- dogenous rhythmicity in melatonin secretion from the pineal gland under continuous dark (DD) conditions in vitro has been reported in three teleost species, the pike (Esox lucius) [16], the gold-fish (Carassius auratus) [17, 18], and the white sucker (Catostomus commersoni) [19]. These results suggest that melatonin secreted from the pineal gland is playing an important role as an internal zeitgeber in controlling bioligcal rhythms in fishes. Photoperiod is considered to be the most impor- tant environmental factor which modulates mela- tonin rhythms. Reports, however, on the effects of long or short photoperiod on circulating melatonin thythms in fishes were restricted to the rainbow trout Oncorhynchus mykiss [20] and the common carp Cyprinus carpio [12], and no report concern- ing endogenous rhythms of melatonin in vivo 1048 under continuous dark (DD) or continuous light (LL) conditions is available. Melatonin and the enzymes responsible for melatonin biosynthesis (i.e. serotonin N- acetyltransferase and hydroxyindole-O- methyltransferase) have been reported to localize in the retina (for review, see [21]). These results indicate that melatonin is synthesized not only in the pineal gland but also in the retina. Pinealec- tomy and ophthalmectomy experiments revealed that contributions of the pineal gland and the retina to circulating melatonin rhythms exhibited inter-species differences. In the Japanese quail, for example, the retina contributes to the blood level of melatonin and daily cycles of circulating melatonin in pinealectomized animals [22]. In the chicken, on the other hand, pinealectomy abo- lished circulating melatonin rhythms [23]. In case of teleost fish, melatonin synthesis in the pineal gland and the retina have been reported in several species [11, 13, 16-19, 24-26], but how these two tissues contribute to the circulating melatonin level is not fully understood. The present study was conducted to examine the effects of photoperiod on circulating melatonin rhythms, and to confirm the main source of circu- lating melatonin in the goldfish. MATERIALS AND METHODS Experimental _ fishes old goldfish (Carassius auratus) were purchased from a local dealer. They were reared in indoor stock tanks at 24°C under natural photoperiod at The Fisheries Laboratory, The University of Tokyo (Maisaka, Shizuoka, Japan) until used. Fish were fed com- mercial trout pellets ad libitum. Two-year Experiment 1 Ninety male goldfish weighing 50-150 g were used in this experiment. In Octo- ber, 45 fish were transferred into each of 2 indoor experimental tanks, and acclimated under LD 16:8 (lights on 0400-2000 hr) or LD 8:16 (lights on 0800-1600 hr) at 24°C for 2 weeks. Blood samples were taken at 2—4 hr intervals (n=5) for twenty-four hr. Experiment 2 Three hundred goldfish weighing H. Kezuka, M. Iico et al. 38-174 g were used in this experiment. In July, 150 fish were transferred into each of two indoor experimental tanks, and acclimated under LD 12:12 (lights on 0600-1800 hr) at 24°C for 2 weeks. Blood samples were taken at 1200 hr on Day 0 (57-97 g females, n=7; 53-110 g males, n= 5) and at 0400 hr on day 1 (47-112 g females, n=6; 47-100 g males, n=5) to examine sexual differ- ences in plasma melatonin levels. The light condi- tions were changed to DD or LL conditions from 0600 hr on day 1. Blood samples were taken every 4 hr (n=5) at 0800 hr on day 1 to 0800 hr on day 3 (days 1-3), at 1200 hr on day 7 to 1200 hr on day 8 (days 7-8), and at 1200 hr on day 14 to 1200 hr on day 15 (days 14-15). Experiment 3 Forty-two glodfish weighing 72- 154g were used in this experiment. After the acclimation under LD 12:12 (lights on 0600-1800 hr) at 24°C for 3 weeks in August, they were pinealectomized (PINX, n=9), sham- pinealectomized (Sham, n=8), ophthalmecto- mized (EYEX, n=8), or pinealectomized and ophthalmectomized (PINX+EYEX, n=9). Pinealectomy and sham-pinealectomy were accomplished according to the method of De Vlaming [27] and ophthalmectomy was performed following the procedure of Fenwick [28]. Animals were identified by fin-clips. Intact control (IN- TACT, n=6) were only fin-clipped. One week after operation, blood samples were taken at mid- dark (2300-0100 hr) and mid-light (1100-1300 hr, 36 hr after the sampling for middark). Sample collection and RIA Blood samples were taken according to the procedure by Kezuka et al. .{12] under with 0.06% 2- phenoxyethanol in Exp. | or with 0.05% ethyl-p- aminobenzoate in Exps. 2 and 3. The anesthesia did not interfere with the RIA. Blood samples were centrifuged at 3000 rpm for 20 minutes, and anesthesia plasma was stored at —20°C until the assay. Melatonin levels in the plasma were measured by the RIA after a partial purification with Sep- pak Cys cartridge as previously described and validated for the goldfish plasma [12]. The mini- mum detectable level of the RIA was 32 pg/ml plasma in these experiments. Plasma Melatonin Rhythms in Goldfish Statistics The difference of means was analyzed by ANOVA and Duncan’s multiple range test, or by paired (test. RESULTS Circulating melatonin rhythms under long or short daylength Plasma melatonin levels exhibited distinct daily rhythms under both long (LD 16:8) and short LD 8:16) daylength as shown in Figure 1. The lowest levels were seen at 1900 hr under LD 16:8 and at 1500 hr under LD 8:16. Levels were observed to increase after lights-off, and remained elevated during the scotophase. The highest levels were 1200 LD 16:8 hy 900 600 # ie 3004 12 16 19210 35 8 12 LD 8:16 MELATONIN (pg/ml) b (=) (=) 12 1517 20 O 4 79 12 CLOCK TIME (hr) Fic. 1. Circulating melatonin rhythms under long (LD 16:8, upper) or short (LD 8:16, lower) photo- period. Each point represents the mean+SE (n= 5). Solid bars and open bars along the X-axis indicate the scotophase and the photophase, respec- tively. Significance: under LD 16:8, +, compared with the values during the photophase (1200, 1600 and 1900 hr on Day 1 and at 0500, 0800 and 1200 hr on Day 2); under LD 8:16, +, compared with the values at 1500 hr on Day 1 and at 0900 and 1200 hr on Day 2; *, compared with the values at 1200 and 1700 hr on Day 1. Levels of significance: one symbol, P<0.05; two symbols, P<0.01. 1049 observed at 0000 hr under LD 16:8 and at 0400 hr under LD 8:16. Subsequently, melatonin levels started to decrease, although the fish were still in the scotophase, and returned to the basal levels after lights were turned on. Under LD 16:8, the values during the scotoph- ase (2100 hr on Day 1 and 0000 and 0300 hr on Day 2) were significantly higher than those during the photophase (1200, 1600 and 1900 hr on Day 1 and 0500, 0800 and 1200hr on Day 2) (P<0.01). Under LD 8:16, the values at 2000 hr on Day 1 and 0000 and 0400 hr on Day 2 were significantly higher than those at 1500 hr on Day 1 and 0900 and 1200 hr on Day 2 (P<0.01), and the values at 0000 and 0400 hr on Day 2 were significantly higher than those at 1200 and 1700 hr on Day 1 (P<0.01). The value at 0700 hr on Day 2 was significantly higher than those at 1500 hr on Day 1 and 0900 and 1200 hr on Day 2 (P<0.05). Sexual difference in circulating melatonin levels Plasma melatonin levels in females and males were 154+18 and 154+38 pg/ml at 1200 hr on Day 0, and 460 +48 and 667 +121 pg/ml at 0400 hr on Day 1 (mean+SE), respectively. There was no detectable difference in plasma melatonin levels between sexes either during the photophase (at 1200 hr on Day 0) or during the scotophase (at 0400 hr on Day 1). Therefore, female and male fish were not separately dealt with in the following experiments. Circulating melatonin rhythms under DD or LL conditions Changes in plasma melatonin levels under DD or LL conditions are shown in Figure 2. Under the LD cycles used for the acclimation, melatonin levels exhibited day-night fluctuations: The values during the scotophase (0400 hr on Day 1) were significantly higher than those during the photoph- ase (1200 hr on Day 0, P<0.01). When the fish were transferred into DD conditions, plasma mela- tonin levels exhibited circadian changes during the first 3 days with high levels during the subjective scotophase and low during the subjective photoph- ase. The level at 0400 hr on Day 2 was significantly higher than tose at 1200 hr on Day 1 and those at 1200 on Day 2 (P<0.05), and the levels at 2000 hr 1050 1200 900 600 300 MELATONIN (pg/ml) 12, 0 12 0 12 =O DAY O DAY 1 H. Kezuka, M. Ico et al. ACCLIMATORY PHOTOPERIOD 12 0 1212 0 12 DAY 2 DAY 3 DAYS7-8 DAYS 14-15 CLOCK TIME (hr) Fic. 2. Circulating melatonin rhythms under DD (@) or LL (™) conditions. Each point represents the mean+SE (1200 hr on Day 0, n=12; 0400 hr on Day 1, n=11; under DD or LL, n=S each). Solid bars and open bars along the X-axis represent the scotophase and the photophase of the acclimatory photoperiod, respectively. Signi- ficance: *, compared with the value at 1200 hr on Day 1; +, compared with the value at 1200 hr on Day 1; A, compared with the value at 1200 hr on Day 2. Levels of significance: one symbol, P<0.05; two symbols, P<0.01. on Day 2 and at 0000 and 0400 hr on Day 3 were significantly higher than those at 1200 hr on Day 2 (P<0.05). During Days 7-8, circadian-like changes in plasma melatonin levels disappeared. During Days 14-15, melatonin levels remained at elevated levels. In contrast, under LL conditions, plams melatonin levels remained at low titers throughout the experiment. Effects of PINX and/or EYEX on circulating melatonin rhythms Plasma melatonin levels at mid-dark and mid- light one week after the operation are shown in Figure 3. At mid-dark, melatonin levels in the PINX and PINX+EYEX fish were significantly lower than those in the Intact, Sham, and EYEX groups (P<0.01). No significant difference was observed among mid-light values of all groups. In the Intact, Sham, and EYEX groups, melato- nin levels at mid-dark were significantly higher than those at mid-light (P<0.01). There was no significant difference between values at mid-dark and mid-light in the PINX, whereas in the PINX + EYEX the value at mid-dark was significantly lower than those at mid-day (P<0.01). 600 (J INTACT (ll SHAM = EYEX 3 400 fe PINX = PINX+EYEX 2 e = w 200 = 0 MID-DARK MID-LIGHT Fic. 3. Plasma melatonin levels at mid-dark and mid- light in the INTACT (n=6), SHAM (n=8), EYEX (n=8), PINX (n=9), and PINX+EYEX fish (n= 9). Each point represents the mean+SE. _ Signi- ficance: *, compared with the value in Sham at mid-dark; s+, compared with the value at mid-light in each group. Levels of significance: two symbols, P <0.01. DISCUSSION In the goldfish, plasma melatonin levels exhi- bited clear daily rhythms both under long and short daylength; low during the photophase and Plasma Melatonin Rhythms in Goldfish 1051 high during the scotophase with peak near mid- dark phase. Our results were basically the same as those reported in other vertebrate species [1-13]. These results indicate that daily fluctuations of melatonin in the body fluid are a common phe- nomenon in vertebrates and that photoperiod is one of the most important factors controlling mela- tonin rhythms. Under DD conditions, plasma melatonin levels exhibited endogenous rhythms during Days 1-3; high melatonin levels were observed during the subjective scotophase and low titers were seen during the subjective photophase. This suggests that circulating melatonin rhythms are driven by a circadian oscillator. These rhythms disappeared during Days 7-8 and 14-15. Several explanations can be presented for this observation such as: large individual variations in free-running period, damp- ing of oscillation, or desynchronization of oscilla- tors under DD conditions [29]. In contrast, under LL conditions, plasma melatonin concentrations remained at low levels and failed to exhibit daily rhythms. This indicates that exposure to light has a strong inhibitory effect on melatonin secretion. Similar results were previously observed in organ culture experiments of the goldfish pineal gland in vitro [17, 18]: under LD cycles, the pineal gland secreted melatonin during the scotophase; circadian rhythms of melatonin secretion were observed under DD conditions; and melatonin secretion is suppressed under LL conditions. The coincidence of these in vivo and in vitro results support the idea that melatonin secreted from the pineal gland contributes to the blood melatonin rhythms. Because melatonin synthesis has also been re- ported in the retina of some vertebrates including fishes in addition to the pineal gland [5, 8, 13, 21- 25, 30-33], we examined effects of pinealectomy and/or ophthalmectomy on circulating melatonin levels to confirm the main source of circulating melatonin in the goldfish. In the PINX and PINX +EYEX groups, high values at mid-dark dis- appeared, whereas in the EYEX group significant- ly higher values were maintained at mid-dark. These results clearly indicate that the pineal gland, and not the retina, is the main organ which sec- retes melatonin into the circulatory system in this species. We cannot, however, exclude the possi- bility that a small amount of melatonin is secreted from the retina. Although the difference is not significant, plasma melatonin levels at mid-dark were lower in the PINX+EYEX group that those in the PINX group. In addition, we recently found in the goldfish that melatonin contents in the eye also show daily fluctuations (ligo and Aida, in preparation). Interestingly, plasma melatonin in the goldfish was still at detectable level after pinealectomy and ophthalmectomy, and the plasma melatonin levels at mid-light was significantly higher than those at mid-dark in PINX+EYEX fish. This residual melatonin may be simply due to secretion from remnants of the pineal tissue after pinealectomy, and/or secretion from other parts of the body. The Harderian gland and the intestine have been suggested at the extrapineal-extraretinal source of melatonin in mammals and birds [31-33]. The pineal gland plays an important role as the photoneuroendocrine transducer in vertebrates, and melatonin secreted from the pineal gland into the blood is thought to serve as an internal zeitge- ber. In seasonally breeding mammals, melatonin mediates photoperiodic information and the mod- ulation of melatonin secretory profiles is involved in the determination of the reproductive season (for review, see [34]). In temperate-zone fishes, photoperiod is one of the most important factors determining the spawning season [35-37]. Photo- period controls daily melatonin cycles in teleost fishes [10-13, 20] as well as in mammals [1-3], however, the relationship between seasonal repro- duction and melatonin rhythms is still unknown. Further investigations will reveal the exact role of the pineal gland and circulating melatonin rhythms in teleost fish. ACKNOWLEDGMENTS We express our thanks to Dr. C. S. Tamaru, The Oceanic Institute, Hawaii, for reading the manuscript. This study was supported in part by a grant-in-aid (Bio Media Program) from the Ministry of Agriculture, Fore- stry, and Fisheries. 9 10 1052 REFERENCES Pelham, R. W., Vaughan, G. M., Sandock, K. L. and Vaughan, M. K. 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(1980) Melatonin in the blood and cerebrospinal fluid of the green sea turtle (Chelonia mydas). Gen. Comp. Endocrinol., 40: 180-187. Vivien-Roels, B., Arendt, J. and Bradtke, J. (1979) Circadian and circannual fluctuations of pineal in- dolamines (serotonin and melatonin) in Testudo hermanni Gmelin (Reptilia, Chelonia). Gen. Comp. Endocrinol., 37: 197-210. Delgado, M. J. and Vivien-Roels, B. (1989) Effect of environmental temperature and photoperiod on the melatonin levels in the pineal, lateral eye, and plasma of the frog, Rana perezi: Importance of ocular melatonin. Gen. Comp. Endocrinol., 75: 46- 313), Gern, W. A. and Norris, D. O. (1979) Plasma melatonin in the neotenic tiger salamander (Ambys- toma tigrinum): Effects of photoperiod and pinealectomy. Gen. Comp. Endocrinol., 38: 393- 398. Gern, W. A., Owens, D. W. and Ralph, C. L. (1978) Plasma melatonin in the trout: Day-night change demonstrated by radioimmunoassay. Gen. Comp. Endocrinol., 34: 453-458. Falcon, J., Guerlotte, F., Voisin, P. and Collin, J. P. (1987) Rhythmic melatonin biosynthesis in a photo- receptive pineal organ: A study in the pike. Neuroendocrinology 45: 479-486. Kazuka, H., Furukawa, K., Aida, K. and Hanyu, I. (1988) Daily cycles in plasma melatonin levels under long or short photoperiod in the common carp, Cyprinus carpio. Gen. Comp. Endocrinol. , 72: 296- 302. 13 14 16 17 20 H. Kezuka, M. [ico et al. Zachmann, A., Knijff, S. C. M., Ali, M. A. and Anctil, M. (1991) Effects of photoperiod and diffe- rent intensities of light exposure on melatonin levels in the blood, pineal organ and retina of the brook trout (Salvelinus fontinalis Mitchill). Can. J. Zool., in press. Kavaliers, M. (1979) The pineal gland and circadian organization of teleost fish. Rev. Canad. Biol., 38: 281-292. Tabata, M., Minh-Nyo, M. and Oguri, M. (1991) The role of the eyes and the pineal organ in the circadian rhythmicity in the catfish Silurus asotus. Nippon Suisan Gakkaishi 57: 607-612. 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(1987) Critical daylength and temperature level for photoperiodism in gonadal maturation of goldfish. Exp. Biol., 47: 89-94. if ' : r i? 4 oo ; ie 7 vt re ih an | ets ‘ ui 7 { a Se mer Te ue ‘i 4 = Be ‘ . irk halal yet hs r snips pat fine le cae yt tit 1a} deen! ee lt. i Das ipemitir nesta — Dien Ni 4. Hint. cer A aldo obL ay ; 3 weeritl be bine, Fareeetbertine iy! te" era ny mee er a? eu sti iahiidh’ abl bo whined a ee a tt! Tay eta sth}, 40F op sneqiart be siihwatew bignwietiert “Suey [)atbaddk eee nebiee in} 4 brit ; ; : se ou man 7M Geer cipal rf areas bi babd itis PAID Me Henan ay eet F {b ety (ue Cee aS tr) promi: eae sey nrsct ¢ © iy aru Andis | Ved M net a yaviY HOES ; | ; ‘ Lol % j Ai tet a EDS es (tye S in he ee Ui ae that ce a ify sb Mowe 2a t t : >» ST ee Li ah PA ean ad { } i ithe bee, 254 jeer buhay ree reobat thatt-J a ; ri auslieahs . Aisnali ‘, wi fig Pcieeimh: othe b ai ‘ ; : Latino’ te weet a et ‘ ys i rales) Gee idee Fabio 22%) ; = i | rt | { U} ' \ } | } | | rn | | AI u ' me h ZOOLOGICAL SCIENCE 9: 1055-1060 (1992) © 1992 Zoological Society of Japan Intermediary Metabolism in Castrated/Thyroidectomized Calotes versicolor: Regulation by Thyroxine and Testosterone VALSA S. PETER and OOMMEN V. OOMMEN Department of Zoology, University of Kerala, Kariavattom, Trivandrum 695 581, India ABSTRACT—Thyroidectomy did not change blood glucose level and acid phosphatase (Ac. Pase; liver and kidney) activity, and castration reduced the same in Calotes versicolor, when compared to their respective sham-operated controls. Castration or thyroidectomy decreased the activities of glucose-6- phosphatase (G-6-Pase; liver and kidney), glutamic oxaloacetic transaminase (GOT; liver and heart), glutamic pyruvic transaminase (GPT; liver) and urea (liver and kidney) concentration. Thyroidectomy markedly elevated hepatic cholesterol level and castration did not change its concentration. Adminis- tration of thyroxine (T4) to castrated and testosterone (T) to thyroidectomized animals stimulated the activities of G-6-Pase, Ac. Pase, GOT, GPT, the concentrations of glucose and urea, and decreased liver cholesterol concentration. It is suggested that both T, and T are effective in stimulating intermediary metabolism in lizards, irrespective of the removal of either thyroid or gonad. INTRODUCTION The relation between the activity of thyroid gland and reproduction has been reported in cer- tain species of reptiles [1, 2]. Testosterone restores the decrease in oxygen consumption of isolated tissues, following thyroidectomy, in the lizard Ca- lotes versicolor and the snake, Natrix piscator [3]. Histological studies revealed that gonadal regres- sion after thyroidectomy and thyroid hyperplasia after gonadectomy are regained to normal by replacement therapy of thyroxine to thyroidecto- mized and testosterone propionate to gonadecto- mized C. versicolor [4]. Administration of differ- ent doses of thyroxine to thyroidectomized lizards [5] and testosterone to castrated animals [6] pro- vide evidence that these hormones play an impor- tant role in intermediary metabolism of C. versico- lor. However, little is known about the thyroid- gonad interrelationship on reptilian intermediary metabolism. In this paper, we report the effects of administration of testosterone to thyroidectomized and thyroxine to gonadectomized lizards on the Accepted June 29, 1992 Received December 12, 1990 biochemical constituents and the enzymes associ- ated with intermediary metabolism in different tissues of the lizard, C. versicolor. MATERIALS AND METHODS Adult male Calotes of 24+4 g body wt captured from the local areas of Trivandrum during December/January were housed in metallic wire- netted cages. The animals were provided with minced beef liver and fresh water given ad libitum and were fasted for two days prior to sacrifice. After a week of acclimation to the laboratory conditions (28+2°C and 12L:12D), the animals from each of the two sets were divided into four groups of five animals each. Group one animals in both first and second sets served as sham-operated, hormone vehicle (propylene glycol/alkaline saline) injected controls. Animals in groups II, III and IV in the first set were thyroidectomized as described elsewhere [5] and groups 2, 3 and 4 in the second set were castrated under open ether anaesthesia. A small incision was made on the left dorsolateral side of the animal, just above the pelvic girdle, and the two testes were removed in toto in asceptic conditions. While group II animals 1056 served as thyroidectomized controls, groups II and IV were administered with 25 and 50 ug of testos- terone (T) respectively. Animals of group 2 served as castrated controls and groups 3 and 4 were administered with 5 and 10 ug of thyroxine (T4), respectively. Hormone administration was started five days after operation, intramuscularly, on alternate legs, between 7.00 and 7.30 A.M. The daily dose of hormone administered was in 0.1 ml of the respec- tive vehicle and treatments were for a period of five days. L-thyroxine (Sigma Chemical Co.) was dissolved in alkaline saline (pH 9.0), and testoster- one (Sigma Chemical Co.) was dissolved in prop- ylene glycol (BDH, India). Twenty-four hr after the last injection, blood was drawn from the heart of anaesthetised animals which were then sac- rificed by decapitation. Liver, kidney and heart were removed, chilled and a 10% homogenate was prepared in 0.25 M sucrose medium. After sub- jecting the homogenate to differential centrifuga- tion (10,000 g) for 10min at 4°C, the post- mitochondrial supernatant was collected. The specific activities of glucose-6-phosphatase (G-6-Pase) [7, 8], acid phosphatase (Ac. Pase) [9], glutamic pyruvic transaminase (GPT; also called alanine aminotransferase) and glutamic oxaloace- tic transaminase (GOT; also called aspartic amino- transferase) [10] and the estimation of the bioche- mical constituents, glucose, [11], urea [12] and cholesterol [13] were colorimetrically done. The details of assay procedure are described elsewhere [S]. Data collected from five animals in each group were analysed by one-way classification of ANO- VA followed by multiple range test [14, 15]. RESULTS Thyroidectomy did not change the blood glucose level (Fig. 1), and castration reduced the same (Fig. 2), when compared to respective sham- operated lizards. Castration and thyroidectomy decreased the activities of G-6-Pase (liver and kidney), Ac. Pase, (liver and kidney), GOT (liver and heart), GPT (liver) and urea concentration (liver and kidney), except that of Ac. Pase, which remained unaltered in thyroidectomized speci- V. S. PETER AND O. V. OOMMEN mens when compared to their respective sham- operated lizards (Figs.1, 2). Thyroidectomy markedly elevated hepatic cholesterol level (Fig. 1), and castration had no effect (Fig. 2). Injections of 25 or 50 ug of T in thyroidecto- mized lizards stimulated the activities of G-6-Pase, Ac. Pase, GOT, GPT, and the concentrations of glucose and urea; markedly decreased hepatic cholesterol when compared to thyroidectomized lizards (Fig. 1). The lower dose of T (25 yg) did not stimulate Ac. Pase activity (Fig. 1). Administration of 5 or 10 ug of T4 to castrated lizards significantly increased the glycemic level and urea concentration when compared to cas- trates (Fig. 2). The activity of G-6-Pase was signi- ficantly stimulated by the administration of 5 and 10 ug of Ty whereas only 10 4g dose stimulated the activities of Ac. Pase, GOT and GPT (Fig. 2). Ten vg Ts had a hypocholesterolemic effect, while 5 ug dose had none in castrated lizards (Fig. 2). DISCUSSION Earlier studies have shown that thyroid and testes have well known seasonal variations in their activity in C. versicolor [4, 16]. The hyperglycemia appeared after Ty, administration to castrates may be due to the action of exogenous Ty. The hyperglycemia after T, or T administration in castrated or thyroidectomized lizards indicates that in the absence of T, T, may increase blood glucose level and vice versa. This suggests an interrela- tionship of thyroid and gonad probably mediated through hypothalamic-pituitary axis. The main effects of thyroid hormones on glycemic level seem to be hyperglycemic [17]. Recently, Jacob and Oommen [5] have reported that thyroidectomy does not change the glucose level, and castration produces a hypoglycemic effect in C. versicolor [6]. Josekumar and Oommen [4] have suggested in C. versicolor that gonadectomy results in hyperplasia of the thyroid follicle which is characterized by high output of thyrotropin (TSH) due to low level of Ty in blood. They have also shown that thyroidectomy in C. versicolor results in degenera- tion of testis, the degree of which varies in diffe- rent periods of the year [4]. In reptiles, thyroidec- tomy has been reported to have a direct effect on Thyroid Gonad Relationship in Calotes 1057 GLUCOSE (Blood) G-6-Pase 240 r 5 S20 ie chy © chy we 2 Bo = 2 9° © 160 =) 6, 210) x oa a > 1% AL, a0 ae & = u 2 S) = Ofo a gS ai) (efo) (eS = =& 80 il Ac. Pase x % Liver Kidney ug/100mg wet tissue i) ive) S o rn nmol pyruvate liberated/min/mg protein (oe) a (=) (=| UREA a ~ 2 390 ae. = ao (cd) cE 20 o§ HE ® 310 aS = B ! oO oie ‘4 Ow Oo my © > ka ou 10 ay A 2) oo =| S as 7 230 Sie ole) ™~ iol) el 150 LIVER CHOLESTEROL 330 150 SO Tx TxX+ Tx+ SO Tx Tx+ TX+ 25ugT 50ygT 25pgT 50pgT Fic. 1. Effect of administration of testosterone (T) on certain enzyme activities and biochemical constituents in thyroidectomized (Tx) Calotes versicolor. Each column is mean+S.D. of five animals. Statistics according to multiple range test. SO: Sham-operated. *P<0.05 **P<0.01. 1058 130 mg/100ml1 blood 400 40 ug/g wet tissue 270 pg/100mg wet tissue Fic. 2. SO V. S. PETER AND O. V. OOMMEN GLUCOSE (Blood) Es a UREA LIVER CHOLESTEROL CC C+5pg C+10ug an cl 4 50 G-6-Pase ™s~ co) Os oS u oe 30 42) ae ce Ee = : i Oe 10+ /2fe = 2 = =] & A P _B 45 c.Pase oO Ba. oO s &_ 20 # oe i Be Tesl55| ne PS 40 —} () oo = eine blll Q “4 ie GPT (Liver) * > ol = on doa pany ol i=) nmol pyruvate liberated/min/mg protein oo = S&S (je) —\ cC C+5pg C+10p¢g aw ae 4 J Effect of administration of thyroxine (Ty) on certain enzyme activities and biochemical constituents in castrated (C) Calotes versicolor. Each column is mean+SD of five animals. Statistics according to multiple range test. SO: Sham-operated CC: castrated control. *P<0.05 **P<0.01. Thyroid Gonad Relationship in Calotes Leydig cells index which is highly sensitive to gonadotropins [18]. Hyperglycemia after Ty or T administration may be due to increased rate of gluconeogenesis as G-6-Pase is related to homeostatic regulation of blood glucose. Decreased activity of this enzyme after thyroidectomy or castration gives evidence for the reduced rate of gluconeogenesis. It has been shown that Ty injection to thyroidectomized and T to castrated lizards produce an increased G-6-Pase and Ac. Pase activities [5-7]. Van Rees et al. [19] have reported that castration decreases both pituitary and serum TSH levels whereas testosterone propionate increases TSH levels in castrated male rats. Ac. Pase, a lysosomal enzyme which hydrolyses phosphate esters in an acid environment is associ- ated with synthetic activities depending upon the physiological state of the tissues involved in the process. The increased activity of Ac. Pase by T or T, in thyroidectomized and castrated lizards re- spectively, is an indication of increased lysosomal activities in liver and kidney. A significant stimula- tion in the activities of the two transaminases, GOT and GPT after injections of T, to castrated and T to thyroidectomized animals may suggest decreased proteolysis, as these transaminases are concerned with amino acid catabolism and gluco- neogenesis [20]. It is also likely that in the absence or presence of T,/T either one hormone or both may participate in increasing transamination and gluconeogenesis. The increased concentration of urea, following administration of T4 and T to castrated and thy- roidectomized lizards respectively, illustrates that both hormones are equally effective in increasing urea concentration in this high energy demanding poikilotherm. The decreased cholesterol concen- tration after T, and T administration to castrated and thyroidectomized animals reveals the lipolytic action of these hormones in lizards. Thus it seems that both T, and T promote lipid breakdown, facilitating availability of substrates for oxidation in this metabolically active animal. Stimulation of oxidative metabolism by thyroid and gonadal hor- mones has been reported in reptiles by Oommen and Sreedeviamma [21]. The present investigation suggests the biochemi- 1059 cal influence of testosterone in the absence of thyroid function and thyroxine in the absence of testicular function. The study helps to confirm the previous suggestion [23] that poikilotherms de- pend on androgens to a considerable extent for the maintenance of intermediary metabolism and also confirms the earlier reported histological effects of thyroid ablation on gonadal function and vice versa [4]. It seems that thyroid and testis in C. versicolor exhibit a direct interrelationship which may in- volve gonadotropins and thyrotropins. The in- volvement of hypothalamo-hypophysial axis in the modulation of thyroid and gonadal function has been known in vertebrates. The hypophysial gly- coprotein hormones, TSH, FSH and LH in some cases overlap their biological activities. It has been proposed that thyroid function evolved parallel with endocrine control of reproduction and that the basic function for thyroid hormones is associ- ated primitively with maturation of gonads [22]. Thyroxine exerts a negative feed back on both TSH producing cells and gonadotropic cells; con- versely, gonadal hormones influence both these pituitary cells [24]. The control over TSH and gonadotrops remain closely linked and there are common pathways to the control of both these cell types in brain of teleost [25]. Further, in teleost fishes the control of TSH is evolved from the control of original gonadotropins which indicate a close relationship between thyroid function and reproduction. 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ZOOLOGICAL SCIENCE 9: 1061-1066 (1992) Immunocytochemical Detection of Prolactin and Growth Hormone Cells in the Pituitary during Early Development of the Japanese Eel, Anguilla japonica Emi ARAKAWA, ToyoJI KANEKO, KATSUMI TSUKAMOTO and TETsuyA HIRANO Ocean Research Institute, University of Tokyo Nakano, Tokyo 164, Japan ABSTRACT—The occurrence and development of prolactin (PRL) and growth hormone (GH) cells were investigated in the Japanese eel by means of an immunocytochemical method. Both PRL and GH cells were detected in the pituitary in all the specimens of the leptocephali, even in the smallest (10.0 mm in total length). In the leptocephalus, the mean percentage of PRL-cell area to the whole pituitary area (% PRL) was 6.5+1.2%, the % GH being 15.5+2.0%. Both % PRL and % GH exhibited a tendency to decrease as leptocephali grew larger. In the glass eel, caught just before upstream migration, the % PRL was 12.6+0.5%, almost twice as much as that in the leptocephalus, whereas the % GH was 16.7+1.3%, similar to the value in the leptocephalus. The activation of PRL cells in the glass eel suggests that PRL has an osmoregulatory role in freshwater adaptation during the upstream migration. Our findings also suggest that GH is important for larval growth and, possibly, osmoregula- © 1992 Zoological Society of Japan tion in seawater during their early life stages. INTRODUCTION Prolactin (RPL) is well known as an important hormone for freshwater adaptation in many eury- haline species [1, 2]. Activation of PRL cells or increase in plasma PRL has been observed after transfer from seawater to fresh water. On the other hand, there is a large body of evidence that growth hormone (GH) is involved in seawater adaptation especially in salmonids. Increase in plasma GH after transfer from fresh water to seawater has been reported in several salmonid species [3-9]. The eels belong to the catadromous fishes; they experience both seawater and fresh water during their life-long migration. Ecological studies have shown that they spawn eggs offshore and leaf- shaped larvae (leptocephali) drift in the current toward the coasts. After metamorphosis, juveniles (glass eels) migrate upstream and stay in the river Accepted July 20, 1992 Received June 20, 1992 or the lake for 5-10 years; they grow in fresh water and return to the ocean for spawning. However, early developmental stages of the Japanese eel, Anguilla japonica, have hardly been studied, be- cause of very limited availability of eggs and leptocephali. Furthermore, the spawning area of the Japanese eel had been unknown until quite recently. In June—Jyly 1991, the expedition of the Haku- ho-Maru, a research vessel of the Ocean Research Institute, University of Tokyo, succeeded in sam- pling more than 900 pre-leptocephali and lep- tocephali, and determined the spawning area of the Japanese eel to be in the North Equatorial Current west of the Mariana Islands [10]. The pre-leptocephali and leptocephali captured ranged from 7.9 to 34.2 mm in total length, much smaller and thus younger than those that had ever been collected. In order to clarify the development of osmoregulatory mechanism during the migration of the Japanese eel, we examined the occurrence and development of both PRL and GH cells by 1062 means of immunocytochemistry, using valuable samples of the preleptocephali and leptocephali, together with glass eels just before upsteam migra- tion to the river as well as sexually immature, cultured eels. This is the first report on the identification of PRL and GH cells during the early developmental stages of the Japanese eel. MATERIALS AND METHODS Pre-leptocephali and leptocephali of the Japanese eel (10.0-30.0 mm in total length), esti- mated to be 12-48 days old, were collected in the area west of Mariana Islands (salinity: 34.5%c) in June-July 1991 [10]. In the present paper, the term leptocephalus designates both pre- leptocephalus and leptocephalus for convenience. Glass eels of the same species (55-60 mm) were caught on the coast of Taiwan (salinity: 30%c) in November 1991. Sexually immature, cultured eels, weighing about 200 g, were obtained from a commercial dealer in Tokyo. Head portions of leptocephali and glass eels and pituitaries of cultured eels were fixed for 24h in Bouin’s solution and preserved in 70% ethanol. Later, they were embedded in paraplast, and sagittal sections were cut serially at 4 um thick- ness. To identify PRL and GH cells in the pituitary, the sections were stained immunocy- tochemically according to the ABC method [11] using commercial reagents (Vectastain ABC Kit, Vector Laboratories). Briefly, the sections were sequentially incubated with (1) 0.6% hydrogen peroxide (H,O3>) for 30 min to inactivate endoge- nous peroxidase activity, (2) 2% normal goat serum for 30 min to reduce non-specific binding, (3) the specific antisera against eel PRL [12] and eel GH [13], overnight at 4°C, (4) biotinylated anti-rabbit IgG for 30min, (5) avidin-biotin- peroxidase complex (ABC) for 1 h, and (6) 0.02% 3,3’-diaminobenzidine tetrahydrochloride contain- ing 0.005% HO, for 4-5 min. To determine the optimal dilution of the anti- sera, preliminary experiments were conducted, where the pituitary of the cultured eel was im- munocytochemically stained with serial dilutions of the antisera against PRL and GH. The best staining was obtained at a dilution of 1: 16000 in E. ARAKAWA, T. KANEKO et al. both PRL and GH cells. In the case of PRL cells, however, a lower dilution of 1:8000 was also employed, since the RPL cells in the leptocephalus were stained rather faintly at the dilution of 1: 16000. The specificity of the immunoreaction was confirmed by preabsorbing the antisera with re- spective antigens. For the quantitative analysis, percentages of PRL- and GH-cell areas to the whole pituitary area (% PRL and % GH) were estimated as follows: Serial sections at intervals of 16-20 um in leptocephali and glass eels and about 100 «m in cultured eels were immunocytochemically stained with either anti-PRL or anti-GH sera. The areas of PRL and GH cells and the whole pituitary were measured on microphotographs with a tablet digi- tizer. The % PRL and % GH were calculated as percentages of the total PRL- and GH-cell areas, respectively, to the total pituitary area. Significant differences were determined by Student’s ftest or Cochran-Cox test after F-test comparison of variance. RESULTS In the leptocephalus, the pituitary appeared as a cell cluster or mass, located beneath the hypotha- lamus, and was barely distinguishable as a defini- tive organ (Fig. 1A, B). Both PRL and GH cells were detectable in all specimens of leptocephali (10.0-30.0 mm) by immunocytochemistry. Com- pared with the cultured eel, however, PRL cells were faintly stained, while GH cells were similarly stained. PRL cells occurred in the rostral pars distalis in the pituitary (Fig. 1A), separated from the location of GH cells in the proximal pars distalis (Fig. 1B). The mid-sagittal section con- tained less than 5 PRL cells and 10-20 GH cells. The PRL cells did not form follicular structures, as typically seen in the adult form of the eel pituitary. The mean value of the % PRL was 6.5+1.2%, while the % GH was 15.5+2.0%, 2.4 times higher than the % PRL (Table 1). Both % PRL and % GH exhibited a tendency to decrease as lep- tocephali grew larger (Fig. 2). The pituitary of the glass eel was morphological- ly more comparable to that of the cultured eel than that of the leptocephalus (Fig. 1C, D). The pituit- Development of PRL and GH Cells in Eel 1063 Fic. 1. Mid-sagittal sections of the pituitaries in leptocephalus (A, B; 19.7 mm in total length), glass eel (C, D) and cultured eel (E, F) of the Japanese eel, stained with anti-eel PRL (A, C, E) and anti-eel GH (B, D, F). HT, hypothalamus; III, third ventricle. Anterior to the left. A and B, X347; C and D, x 126; E and F, x53. ary in this stage appeared pendent from the hypothalamus, whereas that of the leptocephalus was embedded partly in the hypothalamus. The midsagittal section contained about 30 PRL cells, gathering at the ventral edge of the rostral pars distalis, without forming follicular structures (Fig. 1C). The intensity of immunoreaction in PRL cells were still weaker than that of the cultured eel. About 40 GH cells cultured in the ventral part of the proximal part distalis (Fig. 1D). The % PRL was 12.6+0.5%, significantly higher than that in the leptocephalus, whereas the % GH (16.7+ 1.3%) was similar to that in the leptocephalus (Table 1). In cultured eels, PRL cells were located at the antero-ventral edge of the rostral pars distalis, mostly forming follicles (Fig. 1E), whereas GH cells occupied a large part of the proximal pars distalis (Fig. 1F). The % PRL in cultured eels (11.8+1.7%) was comparable to that of glass eels. In contrast, the % GH was significantly increased tO23:: fae 18 Ze: 1064 TasBL_e 1. Percentages of RPL-cell area (% PRL) and GH-cell area (% GH) to the whole pituitary area in different developmental stages of the Japanese eel Percentage Developmental stage of cell area Leptocephalus 6.5+1.2 (6) 12.6+0.5 (6)**11.8+1.7 (6)* 15.5+2.0 (5) 16.7+1.3 (6) 23.741.8 (6)** Glass eel Cultured eel % PRL % GH Values represent means+S.E. (n). * ** and ", significantly different from the values of leptocephali (*P<0.05, **P<0.001) and glass eels ("P<0.05). fo) 20 is} 2 w ae, fe) 8 e 32 10 A i e e e e (@) —————— —————— 10 20 30 Total length (mm) Fic. 2. Relationships between percentages of PRL- and GH-cell areas (% PRL, @; % GH, ©) and body length in leptocephali of the Japanese eel. DISCUSSION In the present study, we examined for the first time the occurrence and development of PRL and GH cells in the Japanese eel, using leptocephali as well as glass eels and sexually immature, cultured eels. This study was made possible by the success of collecting a large number of leptocephali and consequent discovery of the spawaning area of the Japanese eel by the expedition of the research vessel Hakuho-Maru [10]. Both PRL and GH cells were detectable in all the leptocephali examined, even in the smallest specimen (10.0 mm) which was estimated to be 12 days after hatching. Considering the growth- promoting action of GH, it is not surprising that GH cells are present in this early developmental stage. It is of particular interest, however, that PRL cells also exist in the stage of the eel living E. ARAKAWA, T. KANEKO et al. offshore, since PRL is supporsed to be the most important hormone for freshwater adaptation in euryhaline teleosts [1, 2]. The occurrence of PRL cells in the early stages of the life has reported in a marine fish, black sea bream (Acanthopagrus schlegeli) [14] as well as coho salmon (Oncorhyn- chus kisutch) [15] and rainbow trout (O. mykiss) [16]. Our finding and others suggest that PRL is involved in some other functions than freshwater adaptation, such as larval growth; in fact it is generally accepted in amphibian that PRL is in- volved in larval growth [17]. Both % PRL and % GH similarly decreased as leptocephali became larger (Fig. 2), implying that PRL and GH cells develop earlier than other cell types in the pityuitary. PRL cells were more faintly stained than those in cultured eels. Although the intensity of immunocytochemical reaction does not necessarily reflect the activity of endocrine cells, PRL cells in leptocephali seem less active. This is supported by the face that the number of PRL cells, as well as the % PRL, was less in the leptocephalus in comparison with GH cells. The % PRL increased significantly in the glass eel compared with that in the leptocephalus, although PRL cells were still faintly stained when compared with those in the cultured eel in fresh water. In cultured eels, the % PRL was similar to that in glass eels. The increase in the % PRL is well timed to upstream migration of the glass eel. Increase in PRL cells prior to migration is also observed in mullet (Mugil cephalus) {18, 19] and black sea bream [14]; they migrate from seawater to brackish water. In black sea bream migrating from offshore to inshore during the final phase of postflexion stage, Kimura and Tanaka [14] re- ported that PRL production was stimulated during the development from yolk-sac larva to juvenile, coinciding with their inshore migration. In our study, leptocephali were collected in the seawater (34.5%c), while glass eel were collected in the coastal water with a little lower salinity (30%c); the difference in environmental salinity is minor. Thus, the increase of PRL cells in the glass eel seems to be endogenously prepared in advance for freshwater adaptation or migration to the river. The % GH was consistently higher than the % Development of PRL and GH Cells in Eel PRL in all the leptocephali examined (Fig. 2), and the high level was maintained in glass eels. The abundance of GH cells implies their high cellular activity, and suggests that the importance of GH for larval growth and, possibly, osmoregulation in seawater. The % GH was further increased in the cultured eel, although they were in fresh water. The pituitary was distinguishable even in the smallest leptocephalus, and became morphologi- cally similar to the adult form of the gland in the glass eel. In the cultured eel, PRL cells typically formed follicular structures, the functional signi- ficance of which is unknown. During early de- velopmental stages from leptocephalus to glass eel, however, PRL cells existed only as clusters of cells without forming follicles, indicating that the formation of follicular structures occurs in a later stage, presumably around the period of migration to fresh water. In the present study, the activity of PRL cells seemed to be enhanced in the glass eel, just before upstream migration. On the other hand, GH cells remained rather active during all stages of the leptocephalus and glass eel. These findings are in consistent with the notion that PRL is responsible for freshwater adaptation, and GH for seawater adaptation as well as growth. However, further studies are required to understand the whole aspects of osmoregulatory control by PRL and GH during the early life stages of the eel. ACKNOWLEDGMENTS We are grateful to Professor P. P. Hwang, Institute of Zoology, Academia Sinica, Taiwan, for providing glass eels. We thank the crew of the Hakuho-Maru and the scientists aboard. This study was supported in part by grants-in-aid from the Ministry of Education, Science and Culture, and also from Fisheries Agency, Japan. REFERENCES 1 Clarke, W. C. and Bern, H. A. (1980) Comparative endocrinology of prolactin, In “Hormonal Proteins and Peptides”. Ed. by C. H. Li, Academic Press, New York, Vol. 8, pp. 105-197. 2 Brown, P. S. and Brown, S. C. (1989) Osmoregula- tory actions of prolactin and other adenohypophy- sial hormones, In “Vertebrate Endocrinology: Fun- damentals and Biomedical Implications”. Ed. by P. 10 11 12 13 14 15 1065 K. T. Pang, M. P. Schreibman, Academic Press, New York, Vol. 2, pp. 45-84. Sweeting, R. M., Wagner, G. F. and MacKeown, B. A. (1985) Changes in plasma glucose, amino acid nitrogen, and growth hormone during smoltification and seawater adaptation in coho salmon, Oncorhyn- chus kisutch. Aquaculture, 45: 185-197. Hasegawa, S., Hirano, T., Ogasawara, T., Iwata, M., Akiyama, T. and Arai, S. (1987) Osmoregula- tory ability of chum salmon, Oncorhynchus keta, reared in fresh water for prolonged periods. Fish Physiol. Biochem., 4: 101-110. Boeuf, G., Le Bail, R. Y. and Prunet, P. (1989) Growth hormone and thyroid hormones during Atlantic salmon, Salmo salar L., smolting, and after transfer to seawater. Aquaculture, 82: 257-268. Collie, N. L., Bolton, J. P., Kawauchi, H. and Hirano, T. (1989) Survival of salmonids in seawater and the time-frame of growth hormone action. Fish Physiol. Biochem., 7: 315-321. Ogasawara, T., Hirano, T., Akiyama, T., Arai, S. and Tagawa, M. (1989) Changes in plasma prolactin and growth hormone concentrations during freshwa- ter adaptation of juvenile chum salmon (Oncorhyn- chus keta) reared in seawater for a prolonged period. Fish Physiol. Biochem., 7: 309-313. Hirano, T., Ogasawara, T., Hasegawa, S., Iwata, M. and Nagahama, Y. (1990) Changes in plasma hormone levels during loss of hypoosmoregulatory capacity in mature chum salmon (Oncorhynchus keta) kept in seawater. Gen. Comp. Endocrinol., 78: 254-262. Sakamoto, T., Ogasawara, T. and Hirano, T. (1990) Growth hormone kinetics during adaptation to hyperosmotic environment of rainbow trout, Salmo gairdneri. J. Comp. Physiol. B, 160: 1-6. Tsukamoto, K. (1992) Discovery of the spawning area for Japanese eel. Nature, 356: 789-791. Hsu, S. M., Raine, L. and Fanger, H. (1981) Use of avidin-biotin-peroxidase complex (ABC) in im- munoperoxidase techniques: A comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem., 29: 577-580. Suzuki, R. and Hirano, T. (1991) Development of a homologous radioimmunoassay for eel prolactin. Gen. Comp. Endocrinol., 81: 403-409. Kishida, M. and Hirano, T. (1988) Development of radioimmunoassay for eel growth hormone. Nippon Suisan Gakkaishi, 54: 1321-1327. Kimura, R. and Tanaka, M. (1991) Prolactin pro- duction during larval and early juvenile periods of euryhaline marine fish, black sea bream, Acantho- pagrus schlegeli. Nippon Suisan Gakkaishi, 57: 1833-1837. Leatherland, J. F. and Lin, L. (1975) Activity of the putuitary gland in embryo and larval stages of coho 17 1066 salmon, Oncorhynchus kisutch. Can. J. Zool., 53: 297-310. Nozaki, M., Tatsumi, Y. and Ichikawa, T. (1974) Histological changes in the prolactin cells of the rainbow trout, Salmo gairdneri irideus, at the time of hatching. Annot. Zool. Japon., 47: 15-21. White, B. A. and Nicoll, C. S. (1982) Hormonal control of amphibian metamorphosis. In “Meta- morphosis”. Ed. by L. I. Gilbert, E. Frieden, Ple- num Press, New York, pp. 363-396. E. ARAKAWA, T. KANEKO ef al. 18 Abraham, M. (1971) The ultrastructure of the cell types and of the neurosecretory innervation in the pituitary of Mugil cephalus L. from fresh water, the sea, and a hypersaline lagoon. Gen. Comp. Endocri- nol., 17: 334-350. Blanc-Livni, N. and Abraham, M. (1970) The influence of environmental salinity on the prolactin and gonadotropin-secreting regions in the pituitary of Mugil (Teleostei). Gen. Comp. Endocrinol., 14: 184-197. ZOOLOGICAL SCIENCE 9: 1067-1080 (1992) Innervation of the Caudal-Fin Muscles in the Teleost Fish, Medaka (Oryzias Latipes) Yun Isaikawa! Department of Anatomy, Faculty of Medicine, University of the Ryukyus, Okinawa 903-01, Japan ABSTRACT—The peripheral pattern of the spinal nerves in the caudal fin and the spinal motoneurons that innervate the caudal-fin muscles were described in the adult medaka. The peripheral nerves were immunochemically stained by using antibodies to neurofilament proteins. To label the spinal motoneurons retrogradely, horseradish peroxidase was applied to each caudal-fin muscle. The somata of motoneurons innervating the caudal-fin muscles were distributed over the whole area in the motor column from spinal segment 25 to 28 of the ipsilateral spinal cord. Most motoneurons were small (6-15 micra in diameter), but larger ones (more than 20 micra in diameter) were sometimes found. The axon of each motoneuron, rather than entering the nearest ventral root, ran caudad in the anterior funiculus of the spinal cord for the length of several spinal segments before entering a single ventral root. The axon that starts from the anterior segment of the spinal cord entered the anterior ventral root. Each caudal-fin muscle was innervated by several ventral roots of the spinal nerves from 27 to 31B. The anterior caudal-fin muscles were innervated by the anterior spinal nerves. Thus, motoneurons that innervate caudal-fin muscles are organized somatotopically along the cranio-caudal axis of the spinal © 1992 Zoological Society of Japan cord. INTRODUCTION The medaka (Oryzias latipes) is suitable for observing in detail the nerve pattern in whole mounts, since the tail regions are thin and almost transparent in the embryonic stages [1-3]. Moreover, some inbred strains of the medaka have become available for experimental work [4], and more than 60 mutant strains including several morphogenetic ones have been found in the meda- ka [5, 6]. The first transgenic fish has been produced successfully in the medaka [7]. Hence, this teleost fish affords good material for ex- perimental studies in developmental neurobiology of vertebrates [see also 8-12]. Anatomical study is an essential step in investi- gating the development of the nervous system in the medaka. However, little information on the neuroanatomy of the medaka is available. The present study began as an effort to understand how Accepted June 22, 1992 Received November 6, 1991 ' Present address: Division of Biology, National Insti- tute of Radiological Sciences, Chiba 263, Japan. the muscle nerves develop in the caudal fin. This paper describes the anatomy of the nerve-muscle system in the caudal region of the adult medaka. Special attention is focused on the innervation of one of the caudal-fin muscles, the middle interra- dial muscle (MIR muscle), since its development will be described in detail in forthcoming papers. MATERIALS AND METHODS Materials The d-rR strain of the medaka (Oryzias latipes) was given by Dr. H. Tomita (Nagoya University) in March 1986. The fish of this color mutant strain shows the wild-type morphology, except for the mutations in amounts and distribution of several kinds of pigment cells [5]. The fish have been kept in our laboratory in plastic aquaria and fed a diet of Tetra-min (Tetra, West Germany). Adult fish and young adult fish (15-30 mm in total body length) of the d-rR strain were used. At least three fish were used in each labeling experiment and in each staining (see below). Fry (7-15 mm in total 1068 Y. ISHIKAWA body length) were also used to show general structures of the medaka, since the gross patterns of skeleton, muscles, and peripheral nerves in the caudal region of the adult fish are almost identical, except for their sizes, to those of the fry longer than 7 mm in total body length [2]. Retrograde Labeling with HRP Adult fish were anesthetized in 0.01-0.03% MS 222 and secured to a plastic plate with a gluey tape. The unilateral skin above the muscle to be ex- amined was cut and the underlying muscle tissue was macerated with a sharpened steel needle. Small crystals of horseradish peroxidase (HRP, Toyobo, Osaka, Japan; Lot No. 1417) were put on the wound. The fish was put in fresh water in such a way that the head and gill regions were in the water while the wounded tail region was above the water surface to avoid dilution or leakage of the HRP. After 30 min, the fish was washed in fresh water, released into the balanced salt solution [8], and allowed to survive for 24—48 hr. After that, the fish was anesthetized heavily and killed by decapitation. The caudal part of the body was put in a solution of 2 or 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). In the fixative, the spinal cord (including the proximal portions of the ventral roots) and the vertebral column were isolated from the other tissue. The spinal cord and the vertebral column were fixed in fresh fixative overnight at 4°C. After washings over a period of several days with phosphate- buffered saline (PBS) at 4°C, HRP was histo- chemically detected in whole mounts by the methods of Nordlander [13] and Hanker et al. [14]. The spinal cord was cleared with glycerin and observed in whole mount. Celloidin Sections of the HRP-labeled Spinal Cord In order to examine in detail the HRP-labeled cells in the spinal cord, the celloidin-sectioned specimens were prepared from the spinal cords which had been reacted as described above. After washing in PBS, the reacted spinal cords were fixed again in 4% paraformaldehyde in 0.1M sodium phosphate buffer (pH7.4). They were dehydrated, embedded in celloidin, and sectioned serially (22 micra in thickness). Nissl staining was performed in alternate sections by staining with thionin. Labeling with Dil In some caudal-fin muscles, 1,1’-dioctadecyl- 3,3,3’-3’-tetramethylindocarbocyanine perchlorate (Dil, Molecular Probes, Junction City, Oregon) was used to label retrogradely the motoneurons [15]. Young adult fish were fixed in 4% para- formaldehyde in 0.1 M sodium phosphate buffer (pH 7.4) for 4h. After washing in PBS, a lesion was made on the muscle to be examined with a small scalpel or a sharpened steel needle, and a small particle of Dil was put on the wound. The fish was placed in a moist chamber and kept moist for 10-48 h at room temperature (28°C). After the Dil was rinsed off in PBS, the spinal cord with the vertebral column was isolated. It was mounted in PBS and examined under a fluorescent microscope equipped with a set of filters appropriate for rhodamine fluorescence microscopy. Nerve Staining Nerve fibers were stained in whole mounts using anti-neurofilament protein monoclonal antibodies (all neurofilament proteins, 70 K+ 160 K+210 K, Maruzen Oil Biochemical or Cosmo Bio Co., LTD., Tokyo) according to a previously reported method [1, 16]. Muscle Staining The muscle fibers and myotubes were immuno- histochemically stained in whole mounts using a monoclonal antibody against chicken troponin T according to the method of Ishikawa [2]. Double Staining of Muscle and Nerve The nerve staining and the muscle staining were combined so that nerve and muscle could be visualized simultaneously in the same specimens. The muscle fibers and myotubes were first reacted with the anti-troponin T antibody and the HRP-labeled secondary antibody in whole mounts as described above. They were stained with peroxi- dase reaction using 3,3’-diaminobenzidine (DAB) as a substrate. The stained specimens were washed in 0.1 M glycine-HCl buffer (pH 2.4) overnight at room temperature (28°C) to remove the anti- Innervation of Caudal Muscles in Medaka 1069 troponin T antibody and the HRP-labeled anti- body. The DAB reaction product (brown in color) remained on the muscle tissue. After washing in PBS, the nerve fibers of the specimens were then reacted with anti-neurofilament protein antibody and the HRP-labeled secondary antibody. They were stained with peroxidase reaction, this time, using 4-Cl-1-naphthol as a substrate to obtain a blue reaction product. The specimens were cleared with glycerin and immediately observed. Staining of Acetylcholine Receptors For observation of neuromuscular junctions, acetylcholine receptors (AChR) were stained in whole mounts by the indirect HRP-labeled anti- body method, using erabutoxin b [17], one of the curaremimetic toxins from snake venom, and an anti-erabutoxin b polyclonal antibody [cf. 18]. Nomenclature We followed Ishikawa [2] in the nomenclature of B Fic. 1. immunochemically using anti-neurofilament protein antibodies. bones and muscles in the caudal region of the medaka. There was a problem in numbering the spinal nerves since the two cranialmost spinal nerves did not emerge from the vertebral column but instead, emerged from the skull in the medaka (see re- sults). In the present paper these two spinal nerves were referred to as occipito-spinal nerves accord- ing to Ray’s description [19] of the cranialmost spinal nerves of a teleost fish (see refs. [20], [21] for detailed discussion on the nomenclature of the rostral spinal nerves in fish; see ref. [22] for an alternative numbering of the spinal nerves of the medaka). RESULTS Spinal Nerves and Caudal-Fin Muscles Prior to describing the innervation of caudal-fin muscles, we provide brief accounts of the ana- 15 Occipito-spinal nerves (OS) and spinal nerves (numbered) of a fry (A and B). The nerves were stained The left lateral view of the whole-mount specimen is shown. The head is tilted dorsally. PF, pectoral fin. Scale bar=0.1 mm. 1070 tomical features of the spinal nerves and caudal-fin muscles. Two pairs of the occipito-spinal nerves and 32 pairs of the spinal nerves were usually observed in the medaka (Fig. 1). The occipito-spinal nerve 1, which is the first postvagal nerve, emerged through a foramen in the skull. The occipito-spinal nerve 2, the second postvagal nerve, emerged im- mediately caudal to the skull. Spinal nerve 1, the third postvagal nerve, passed through a foramen in the first vertebra. Four cranialmost nerves, name- ly, the occipito-spinal nerve 1, the occipito-spinal nerve 2, the spinal nerve 1, and the spinal nerve 2 formed the cervico-brachial plexus at the base of the pectoral fin. In the caudalmost region, two pairs of spinal nerves (31A and 31B) emerged through the foramina in the same caudalmost vertebra, namely the 31st vertebra. — V Fic. 2. Y. ISHIKAWA Figure 2 shows the main branches of a typical spinal nerve in the tail region. The ventral root (VR) was larger than the dorsal root (DR), and extended through its own formen in the vertebra. Outside the vertebral column the lower part of the ventral root turned downward to form the ramus ventralis, while the upper part turned dorsad to join the spinal ganglion which formed on the dorsal root. The ramus dorsalis originated near this ganglion. The ramus ventralis coursed ventro-caudally in the ventral region of its own segment and pro- jected many side branches. The largest side branch projected laterally from the ramus ventralis to form a lateral branch at the level of the horizon- tal septum. The lateral branch ran toward the lateral surface of the segmental muscle and bifur- cated dorsally and ventrally near the posterior RD SEGMENTAL MS <—— MUSCLE —- ; {SPINAL CORD Drawing of the peripheral pattern of a typical spinal nerve (spinal nerve 25) in the tail region of a fry. The nerve of the fry were stained immunochemically using anti-neurofilament protein antibodies, and the main nerve branches were traced. The left lateral view is shown. A, nerve bundles in the anterior funiculus; DL, nerve bundles in the dorsal portion of the lateral funiculus; DR, dorsal root; HS, horizontal septum; LB, lateral branch; MS, myoseptum; RC, ramus communicans; RD, ramus dorsalis; RV, ramus ventralis; SG, spinal ganglion; V, developing vertebral column, VR, ventral root. Scale bar=0.1 mm. Innervation of Caudal Muscles in Medaka lateral line nerve in the horizontal septum. The bifurcated branches ran near the anterior myosep- tum. The ramus dorsalis coursed dorso-caudally in the dorsal region of its own segment. It received a small branch (ramus communicans) from the ramus dorsalis of the next anterior spinal nerve. The supporting skeleton of the caudal fin was overlaid with two groups of caudal-fin muscles, namely, the anterior caudal-fin muscles and the posterior caudal-fin muscles (Fig. 3). In the anterior caudal-fin muscles, a pair of deep dorsal flexors (DDF) and a pair of deep ventral flexors (DVF) were present in the dorsal and ventral regions, respectively. There might be superficial dorsal flexors and superficial ventral flexors on the surfaces of these two muscles. However, it was difficult to discriminate between the caudal superficial muscles and the caudal deep is Fic. 3. 1071 muscles. Hence, in the present study, DDF and DVF are defined as containing in entirety both the deep and superficial muscle layers. A pair of the hypochordal longitudinal muscles (HLM) was situ- ated between the two hypural plates. In the posterior caudal-fin muscles, 13 to 15 pairs (adult fish) or 8 pairs (young adult fish) of the interradial muscles were observed beteen the fin rays. The centrally positioned muscle is called the middle interradial muscle (MIR muscle). The MIR muscle was located between the HLM and the caudalmost pit organ. Labeling with HRP In order to estimate the diffusion of HRP from the wound, we performed a series of experiments in which HRP activities were reacted in the whole specimens prior to the isolation of spinal cords. St passes Ba Anterior (ANT) and posterior (POST) caudal-fin muscles in a young adult fish. The muscles were stained immunochemically using anti-troponin T antibody. The left lateral view of the whole-mount specimen is shown. The vertebrae (V30 and V31) and hypural plates (H1 and H2) are also shown. DDF, deep dorsal flexor; DVF, deep ventral flexor; HLM, hypochordal longitudinal muscle; LIR, lower interradial muscle; MIR, middle interradial muscle; UIR, upper interradial muscle; P, caudalmost pit organ. Scale bar=0.1 mm. 1072 From the results, it appeared that HRP did not diffuse from the site of the application (Fig. 4). However, in the case of the interradial muscles, we could not rule out a diffusion of HRP to the same muscle in the opposite side: In some cases, the interradial muscles in the opposite side turned brown after the reaction. In such cases, there were several lightly stained neurons in the contralateral sides of the spinal cords that may have been labeled by a small amount of diffused HRP. In the present study, therefore, only darkly stained Golgi like cells were considered to be labeled motoneurons innervating the HRP-applied muscle. Fic. 4. HRP-positive area in the caudal fin of an adult fish after applying HRP to the MIR muscle (arrow). Note that the HRP activity is restricted in the MIR-muscle region. Left lateral view of the whole- mount specimen is shown. For abbreviations, see Figure 3. Scale bar=1 mm. At least three animals were used for the labeling experiments of each muscle. The results in each muscle were generally consistent in all experi- ments. Labeled Motoneurons The somata of the labeled motoneurons were either spherical or ellipsoidal and found in the Y. ISHIKAWA ipsilateral side of the spinal cord (Fig. 5). There appeared to be two populations which were differ- ent in the soma size of the labeled motoneurons (Figs. 6B, 6C and 7), The smaller ones (small motoneurons, Fig. 6B), to which most of the labeled motoneurons belonged, were 6-15 micra in diameter, while the larger ones (large motoneurons, Fig. 6C) were few in number and were more than 20 micra in diameter. The anteri- or caudal-fin muscles (DDF, HLM, and DVF) were innervated by both the small and large motoneurons, whereas the posterior caudal-fin muscles (interradial muscles) were innervated only by the small motoneurons (Fig. 7). The labeled small and large motoneurons usual- ly had two primary dendrites, one running dorso- rostrally and the other dorso-caudally; both den- drites ran in the lateral funiculus of the spinal cord, and branched along their entire courses (Figs. 5B and 6C). The cranio-caudal extension of the dendrites was large in the motoneurons filled from the anterior caudal-fin muscles and small in those filled from the posterior caudal-fin muscles. An axon emerged from the ventral portion of the soma as a single tapering process that extended ventro-caudally and ipsilaterally in all motoneu- rons (Figs. 5A and 6A). Axon collaterals were sometimes observed in the initial part of the axon (Fig. 6B). The axon did not emerge immediately via the nearest ventral root, but ran caudad in the anterior funiculus of the spinal cord for several (2— 4) segment lengths before entering a single ventral root (Fig. 6A). The axon of the motoneuron that lies in the more cranial segment of the spinal cord ran the more ventral course in the anterior funicu- lus of the spinal cord and emerged from the more cranial ventral root. Location of Labeled Motoneurons in the Spinal Cord The spinal motor column in the cross plane was revealed by plotting the locations of the somata of the HRP-labeled motoneurons in a schematic cross plane of the spinal cord (Fig. 8). The HRP-labeled motoneurons filled from each muscle were found over the whole area of the motor column: No restricted localization of any specific groups of the motoneurons was found in the motor column in Innervation of Caudal Muscles in Medaka 1073 Fic. 5. HRP-labeled motoneurons (arrows) in the adult spinal cords, after applying HRP to the MIR muscle (A) and the HLM (B and C). The left lateral views of whole mount specimens (A and B), and the cross section (22 micra in thickness) of the spinal cord embedded celloidin (C) are shown. The cross section is Nissl-stained to show the gray matter in the spinal cord. The dendrites are indicated by arrowheads. The vertebae are numbered. CC, central canal; MF, Mauthner fibers. Scale bars in panels A and B=0.1 mm. Scale bar in panel C=20 um. the cross plane of the spinal cord. In the sagittal plane of the spinal cord, the somata of all labeled motoneurons were distrib- uted from spinal segment 25 to spinal segment 28 of the spinal cord (Fig. 9). Each motor pool of the anterior caudal-fin muscles stretched over approx- imately two spinal segments, and each motor pool of the posterior caudal-fin muscles extended over about one spinal segment. For example, the motor pool of the MIR muscle was usually (16/20) lo- cated in spinal segment 27 and occasionally (4/20) distributed from spinal segment 27 to the anterior one-third of spinal segment 28. The motoneurons were not evenly distributed in a single motor pool but tended to be clustered (Fig. 5A). The position of each motor pool was partially overlapping in the spinal cord (Fig. 9). The motor pools of the anterior caudal-fin muscles were lo- cated from spinal segment 25 to spinal segment 27, while those of the posterior caudal-fin muscles were distributed from spinal segment 26 to spinal segment 28; thus, the center of the motor pools of the anterior caudal-fin muscles was located more cranially than that of the posterior caudal-fin mus- 1074 Y. ISHIKAWA B sets Fic. 6. C Drawing of HRP-labeled motoneurons in the adult spinal cords, after applying HRP to the MIR muscle (A and B) and to the HLM (C). In panel A, the left lateral view of the whole mount specimen is shown. The spinal nerves (ventral roots) are numbered. In panels B and C, the typical “small” (B) and “large” (C) motoneurons in cross sections of the spinal cords are shown. In panels B and C, the axons (large arrows), the dendrites (arrowheads), the axon collateral (small arrow), the central processes of the spinal ganglion cells (CP), the anterior funiculus (A), and the lateral funiculus (L) are indicated. CC, central canal; MF, Mauthner fibers. Scale bar in panel A=0.1 mm. Scale bar in panel B=20 ym, valid for panels B and C. cles. The number of motoneurons in each motor pool filled from a single caudal-fin muscle varied from 1 to 30 (Fig. 9). Innervation of Caudal-Fin Muscles By retograde labeling with HRP (Fig. 6A) or Dil (data not shown), the motor axons that project to each caudal muscle were revealed. Thus, the spinal nerves that contribute to the innervation of each caudal-fin muscle were identified (Fig. 10). The DDF was innervated by the rami dorsales from the ventral roots of spinal nerves 28, 29, 30, and 31 (mainly 30 and only slightly 31). The HLM was served by the rami ventrales from the ventral roots of spinal nerves 28, 29, and 30 (mainly 30). The DVF received the rami ventrales of the ven- tral roots of spinal nerves 27, 28, and 29 (mainly 28 and 29). All interradial muscles were supplied by the rami ventrales of the ventral roots of spinal nerves 30 and 31 (mainly 31). Muscle Nerve to the MIR Muscle The peripheral branching pattern of the spinal nerves was examined in detail in the caudal fin (Figs. 11A and 11B). A characteristic pattern, the entire shape of which is like that of a fan-shaped Fic. 7. Fic. 8. Innervation of Caudal Muscles in Medaka A DDF+HLM+DVF NUMBER Oo 15 10 (0) 5 10 15 DIAMETER Size distribution of labeled somata of the motoneurons in adult spinal cords, after applying HRP to the anterior caudal-fin muscles, DDF+ HLM-+DVFE (A) and the posterior caudal-fin mus- cles, IR (B). All measurements were made on the drawings of the HRP-labeled motoneurons in cel- loidin sections and were not corrected for shrinkage. The average diameter of an individual cell was calculated as the average of the diameter across the shortest and longest axis of the soma. The ordinate indicates the number of motoneurons. The abscissa indicates the average diameter. 20 pm MF Schematic drawing of a cross section of the caudal spinal cord, showing the motor column (shaded area) at the right side. The gray matter (dotted area), central canal (CC) surrounded by ependymal cells, and Mauthner fibers (MF) are also shown. A, anterior funiculus; L, lateral funiculus. NUMBER Fic. 9. 1075 12- DDF BF 4b 8 HLM AL —_ +—+ a 12 - 7 DVF 8 | a |e =| a U6 ] 2 ie + +——_}+_+—_+—_ + = + + | (5 ZA US : +——+ +——+—_+ + + + + A= 4+ U4 : ‘oe eeece Leal SEGMENT Histograms showing the distribution pattern of labeled motoneurons along the cranio-caudal axis of the caudal spinal cord, after applying HRP to each caudal-fin muscle of adult fish. A typical result is shown for each muscle. The ordinate indicates the number of labeled motoneurons. The abscissa indi- cates the spinal segments, each of which is divided into three equal parts; anterior (a), middle (m), and posterior (p). L1—6, lower interradial muscles; U1- 6, upper interradial muscles. For other abbrevia- tions, see Figure 3. 1076 Y. ISHIKAWA Fic. 10. Schematic drawing of muscle nerves to each caudal-fin muscle of the adult fish. The spinal nerves (ventral roots) are numbered. The main innervating nerves are indicated by thick lines. For abbreviations of muscles, see Figure 3. Innervation of Caudal Muscles in Medaka 1077 Fic. 12. Drawing of the caudal-fin-motor nerve (MN) and the caudal-fin-sensory nerve (SN) in the left lateral view of the tail region of a young fish. The spinal nerves (rami ventrales) are numbered. The muscle nerve branches in the MIR muscle are indicated by arrowheads. For abbreviations, see Figure 11. Scale bar=0.1 mm. tree, was found in this region (Figs. 10 and 12): The rami ventrales of the ventral roots of several caudalmost spinal nerves ran ventro-caudally to form a nerve plaxus at the ventral edge of the vertebral column. From the nerve plexus, a thick nerve bundle (caudal-fin-motor nerve) projected caudad. The caudal-fin-motor nerve ran under- neath the dorsal part of the HLM toward a space between two central fin rays. The caudal-fin-motor nerve bifurcated dorsally (upper branch of the caudal-fin-motor nerve) and ventrally (lower branch of the caudal-fin-motor nerve) at the posterior border of the HLM. The two nerve branches ran along the posterior ends of the hypural plates. From these branches, nerve twigs projected and entered the interradial muscles to form the neuromuscular junctions (Fig. 11C). On the other hand, one or two tightly packed nerve bundles (SN in Figs. 11A and 12) also pro- jected from the nerve plexus. The nerve bundles took a course similar to that of the caudal-fin- motor nerve. However, the distal nerve branches did not stay in the region of the interradial mus- cles, but extended further along the fin rays to the caudal end of the caudal fin. The nerve bundles were named the caudal-fin-sensory nerve, because putative sensory axons in the caudal fin should be contained in the bundles. Thus, the courses of the muscle nerves to the interradial muscles were revealed entirely. For example, the muscle nerve to the MIR muscle started from the motoneurons in spinal segment 27 of the spinal cord (Fig. 9), extended caudad in the spinal cord, emerged from the spinal cord via the rami ventrales of the ventral roots of the spinal nerves 30, 31A, and 31B, ran further ventro- Fic. 11. interradial muscles in an adult fish (C). Double staining of muscles and nerves in the caudal fin of a young adult fish (A and B) and AChR staining of The muscles and nerves are immunochemically stained using anti-troponin T antibody and anti-neurofilament protein antibodies, respectively. The AChR is stained immunochemically using erabutoxin b and its antibody. The left lateral views of the whole mount specimens are shown. In panels A and B, the photographs were taken in slightly different fields at different focus planes. The neuromuscular junctions in the MIR muscle (MIR) are observed along the courses of the muscle nerve branches in the MIR muscle (arrowheads). F, central fin rays; LMN, lower branch of the caudal-fin-motor nerve; MN, caudal-fin-motor nerve; SN, caudal-fin-sensory nerve; UMN, upper branch of the caudal-fin-motor nerve. Scale bar in panel A=20 ym, valid for panels A and B. Scale bar in panel C=0.1 mm. 1078 caudally for a long distance (about 600 micra), and finally entered the MIR muscle via nerve branches of the upper and lower caudal-fin-motor nerves (Fig. 12). DISCUSSION The present study has provided for the first time an anatomical description of the innervation of the caudal-finmuscles of the adult medaka. We found that there was a characteristic pattern of peripheral nerves in the caudal fin and that the motoneurons were organized somatotopically along the cranio- caudal axis of the caudal spinal cord. The caudalmost musculature in the medaka are derived ontogenetically from several caudalmost myomeres [10]. Also, from phylogenetic studies, it has been pointed out that the caudal-fin muscles of fish are somitic in origin, being derived from caudal myomeres [23]. It might be reasonable to assume, accordingly, that the caudal motoneurons of the medaka are essentially similar to those found in the spinal motoneurons innervating the trunk segmental muscles of the teleost fish. However, as discussed below, the present results indicate that not only similarities but also differ- ences are present between the caudal motoneurons and the trunk motoneurons. Innervation and Axonal Pattern The dorsal caudal-fin muscle, the DDF, is obviously a caudalmost component of the epaxial musculature of the trunk muscle system, while the ventral caudal-fin muscles, the DVF and HLM, are caudalmost components of the hypaxial muscula- ture (Fig. 3). The interradial muscles are the hypaxial muscles as well, because they differenti- ate in the hypochordal region of the developing caudal fin [2]. Only DDF is innervated by the rami dorsales, while the other muscles are served by the rami ventrales of the ventral roots (Fig. 10). These observations are consistent with studies of trunk segmental muscles in the teleost fish; the studies indicate that the ramus dorsalis and ramus ventral- is innervate the epaxial musculature and hypaxial musculature, respectively (see ref. [24] for a re- view; see also Fig. 2). In the present study, a motor axon ran longitudi- Y. ISHIKAWA nally and caudally for a long distance in the caudal spinal cord, bypassing several ventral roots before entering a single ventral root (Fig.6A). This axonal pattern in the caudal spinal cord differs from those reported in the trunk spinal cords of the teleost fish. In the trunk spinal cord of the adult goldfish, Fetcho [25] showed that most motor axons ran caudally in the spinal cord and entered the nearest single ventral root, although some motor axons bypassed one root before exiting from the cord. In the rostral trunk region of a larval teleost fish (zebrafish), Myers [26] reported that a single axon of each motoneuron entered the nearest single ventral root, and the motor axons within the given ventral root were all derived from motoneuron somata within a single spinal seg- ment. Motoneuron Types The morphology of the trunk motoneurons has been studied in detail in the adult teleost fish using modern techniques ({25], [27], [28]; see ref. [24] for a review). All authors agree that there are two groups of motoneurons; small motoneurons that locate in the ventro-lateral portion of the motor column and larger motoneurons that occupy the dorso-medial part of the motor column. Raamsdonk et al. [27] and Fetcho [25] showed that the differences in the location and morpholo- gy of the two types of motoneurons were corre- lated with differences in the functionally different muscle fiber types they innervate. These investiga- tors reported that “red motoneurons” which inner- vate the superficial slow (red) muscles are small in their soma size and located in the ventro-lateral part of the motor column; at least some of the “white motoneurons” which innervate the deep fast (white) muscle, were larger and lay near the central canal. Several authors proposed that the two classes of motoneurons also had different developmental his- tories [25, 28]. According to these authors, the small motoneurons differentiate later than the larger motoneurons; the small motoneurons and the larger ones were referred to as “secondary motoneurons” and “primary motoneurons”, re- spectively. In accordance with the above results reported by Innervation of Caudal Muscles in Medaka other investigators, two classes of soma size were found in the spinal motoneurons innervating the trunk segmental muscles of adult medaka (Ishikawa, unpublished observations): The small motoneurons were about 8-16 micra, while the larger ones were about 20-30 micra in diameter. In the present study too, we observed that there were two populations of soma size in the HRP- labeled motoneurons (the “small motoneurons” and the “large motoneurons”), although there was not a very distinct dichotomy of motoneuronal types (Figs. 6B, 6C and 7). However, no “large motoneurons” were found in the motor pools of the posterior caudal-fin muscles; only the “small motoneurons” were pre- sent. The “small motoneurons” were located over all areas of the motor column including the medial area near the central canal of the spinal cord. Moreover, the “large motoneurons” in the present study were distributed in the lateral area of the cross plane of the motor column (Fig. 6C). Hence, in the motoneurons of the caudal spinal cord of the medaka, it seems inappropriate to apply simply the designation of “white and red” types or “primary and secondary” types of motoneurons which have been reported for the trunk motoneurons. The “large and small” motoneurons in the pre- sent study may reflect the time sequence of de- velopment of motoneurons. It is generally accepted that large neurons are produced before small ones in any part of the nervous system [29, 30]. The caudal-fin muscles differentiates later than the trunk segmental muscles [2]. In particu- lar, the posterior caudal-fin muscles develop much later than the anterior caudal-fin muscles. If the motor pools develop in the same sequence as the groups of muscles, the posterior caudal-fin muscles would be innervated by the “small motoneurons”. This hypothesis can explain the fact that only the “small motoneurons” are present in the motor pools of the posterior caudal-fin muscles. ACKNOWLEDGMENTS This paper is dedicated to my late father, Kazuo Ishikawa, who had warmly supported my interest in science. I am grateful to Dr. Hideo Tomita for supplying the medaka. I also thank Mr. Yoshinari Fusa and Miss Katsumi Chinen, for their technical assistance. This 1079 study was supported by a Grant-in-Aid for Scientific Research on Priority Area (molecular basis of neural connection), Ministry of Education, Science and Cul- ture, Japan. REFERENCES 1 Ishikawa, Y., Zukeran, C., Kuratani, S. and Tanaka, S. (1986) A staining procedure for nerve fibers in whole mount preparations of the medaka and chick embryos. Acta Histochem. Cytochem., 19; 775-783. 2 Ishikawa, Y. (1990a) Development of caudal struc- tures of a morphogenetic mutant (Da) in the teleost fish medaka, (Oryzias latipes). J. Morph., 205: 219- 232. 3 Ishikawa, Y. 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ZOOLOGICAL SCIENCE 9: 1081-1085 (1992) [COMMUNICATION] © 1992 Zoological Society of Japan Sexual Development of Immunocompetence in the Toad, Bufo regularis ABDEL HAKIM SAAD Zoology Department, Faculty of Science, Cairo University, Cairo 12613, Egypt ABSTRACT—The kinetics of primary anti-rat erythro- cytes (RRBC) antibdody were compared in juvenile and adult toads Bufo regularis. The antibody titers were measured by haemagglutination assay, and antigen spe- cific rosette-forming cells were enumerated in the spleen using immunocyto- adherence assay. Both adults and juveniles responded to RRBC, with adult being always the highest responder. Among the adult toads, slight differences between male and female could be observed. However, juvenile toads did not demonstrate sex- associated immune differences. INTRODUCTION Immunocompetence is a complex physiological process that develops during ontogeny and is maintained throughout animals’s life cycle. Anu- ran amphibians are excellent models for immuno- logic studies from the perspectives of both evolu- tionary and developmental biology [1, 2]. In comparison with amniotes, there is an obvious advantage in using the amphibian models for studying the ontogeny of immune responses, since amphibians develop as free-living embryos and larvae thus allowing experimental manipulation from the earliest stages of immune system develop- ment [3, 4]. However, little attention was paid to the immune response of anuran amphibians during sexual maturity and ageing [5, 6]. Here, we examine the importance of major biological vari- ables such as sex and age on the humoral immune response of the toad, Bufo regularis. In addition, Accepted July 4, 1992 Received January 7, 1992 the role of sex hormones played in the immune response of amphibians is discussed. MATERIALS AND METHODS Animals and housing Male and female toads, Bufo regularis (Anura, Bufonidae), were obtained from Abu Rawash area in the vicinity of Cairo (Egypt). Toads were kept in glass aquaria with tap water in a sunny animal room at ambient tempera- ture of 20-27°C during spring and provided with granulated trout feed ad libitum. Cell preparation Individual spleens were ex- cised carefully and gently teased apart with fine forceps over stainless-steel meshes in ice-cold, serum-free Leibovitz (L-15) medium (CIBCO, Grand Island, N.Y., USA). Tissue debris were decanted, and cells were washed twice with ice- cold medium by centrifugation (400 xg, 5 min). In the last wash, pellets were counted and their viability was assessed by the trypan blue exclusion test. Histological procedure After dissection, the spleen was fixed in 10% buffered formalin, dehy- drated in alcohol and embedded in paraffin. Eight yem-thick sections were stained with haematoxylin and eosin. Antigen and immunization Rat erythrocytes (RRBC) were obtained from at least three ani- mals. Pooled heparinized blood was centrifuged 1082 (400g, 5 min) and the cells were washed thrice with phosphate-buffered saline (PBS), pH7.2 before use. Preliminary experiments disclosed that 0.4 ml of 10% RRBC is an optimum dosage which induced maximum antibody (Ab) response when given intraperitoneally (i.p.). Control unimmu- nized toads were injected i.p. with 0.4 ml PBS, pH 7.2. Immunized and control blood samples were allowed to clot for 2 hr at room temperature and then overnight at 4°C. After centrifugation (650 x g, 15 min), individual sera were heat-inactivated at 56°C for 30 min and stored at —20°C until use. Assay of humoral immune response Groups of animals were decapitated at specific intervals after immunization and a single spleen cell suspension was prepared as described previously. Antigen specific rosette-forming cells (RFC) were enumer- ated in the spleen using immunocyto-adherence assay as described by Saad and Ali [7]. On the basis of actual number of cells for each sample, the RFC per 10° spleen cells was computed. Serum Ab titers were determined in standard haemagglutina- tion assay as described previously [7]. Ab titers were expresed as log reciprocal of the highest dilution showing microscopic agglutination. Statistical analysis Student t-test was used to determine levels of significance between control and experimental groups. Diferences were consi- dered to be significant at P values <0.05. RESULTS AND DISCUSSION Field studies have shown that the growth rate of anuran amphibians, as determined from snout- vent (SV) length, can be used as an indicator of growth and sexual maturity [8, 9]. As depicted in Figure 1, the total number of splenic lymphocytes recovered from males and females appear to increase with growth. At SV-length greater than 95mm, the lymphocyte population starts to de- cline, possibly reflecting an increase in animal age and approaching senescence. However, no sex- related differences were observed between male and female toads. comparatively the variations in the kinetics of immune response between two different SV- For simplicity, we analyzed A. H. Saap (Xx 10°) - Yj, %2 = 1 e mM ¢¢ ) [e) Ios jae = > —_ 6 Ww (e) Ow 4 Lu oo = = 2 (30 -55) (60-75) (80-95) (> 95) SNOUT- VENT LENGTH (mm) Fic. 1. Total number of viable splenic lymphocytes obtained from female and male toads, B. regularis of varying snout-vent length. Each column represents the mean number of 10-15 separate animals. Ver- tical bars indicate standards error of the mean. length: 30-55 mm (i.e. juvenile toads) and 80-95 mm (i.e. adult toads). The main issue raised in our study is that the response to RRBC in B. regularis appeared to be highly age-dependent but not sex-dependent. Juvenile animals showed a lower RFC and Ab titers. A considerable number of RFC were detected in male and female toads on the first four days after RRBC injection. Thereaf- ter, a sharp decline occurred in the number of RFC during the subsequent periods. The shape of the curves was otherwise quite similar (Fig. 2A). However, despite the fact that the peak of RFC response to RRBC was somewhat delayed and diminished in adult males in comparison with adult females, yet sex-associated differences at some time point were not significant (Fig. 2B). The kinetics of the primary anti-RRBC Ab response are depicted in Figure 2. It can be seen that, in adult toads, primary response appeared to be more vigorous, and total Ab titers were significantly higher (P<0.001) than those of juvenile toads (Fig. 3A). Also, with adult female toads the peak of primary Ab response to RRBC is somewhat delayed in comparison with adult male toads (Fig. 3B). It is concluded that juvenile toads have the competence to respond to RRBC but their humor- Sex, Age and Immunity of Toads 1083 (x104) NUMBR OF RFC Oo 4 8 12 16 DAYS POST IMMUNIZATION Fic. 2. Kinetics of rosette-forming cells (RFC) response in the spleen of female (@—@) and male (O—©) toads, B. regularis. Juvenile (A) and adult (B) toads were immunized on day O with 0.4 ml 10% RRBC suspension. Each point represents the mean response of 3-5 separate animals and the vertical bars indicate standard error of the mean. =0.05) has been substanti- ated by recent experiments with the equally potent opioid (D-Ala*) deltorphin I [13]. Preincubation of human granulocyte membranes with DALCE (a select detla-opioid antagonist) revealed a binding capacity of this opioid, as well as that of DAMA, distinctly different from that of other opioids. In comparing the modulatory effects of various Comparative Neuroimmunology opioids on certain growth processes, Zagon et al. [14] likewise found Met-enkephalin to be more potent than the rest. This pentapeptide had a strong inhibitory effect on the growth of neuro- blastoma cells in tissue culture. The special recep- tor proposed to be selectively involved was found in abundance in the cerebellum of infants and in a human brain tumor, but not in the cerebellum of normal adults. 2. Immunosuppressive effects In contrast to most of the neuropeptides tested thus far two, derived from pro-opiomelanocortin, i.e., ACTH and MSH, are immunosuppressive. When tested in appropriate concentrations, they have been shown to inactivate immune cells that are either spontaneously active (5-10% in un- treated preparations) or have been experimentally activated by other signal molecules known to have a stimulatory effect [11]. Part of this activity of ACTH seems to be indirect, i.e., by its conversion to MSH under the influence of the special en- dopeptidase (NEP) mentioned earlier. These observations have been made in experiments with vertebrate and invertebrate immune cells. Information on additional immunoregulatory effects brought about by various neuropeptides is primarily based on mammalian studies. Such effects include antibody production, histamine re- lease from mast cells, cytokine activity, vasodila- tion in inflammatory area, proliferation of lympho- cytes, and phagocytotic activity. Cytokines In the search for basic immunoregulatory princi- ples shared by invertebrates and vertebrates, the results of pilot studies on the presence and activity of cytokines in molluscs and echinoderms promise to be as rewarding as those on neuropeptides. The hemolymph of Mytilus was found to contain im- munoactive interleukins (IL-1 and IL-6) as well as tumor necrosis factor (TNF-a). Its immunocytes respond to the respective mammalian cytokines in vitro and in vivo in a manner comparable to that of human granulocytes [15]. Like in mammals, IL-1 appears to participate in the internal regulation of the immune system of Mytilus in part indirectly, i.e., by its stimulatory effect on the formation of 1099 TNF. Moreover, IL-1 and TNF were demon- strated to be present in the hemolymph of Leucophaea (YT. K. Hughes, Jr., Personal com- munication). A molecule with the biochemical and biological characteristics of mammalian interleukin 1 has also been reported to be produced by an echinoderm, Asterias forbesi [16]. Mode of operation of immunoregulatory substances Neuropeptides, cytokines and additional factors (including special enzymes) interact to form an efficient immunoregulatory network. Depending on their concentration and additional determining factors present, certain messenger molecules may be either immunostimulatory or -inhibitory. In addition to its immunosuppressive function, men- tioned earlier, ACTH may stimulate the activity of B-lymphocytes and the phagocytotic capacity of molluscan immunocytes [17]. Its own production, as well as that of endorphins, by immune cells can be induced by another neuropeptide, corticotropin releasing hormone [18]. Conversely, the synthetic opioid DAMA has been shown to stimulate the formation of an IL-1-like molecule in human and Mytilus immune cells [11]. One way in which the degree of cellular activa- tion is kept within appropriate limits is by enzy- matic degradation of some of the signal molecules involved [10]. The observation that the local concentration of Met-enkephalin (and of the re- lated heptapeptide Met-enkephalin-Arg®-Phe’) is downregulated by NEP also applies to the inverte- brate Mytilus [11]. Tests with two major com- pounds resulting from the hydrolysis of the hep- tapeptide showed that they antagonize its effect, presumably by competing for part of the same receptor sites as those used by the heptapeptide. A balancing mechanism is thus provided by the enzymatic generation of antagonistic fragments from the agonist’s molecule. Interference with the normal operation of the immunoregulatory network may occur in various ways and may have important biomedical conse- quences. In schistosomiasis, immunosuppression, attributable to the release by an invertebrate para- site of immunosuppressive signal molecules re- sembling those of the host, appears to influence 1100 the course of the disease [19]. Similarly, the survival of the human immunodeficiency virus in patients seems to be supported by its ability to stimulate the production of ACTH by the host [20]. An involvement of neuropeptides in the re- sponse to stressful conditions has been demon- strated not only in vertebrates, but also in the mollusc Mytilus [21]. The animal’s immune/de- fense system can be alerted, for example, by mechanical interference with the valves of the in-current syphon. This leads to a significant rise in the number of “activated” immunocytes, pre- sumed to be due to the release of an endogenous opioid-like material from the brain. In conclusion, the rewards gained from a broad- ly based comparative approach to the study of neuroimmunological phenomena reach beyond the elucidation of commonalities between vertebrates and invertebrates. They provide information on the evolutionary history of a basic biological phe- nomenon and open new vistas in its exploration. REFERENCES 1 Scharrer, B. (1991) Adv. Neuroimmunol., 1: 1-6. Blalock, J. E. (1989) Physiol. Rev., 69: 1-32. 3 Hughes, T. K., Jr., Smith, E. M., Barnett, J. A., Charles, R., and Stefano, G. B. (1991) Cell Tissue Res., 264: 317-320. 4 Stefano, G. B., Leung, M. K., Zhao, X. and Scharrer, B. (1989) Proc. Natl. Acad. Sci. USA, 86: 626-630. 5 Schon, J. C., Torre-Bueno, J. and Stefano, G. B. (1991) Adv. Neuroimmunol., 1: 252-259. 6 Scharrer, B., Stefano, G. B. and Leung, M. K. B. SCHARRER 10’ 11 12 13 14 15 16 17 18 19 20 21 (1988) Cell. Mol. Neurobiol., 8: 269-284. Leung, M. and Stefano, G. B. (1984) Proc. Natl. Acad. Sci. USA, 81: 955-958. Stefano, G. B., Cadet, P. and Scharrer, B. (1989) Proc. Natl. Acad. Sci. USA, 86: 6307-6311. Shipp, M. A., Stefano, G. B., D’Adamio, L., Switzer, S. N., Howard, F. D., Sinisterra, J., Schar- rer, B. and Reinherz, E. (1990) Nature, 347: 394— 396. Shipp, M. A., Stefano, G. B., Switzer, S. N., Griffin, J. D. and Reinherz, E. L. (1991) Blood, 78: 1834-1841. Stefano, G. B., Shipp, M. A. and Scharrer, B. (1991) J. Neuroimmunol., 31: 97-103. Jankovic, B. D., Levi¢, Z., Nikoli¢, J. and Strojisa- vijevic, N. (1989) Int. J. Neurosci., 48: 155. Stefano, G. B., Melchiorri, P., Negri, L., Hughes, T. K. and Scharrer, B. (1992) Proc. Natl. Acad. Sci. USA (in press). Zagon, I. S. and McLaughlin, P. J., (1989) Brain Res., 480: 16-28. Hughes, T. K., Jr., Smith, E. M., Barnett, J. A., Charles, R. and Stefano, G. B. (1991) Dev. Comp. Immunol., 15: 117-122. Beck, G. and Habicht, G. (1991) Mol. Immunol., 28: 57-584. Ottaviani, E., Petraplia, F., Montagnani, G., Cos- sarizza, A., and Monti, D. (1990) Regul. Peptides, 27: 1-9. Simith, E. M., Morrill, A. C., W. J. Ill, and Blalock, J. E. (1986) Nature, 321: 881-882. Duvaux-Miret, O., Stefano, G. B., Smith, E. M. Dissous, C. and Capron, A. (1992) Proc. Natl. Acad. Sci. USA, 89: 778-781. Smith, E. M., Hashemi, F. and Hughes, T. K., Jr. (1991) FASEB J., 5: 1486 Stefano, G. B., Cadet, P., Dokun, A. and Scharrer, B. (1990) Brain, Behav., Immun., 4: 323-329. ZOOLOGICAL SCIENCE 9: 1101-1111 (1992) REVIEW Rhythmic Processes in Lower Vertebrate Embryogenesis and Their Role for Developmental Control Yuriy N. GORODILOV Biological Institute of St. Petersburgh University, Oranienbaum Chaussee 2, Stary Peterhof, St. Petersburgh 198904, Russia ABSTRACT—A great number of various rhythmic processes (biochemical, biophysical, cytological) during an animal embryogenesis is described up to now. Many of them are revealed in the course of cleavage. The duration of a single cycle of these processes is usually equal to the duration of a single cleavage division (Zo). The somitogenesis is demonstrated the feature of rhythmic process too. We studied thoroughly increase of somites in many fish species, particularly in slow-developing salmon fishes allowing most exact measurements. It was shown that the formation of somites, next one after another, occurred (at constant conditions) strictly rhythmically with the same interval (ts). The last is specific for each species. It turned out that in embryos of the same species at the resemble temperature interval z, was equal to. These data permit to suppose that both embryonic processes are governed by the same intrinsic rhythm, peculiar to all cells of the embryo. This general conclusion is sustained by the data showing that in some salmon hybrids when the reorganization of the rhythms occurred a similar shift in the frequency of the ones for both processes was observed. It is possible that the autonomous changes in the competence of amphibian ectoderm for induction are also governed by the same endogenous rhythm. The endogenous rhythm described could be responsible for switching on/off various specific programmes of development, and also for the coordination of numerous developmental events of embryogenesis. It is supposed that various programmes switched on/off by the same rhythmic signal © 1992 Zoological Society of Japan can be performed independently. INTRODUCTION In adult organism, temporal coordination of various aspects of life processes is usually accom- plished by means of periodic external phenomena like day and night, seasons, etc. Do organisms have any internal time control mechanism during the embryogenesis? This period is remarkable for relative concise and extraordinary eventfull processes of dif- ferentiation and morphogenesis. One is tempted to ask how the activity of enormous numbers of genes is organized, not forgetting that gene ex- pression is only the first step in the multistage Received August 14, 1992 process when the realization of genes in characters occurs. It has been generally believed that the develop- ment of an organism is a chain of consecutive stages: “Development seems to occur in a stepwise fashion, the completion of each step setting the stage for the next” [1]. If this is the case, the duration of a process depends on environmental factors (temperature, oxygen, pH, etc.), that’s say the time is derivative of those ones, and does not play the role of the primary factor of development. The proponents of this point of view believe that the duration of different embryonic processes is usually not stirctly constant exploring it by the imperfections of control of each process. This hypothesis provides the basis for an opinion that 1102 the development of embryos from one batch is not simultaneous, as it is shown by non-simultaneous hatching of embryos. According to Crisp [2] the duration of embryogenesis is one batch estimated at this stage can differ by 20%. But hatching depends on a set of environmental factors and can occur at different stages of development [3]. The point of view that embryogenesis is a pro- cess with relatively variable time limits is beginning to change actually. This is the consequence of more appropriate methods using allowing to esti- mate quantitative aspects of embryonic develop- ment and of a series of direct experiments which are studying the time factor in the process of development. Several rhythmic processes in embryos indicate the existence of some embryonic time control mechanism or “biological clock” [4]. Several of these rhythmic processes showing similarities in particlar aspects are described below. RHYTHMIC PROCESSES DURING CLEAVAGE Cleavage is the earliest period of the embryogenesis, but several rhythmic processes, including cell divisions, are observed already at this stage. These cleavage cell divisions occur synchronously in 10-12 cycles [5—6] with approxi- mately equal time interval between similar phases of mitosis. This interval was designated To [7]. As it has been recently shown, rhythmic changes also occur during this period in biochemical and biophysical parameters. The most interesting ones are periodic changes of rigidity in the superficial layer of the egg cytoplasm [8], flattening-rounding of the egg and surface contraction waves and other changes [9-11] (for review of these and other changes see [12-14]). These processes occur at time intervals, which are approximately equal in duration to the cell cycle duration in cleavage. They occur both in intact and enucleated eggs and in egg fragments not containing nucleus. Besides, it has been shown that the changes of an electric activity of embryonic membranes occur also with a period equal to the cell cycle [15-16]. Y. N. GoropiLov THE TIME CONTROL IN EMBRYOS AND SINGLE EMBRYONIC CELLS AT STAGES FOLLOWING CLEAVAGE How is the time controlled in embryo cells after cell divisions are desynchronized during blastula- tion? The following answer on this question is possible: since the cyclic changes of some indices remain after the suppression of cleavage [12-14] it could be supposed that the same occurs when the cell cycle reorganization and the loss of rhythm by cell divisions takes place in intact embryos, namely rhythm of another processes retain. Some data may be presented, which prove the existence of temporal control in the embryos and their cells following the cleavage. It has been ascertained in series of experiments that the beginning of blastulation and gastrulation in different groups of animals does not depend on the nuclear-cytoplasm ratio or on the number of divisions, but on the duration of development [17- 19]. It has been shown also that the time of realization of different processes in either anlages of embryo programmed in the cells and the sche- dule of their development (with one moment or another) occurs independently from neighbouring anlages. For example, at the fusing of starfish embryos at different stages of development, in spite of the formation of highly permeable contacts between them, every embryo starts the gastrula- tion by own schedule [20]. The attempts to change the time of beginning of gastrulation using prema- ture processing of embryos with the mesoderm- inducing factors have been unsuccessful [21]. The latter authors think that each cell in the early embryo contains a copy of the schedule controlling the correct initiation of developmental events, whether or not that cell has been instructed to participate in each event. Grainger and Gurdon [22] have shown that the loss of ability of mesodermal induction in ectodermal cells does not depend on cellular interactions and cell divisions: isolated cells lost their competence simultaneously with intact embryos. Further it has been demon- strated [23] that in Xenopus the cultured ectoderm undergoes the set of successive changes: first it is competent to form mesoderm, then neural tissue and finally placodal tissue. The authors conclude Rhythmic Processes in Embryogenesis that the competence of ectoderm changes over developmental time, and that no tissues interac- tions are required for these changes in compe- tence. It seems that the time control mechanism keeps at later stages and has certain significance for morphological differentiation of cells already in the separate anlages of organs. So, the ability of chick embryo limb bud cells to differentiate into distal or proximal region of the wing is dependent on the duration of a period which the cells spend in the growth zone [24]. It seems that switching on/off the synthesis of several specific enzymes and chemical substances is also controlled by an intracellular embryo clock. A tissue-specific enzyme acetylcholinesterase (ACh E) is found in muscle cells of Ascidia embryos [25]. If at the stage of 32 blastomeres ascidian eggs were treated with cytochalasin (which inhibits cytokinesis, but does not suppress nuclear divisions) or colkhicine (which inhibits nuclear divisions) in these seemingly undeveloping embryos ACh E activity appeared at the same moment as in intact embryos [12, 25, 27]. Caplan [27] also pointed to the existence of a biological clock in the synthesis the specific progeoglycans in chondrocytes both in embryonic organs and iso- lated cells. SOMITHOGENESIS AS A RHYTHMIC PROCESS The axial zone in vertebrates is subject to seg- mentation the first step of which is separation of parachordal mesodermal layers in rostro-caudal direction and formation of somites. They are formed in pairs, by one on each side of chorda. Their total number ranges from several tens to several hundreds of pairs. As has been shown earlier, the formation of somites is a relatively rhythmic process [28-30]. We studied somitogenesis in several species of teleosts at different temperatures. Teleosts embryos represent a convenient model for live observations since they are transparent. In the laboratory the fertilized eggs of different fish species placed into special apparatus for in- cubation of salmon (and also other) fishes. During 1103 incubation normal conditions of spawn develop- ment (oxygen concentration, flowing water, con- stant temperature (+0.1°C) maintained carefully. Within the optimal temperatures (for salmon eggs 2-10°C) our incubators are a success to provide the survival of 95-98% embryos up to hatching [31, 33]. In the course of somitogenesis we took out the samples of eggs for counting of somite pairs from the same batch from time to time. For the sake of this the chorions were removed microsurgically in a twofold Holtfreter solution, the yolk was drawn off and embryos were carried by pipette on the preparation glass. Then we counted the number of somite pairs in every embryo under microscope (enhance 7X8). The production of 5-10 living embryo preparations and the counting of somites in them usually took no more than 30-50 min. Numbers of somite pairs in each sample under investigation were averaged and plotted on the grafic of increase of somite pair sum in dependence on the incubation duration. In a series of observations of embryos from one batch it was found that at constant temperature the total number of somites is a linear function of incubation time (Fig. 1; see also [3, 31-34]). This tule is retraced from the laying of first somite pair up to 35 pairs in perca [34], 57 pairs in pike [32], 60 pairs in Atlantic salmon [34] and 64-65 pairs in chum (Fig. 1). It is only the last several pairs in each species that are formed slower. The fact that dependence of the somite pair number upon the incubation time has the strictly linear character means that formation of somites, one after another, occurs with the one and same temporal interval. This circumstance allows to calculate a given interval as the time of formation of one somite pair Zs: Ca Ei CSSamasen 1S n2—Ny were 7, and rf are the moments when somite pairs nN, and np respectively started to form. The more is the difference nj—n,, the more exact estimation of ts can be obtained. It is necessary to point out the high developmen- tal synchroneity that is observed in embryos of all species and all batches during the period of somi- 1104 Somite pairs © 2 60 A / 5 1 3 ee 50 FF See eer, chef fs) 20 cs) / 10 | © +/ ®@ | fotidits 16 eooltermeda Ip gone 0 200 400 600 hour Fic. 1. Increase of the number of somites in chum Oncorhynchus keta Walbaum during axial seg- mentation depending on incubation time at various temperature regimes: 11.3°C (1), 6.0°C (2), 3.7°C (3). Time since the beginning of somitogenesis observations (hours) is marked on the horizontal axis, the number of somite pairs is marked on vertical axis. togenesis. The synchroneity keeps at the same level at the beginning and at the termination of somitogenesis period. For example, we reveal the data of two plots in Table 1 received from the Atlantic salmon embryos from Kola river popula- tion at temperature of 0.4°C. The average value of somite pair number of 12.8 for the first sample had been received from studying of 7 embryos: 6 ones among them had 13 pairs each and one had 12 pairs. After 840 hr (35 days) we have counted somites of 6 embryos from another sample of the same batch. They were 56, 57, 57, 57, 57, and 57 (56.8 in average). We observed the similar level of synchroneity in other instances that can be seen from the meanings of statistic deviation from aver- age numbers of somite pairs (Table 1 and 2). Y. N. GoropILov Thus the somitogenesis at constant conditions appears to be the strictly rhythmic process which is controlled with extraordinary high precision. The data of Table 1 and 2 proves that rg is a constant temporal characteristic for all embryos of a given species independently on the time and place of spawn sampling. Deviation of ts in parallel sam- ples is usually less than 1% or in some cases 0.1%, that is the absolutely unusual phenomenon for biological processes. If embryonic development is controlled by any cyclic process, the last one should have the same characteristics in all embryos of a species, but these characteristics can be different in various species, as we see in somitogenesis. For example, in chum and Atlantic salmon at 6.0°C the duration of each cycle of somitogenesis is 368 and 401 min respectively (Table 1 and 2), in pike at 9.9-10.0°C it is approximately twice shorter than in chum (Table 2). It would be said that the embryos of each species have their own time counting off that determine specine-specific “own time” of develop- ment. THE RHYTHMS OF PERIODIC PROCESSES IN TELEOSTS AND AMPHIBIANS AND THEIR INTERCONNECTIONS If we compare the duration of the cycles of cleavage divisions to and that of somitogenesis Ts it is possible to see that at the particular tempera- ture they are similar. We can compare two curves of dependence of these parameters from the in- cubation temperatures received for Atlantic sal- mon (Fig. 2). Similar proportions between 7 and Ts are observed in other species. It can be easily traced from the comparison of to and Ts values in species having different “own time”. As it can be seen from Table 3, species with higher cleavage rate (rainbow trout) also have higher somitogene- sis rate and vice versa (Atlantic salmon). In Fig. 3 the duration of the first five cleavage divisions determined cytologically [6] and that of the formation of any five somites at the same temperature (4.8 °C) is compared. Here it can be clearly seen that the cleavage rhythm that was lost during the subsequent asynchronous cell divisions seems to reappear during somitogenesis after some Rhythmic Processes in Embryogenesis TABLE 1. 1105 Determination of time of somite formation (zs) in Atlantic salmon (Salmo salar L.) embryos from different populations during egg incubation at several constant temperature No. of somites ae tare gg ation ca of Ree No. of embryos pouites a ae CO) vey) euser (hr) onset end pene (min) Kola! 5/XI 840 aoe ee 44.0 1146 a Neva? 28/XII 818 a eee 42.8 1147 Kola 9/XI 580 cian ee 48.2 2 i Neva 6/XII 581 St see 47.9 720 Kola 4/X1 291 are ie 43.7 400 6.0 Neva 17/X1 335 ee mene 49.9 403 Salatza? 14/X1 306 coat’ eres G0 309 Kola 28/X 164 —_ =e 43.6 225 9.7 Neva 11/XI 164 ae Se 43.6 226 Salatza 9/XI 163 ae a 43.8 222 ' Barents sea basin (69° N.L.) ? Baltic sea basin (60° N.L.) 3 Baltic sea basin (57° N.L.) hundreds hours. Incomplete coincidence of to and zg in investi- gated fish species at several temperatures may be stipulated by different factors. Firstly, the dura- tion of different cleavage cycles, evaluated either by cytokinesis and mitotic index, are not always coincided [37]. Secondly, the activity of inner oscillator is registered only through external pro- cesses that can not fully adequately reflect this inner rhythm. It should be noted that in all fish species studied T and zs give the ratio of 1:1, but this ratio is different in common frog Rana temporaria: at 15°C the duration of cleavage cell cycle in frog is 70 min [38] and of the formation of one somite pair is 140 min [39], i.e. the zo/zs ratio is 1:2. Conse- quently, for the similar event in reality in one case (fishes) the time equal to Zo is spent, in other case (amphibia) is twice as much. The existence of divisible ratio seems to be caused by the rhythmic processes under discussion submitted to endogenic rhythm generated in embryo with the main period approximately equal to zo. In dependence on complexity and specificy of the process, it manages in one, two and more periods of endogenic rhythm. It can be supposed that the duration of morphogenesis of other organs and that of the period of postulated endogenic rhythm are relating as divisible numbers. COMPARABLE TRENDS OF CHANGES OF EMBRYONIC RHYTHMS IN INTERSPECIES HYBRIDS We studied the embryonic rhythms in hybrids between the Pacific salmon species: pink 1106 TABLE 2. Y. N. GoropDILov Determination of time of somite formation (zs) in chum (Oncorhynchus keta Walbaum) embryos from different populations and pike (Esox lucius L.) embryos from different accessions during egg incubation at several constant temperatures No. of somites 5 P lati 5 , Rest ce species cen) Date Dalat of | No:ofembyos’ (Sone eae (CS) of accession ONSe! (hr) onset end (min) chum —_Naiba! 11/X-81 336 aneee ae 54.5 370 6.0 chum Zavetinka? _14/X-86 319 ihe a 51.9 369 gum Tae 26/X-86 260 ag a 42.7 364 chum Zavetinka 14/X-86 122 Beste eel 36.3 201 i dima | Then 26/X-86 118 eee neh 34.7 204 pike 1 29/1V 90.5 a ee 48.3 112 ue pike 2 2/V 78.0 o2et2 2 43.3 108 pike 1 2/V 54.8 —— ae 40.6 81 oe pike 2 2/V 57.4 ages sent 43.7 79 Ww N = (Onchorhynchus gorbuscha) and masu or cherry salmon (O. masu). It was found out that masu has a higher rate of somitogenesis than pink at all temperatures (Table 4). The comparison of Ts parent species and hybrids (Table 4) showed that the rate of somitogenesis in hybrids deviated from both parents but was closer to that of male parents. In 1989-1990 we could not obtained masu females, so we compared Tg only in pink and in the hybrid where masu was a male parent. At all four temperature regimes tz in the hybrid was shorter than in the maternal species. If cleavage and somitogenesis rhythms have anything in common, the shift in rhythm of one process in a hybrid should be followed by an adequate shift in the other. That was the reason why we studied cleavage rate at one temperature (10.1°C) in masu, pink and their hybrids (Table 5). In masu cleavage, as well as the somitogenesis, The river on the Western coast of island Sakhalin, entering the Sea of Okhotsk (47.5° N.L.). The river on the eastern coast of island Sakhalin, entering the stream of Tartary (47 N.L.). The river in the north of island Sakhalin, entering the Sea of Okhotsk (52° N.L.). is completed faster than in pink, that additionally confirm the existence of correlation between the rates of these processes in the same species. The early three cleavage divisions (II-IV) in masu eggs take 83 min less than in pink ones, i.e. t) in masu at 27-28 min shorter than in pink. The ro duration determined as the average time of three cleavage cycles: (II-IV) in all variants practically coincided with to, measured as one cycle duration between cytokinesis of the first and the second divisions. The measurement of zt» in hybrids demonstrates pronounced the shift in the development rate of maternal eggs owing to the influence of father genome: in pink eggs the cleavage rate is enhanced when they were fertilized by the masu males. On the contrary, in masu eggs fertilized by the pink males the cleavage rate became slower (Table 5). Consequently the rhythms of cleavage and somi- togenesis in hybrid embryos change by the similar Rhythmic Processes in Embryogenesis 1107 0 2 4 6 8 10 12 14 "C Fic. 2. Duration of cell cycles during cleavage (79) and of the formation of one somite pair during linear somitogenesis (zs) in Atlantic salmon embryos de- pending on the temperature of incubation. Horizon- tal axis, temperature. Vertical axis, duration. way: if one process is accelerated, the other pro- cess 1s accelerated too, and vice versa. It should be noticed that the inheritance of the cleavage rate in hybrids of considered variant is evidently influenced by the male parent. It breaks up the old presentation that the cleavage rate is dependent on the egg cytoplasm only (for review see [13]). Thus the evident similarity in the rhythms of cleavage and somitogenesis, taking place at the different periods of embryogenesis, definitely proves the existence of common biolog- ical time in embryos for these periods, and perhaps for other periods of embryo development as well. AGE-RELEVANT CHANGES IN COMPETENCE Competence is defined as the ability of embryo tissues to respond to an inductive signal that activates particular developmental programmes [41-42]. It has been found that the competence of the embryo tissues changes with time, following a strict programme. Holtfreter [43] observed the changes in the ability of isolated ectoderm of the early gastrula cultivated for different periods in a salt medium to form different rudiments. It is important that these changes occur in a stepwise and autonomous- ly. This was confirmed by recent studies. It was shown that mesodermal competence is lost not only in isolated ectoderm, but in dissoci- ated division-arrested cells on the same time sche- dule as in whole embryos [22]. Loss of neural competence, and the gain and loss lens compe- tence can all occur in isolated ectoderm. The major conclusion is that these changes in compe- tence take place as a result of intrinsic and auto- nomical processes, occuring within the cells of TABLE 3. Comparison of time (min) of cell cleavage ( 7) and somite formation (Ts) at identical temperatures in embryos of three salmon species’ (Salmonidae) Species ane Chum Rainbow’ Temperature To Ts T Ts To Ts (Ke 3.0 657 670 570 654 514 S27) 4.0 558 555 480 538 433 431 5.0 479 470 408 452 367 357 6.0 415 397 350 374 314 300 7.0 364 328 303 317 270 254 8.0 321 284 265 270 234 219 9.0 234 232 +° 16.7 3.9 V3) 16.9 20.9 4.6 ' The value of to and rs are obtained by nonlinear regression [35] from the own data. The value of to for rainbow trout embryos is calculated from the data of Ignatieva [36]. 1108 MI Y. N. GorRoDILov | 100 34 42 50 hr 584 592 600 Fic. 3. 608 616 624 hr Comparison of the duration of cell cycles of the first five cleavage divisions and of the formation of five successive somites (from 19th to 23rd) during linear somitogenesis in Atlantic salmon embryos at constant temperature. Horizontal axis (a, b), time of incubation at 4.8°C from the moment of fertilization. Vertical axis, (a) changes of mitotic index (MI) (data of Gorodilov and Lilp, 1978). TABLE 4. Time of somite formation (zs) in pink and masu embryos (Pacific salmons from genera Oncorhynchus) and their hybrids at identical incubation temperature Value of ts (min) Vener Temperature b ti of incubation 2 pink 2 masu obsenvallon (C, +0.05) pink masu x x masu pink & 1987 125) 148 137 — 149 1988 6.6 327 310 314 — 1989 4.6 481 — 458 — 1989 6.4 344 — 333 — 1989 12.4 150 = 140 — 1990 5.9 379 = 368 _ ectoderm [23]. Besides, in some experiments it was observed that inducing and reacting abilities of developing tissues and cells gain or loss abruptly [23, 44-45]. As Gurdon [41] has emphasized, in the analysis of induction it is important to be able to analyse a rapid response that can be observed in single cells. It may be presumed that the changes in development occur in discrete steps the duration of which is equal or proportional to zo and ts. THE ROLE OF DEVELOPMENTAL CLOCK IN THE REGULATION OF EMBRYOGENESIS The evidence of rhythmic processes occuring at various stages of embryogenesis definitely indi- cates the presence of a time control mechanism in the embryos. The time count probably begins at the moment of fertilization and might continue Rhythmic Processes in Embryogenesis 1109 TABLES. Determination of earlier cleavages duration and to values in embryos of pink, masu and their hybrids at temperature 10.1°C Time. (min)’ from insemination up to Duration of one cell Species Numbecot beginning in 50% embryos cytokinesis of cycle to (min) et the cubis first second forth Fe ee Sony Os aes cleavage cleavage cleavage Tu T1 aa T] TIv pink 113 Se3)3E 2.3) 765 +2.8 1240+3.5 232+1.8 236+1.2 masu 125 SiS)ae2,/ 780+1.9 1199+3.6 205+1.6 208+1.3 2 pink Xx 109 585+4.9 802 +3.8 1245 +2.7 217+1.7 220+1.4 masu 9 masu x 116 573 +4.0 790 +3.5 1250+3.3 NG Pew 226+1.6 pink ff " The deviations of the data was estimated by the Berehns formula [40] until the end of embryogenesis. We consider this mechanism to be responsible for the general sche- dule of development. It keeps all processes within particular time limits. This system can be imagined as a kind of metronom with oscillation period equal or proportional with ration 1:1, 1:2, 1:3, etc. to the duration of different processes, steps and stages of differentiation and morphogenesis, oscillations of which influence all embryonic cells. We suggest that in the egg the signals which are received by all embryonic cells are generated periodically and serve to switch on/off expression of genes (or clusters of genes), programmes and subprogrammes of differentiation and mor- phogenesis in various types of cells and parts of the embryo depending on the serial number of a particular signal. This endogenic pacemaker with regular time intervals triggers the development of cells and multicellular structures in different pre- set directions. We tried to show this scheme of development in Fig.4. The time schedule of the development beginning from fertilization with (zo or Ts if these intervals are equal) as a time unit is shown on the left. The stages of development of some embryonic structures, parts, organs which are started or finished simultaneously, i.e. at one and the same controlling signal of a clock are indicated at a horizontal line. This scheme is based on our data on embryo development in salmon fishes. For example [3, 33] during the first zt intervals the cleavage divisions occur, after 12 rz from the start of development blastula with enveloping and deep cell layers and periblast is formed, after 56 r the axial organs are formed and somitogenesis begins, etc. By 350 +t embryogenesis is completed in general and morphologic development is mostly over. According to this scheme the developmental programmes for various differentiations are re- leased in time by the embryo clock so that each of them is switched on according to the serial number of a clock signal. Under such condition, various programmes can be completed independently. This would explain the fact that some cyclic pro- cesses in embryonic cells continue when cyto- kinesis or nuclear divisions are suppresed [26]. This can be also the cause when isolated cells and tissues of cartilage [27] or of amphibian ectoderm [22, 23] kept their developmental schedule by the same way as if it had been happened in the intact embryos. At the same time some processes are, of course, interdependent due to induction, hormo- nal and other interactions in the embryo. What happens if the formation of a structure is not completed by the time of a new signal? Prob- ably, in the case of vitally importnat organs (heart, brain, kidneys) it is lethal to the organism, but in the case of less important organs the resulting abnormality can be retained and at the new time signal the organ enters the next stage of develop- ment. For example, when deformations were produced in somites by heat shock, further de- velopment of somites continued normally and with 1110 i) MEIN SS PININASS : Y. N. GoropILov Number of cells TAINS : enveloping deep cell 2000 D cell layer layers periblast peri- germ ring outgerm germ 25000 derm cells cells cells 33 Ist ecto- neural noto- meso- somite ento- 56 derm cord chord derm pair derm 18th prone- brain spinal otic somite heart gut phros 3 cord cord vesicles pair anlage anlage anlages 30th differentiation fore- hind- heart gill somite of gut of pro- 85 brain brein tube anlages pair nephros Fic. 4. The scheme of salmon embryos development using z as a time unit (see explanation in the text). normal rate [46, 47]. Thus, the time factor in animal embryogenesis is not only one of the coordinates of the process of organism formation, but it is a primary factor actively monitoring the development with the help of periodically generated endogenic rhythms. The latte provides the mechanism which measures the embryo inner time and thus defines the order of functioning of embryo cells and multicellular struc- tures. In contrast with the formed adult organisms in which timing is based on periodic astronomic pro- cesses and hence is common in all species, we think the time in embryos is counted by a species-specific temperature-dependent internal periodic process (of unknown nature). After completion of embryogenesis the individuals gradually acquire outer universal rhythms which direct their living. ACKNOWLEDGMENTS I would like to thank Ms. Tamara N. Svimonishvili for the help in the experimental work, Prof. G. V. Lopashov and Prof. L. N. Seravin for useful theoretic discussions and critically reading the manuscript. REFERENCES 1 Smith, M. J. (1968) In “Towards a theoretical biology, 1, Phenomena”. Ed. by C. H. Waddington, Edinborough, pp. 120-124. Crisp, D. T. (1981) Freshwater Biol., 11: 361-368. Gorodilov, Y. N. (1983) Proc. State Res. Inst. of Lake and River Fishing (Russ.), 200: 107-126. 4 Goodwin, B. S. (1976) Analytical physiology of the cells and developing organisms. 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(1978) J. Embryol. Exp. Morphol., 45: 283-294. Cooke, J. and Elsdale, T. (1980) J. Embryol. Exp. Morphol., 58: 107-118. J. Theor. (1964) Biometrical Methods. j 2 moaned a) veneer) onnitivad 2 eee ~evnteate OC r& (TORTy A ow fisted heh 4 teukeprerte : nto t et ime nn ’ (4t0i) 2 init’ bas. eb 5 asl & vCr ae 1a AP vr Abii wd) (init Mamie Di 4 ie bet tees Mall job—tok tT ACL GR pae ee ee poten. (LAO Eo Oil ile 4 bn . PP . ia os ‘ Pas ‘ “a= Ay ant PLE rie ' i 1 ” ag nee oan? 7. siz) OA & ud { i) oi iwi olhivaval r F jot > (h0C1) 4 Sie chitie if 3 e ? ho iol Uae + Pe ote all Lyi rey ¥ heb * ky : at ZOOLOGICAL SCIENCE 9: 1113-1126 (1992) © 1992 Zoological Society of Japan REVIEW What is a Classification? A Case Study in Insect Systematics: Potential Confusion before Order Kunio Suzuki! and Davip G. FurtH? "Department of Biology, College of Liberal Arts, Toyama University, Gofuku, Toyama City, 930 Japan; Museum of Comparative Zoology, Harvard University Cambridge, Massachusetts 02138, U.S.A. ABSTRACT—A case study is presented of the Leaf Beetle genus Orthaltica (Coleoptera: Chrysomeli- dae) as an example of a group involved in a variety of taxonomic problems and controversies demonstrating many of the technical and philosophical complexities of systematics that often make the derivation of a classification difficult and confusing. The history of Orthaltica is reviewed providing discussion and illustration of the concepts of: multiple synonymy and homonymy; the genus and subgenus; lumping and splitting; and a systematic character, especially morphological, and its application in formulating higher classifications. Based on morphological characters Orthaltica provides a good vehicle for considering the classification of the two largest subfamilies of the Leaf Beetles, the Galerucinae and the Alticinae (approximately 500 genera each). The primary difference between the Galerucinae and the Alticinae is the absence or presence, respectively, of the jumping organ (metafemoral spring), although some secondary differences exist in varying degrees. These two chrysomelid subfamilies are so diverse and relatively unstudied systematically that deriving a higher classification or speculating on their phylogeny, including their derivation from one (monophyletic) or many (polyphyletic) lineages, is premature and needs a considerable amount of study of many characters. INTRODUCTION One purpose of systematics is to recognize the existence of all organisms on the earth, to describe and order them, and to establish a good classifica- tion system. A well-organized classification system may be useful in deepening our understanding of the organic world. If we have no reliable system established by systematists, we cannot find any order in the biologically diverse organic world. This can be easily understood if we go to a zoo, a botanical garden or a natural history museum. If animals and plants are displayed without any con- sideration of their inter-relationships, we know only that these various organisms exist. The inter-relationship among organisms is the key to Received August 19, 1992 organizing them. What is the “inter-relationship” among organisms? The authors use this term in the sense of evolutionary relationship; that is, they assume that all organisms on the earth have evolu- tionary relationships, and that they are descended from the first organism about 4 billion years ago. One purpose of systematics is to organize all organisms by such evolutionary relationships, and even though exact evolutionary history may be impossible to reconstruct completely, we can use systematic methods to make limited approxima- tions. The authors do not adopt the viewpoint of numerical taxonomy (phenetics) which generally ignores historical perspectives. The authors also do not completely follow cladistics, a type of systematics which often forces taxonomists to make premature choices based on a restricted methodology oriented toward producing phy- 1114 logenetic (lineage) results. In this context the authors follow more the methodology of ‘evolu- tionary systematics’ (sensu Mayr, 1963 [1]). Evo- lutionary relationships may be recognized based on close analysis of systematic characters in a broad sense. Much of this paper will address the question “what is a systematic character?” and its application. What is a phylogenetic classification? Most of the problems discussed here are interconnected and relate to this question. To answer this ques- tion is not a simple matter, as with a dictionary difinition, but rather a complex multi-faceted issue with arguably different viewpoints concerning both present and past relationships of organisms. We attempt here to recognize the various aspects and problems in answering such a question. The term “phylogenetic classification” in this question refers to a classification of a particular group which will reflect evolutionary history. Readers should note that the authors use the term “phylogenetic” in- stead of “natural” classification. This is a con- scious usage in order to avoid confusion because the term “natural classification” has also been used by numerical taxonomists in a restricted sense to mean a clustering of groups based on overall similarities of many characters. The authors’ usage of this term (natural classification) actually agrees more with its usage by cladists. One goal of systematics, and the taxonomists who practice it, is to establish a better phylogenetic classification system which will be acceptable to more system- atists and will result in a more consistant under- standing and usage of scientific names for taxa. One of the intentions of this paper is to demons- trate to other non-systematist biologists and other scientists some of the issues, problems and logic present in the field of taxonomy/systematics. Although the examples used from insect (beetle = Coleoptera) systematics are at times somewhat complicated and some problems are left unre- solved, nevertheless, they illustrate some of the fundamental complexities of biological system- atics. In this paper the authors discuss some of their opinions about phylogenetic systematic proc- edure (theory and methods) based on recent stu- dies. As with most basic systematic research, the authors are studying a specific group, the Leaf K. SUZUKI AND D. G. FurtH Beetles (Chrysomelidae) from a particular view- point. In systematic studies there are different problematic situations with theoretical and/or methodological viewpoints that differ from syste- matist to systematist and from taxon to taxon. This is occasionally an important obstacle to com- munication among systematists. Using these spe- cific studies, the authors attempt here to present their awareness of the issues in systematics. The authors would also like to help correct or improve the present unfortunate situation which underesti- mates the role and importance of systematic study as compared with other experimental biological disciplines. Such an attitude is based on scientific ignorance and/or a glaring misunderstanding or prejudice about the practice and significance of systematics. Unfortunately there are even some systematists who do not understand the signi- ficance of systematics. This is all the more reason for a clear and consistent explanation of biological systematics and its role in basic science. In order to stimulate further development of biosystema- tics, it is essential to cooperate with other biologic- al disciplines. This has been occasionally pointed out but has not been practiced effectively. One possible reason for this scarcity of interdisciplinary cooperation may be the lack of mutual awareness of the issues existing between systematics and those other disciplines. Systematists are at least partially to blame for this because they have not often stated their awareness of such interdisciplin- ary issues or the significance of systematics as a basic empirical science, and as a stepping stone to other modern aspects of biological sciences. The authors have recently conducted research together concerning several topics in insect sys- tematics. There are even differences in theoretical and/or methodological approaches between the authors even though they both have studied the same insect group for many years. However, it has become increasingly apparent through discussion and mutual criticism that both authors have a common recognition of the above-mentioned problems of cooperative efforts in systematics. Through this mutual and reciprocal interaction the authors have managed to complete many systema- tic studies within a relatively brief period that could not have been accomplished in the same What Is a Classification? broad-spectrumed way, if each of us had studied the same topics individually. The authors hope that such cooperative studies will serve as an example to systematists studying different groups or using different methodologies, and to biologists of different disciplines who might mutually augu- ment their individual research through cooperative discussion and studies. In this paper, we are going to focus on a problem which we call the “GA Problem”. G and A refer to the chrysomelid subfamilies Galerucinae and Alticinae, respectively. The core of the “GA Problem” may be summarized as follows: “How can we classify the Galerucinae and Alticinae?” or more specifically “How should the Galerucinae and Alticinae be defined?” —this form of question may show that this problem is one of group/taxon recognition or establishment. Most systematists have accepted the view that the family Chrysomeli- dae should be classified into 19 subfamilies; Gale- rucinae and Alticinae are the largest. Various opinions have been proposed concerning the sys- tematic position or phylogenetic relationships of these two groups (e.g. Crowson, 1955, 1982 [2, 3]; Lawrence & Britton, 1991 [4]) but it is generally accepted that they are closely related to each other. However, within these two groups there are several taxa (mostly genera) whose true systematic position (even subfamily placement) has not been decided. So that, the above question may be better expressed as: “How should the Galerucinae and Alticinae be distinguished?”. There is a variety of possible ways to express this problem depending on differences in the awareness of the issues and/or which points are emphasized. Var- ious practical aspects derived from this GA Prob- lem may be included in this type of question. The authors will introduce these aspects using exam- ples wherever possible because they involve many typical problems encountered in systematic study of other organisms. Systematics has, of course, an essential purpose to deepen our recognition of a particular toxon. However, if we are satisfied with only this aspect of systematics one might ask “why is systematics needed?”, especially when such a huge number of organisms exists on the earth. In other words, if we say that a purpose of systematics is to know all 1115 organisms, is this not an endless task, how can this be sensibly planned and how can this survey of organisms be justified? Such questions are often used as arguments against systematists; Sibatani (1960) [5] asked such an essential question to systematists in Japan. Some systematists may reply that the people who laugh at such a biotic inventory do not know the world and they cannot understand the significance of organic diversity on our earth. Yet systematists are convinced that giving meaning to organic diversity on the earth from various aspects should be one of the most important priorities in biology. Even though systematics has its own issues and perspectives, it also gives a kind of a bird’s eye view to other biological disciplines. The bird’s eye view which systematists offer resembles a topo- graphical map. The precision (scale) of the map depends on the area (organic group). If we do not have any map, we cannot walk even one step. At the same time, even if we have a map we may lose our way because of disagreement between the map and our present spot. We must make an effort to improve the map in order to reflect our present position; this analogy resembles the relationship between systematics and other biological disci- plines. The opinion that systematics is a synthetic discipline in biology may have originated from the viewpoint that both systematics and other biologi- cal disciplines should be mutualistic. survey of our present position will be necessary in order to improve the map to the point of maximum usefulness. The opinion that we do not need so many kinds of organisms for biological research shows gross misunderstanding of science. Biolog- ical inventory studies have limitations and, be- cause of rapid destruction of natural ecosystems by humans in recent years and the resulting extinction of many organisms, there is an intense crisis and urgency that systematists realize more than any scientists. Systematists are the only ones who can record the great biological diversity that exists and that which is being lost forever. Therefore, system- atists should play a leading role in alerting others (scientists, politicians, etc.) to the current crists and in devising plans for conservation of this organic diversity. Extensive 1116 GA PROBLEM Orthaltica: History and Confusion In order to consider “GA Problem” (Galeruci- nae— Alticinae Problem) the authors have chosen the genus Orthaltica as a good example to demon- strate many of the problems of systematics, begin- ning with a brief historical review. The authors would like to give an explanation of the present problem based on Scherer (1974) [6] and using the following table (list of synonymous names) from his article: Genus Orthaltica Crotch, 1873 Orthaltica Crotch, 1873:69 (type-species: Crioceris copalina Fabricius; N. America); Horn, 1889 :236, 247; Blatchley, 1910: 1206, 1215; Heikertinger, 1924-25 (1925): 65; Arnett, 1963 :914, 938. Leptotrix Horn, 1889 : 236, 249 (type-species: L. recticollis LeConte; N. America (nec Menge, 1868; Araneae) see Leptotrichaltica. Livolia Jacoby, 1903:15 (type-species: L. sulci- collis Jac.; Africa); Scherer, 1961 :268: 1969: 10, 19, 118, 242; 1971: 1-37. New Synonym. Leptotrichaltica Heikertinger, 1924-25 (1925): 68 for Leptotrix Horn. New Synonym. Micrepitrix Laboissiére, 1933 : 205 (type-species: M. coomani Lab.; Tonkin); Gressitt, 1955 :35 (Alticinae); Gressitt & Kimoto, 1963: 404, 575; Samuelson, 1965 :215; Scherer, 1969 : 10, 19, 98; 1971: 10 (as synon.) Serraticollis B. E. White, 1942 : 17 (type-species: S. rhois White; Calif.); Arnett, 1963 :938 (as synon. ) The essence of the history of systematic treat- ment of Orthaltica is compiled in the above table. A trained systematist can easily understand not only the formal meaning but also the fact that beyond the nomenclature this group may contain many systematically difficult problems. The main information that should be deduced from this table may be arranged as follows (supplemental com- ments in brackets). This genus was established by Crotch (1873) [7] based on Crioceris copalina described by Fabricius K. SUZUKI AND D. G. FurtH from North America. [The genus Crioceris cur- rently belongs to the subfamily Criocerinae. Crotch considered O. copalina as a member of a previously unknown genus currently in the Altici- nae. However, even by Crotch’s time higher classification of the family Chrysomelidae had been only gradually and not well established. Chapuis (1874) [8] was the first to propose a higher classification system; this was the basis of our modern system. However, not until Jacoby (1908) [9] was an actual subfamily system established. So that, we should understand that Crotch regarded the species copalina as a member of a close relative of many genera which are included in the Alticinae today]. After Crotch, Horn (1889) [10], Blatchley (1910) [11], Heikertinger (1925) [12], and Arnett (1963) [13] followed his treatment. [This also means that all of them recognized the genus Orthaltica as valid]. Until now the following have been considered synonyms of Orthaltica: Leptotrix: This genus was established by Horn (1889) [10] based on the species recticollis de- scribed by LeConte from North America. Howey- er, this genus name (Leptotrix) was a homonym, in other words, the name was already preoccupied by Menge (1868) [14] as a name of a spider genus (see also Leptotrichaltica). [Here, some readers may think that Horn should have recognized the inde- pendence of Orthaltica from Leptotrix within his 1889 article [10]. This suggests the following two possibilities: (1) Horn positively recognized the independence of both genera or (2) he established Leptotrix because he did not recognize the identity of Orthaltica. In order to judge which possibility is probable, we have to examine Horn’s 1889 paper [10]. In this paper, Horn pointed out that Orthalti- ca was similar to other genera like Crepidodera and Pseudoepitrix. He then described Orthaltica melina as a new species and, following the descrip- tion of O. melina, he established Leptotrix and pointed out that this new genus resembles Orthalti- ca and Pseudoepitrix but did not belong to any genus of the tribe Crepidoderides. Subdivision of Alticinae into tribes has been partially attempted by Leng (1920) [15] and Bechyné and Bechyné (1975) [16] but remains very confusing and invalid. According to the above facts, the authors judge What Is a Classification? that Horn took the first possibility mentioned above. Moreover, we should pay attention to the fact that in Horn’s era a genus was likely to have been defined more typologically than in recent times. In other words, a genus was established based on a comparison with type-species and, in general, it was likely to be more subdivided. Of course, this may vary from worker to worker and from group to group]. Livolia: This genus was established by Jacoby (1903) [17] based on a new species sulcicollis from Africa. Scherer (1961, 1969, 1971) [18, 19, 20] followed this but synonymized it with Orthaltica in his 1974 article [6]. Leptotrichaltica: Heikertinger (1925) [12] gave a new name to Leptotrix Horn because the exis- tence of a homonym (mentioned above); this genus was also synonymized with Orthaltica by Scherer (1974) [6]. Micrepitrix: This genus was established by Laboissiére (1933) [21] based on the type-species M. coomani from Tonkin. [Laboissiére described this genus as a member of the Galerucinae but Gressitt (1955) [22] transferred it to the Alticinae. After that, Gressitt & Kimoto (1963) [23], Samuel- son (1965) [24], and Scherer (1969) [19] followed this, but Scherer synonymized it with Orthaltica in his 1974 paper [6]]. Serraticollis: This genus was established by White (1942) [25] who described the type species (S. rhois) from California. After that, Arnett (1963) [13] synonymized this genus with Orthalti- ca. |Though his opinion cannot be determined from this synonym list, this was suggested to Arnett by J. A. Wilcox, an authority of the Gale- rucinae]. The items mentioned above are the things which are summarized in Scherer’s (1974) [6] synonym list. One should examine further each of the previous worker’s opinions; this is an impor- tant routine for many systematists. The authors will now examine some of the details of the confusion in the systematic treatment of Orthaltica. Scherer (1974) [6] synonymized five genera with Orthaltica. In considering whether his treatments are reasonable or not one has to ex- amine all the literature involved and often to examine original type-specimens, depending on the situation. Seeno and Wilcox (1982) [26] listed 1117 Leptothrix Heikertinger et Csiki (1940) [27] as a synonym of Orthaltica; however, such a differece in spelling between Leptotrix and Leptothrix is not significant. Because Leptotrix is a homonym of a spider genus, the species belonging to it should be automatically transferred to Leptotrichaltica estab- lished by Heikertinger (1925) [12] as a new name for Leptotrix. This is merely follows proper sys- tematic treatment in accordance with the interna- tional rules of zoological nomenclature. We have to consider the other four genera Livolia, Micr- epitrix, Leptotrichaltica and Serraticollis, the first three of which were synonymized with Orthaltica by Scherer (1974) [6]. 1. Livolia: When this genus was established, Jacoby (1902) [28] mentioned that this genus may be transitional between the Alticinae and Galerucinae. In his revisional study of this genus, Scherer (1971) [20] treated the following 20 species, which included seven known and 13 new species. Also in this 1971 paper Scherer synonymized Micrepitrix with Livo- lia species ([20]; see also Scherer, 1981 [29]). In the list below “nov. comb.” (new combination) means a new change of genus assignmnet of the species in question, “nov. spec.” (new species) a new species is described and in brackets is the original locality (type-locality). vestita (Baly, 1877) nov. comb. [W. Australia] sulcicollis Jacoby, 1903 [Mashonaland: Salis- bury] “africana nov. spec. [W. Africa] coomani (Laboissiére, 1933) nov. comb. [China: Tonkin; Hainan I.] carolina (Chuaj6, 1943) nov. comb. [Yap; Palau] minuta (Jacoby, 1887) [Ceylon] *minor nov. spec. [Singapore] *fulva nov. spec. [W. Sarawak] *nigripennis nov. spec. [Singapore] *sarawakensis nov. spec. [W. Sarawak] “parva nov. spec. [Singapore] *malayaensis nov. spec. [Malaya] *perakensis nov. spec. [Malaya] *assamensis nov. spec. [India: Assam] *serraticollis nov. spec. [Burma] [=impres- siceps: Scherer, 1974] *tenasserimensis nov. spec. [S. Burma] 1118 *ceylonensis nov. spec. [Ceylon] minutiuscula (Csiki, 1940) [Sumatra] *Jaticollis nov. spec. [Singapore] laboissierei (Chen, 1935) nov. comb. [E. China: Kiangsi] Of these 20 species, we exclude here the 13 species with asterisks described by Scherer in this paper [20] from our present discussion because they were originally described as members of Livolia; we will examine the seven remaining species. Naturally in such cases systematists have to examine all the original literature involved, even though in a practical sense this is sometimes quite difficult for taxonomists who do not have easy access to good libraries. vestita: This species was described by Baly (1877) [30] as a member of Crepidodera. This means that in Scherer’s opinion at least some species which belong to Livolia have been mixed in Crepidodera (see also Scherer, 1982 [29]). sulcicollis: This is the type-species of Livolia. Thus, as far as the genus continues to exist this species plays an important representative role. coomani: This species was described by Laboi- ssiére (1933) [21] as a member of Micrepitrix. If we regard this species as a member of Livolia, this means that at least some species of Livolia have been mixed in Micrepitrix. If we agree with Scherer’s (1971) [20] opinion that Micrepitrix is a synonym of Livolia, this problem is eliminated immediately; this is discussed below. carolina: This species was described by Chuj6 (1943) [31] as a member of Epithrix (= Epitrix). If we regard this species as a member of Livolia, this means that some species have been mixed in Epitrix. Gressitt (1955) [22] treated this species as a member of Micrepitrix. Therefore, as in the case of coomani, at least some species which should belong to Livolia have been mixed in Micrepitrix. Here, we experience the following two derived problems: (1) what kind of systematic relationship is there between Epitrix and Livolia and (2) how should we treat the systematic position of carolina. For the first problem, one possible answer may be that if Micrepitrix should be regarded as a synonym of Livolia, we have to consider systematic treat- ment of Epitrix separately; that is, because Epitrix K. SuzuKI AND D. G. FurtH is a very big group, even if there are systematic changes for some species treated as members of this genus, all other species are not necessarily transferred to Livolia. For the second problem, as with other species which have been treated as members of Micrepitrix, the true systematic posi- tion of this species cannot be determined automati- cally. minuta: This species was described by Jacoby (1887) [32] as a member of Crepidodera and, thus, the same situation as in vestita can be pointed out. Scherer (1969) [19] already pointed out that this species should belong to Livolia. minutiuscula: Concerning systematic treatment of this species, there is a problematic history. The author of this species is Csiki (1940) [33] in Heiker- tinger and Csiki (1940) [27]. But, the taxon corresponding to this species was first described by Jacoby (1895) [34] under the name of minuta as a member of Crepidodera. But, as above, Jacoby (1887) [32] already used the name minuta as a member of Crepidodera. That is, Jacoby produced a new homonym to his other species in the same genus! This is a very unusual example of a homonym. By discovering this fact, Csiki (1940) [33] became the author of this species without describing even one line about this species. laboissierei: This species was described by Chen (1935) [35] as a member of Micrepitrix. This is the same situation as in the above cases of coomani and carolina. Scherer (1974) [6] changed the name of his L. serraticollis to impressiceps because of homonymy; that is, the name was preoccupied by Samuelson (1965) [24] as a name for one of three species of Micrepitrix from New Guinea. Such homonymy produces a quite confusing history that seems to be mysterious for workers of other fields of biology. The authors summarize above items as follows. In analyzing Scherer’s (1971) [21] opinion, the systematic relationships between three genera (Crepidodera, Epitrix and Micrepitrix) and Livolia are: (1) in the first two genera, only some species belonging to Livolia had been mixed; so that, their systematic assignment to a genus needed to be changed and; (2) Micrepitrix should be regarded as a synonym of Livolia; so that, all species described as members of Micrepitrix should be automatically What Is a Classification? transferred to Livolia. After all these changes, Scherer (1974, 1982b) [6, 29] ultimately regarded Livolia as a synonym of Orthaltica. 2. Leptotrichaltica: Concerning the systematic treatment of this genus, we completely agree with Scherer’s opinion that it should be regarded as a synonym of Orthal- tica. That is, Leptotrix (=Leptotrichaltica) recti- collis described by Horn (1889) [10] can be treated as a member of Orthaltica according to the original description. 3. Micrepitrix: Sherer (1971) [20] synonymized this genus with Livolia. Samuelson (1973) [36] followed his opin- ion. Because of several reasons mentioned below, the authors would like to treat Livolia and Micre- pitrix as independent genera, not as synonyms. 4. Serraticollis : This genus was treated by Arnett (1963) [13] (following Wilcox’s opinin) as a synonym of Orthaltica. The authors agree that White’s (1942) [25] original description of this genus can be com- pletely adopted into Orthaltica. Genus vs. Subgenus/Lumping vs. Splitting. Scherer (1971, 1974, 1982, 1988) [20, 6, 29, 37] synonymized Livolia and Micrepitrix with Orthalti- ca. The first two genera have a symmetrical aedeagus, whereas Orthaltica as an asymmetrical one, which is extremely rare in the Alticinae. Of course, Scherer recognized this fact and main- tained that such differences should be regarded as the characters of a subgenus level. The basis of his assertion seems to be his special concept of a genus. In his 1973 paper [38], Scherer pointed out “how reliance on topological criteria and over- ranking of lesser units can destroy the phylogenetic image of a genus.” He also regretted that many systematists are likely to treat closely related spe- cies groups as independent genera based only on morphological characters. According to Scherer, Livolia and Micrepitrix should not be separated from one another (the latter, which was estab- lished more recently than the former, is regarded as a junior synonym) and neither should be distin- guished from Orthaltica at genus level. Thus, this Livolia-Micrepitrix complex is regarded as a synonym of Orthaltica and should be distinguished 1119 from Orthaltica in the strict sense (Orthaltica (s. str.)) only at the subgeneric level as Orthaltica (Livolia). In the case of Orthaltica, if one makes a classification based on Scherer’s broadened con- cept, he would be considered a “lumper” (i.e. joining groups together); in contrast to this, many systematists are “splitters” (i.e., dividing groups apart). Occasionally the distinction between the so-called “lumper” and “splitter” has been discus- sed in systematics; it is, of course, a somewhat relative and subjective matter. The authors would like to point out the follow- ing two problems. First, Scherer (1973, 1982, 1988) [38, 29, 37] suggests that taxonomists use a broadened interpretation of the genus by “lump- ing” subunits which share common ecology and/or historic-zoogeography into subgenera rather than “splitting” them into several genera. Although Scherer agrees that the genus should include spe- cies of common ancestry (monophyletic), he con- siders that interpretation of the “gap” separating higher categories is a critical aspect of defining generic level ranks (i.e. genera or subgenera). However, even as Scherer partially admits, the information (e.g. ecology, historic-zoogeography, etc.) mecessary to define a genus is not always available for many groups; on the contrary, it is often quite limited. Although the authors agree in principle with Scherer’s (1973) [38] definition of the genus concept, we suggest that some of his examples of genera (especially Orthaltica) are too broadly defined (“lumped”) and may be as difficult to interpret evolutionarily as with genera which he claims are over-split. Secondly, several items that may lead to diffe- rent systematic treatment should be considered. Synonymyzing of genus A with genus B will result in automatic transfer of the species members of A to B. This also means automatic extension of category A. In the present case, if Livolia and Micrepitrix are synonymized with Orthaltica, this automatically extends Orthaltica into having a very wide geographical range. On the contrary, if we regard these three as independent genera, we understand their distribution as follows: Orthaltica from North America, Livolia from Africa, and Micrepitrix from Southwest Asia to Pacific. Of course, comprehension of such facts are important 1120 for us and although some may consider that such a divided treatment of ranking in a taxon is essential- ly not important, this demonstrates a gross mis- understanding. For example, when we consider phylogenetic relationships at the genus level, from the lumper’s viewpoint (Scherer, 1974, 1988) [6, 37] the differences among these three groups (Orthaltica, Livolia, Micrepitrix) may be regarded as intra-generic variation, whereas for the splitters they may be viewed at the inter-generic level. This may be deduced by an ordinary comparative method. Suzuki (1984, 1989a) [39, 40] has pointed out that the ranking of any taxon must strongly influence the phylogenetic consideration of that group. Lumping and splitting have both good and bad points. Actually, one should judge individual cases of taxon ranking according to the differences in the effect on the understanding of the groups in question. Our conclusion about this problem is that in a group for which there is a wealth of information, Scherer’s (1973) [38] broadened treatment of the genus (using subgenera) may help the evolutionary understanding; however, in cases where there is not much information about the group in question, Scherer’s lumping treatment can have a negative effect on the evolutionary understanding. Unlike some systematists the au- thors do not think that splitting is an improper systematic practice. Rather we recognize the na- ture of each of the groups which we study and prefer to enhance the knowledge of each group by finding and analyzing differences between/among them. Essential characteristics and relationships of a given group can be obscured by the lumping treatment. Concerning the Livolia-Micrepitrix-Orthaltica problem discussed in this paper, unfortunately there is currently only limited information about them. A lumping treatment of these genera into Orthaltica would indicate their general similarity but the individual characteristics and differences of each of three groups would be hidden. Therefore, at this relatively initial stage, based on differences in the aedeagus and geography and in the interest of clarify of these entities for future evolutionary analysis, the authors prefer to treat Livolia, Micr- epitix, and Orthaltica as three independent genera. K. Suzuxi AND D. G. FurtH In the phylogenetic sense of cladistics, it is impor- tant to establish that each genus has evolved from a single ancestral lineage or clade, i.e. monophyle- tic. The above examination of the problematic and complex situation of the genus Orthaltica has gra- dually unfolded to the authors and based on it we now begin to approach the core of GA Problem. Concerning the higher systematic position of Orthaltica, there is a difference of opinion among some chrysomelid systematists, i.e. even its sub- family placement (Furth, 1985, 1988 [41, 42]; Reid, 1990, 1992 [43, 44]). Recognition of Higher Taxa in Chrysomeloidea. Mainly based on extensive comparative morpho- logical study of the internal reproductive systems of both sexes, Suzuki (1988) [45] pointed out that the family Chrysomelidae cannot be regarded as a monophyletic group in relation to the two other familes Cerambycidae and Bruchidae of the super- family Chrysomeloidea. Suzuki's first essential question about chrysomeloid phylogeny concerned the fact that, surprisingly, the Chrysomelidae (Leaf Beetles) cannot always be clearly distin- guished from the Cerambycidae (Long-horned Beetles) and Bruchidae (Seed Beetles); he natural- ly assumed that there must be greater differences among higher categories than among lower ones. The classification at subfamilial level in these three families has been considerably well established and consistent, although there are still differing opin- ions. There are, however, relatively few obvious differences that separate these three familes from each other. The assignment of a given species to any of the three families is automatically determined by the fact that the species belongs to a particular sub- family within them; a kind of inverted funnel system. Suzuki (1984, 1989a) [39, 40] pointed out that this type of problem is one of essential weak points in the Linnaean hierarchical classification system and its concomitant hierarchic system of category names. In most entomology textbooks, one can find some characteristics to separate these three families, and actually one can classify most specimens/taxa into families, but there are several exceptions. In a recent popular book, White (1983) [46] mentioned that “(the family Chry- What Is a Classification? somelidae) cannot be readily characterized; family members are very diverse and have no distinctive characters in common.” This is a very basic statement, but if true, how should we answer the question “what is a Leaf Beetle or a Long-horned Beetle or a Seed Beetle?” Actually beetle workers have not really answered this question. For the phylogenetic relationships in this superfamily, the first author will give a general interpretation else- where (Suzuki, in press [47]). The present GA Problem is a smaller version of the same kind of problems mentioned above; that is, GA Problem involves the questions “what is Galerucinae?” or “what is Alticinae?” or “how can one separate the Galerucinae and Alticinae from each other?”. There are probably similar situations in many groups; however, in this case our current system of recognition for these two subfamily groups is rel- atively advanced. Relationship of the Galerucinae and Alticinae, and the MS Organ. Howe have we separated the Galerucinae and Alticinae so far? Actually there is a diagnostic character, as indicated by the name “flea beetle,” the members of the Alticinae can jump using a well developed hind leg. They have a special jumping organ, which has been known as Maulik’s organ or metafemoral spring (MS) (Furth, 1982) [48] in their hind femora (Fig. 1, [49]). In fact, the scientific name of the type genus (Altica Fabricius) is derived from the Greek ‘haltikos’ which means good at jumping (Furth, 1988) [42]. Most chry- somelid systematists have regarded the presence of this MS organ as a diagnostic character by which they can separate the Alticinae from the Galeruci- nae. However, there are several potential excep- tions to which this rule cannot be applied. The genus Orthaltica represents just such an exception- al group, because it lacks the MS organ in the hind femora. The authors confirmed this fact in the three North American species. If we follow the tule strictly, the genus Orthaltica should belong to the Galerucinae. Based on this fact Scherer (1974) [6] and J. A. Wilcox (personal communication) maintained that Orthaltica was an exceptional alti- cine genus lacking MS organ. But Furth opposes this point and has asserted that the genus belongs 1121 Fic. 1. The MS organ (Metafemoral Spring) of Nonar- thra postfasciatum (Fairmaire, 1889) (Coleoptera, Chrysomelidae, Alticinae). (a) Posterior view of loft leg including the MS organ. (b) Enlarged MS organ. Scale: 0.5mm. Taken from Suzuki and Furth (1990b) [49]. to the Galerucinae (Furth, 1985, 1988, 1989) [41, 42, 50]. Before proceeding, the authors would like to point out the following facts: 1. Besides Orthaltica, both Livolia and Micr- epitrix also lack the MS organ (Furth & Suzuki, in preparation). 2. The genus Eubaptus, the only member of the subfamily Eubaptinae (Bruchidae), has the MS organ (Teran, 1964, 1967) [51, 52]; the authors have also confirmed this fact. The authors ex- amined many other bruchids but could not find this organ in any other groups (Suzuki & Furth, 1990b [49]). In addition, the authors have examined some Sagrinae species (Chrysomelidae), which have been occasionally considered as close rel- atives of the Bruchidae, and the genus Rhaebus (Bruchidae, Rhaebinae), which has been treated as a group of the Sagrinae, but has been transfer- red to the Bruchidae (Crowson, 1946 [53]; King- solver & Pfaffenberger, 1980 [54]; Borowiec, 1987 [55]); the authors could not find the MS organ in either group. 3. There are species of the Rhynchaeninae (superfamily Curculionoidea, family Curculioni- dae) that have the MS organ. Maulik (1929) [56] 1122 mentioned that the metafemoral organ existed in one species of the genus Rhynchaenus but that it differed from that in the Alticinae. Pomorski (1978) [57] first described this metafemoral organ for another species of Rhynchaenus. Furth has recognized a considerable intrasubfamilial (in- tergeneric) morphological variation and disco- vered seven different MS morpho-groups in the Alticinae genera (Furth, 1980, 1982, 1985, 1988) [58, 48, 41, 42]. The MS organ in the Rhynchaeni- nae is quite similar to that in the Eubaptus and to the most simple morpho-group in the Alticinae (Furth & Suzuki, 1992 [59]). The above facts indicate that the MS organ has evolved independently in three phylogenetically separated groups. Furth and Suzuki (1992) [59] have discovered that it also exists in other coleo- pterous groups. This suggests that these groups may not have acquired this organ in their hind femora in the same way, i.e., evolution of this organ may have been regulated by the same de- velopmental constraints but under different selec- tive pressures. At the same time we must also contemplate the reason why the MS organ did not develop in other groups. In fact, there are various coleopterous groups which have a developed jumping ability in different ways (cf. Pomorski, 1983 [57], Furth & Suzuki, 1992 [59]). The MS organ evolved from the sclerotization of the metafemoral tibial extensor tendon (Furth & Suzu- ki, 1990b) [61]. There are several non-jumping beetle groups with well developed (swollen) hind femora (Furth & Suzuki, 1990b) [61]; therefore, the enlargement of hind femora is not always concerned with a jumping function. The authors have examined comparatively many insect orders as well as beetles with enlarged hind femora from the viewpoint of leg morphology (Furth & Suzuki, 1990a, 1990b) [60, 61]. The Galerucinae and Alticinae have long been treated as independent subfamilies. Suzuki (1988) [45] pointed out that there are few essential differ- ences between them concerning much of their internal reproductive systems; however, there are some general differences between them in the aedeagus (male) and the spermathecae (female). For example, the aedeagus is usually asymmetrical in Galerucinae and almost always symmetrical in K. Suzuki AND D. G. FurtTH Alticinae, the transverse spermathecal folds on the spermathecal capsule are quite consistant in the Galerucinae but variable in the Alticinae and the bending of the basal part of the spermathecal capsule (cf. Suzuki, 1988) [45] is distinctive in most of the alticine genera. Wilcox (1965) [62] mentioned that: “the Altici- nae can be distinguished from Galerucinae by the sclerotized extensor apodeme [=MS organ] in the posterior femora of the former. Orthaltica and Leptotrichaltica from the United States, Micrepi- trix Laboissiére from the Oriental Region, and Micrantipha Blackburn from Australia seem to be the only exceptions to the rule. They appear to be flea beetles which lack the extensor apodeme.” Wilcox (1975) [63] also listed many genera, long treated as the members of the Galerucinae, which he claimed should be transferred to the Alticinae. He considered the MS as the only character to distinguish the Alticinae from the Galerucinae. It should be mentioned here that recently Reid (1990) [43] synomymized Micrantipha with Orthal- tica as a subgenus of the latter. Reid included all species of Livolia into Micrantipha (Micrantipha was described in 1896 and, therefore, its name takes precedence over Livolia described in 1903). However, even more recently Reid (1992) [44] suggests that his 1990 Micrantipha subgenus (in- cluding the former Livolia and Micrepitrix) should be restored to generic status. This is consistent with the principles of the current paper as stated above and previously (Suzuki & Furth, 1990a, 1990b) [64, 49]. Suzuki (1988, 1989b) [45, 65] emphasized char- acter correlation among the phylogenetically im- portant morphological charactesr. If we adopt the MS organ as the only character to separate the Galerucinae and Alticinae, at least the following three assumptions should be valid: 1. The Galerucinae and Alticinae are each monophyletic groups, we use this term in a cladis- tic sense to show phylogenetic relationships. 2. The MS organ was acquired in the alticine lineage after they diverged from the common ancestor of the galerucine and alticine lineages. 3. In the alticine lineages species did not lose the MS organ secondarily and similarly in the galerucine lineages no species acquired the organ What Is a Classification? 1123 secondarily. These assumptions mean that the MS organ is a completely derived (apomorphic) char- acter of the Alticinae. In other words, the mem- bers of the Alticinae can be recognized by the shared derived (synapomorphic) existence of the MS organ. If these assumptions are valid, we should be able to trace logically the transformation process of the organ based on a comparison of the existing species or groups. However, does the MS organ guarantee us this ability? In other words, does the MS organ reflect a clade (single lineage) or a grade (group of lineages; cf. Huxley, 1957 [66])? If assumptions 2 and 3 are not valid, then we cannot determine definitely whether Orthaltica and the other two genera belong to the Alticinae, an alticine lineage that secondarily lost its MS organ, or the Galerucinae. CONCLUSIONS The diversity of Galerucinae (489 genera and about 6,000 species—Seeno & Wilcox, 1982 [26], Jolivet, 1987 [67]) and the Alticinae (more than 500 genera and approximately 8,000-10,000 spe- cies (Seeno and Wilcox, 1982 [26]; Scherer, 1988 [37]) is so great that our current knowledge of most aspects of relationships within each subfamily and between them is very primitive. The variation is extremely large in body size and form, color and pattern, external and internal morphological char- acters, as well as some important aspects of their biology. Although the MS organ is currently a useful taxonomic morphological character for separating the Galerucinae and Alticinae as well as for distinguishing and grouping Alticinae genera, its evolution within the Chrysomelidae needs much more study. Wilcox (1975) [63] listed 20 genera formerly considered to be Galerucinae which he transferred to other families or chrysomelid sub- families. Most of these were transferred to the Alticinae based solely on the presence of the MS organ. This underlines the historical confusion of these two obviously closely related chrysomelid subfamilies. The authors are beginning a long term study using morphological character correlation, includ- ing the MS organ, to elucidate the classification within the Alticinae and their relationship to the Galerucinae. Only after such a study is well underway can we attempt to answer the questions posed above about the assumptions for the phy- logenetic relationship between the Galerucinae and the Alticinae. Along with the questions raised by assumptions 2 and 3 mentioned above, is the question of assumption 1. In other words, can we be certain that the Galerucinae and Alticinae are monophyletic? If either or both subfamilies have evolved from different (several) lineages (clades), this would be considered paraphyletic (in a cladistc sense) and would not be a valid unit for consider- ing a phylogenetic analysis. However, there may still be value in analyzing such a combination of clades (cluster of groups or grades). This idea of grades was discussed by Huxley (1957) [66] and some systematists maintain that such grades also reflect valid evolutionary processes (see Takagi, 1978) [68]. In fact, in many groups it is difficult to clearly define clades or to be certain of monophly; in other words, whether a group is composed of a clade or several grades. This indicates one of the problems with premature cladistic analysis. However, as Huxley (1957) [66] pointed out, clade and grade agree in many cases. The GA Problem reflects a situation where, based on our limited current state of knowledge, it is not possible to make definite statements about phylogeny or evolutionary relationships. Even though there is theoretically no way at this point in our knowledge of Galerucinae/Alticinae rela- tionships to prove how the MS organ evolved or if it could be secondarily lost, we must rely some- what on what is known from an increasingly large number of genera from both subfamilies. We must proceed to study as many aspects (e.g. morpholo- gy, ecology, genetics, etc.) of the relationship of these two groups as possible with the ultimate goal of confirming or negating the above three assump- tions. However, the authors prefer to take a conservative approach without over-speculation, without lumping, and to consider the Galerucinae and Alticinae as valid and separate subfamiles. ACKNOWLEDGMENTS We would like to thank the National Science Founda- tion (INT9116359)and the Japan Society for the Promo- 1124 tion of Science for grants of the U.S.-Japan Cooperative Science Program that enabled the authors to complete this paper. The previous editor Dr C. Oguro (Toyama University) kindly read our manuscripts and recom- mended us to contribute to this journal. 10 11 12 13 14 15 16 REFERENCES Mayr, E. (1963) “Animal Species and Evolution”., Belknap Press, Harvard Univ. Press, Cambridge, xiv-+797 pp. Crowson, R. A. (1955) “The Natural Classification of the Families of Coleoptera”., Nathaniel Loyd, London, 187 pp. Crowson, R. A. (1981) “The Biology of the Coleo- ptera”., Academic Press, Inc. (London) Ltd., xii+ 802 pp. Lawrence, J. F. and Britton, E. B. (1991) Coleo- ptera. In “The Insects of Australia” (2nd ed.), Ed. by CSIRO, Melbourne Univ. Press, II: 543-683. Sibatani, A. (1960) “Revolution of Biology”., Mis- uzu Shobo Co. 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(1992) The leaf-beetle genus Micro- donacia Blackburn (Coleoptera: Chrsomelidae: Galerucinae): revision and systematic placement. Systematic Entomology, 17. Suzuki, K. (1988) Comparative morphology of the internal reproductive system of the Chrysomelidae (Coleoptera). In “Biology of Chrysomelidae” (xxvi +615 pp.)., Ed. by P. Jolivet, E. Petitpierre and T. H. Hsiao, Kluwer Academic Publishers, Dordrecht/ Boston/London, pp. 317-355. White, R. E. (1983) “Beetles”., Houghton Mifflin Co., Boston, xii+368 pp. Suzuki, K. (in press) Comparative morphology of the hindwing venation of the Chrysomelidae 48 49 50 51 52 53 54 SS) 56 ST 58 59 60 61 62 1125 (Coleoptera). In “Novel Aspects of Biology of Chry- somelidae”., Ed. by P. Jolivet, M. L. Cox and E. Petitpierre. Kluwer Academic Publishers, Dordrecht/Boston/London. Furth, D. G. (1982) The metafemoral spring of flea beetles (Chrysomelidae: Alticinae). Spixiana (Suppl.), 7: 11-27. Suzuki, K. and Furth D. G. (1990b) What is a natural classification? A case study in insect sys- tematics: potential confusion before order. (II). Seibutsu Kagaku (Biological Sciences), Tokyo, 42: 201-209. (In Japanese) Furth, D. G. (1989) Metafemoral spring studies of some Neotropical genera of Alticinae. Entomogra- phy, 6: 497-510. Teran, A. L. (1964) Algunas novedades en el genero Soult a i< apts oi64 wal 7 “le geno wert te one t o- chit) eee 10.33 imi Shee rr? il), MM 2 MilTh Ty vily bo visti i,q nion lifea hwnd yy t Biochemistry 1133 ACTIVATION OF PROPHENOLOXIDASE IN DROSOPH= ILA. VI. ANALYSIS OF ACTIVATING SYSTEM. T. Fukumitsu, K. Fujimoto, K. Masuda, M. Tanaka, N. Asada, E. Ohnishi. Biol. Lab., Fac. of Sails 4 Okayama University of Science, Okayama. In insect, phenoloxidase in hemolymph occurs as an inactive proenzyme and is activated upon bleeding. We have presented evidence showing that the activating enzyme ( PPAE ) is a serine protease. Nature of the activation reaction has been analyzed with respect to effects of salts, pH dependency and other factors. Kinetic experiments revealed that the PPAE was rapidly inactivated during the activation reaction. ACTIVATION OF PROPHENOLOXIDASE IN DROSOPH- ILA. VII. CHARACTERIZATION AND ACTIVATION OF Ay. K. heGaneton T. Fukumitsu, K. Masuda, M. Tanaka, N. Asada and E. Ohnishi. Biol. Lab., Fac. of Silo» Okayama University of Science, Okayama. Insect phenoloxidase exists as an inactive percursor (prophenoloxidase; proPO) and the proPO is converted to an active enzyme by an activating system. In Drosophila, it has been reported that proPOs are consisted of three A components: Ai» Ao and A3- We have so far confirmed the two isoforms: A, and A3. Procedure for the purification of A, was improved. It consisted of ammonium sulfate fractionation, DEAE-cellulose, chromatofocusing and phenyl Sepharose column chromatography. Purified Ay migrated as a single band on SDS-PAGE. Using the homogeneous samples, properties of the protein were studied. ACTIVATION OF PROPHENOLOXIDASE IN DROSOPH- ILA. VIII. PURIFICATION AND CHARACTERIZAT- ION OF Az. K. Masuda, T. Fukumitsu, K. Fujimoto, M. Tanaka, N. Asada and E. Ohnishi. Biol. Lab., Fac. of Sci., Okayama University of Science, Okavama. Insect phenoloxidase occurs as an inactive proenzyme (prophenoloxidase; proPO), which is converted to the active enzyme by the activating enzyme. In Drosophila, there are two molecular species of the proP0O, designated as A, and Az. They are distinguishable by ammonium sulfate fractionation and native-PAGE. A3 was purified by ammonium sulfaté fractionation, Sephacryl S-200, DEAE- cellulose and hydroxylapatite column chromatography. The properties of A including molecular weight, isoelectrié point, thermostability, pH stability and substrate specificity of the activated proPO were studied. ACTIVATION OF PROPHENOLOXIDASE IN DROSO- PHILA. IX. ACTIVATION OF A, WITH 2- PROPANOL. N.Asada, T.Fukumitsu, K.Fujimoto, K.Masuda, M.Tanaka and E.Ohnishi. Biol. Lab., Fac. of Sci., Okayama University of Science,Okayama In D. melanogaster, A, component of the prophenoloxidase (proPO) could be activated with both an endogeneous activating system (AMM-1) and organic compounds including alcohols. In the activation of A, with alcohols, 2-propanol was the mos and glycerol was the least effective among the alcohols tested. A, was activated within 2 min after the addition of 2-propanol. Rate of activation and final yield of the PO activity depended on the concentration of 2-propanol. When the concentration of 2- propanol was lowered by dilution, PO activity decreased gradually. Upon re- addition of 2-propanol to this diluted mixture, PO activity re-elevated. Thus the reversibility of the activation of A, in response to the alteration of the concent- ration of 2-propanol could be observed. The maximum level of the PO activity, which had been activated with 2-propanol, was higher than that activated with AMM-1. Optimum concentration of 2-propanol for the rate of activation was 50 %. The activated state of A, showed proper- ties of a tyrosinase-type. The results indicate that the activation of A, with 2- propanol is caused by the reversible con- formational change of the proPO molecule. 1134 Biochemistry ACTIVATION OF LACCASE-TYPE PROPHENOLOXIDASE IN THE CUTICLE OFUENSECT. IK.PROPERTIES OF PROLACCASE IN LARVAL CUTICLE OF SILKWORM, BOMBYX MORI. z H. 1. Yamazaki., Biol., Lab.,Atomi Gakuen Women’s Univ., Saitama. The hardening and darkening mechanism of cuticle has been studied extensively on larval-pupal ecdysis.The laccase,a type of phenoloxidase is found in cuticular matrix and supposed that the enzyme mediates the hardening and darkening process. And the laccase itself is involved in the newly tanned matrix. The laccase is already found in newly formed pupal cuticle as an inactive proform (prolaccase) bound with matrix and it is able to isolate and acti- vate by proteinase. Prolaccase was also found in the cuticle tanning process for larval-larval ecdysis between 4th and 5th istar. The larval prolaccase activated by trypsin and the activation was completely inhibited by 1 M NaCl. The prolaccase could not be activated by pepsin different from the case of pupal prolaccase. The cuticle hardening mechanism is funda- mentally similar between larval-pupal and larval-larval ecdysis. However,it was observed that the peptides involved in the process were slightly different. PROPHENOLOXIDASE ACTIVATING SYSTEM IN CUTICLE OF THE LARVAL SILKWORM, BOMBYX MORI. M. Ashida, Y. Koizumi and P. Brey. The Institute of Low Temperature Science, Hokkaido Universit Sapporo. Three types of cuticular enzymes which are capable of oxidizing phenolic compounds have been reported in the literature. They are laccase and two types of tyrosinase-type phenoloxidase. One of the phenoloxidases is called injury phenoloxidase, because it exerts its action when the cuticle is injured. The injury phenoloxidase is claimed to be present as an inactive form (prophenol- oxidase, proPO) in cuticle, but its presence remains to be demonstrated. We studied the regulation mechanism of phenoloxidase activity in the larval cuticle of the silkworm, Bombyx mori. We developed a method to extract the "injury" Phenoloxidase as an inactive enzyme (cuticular proPO). The cuticular proPO was shown to be activated through a limited proteolysis. As our studies using protease inhibitor indicated that cuticular proPO activating enzyme is also present as an inactive enzyme, cuticular proPO seemed to be activated by the action of a cascade. A method to obtain a homogeneous cuticular proPO and the immuno- cytochemical localization of the enzyme in cuticle were also reported. PURIFICATION OF PRO-BAEEase (a serine enzyme zymogen) IN PROPHENOLOXIDASE CASCADE OF INSECT AND ITS ACTIVATION TO AN ACTIVE FORM. Y.Katsumi and M.Ashida. Biochemical. Lab., The Institute of Low Temp. Science, Hokkaido University,, Sapporo. In insect hemolymph prophenoloxidase has been shown to be activated through the action of a cascade, which is termed prophenoloxidase cascade. Although two zymogens of serine protease, proBAEEase and proPPAE (prophenoloxidase activating enzyme) are known to be activated sequentially when the cascade is triggered by microbial cell wall compornents such as f-1,3-glucan and peptidoglycan, none of them has been prified yet. We developed a method to purify proBAEEase from hemolymph of the silkworm, Bombyx mori. The purification procedures consisted of fractionation by ultracentrifugation and ammonium sulfate and column chromatography on Sephadex G-150, Pheny1-Toyopearl, Heparin-Toyopear], hydroxylapatite, and Mono-Q The purified proBAEEase migrated as a single polypeptide with a molecular mass of 39kDa.in SDS -PAGE. The results of the experiments to partially reconstruct prophenoloxidase cascade using the purified proBAEEase and a fraction obtained during the purification of proBAEEase were reported A STUDY ON THE RELATIONSHIP BETWEEN THE CROSS-LINKING OF INSECT CUTICULAR PROTEINS AND THE BIOSYNTHESIS OF PAPILIOCHROME II. Y. Umebachi. Izumino-machi 2-12-24, Kanazawa. Papiliochrome II is a pale yellow pig- ment in the wing-scales of papilionid bu- tterflies. The structure has been report- ed to be N® -[%-(3-aminopropionylamino- methyl)-3,4-dihydroxybenzy1]-L-kynurenine, in which the aromatic amino nitrogen of L-kynurenine is bonded to the side chain 8 -carbon of N-f-alanyldopamine (Rembold and Umebachi, 1984). This binding can be enzymatically made by the extract of some insect cuticles or colleterial glands ( Yago, 1989; Sugumaran et al., 1990). On the- other hand, N-acyldopamines like N- acetyldopamine and N-§-alanyldopamine are known to function as cross-linking agents in insect cuticle (Andersen, 1985; Kramer and Hopkins, 1987; Sugumaran, 1988). There are two kinds of cross-linking me- chanism: (1) quinone-tanning and (2) #- (or &, B-)sclerotization. In the latter mechanism, amino or imino nitrogen of pro- tein is bonded to the side chain @-(or &, A-)carbon of N-acyldopamine by both phenol oxidase and quinone-methide isome- rase (Saul and Sugumaran, 1989). All av- ailable evidence indicates that both the enzymatic synthesis of Papiliochrome II and the @-sclerotization of cuticular pro- teins come under the same category. Biochemistry 1135 THE ROLE OF PIGMENT BINDING PROTEIN IN XANTHOMMATIN BIOSYNTHESIS IN THE EPIDERMAL CELLS OF THE SILKWORM, BOMBYX MORI. H. Sawada’, M. Tsusué! ,T. Iino? ‘Biological Lab.School of Liberal Arts, Kitasato Univ., Sagamihara Dept. of General Education, Nihon Univ., Tokyo We have already reported that the pigment protein separated by SDS-PAGE had an activity binding with xanthommatin and cinnabarinic acid. However, 3-hydroxy-kynurenine or 3- hydroxy-anthranilic acid did not bind to the protein. These data indicate that the protein has an affinity with phenoxazinone ring of xanthommatin. Biosynthesis of xanthommatin was also investigated. The homogenate of epidermal tissue was fractionated by sucrose density gradient centrifugation. When the substrate 3-hydroxy-kynurenine was added to each fraction, catalytic activity to form xanthommatin localized in the pigment granules fraction. In addition, HPLC analysis revealed the incorporation of 3-hydroxy-kynurenine into the intact pigment granules. These data suggest that the pigment granule has an important role in both biosynthesis and accumulation of xanthommatin. Recently, we obtained polyclonal antibody from the pigment protein which was purified by SDS-PAGE. Furthermore, we are now investigating the cross-reactivity of the antibody with crude extract of epidermis of w2 mutant which is unable to synthesize the pigment. CHARACTERIZATION OF RAT AND HUMAN SEPIA- PTERIN REDUCTASE GENES H.Ichinose, K.Titani, K.Fujitea, T.Nagatsu (Inst.for Comprehen. Med. Sci., Sch.of Med., Fujita Health Univ., Toyoake), and S.Katoh, T.Sueoka (Dept.of Biochem., Sch. of Dent., Meikai Univ., Sakado) . Biologically active biopterins observed in insects and vertebrates are produced by the function of sepiapterin reductase (SPR) . We studied on the characterization otf rat and human genes of SPR. We have isolated a full-length cDNA clone for SPR from a human liver cDNA library by plaque hybridization and analyzed the nucleotide sequence of the cDNA’. We amplified the cDNA for rat SPR by the PCR.Synthetic primers for the amplifica- tion were designed based on the nucleotide sequence of rat SPR* and the amino acid sequence of the mature form of rat SPR*. The clone encoded a protein of 261 amino acids (783 bases) with a calculated Mr of 28047 daltons.A single gap of codon was introduced into the human SPR sequence against rat SPR [1*AGG] . Consensus sequences for NADPH and pterin located in the range near the 5'-end in both SPR. Pterin binding site |GCCGGGTTG- CTGTCG (A-G-L-L-S)] for rat SPR? was revealed as |GCCTCGCTGCTGTCG (A-S-L-L-S) ] 1n human SPR. Estimation of the number of nucleotide sub- stitution was 0.250+ 0.021 (total;JC method) , and 0.262, 0.105, and 0.429 at the 1,2, & 3 base positions of codon, respectively (K3P method) . Human SPR showed a 74% identity in amino acid sequence with that of rat SPR. Rate of amino acid substitution was1.9x10°%. 1Tchinose et al'91,BBRC179,183;7Citron et al "90,PNAS87,6436;*Oyama et al‘90,BBRC173,627. PTERIDINES IN THE YELLOW-COLORED CHROMATO- PHORES OF THE ISOPOD, ARMADILLIDIUM VULGARE. M.Nakagoshi, S. Takikawa and S. Negishi.* Biol. Labo., Kitasato Univ., Sagamihara. *Dept. of Biol., Keio Univ., Yokohama. In A. vulgare the ommochromes in the integument impart a dark gray or brown body color. The male of A. vulgare displays a uniform dark gray color, while the female usually displays a brown color with characteristic yellow markings aligned along the dorsal region. Principal component of the yellow pigment isolated from the yellow markings was already identified as sepiapterin. Morphological investigations revealed that the pattern of yellow-colored chromatophores in the female was externally observable at the dorsal surface of the integument as the yellow markings. In contrast, the yellow-colored chromatophores were not externally observable in the male, since they were covered by an ommochrome chromatophore layer. The yellow-colored chromatophores contained numerous granules in the cytoplasm and the morphological properties of the granules were similar to those of pteridine granules which contain unc acid occurring in the silkworm integument. Based on TLC, HPLC and UV-spectrophotometric analyses, we concluded that blue and violet fluorescent compounds isolated from the chromatophores were biopterin, pterin and isoxanthopterin. Uric acid also accumulated in the chromatophores. The content of both sepiapterin and biopterin in the male was about two times greater than in the female, while the content of both pterin and isoxanthopterin showed few difference between the male and female. The quantitative difference in sepiapterin and biopterin between both sexes Suggests that the activities of various enzymes involved in pteridine metabolism may differ between the male and female of A. vulgare. THE STUDY ON BODY COLOR OF ARMADILLIDIUM VULGARE BY THE FINE STRUCTURE OF EPIDERMIS AND PIGMENT QUANTITY. S.Negishi! Y.Haseqawa! , Y.Katakura? , P.Juchavlt3, G.Martin3. 'Dept.of Biol., Keio Univ., Yokohama, ZDept.of Bioengin., Soka Univ., Tokyo and “~Lab.of Animal Biol. Univ.of Poitier, France. A.vulgare is polymorphic for the body color. Red body color is dominant over the usual black or grey (wild type). Polymorphism may be provided by the difference in the fine structure of pigment granules and/or pigment quantity under the control of proper genes. The study was undertaken to prove this hypothesis. Chromatophores of the red phenotype were filled with pigment granules of filamentous structures such as immature granules + occurring within the same limiting membrane. Xanthommatin content of red A.vulgare is much the same with that of wild type. Epidermis of white A.vulgare 1s very partially pigmented in the large vesicles. This observation suggests that white woodlice provide the enzyme for ommochrome synthesis and its activity may be prohibited though the mechanism is uncertain. These results show that the structure of pigment granules is determined by the corresponding gene to the body color. 1136 Biochemistry A SIMPLE CRITERION FOR PREDICTING WHETHER OR NOT A MYOGLOBIN HAS THE USUAL DISTAL HISTIDINE RESIDUE A. Matsuoka and K. Shikama. Biol. Inst., Tohoku Univ. Sendai Myoglobins from various species can be divided into two groups by an absorbance ratio of the Soret peak of the acidic met-form to that of the oxy-form, namely the Ymet / Yoxy ratio. Values higher than 1.0 (ranging from 1.16 to 1.41) were thus obtained for the myoglobins containing the usual distal histidine, whereas those of less than 1.0 (ranging from 0.79 to 0.84) were the ratio for the myoglobins lacking this residue, such as those from three kinds of gastropodic sea molluscs and two kinds of sharks. On the basis of these Soret absorption spectra, we have also examined the unique structures of a protozoan myoglobin from Paramecium caudatum, an annelid giant hemoglobin from Tylorrhynchus heterochaetus, and an insect hemoglobin from Tokunagayusurika akamusi. Shikama, K. and Matsuoka, A. (1989) J. Mol. Biol. 209, 489-491. ELEPHANT MYOGLOBIN WITH THE DISTAL GLUTAMINE : AN UNUSUAL STABILITY PROPERTY OF OXYMYOGLOBIN T. Tada, A. Matsuoka and K. Shikama. Biol. Inst., Fac. of Sci., Tohoku Univ., Sendai. In the usual mammalian myoglobins, the distal (E7) histidine 1s known to play a key role in the stability properties of the bound dioxygen. Elephant myoglobin, however, lacks this residue and has a glutamine at E7 position. In order to know the effect of the distal residue on the stability, we have isolated native oxymyoglobin (MbO2) directly from the cardiac and skeletal muscle tissues of the African elephant (Loxodonta africana ), and examined the autoxidation rate from MbO?2 to metMb over the wide range of pH 4.5 - 12.3 in 0.1 M buffer at 25°C. The pH profile obtained was similar to that of Aplysia MbO2 bearing the distal valine, but elephant MbOz2 was found to be less susceptible to autoxidation and its extent was almost comparable with sperm whale MbO3. ROLE OF THE DISTAL RESIDUE ON THE REACTION OF METMYOGLOBIN WITH HYDROGEN PEROXIDE : A COMPARATIVE STUDY G. Tajima', A. Matsuoka and K. Shikama’. ‘Dept. of Biol. Sci., Coll. of Gen. Edu., and “Biol. Inst., Fac. of Sci., Tohoku Univ., Sendai. Metmyoglobin reacts with hydrogen peroxide to form ferryl-myoglobin, which can revert back spontaneously to the met-form. This reaction seems to be of physiological importance, since through this cyclic reaction of myoglobin between metMD(III) and ferryl- MDb(IV), H,O., one of the most potent oxidant in vivo, can be decomposed continuously in red muscle tissues in the absence of catalase and peroxidase (Tajima, G. and Shikama, K. (1992) Int. J. Biochem. in press .). We have isolated native metmyoglobins from various species, and examined the mode of reaction with hydrogen peroxide in relation to the kinds of the distal (E7) residue. The spectroscopic results have shown that metmyoglobins lacking the usual distal histidine, such as those from African elephant, shark (Galeus nipponensis), and three species of gastropoda, Aplysia kurodai, Aplysia juliana and Dolabella auricularia, are little or never converted to ferryl-form. EFFECT OF PHOTOIRRDIATED TIN-PROTOPOR PHYRIN ON ARYLSULFATASE ACTIVITY OF RAT BRAIN LYSOSOMES. H. Keino, and T. Banno. Dept. Perinatol., Inst. Dev. Res., Kasugai. "A synthetic heme analogue (tin-protopor phyrin; SnPP) is known to cause serious responses of human babies and suckling rats to photoirradiation. Le alisio suggested that photo-excited porphyrins cause great damage to lysosomal membranes. In this study we report the effects of SnPP plus photoirradiation on a lysosomal enzyme (arylsulfatase; ASase). The crude mitochondria fraction was obtained from the brain of suckling rats. The mitochon- dria-free lysosomes were then isolated from the crude mitochondria fraction by Percoll density gradient centrifugation. ASase activity was markedly reduced by photo-excited SnPP. No reduction of ASase activity was detected under the dark. Photoirradiation never reduces the ASase activity without SnPP. The reduction was prevented by administration of L-ascorbic acid and was reinforced by D920. Photo- excited SnPP may bring singlet oxygen and inaugurate oxygen-free radical reactions. The kinetic study demonstrates that apparent Km value of ASase for 4-nitro- catecholsulfate was calculated to be 0.39mM in both presence and absence of the photo-excited SnPP. Photo-excited SnPP reduced the velocity of ASase activity. Biochemistry 1137 METMYOGLOBIN AUTOXIDATION AND REDUCTION M.Kariya!, K.Machida!, H.Namiki?. 1Dept. of Sports Sci., Sch. of Human Sci., Waseda Univ., Tokorozawa, 2Dept. of Biol., Sch. of Educ., Waseda Univ., Tokyo. In muscles such as the cardiac and the skeltal, myoglobin (Mb) plays a role in maintaining aerobic metabolism, both as an oxygen store and by facilitating oxygen diffusion. The reduced form of Mb is required for this reversible oxygenation to occur. However, myoglobin is easily transformed into metmyoglobin (metMb) by autoxidation. Muscles therefore contain metMb reductase. We presently report a reducing factor in beef heart muscle dif- ferent from the known metMb reductases. The factor was separated between m.w. 1000 and 3000, by ultrafiltrations. The crude sample was then applied to a HPLC column of anion exchanger and frac- tionated. A metMb reducing activity was separated in one fraction. This factor reduced metMb faster with NADPH than with NADH. Some other hemoproteins were as- sayed if they served as electron ac- ceptors. Methemoglobin was hardly reduced, but cytochrome c was reduced. It also reduced an artificial dye 2,6- dichlorophenolindophenol. The pH optimum of this NADPH-diaphorase activity is about 6.0. PHOTO-ACTIVATION OF RESPIRATION IN ABALONE SPERM. E. Tazawa', A. Fujiwara? and I. Yasumasu? ‘Biol. HASTE 5 p Yokohama City Unilvin; Yokohama and ?Dept. of Biol., School cf Education, Waseda Univ., Tokyo. In sperm of the abalone, Norditis discus the respiratory rate was enhanced by light irradiation with the peaks of activating effect of light at 430, 550 and 570 nm. The wavelengths at the peaks of activating effect on respiration correspond to those at peaks of light absorption of cytochrome be Probably, photo-activation of cytochrome b results in augmentation of respiration in abalone sperm. In sperm of sea urchin, starfish and echiuroid, photo- activation of respiration with peaks corresponding to the absorption peaks of cytochrome b was not found, unless eperm were exposed to PMS or DCPIP. Acceleration of electron supply to cytochrome b by these compounds probably makes photo-activation of cytochrome b reaction apparent as an increase in the respiratory rate in these sperm. This study was carried out under the NIBB (National Institute for Basic Biology) Cooparative Research Program for the Okazaki Large Spectrograph (91-502) . HIGH LEVELS OF AL,FE AND MINOR ELEMENTS BOUND TO SKELETAL PIGMENTS IN A CORAL OULASTREA CRISPATA H. Yamashiro. Radioisotope Lab., Univ. of the Ryukyus, Okinawa A zebra coral Oulastrea crispata (Faviidae Scleractinia) has colored skeleton (gray to black). Skeletal Pigments are separated from the skeleton by the treatment of weak acid. Resultant acid-insoluble matter containing Fe has been reported (not quantitative study). The present study was done to measure the amount of Fe and other minor elements bound to skeletal pigments by neutron activation analysis. Highly concentrated Fe (>9500ppm) and Al (>14000ppm) were detected in the acid-insoluble matter of the skeleton. Furthermore, this matter contained high levels of minor elements such as Sc, V, Cr, Br, Ag, I, La, Ce, Sm, Hf, Th and U with the value more than 2ppm. Concentration factors reached x10 7 for Al, Ag, Ce and Sm. These results suggest that the acid-insoluble organic substance of the coral skeleton has a powerful affinity for Al, Fe and many other minor elements. Coral soft tissues seem to be involved in the concentration of these elements either at the time of uptake or calcification. DIABETIC PERIPHERAL NEUROPATHY AND DECREASE IN MICROTUBULE-ASSOCIATED PROTEIN KINASE ACTIVITY. T.Kobayashi , K.HouiZ, Y.Mori2 ,H.Sasaki2 gna SMO ChAC mr ‘Dept. of Biochem. and the 3rd Dept. of Intern. Med., Jikei Univ. School of Med., Tokyo The activity of microtubule-associated protein (MAP) kinase is controlled through phosphorylation of its tyrosine residue by either of insulin receptor or nerve growth factor receptor, and the enzyme is regard- ed to play important roles in cell prolif- eration and neuronal functions. We consid- er that decreased insulin fails to stimu- late MAP kinase activity to the sufficient level even in the presence of nerve growth factor (NGF) and induce diabetic neuropa- thy. We investigated MAP kinase activity in a new insulin-defective strain of rat (WBN/Kob) and found that the activities were remarkably decreased in every neural tissues of the animals at the age of 15 months. Especially, the activity in sciat- ic nerve was only 19% of that of the control. On the other hand, the activities in spleen and in submandibular gland were elevated to 400% and 250%, respectively. We then employed cultured dorsal root ganglion cells isolated from 10 day chick embryo. When they were cultured in the absence of insulin, their neurite elonga- tion, MAP kinase activity and contents of MAP1 and MAP2 were all decreased remarka- bly. 1138 Biochemistry ANALYSIS OF THE PROCESSING OF ALZHEIMER AMYLOID PROTEIN PRECURSOR EXPRESSED IN COS CELLS BY cDNA TRANSFECTION. Wakako Yamao-Harigayal,2, Mihoko Usamil and Kei Maruyama!,2. 1Department of Molecular Biology, Tokyo Institute of Psychiatry, Kamikitazawa, Setagaya, Tokyo 156, and 2Department of Molecular Biology, Tokyo Metropolitan Institute of Clinical Medicine, Honkomagome, Bunkvo, Tokyo 113. One of the pathological features of Alzheimer's disease (AD) is the deposition of 8/A4 protein (%P) as senile plaque. BP is a 42-amino-acid protein and it is derived from a 695-amino-acid precursor, amyloid precursor protein (APP). In normal condition, APP is cleaved in the interior of 8P by the hypothetical protease “secretase,” thus preventing its production. The cause of neuronal degeneration and AD might be the abnormal processing of APP. Since the expression of APP is fairly low, APP was expressed transiently in COS-1 cells by cDNA transfection to study its processing. APP695 and APP770 (with Kunitz-type protease-inhibitor domain) were processed in a similar way to produce 100 kd and 9 kd fragments. Three kinds of mutations of APP, Val717-Ile, Phe or Gly, were reported in familial AD. These mutations had no effect on the processing of APPs expressed in COS-1 cells. This means that these mutations might not be a direct cause of AD. The mutations of the proposed cleavage site of secretase had no effect. Hence secretase seemed to recognize the conformation of APP rather than its amino-acid sequence. The identification of secretase is now under way. THE INHIBITORY MECHANISM TO TRYPSIN OF A 58 KDA INHIBITOR FROM THE HEMOLYMPH PLASMA OF HALOCYNTHIA RORETZI. T. Abe, F. Shishikura, S. Ohtake and K. Tanaka. Dept. of Biol., Nihon Univ. Sch. of Med., Tokyo. A 58 kDa plasma inhibitor inhibited bovine pancreatic trypsin as well as its endogenous enzyme(s) in the hemolymph of an ascidian, H. roretzi. To analyze the inhibitory mechanism of the inhibitor, bovine pancreatic trypsin was used. The trypsin activity was first decreased by the addition of inhibitor in both dose- dependent and time-dependent Manners, whereas after prolonged incubation (over 20 hours) the trypsin activity recovered almost the initial activity of trypsin. On SDS-PAGE analysis the inhibitor (58 kDa) associated with the trypsin (23 kDa) to form a complex (approx. 80 kDa) in reduced and unreduced samples. This complex re- sisted to treatment of SDS but easily dissociated by treatment of a nucleophilic reagent, such as ammonia. These results indicated that the complex was covalently formed by the reaction of the inhibitor and the trypsin and then the inhibitor might be degraded by trypsin. As a similar time course of the inhibi- tion was found in the enzymatic activity of the hemolymph of H. roretzi, this mechanism seems to exist in the inhibito- ry reaction between the inhibitor and cognate plasma enzyme(s). DEXTRAN SULFATE ACCELERATES THE INHIBITORY ACTIVITY OF PLASMA-ENZYME INHIBITOR FROM HALOCYNTHIA RORETZI HEMOLYMPH. F. Shishikura, T. Abe, S. Ohtake and K. Tanaka. Dept. of Biol., Nihon Univ. Sch. of Med., Tokyo. We have tested the abilities of dextran sulfates (DSs; 500 kDa, 50 kDa, 15 kDa, and 5 kDa) and other polyanions including four kinds of glycosaminoglycans (GAGs) to increase the inhibition of plasma-enzyme activity by its endogenous inhibitor purified from the hemolymph of ascidian, H. roretzi. DS whose molecular weight is about 50,°500, 15, or 5 kDa (am@ondengos decreasing activity) dramatically accel- erated the rate of reaction between plasma enzyme and its inhibitor in 50 mM Tris- HCl, pH 8.0, containing 0.5 M NaCl (0.5 M NaCl-Tris buffer). When each GAG mixed with the inhibitor in 0.15 M NaCl-Tris buffer, not only four kinds of DSs but also heparin, chondroitin sulfate B, and fucoidan activated the inhibitor more than those in 0.5 M NaCl-Tris buffer. However, chondroitin sulfate A, C and phosvitin in neither 0.5 M NaCl-Tris buffer nor 0.15 M NaCl-Tris buffer were not effective at concentrations of 1 mg/ml. In the presence of 50 kDa DS, plasma enzyme seems to undergo instantaneous ternary-complex formation with inhibitor and DS. Poly- brene (10 pg/ml) abolished the abilities of GAGs. These results suggest a similar mechanism proposed in the activation of antithrombin III in mammalian blood. PRIMARY STRUCTURE OF SP-25, A SPECIFIC SUBSTRATE FOR ATP-DEPENDENT PROTEASE IN BOVINE ADRENAL CORTEX, AND ITS COMPARISON WITH MER; 5 IN MOUS ERYTHROL UKEMIA S.Watabe!, T.Hiroi H Kohno K. .Akiyama;, H.Kouyama 4 S. Tatunami}, N. Yago 5 Hts Hara;, -Ohnishi T.Suzuki~ and [.Nakazawa Radioisotope Res. Inst. and Ist Dept. of Anatomy, St. Marianna Univ.,Kawasaki, and Dept.of Biol. Toho Univ. Funabashi. We purified SP-25, a specific substrate protein for ATP-dependent protease in bovine adrenocortical mitochondria and determined its amino acid sequence using a protein sequencer after fragmentation and purification of the fragments. By homology search using NBRF data base it was found that SP-25 had 92 % homology with an amino acid sequence coded by a cDNA clone, MER- 5, in mouse erythroleukemia cells. We iso- lated a cDNA clone coding bovine SP-25 using a PCR-amplified cDNA fragmant as a probe. The amino acid sequence deduced from the nucleotide sequence contained a presequence specific to mitochondrial proteins, which had less homology with MER-5 than mature protein region had. Since it was reported that MER-5 increased transiently after induction of differen- tiation in erythroleukemia cells, and that removal of MER-5 mRNA by an antisence RNA inhibited the differention, MER-5 and SP- 25 might have an important role in cell differentiation. Biochemistry 1139 ISOLATION AND CHARACTERIZATION OF PHOSPHOLYPASE A2-LIKE PROTEIN FROM BULLFROG PITUITARY GLAND. H.Hayashi!, M.Sakai?, H.Takasu?, S.Tanaka!, Y.Hanaoka! and S.Kikuyama2. !Inst. of Endocrinol., Gunma Univ., Maebashi and 2Dept. of Biol., Sch. of Educ., Waseda Univ., Tokyo. During the course of isolation and purification of thyrotropin from the adult pituitary glands of bull- frog, Rana catesbeiana, a protein that behaved with thyrotropin until final purification step was found. This protein was finally separated from thyrotropin by hydroxyappatite column chromatography. The molecular mass of this protein was estimated to be 14kDa by SDS gel electrophoresis. The N-terminal 40 residues and some lysylendopeptidase peptides were sequenced. The sequence of this protein has about 40% identity with that of phospholypase A2 from Indian cobra. The antibody against this protein was raised in a rabbit. This antibody did not rec- ognize bullfrog lutropin, follitropin, thyrotropin and their subunits. The contents of this phospholypase A2-like protein in circulating blood of various stages during bullfrog metamorphosis were measured by radioimmunoassay. The plasma level of this protein was low (<15ng/ml) in premetamorphic stages, then rapidly increased at the onset of climax (40ng/ml), and declined at the end of metamorphosis. Immu- nohistrochemical studies using this antibody showed that this protein is synthesized in glyco- protein-producing cells in the bullfrog pars distalis. PURIFICATION AND CHARACTERIZATION OF ACID PROTEINASE (ENZYME IV) FROM BULLFROG GASTRIC MUCOSA T. Inokuchi, K. Kobayashi and S. Horiuchi. Life Sci. Inst., Sophia Univ., Tokyo. Enzyme VI is acid proteinase and exists in both larval fore-gut and adult stomach of bullfrog, Rana catesbeiana. This enzyme was purified from adult gastric mucosa by Hydroxyapatite fast flow type, @ Sepharose fast flow, Con A-Sepharose and Mono Q HR column chromatography. Enzyme IV was divided into four peaks (IV-1, IV-2, IV-3 and IV-4) by Mono Q HR. Mobilities of the four enzymes on PAGE were slightly different from each other. M.W. of IV-2 and IV-3 was estimated to be 50,000 in reducing condition but about two times higher in non-reducing condition by SDS-PAGE. By acid treatment their M.W. decreased. The result showed conversion of pro-enzyme to its activated form, similar to conversion of pepsinogen to pepsin at acidic pH. Enzyme VI-2 hydrolyzed bovine hemoglobin with maximum activity between pH 2-3 but it showed lower activity on BSA, y -globulin or casein than on bovine hemoglobin. From these results we thought that enzyme IV was cathepsin E- type enzyme in anuran. ACTIVATION OF CYSTEINE PROTEINASE OF THE SILKMOTH, BOMBYX MORI. S. Y. Takahashi and Y. Yamamoto. Dept. of Biol., Fac. Liberal Arts, Yamaguchi Univ., Yamaguchi. Cysteine proteinase purified from the silkmoth, Bombyx mori, can be activated during incubation in the acidic medium (DH aah Sn. Activation requires the presence of substrates in the incubation medium and is inhibited by E-64, specific inhibitor for cysteine proteinases. Concomitant with the activation, the purified enzyme (47 kDa) is converted to an intermediate (44 kDa), and finally an active form (39 kDa) which is relatively stable. These results indicate that partial proteolysis of the enzyme is likely to be involved in the activation process. The same pattern changes occur in intact eggs at the beginning of embryogenesis and the activated form of the enzyme can be detected throughout the embryonic life. These facts strongly suggest that cysteine proteinase is stored in the eggs as a proenzyme which is latent and is activated by partial proteolysis during embryogenesis. The regulatory mechanism of the cysteine proteinase and involvement of the proteinase in the yolk protein degradation will be discussed in relation to the embryogenesis of the silkmoth, Bombyx mori. BIOSYNTHESIS OF THE BLOOD GROUP P ANTIGEN-LIKE GalNAc#173Gal#174G1cNAc/Glc STRUCTURE: A NOVEL N-ACETYLGALACTOSAMINYLTRANSFERASE ACTIVITY IN HUMAN BLOOD PLASMA A. Takeya’, 0. Hosomi’, N. Shimoda’ and S. Yazawa’ Dept. of Legal Med., Gunma Univ. Sch. of Med., Maebashi and ?Japan Immunoresearch Laboratories, Takasaki. Human blood group 0 plasma was found to contain a GalNAc-transferase which catalyzes the transfer of GalNAc from UDP-GalNAc to Galfl>4Glc, Galfl-> 4GlcNAc, asialo-«a,-acid glycoprotein and Galfl- 4G1cNAc#173Gal £124Glc-ceramide, but not to Gal?#1-> 3G1cNAc. The enzyme required Mn’* for its activity and showed a pH optimum at 7.0. The reaction products were readily hydrolyzed by §-N-acetyl- hexosaminidase and released GalNAc. Apparent Ka values for UDP-GalNAc, Mn?*, lactose, N-acetyl— lactosamine, and terminal N-acetyllactosaminyl residues of asialo-«,-acid glycoprotein were 0.64, 0.28, 69, 20, and 1.5mM, respectively. Studies on acceptor substrate competition indicated that all the acceptors mentioned above compete for one enzyme, whereas the enzyme is distinguished from an NeuAc«233Gal -],4-GalNAc-transferase, which also occurs in human plasma. The methylation study of the product formed by the transfer of GalNAc to lactose revealed that GalNAc had been transferred to the carbon-3 position of the 8-Gal residue. Although the GalNAc£173Gal structure is known to have the blood group P antigen activity, human plasma showed no detectable activity of Galel74Gal §-1,3-GalNAc-transferase, which is involved in the synthesis of the major P antigen, GalNAc#173Gale]- 4Gal#1+4Glc-ceramide. Hence, the GalNAc#173Gal 61> 4GlcNAc/Gle structure is synthesized by the novel Galf1>4GlcNAc/Gle -1,3-GalNAc-transferase. 1140 Biochemistry A MODIFIED METHOD TO OBTAIN A STABLE TREHALASE FROM ARTEMIA SALINA. Z. Nambu and F. Akiyama-Nambu, Dept. of Biol., Univ. of Occupational and Environm. Health, Japan, Sch. of Nursing and Med. Technol. Kitakyushu. As we reported previously, the trehalase from the nauplii of Artemia salina specifically hydrolyzes &d- trehalose. The activity was found to be inhibited by $-D-glucosides and so we tried to purify the enzyme by introducing p-aminophenyl g-D-glucoside - Sepharose chromatography into our isolation procedure. The enzyme was purified by acetone treatment, DEAE-Sepharose CL-6B, Con A-Sepharose, p-aminophenylppeglucoside- Sepharose, Poly(A)-Sepharose and gel filtration using HPLC with a MTSKgel G3000SW column. The activity of the trehalase was adsorbed on the p-aminophenyl 9Q-D-glucoside — Sepharose column with O.5 mM piperazine-HCl (pH 6.5) and was eluted by 100 mM piperazine-HCl (pH 6.5). The yield of the activity was 1.4 % and the specific activity was 17.7 units/mg protein. The obtained enzyme was shown to be stable in a phosphate buffer pH 7.0 at 4°C for 10 days and the stability was prolonged for 26 days by a treatment of the enzyme with 1 mM dithiothreitol. The p-aminophenyl §-D-glucoside-Sepharose chromatography seems to be effective to some extent for the preparation of the enzyme. A MUSCLE ACTIN GENE FAMILY IN THE ASCIDIAN HALOCYNTHIA RORETZI. T. Kusakabe!, K. W. Makabe?, N. Satoh’. 1Dept. of Zool., Fac. of Sci., Kyoto Univ., Kyoto. ?California Institute of Technology, Pasadena, USA. Our previous studies have shown that at least five muscle-type actin genes form a cluster in the Halocynthia roretzi genome. Some of these genes are specifically expressed in larval muscle cells. In this study, we isolated another muscle-type actin gene, HrMA1 from an H. roretzi genomic library. The HrMA1 gene was fairly different in the nucleotide sequence and structure from actin genes in the cluster Northern blot analysis showed that HIMA1 mRNA was undetectable in zygotes and cleavage stage embryos. HrMA1 mRNA first appeared at gastrula stage and accumulated during later embryogenesis. However, no HrMA1 transcript was observed in adult tissues including body-wall and heart muscles. That is, the expression pattern of HrMA1 was the same as those of actin genes in the cluster A coding region probe derived from an HrMA2 cDNA detected transcripts in adult body-wall and heart muscles but not in non-muscle tissues. Furthermore, the length of transcripts was slightly different between body-walls and hearts. These results suggest that different muscle-type actin genes are expressed in the two different types of ascidian adult muscle. GENOMIC STRUCTURE OF 29 kDa PROTEIN FROM THE ASCIDIAN Halocynthia roretzi. A. Nakamura! and T. Takagi?. !Dept. Pharmacol., Gunma Univ., School of Medicine, Maebashi and Biol. Inst., Fac. Sci., Tohoku Univ., Sendai The body wall muscle of the ascidian Halocynthia roreizi contains a large amount of 29 kDa protein (HR-29), whose the function is still unknown. To elucidate the function and origin of this protein, we have determined the cDNA and genomic structures. The cDNA was composed of 871 bp and encoded 251 amino acid residues. The N-terminal 151 residues contained three homologous repeated sequences and showed no significant homology with other proteins. On the other hand, the C-terminal 100 residues showed homology with small heat shock proteins and alpha- crystallin. Thus HR-29 is composed of two domains. We obtained two genomic clones (HR-29G1, HR-29G2) amplified by PCR. HR-29G2 was slightly different from HR-29 by partial sequence analysis and not analyzed further. HR-29G1 was exactly same as HR-29 and has three introns (I, II, III). The largest intron II (1093 bp) was placed between two domains and included a possible promoter site and the PELHAM BOX-like structure which is known to be important for the expression of heat shock protein gene. Therefore the C-terminal domain is obviously derived from heat shock protein. Although the origin of N- terminal domain is not clear, HR-29 is a fusion protein of two different origins. MOLECULAR CHARACTERIZATION AND DIFFEREN- TIAL GENE EXPRESSION OF ASCIDIAN EMBRYONIC NUCLEAR ANTIGEN HGV2. S. Fujiwara. Dept. of Biol. Fac. of Sci. Kochi Univ., Kochi. We have previously obtained a hybridoma clone which produces the antibody against the antigen, named Hgv2, specific to the nuclei of the oocytes, embryos and larvae of the ascidian Halocynthia roretzi. In the present study, a complete nucle- otide sequence of a cDNA clone encoding the Hgv2 antigen was determined. The amino acid sequence deduced from the cDNA was Similar to amphibian histone-binding protein Nl, which is thought to be in- volved in the chromatin assembly process. Close relation between these two proteins was confirmed by their similarity in the amino acid composition and hydropathy pro- file. Although putative histone-binding domains, which are two acidic residue-rich clusters, do not seem to be conserved, there evidently are two highly acidic regions in the Hgv2 polypeptide. Hgv2 protein may function as a nucleosome as- sembly factor during rapid embryonic cell divisions. While the Hgv2 protein was constantly detected in the nuclei of the embryos and larvae, the amount of the Hgv2 mRNA gradu- ally decreased during embryogenesis. Among the adult tissues, the mRNA was detected only in the branchial sac, except for the gonad which contains oocytes. Biochemistry 1141 TISSUE SPECIFICITY OF ARTHROPOD TROPOMYOSIN J. Miyazaki, K. Yahata, Y. Fujiwara, and T. Hirabayashi. Inst. of Biol. Sci., Univ. of Tsukuba, Tsukuba. Tropomyosin is a protein family composed of many isoforms which were classified into two groups, high-molecular-weight and low- molecular-weight isoforms. We have inves-— tigated tissue specificity of tropomyosin in arthropods. Crustaceans had many tropo- myosin isoforms distributed in a tissue-— specific manner including low-molecular- weight isoforms in non-muscle tissues. Crustacean hearts had isoforms strictly specific to cardiac muscle. However, horseshoe crab tropomyosin showed no tissue specificity, but had low-molecular-weight isoforms in non-muscle tissues. Therefore, different results were obtained between representatives of two groups in arthro- pods. This prompted us to further investi- gate tissue specificity of tropomyosin in other arthropods. In this study, we used the beetle, centipede, and scorpion which belong to three different groups in arthro- pods and obtained the results as follows. 1) Tissue specificity of high-molecular- weight isoforms was recognized in all the examined arthropods except the horseshoe Crab. 2) Low-molecular-weight isoforms were found in non-muscle tissues in all the arthropods except the centipede in which non-muscle tissue was not examined. 3) Only crustaceans had cardiac-specific isoforms. eDNA CLONING OF BOMBYX LECTIN GENE AND ANALYSIS oH ITS EXPRESSION. K. Amanai-_, Suzuki-, S. Sakurai2, T. Ohtaki2. INatl. Inst. for Basic Biol., Gkazalct, Univ. of Kanazawa, Kanazawa, Univ. of Nishougakusha, Shounan. We have reported previously that the hemocyte is the major source of lectin in the hemolmph of the silkworm, Bombyx mori. In whole extracts of hemocytes from 5th instar larvae, 50 kDa protein which react with anti-hemolymph 350 kDa lectin monoclonal antibody have been detected. This protein exhibited the same biochemical properties as the hemolymph 350 kDa lectin. In order to reveal the function and structure of the hemolymph lectin and other lectins in various tissues, cDNA expression library of hemocytes was constructed and, from this library, Bombyx lectin cDNA clones were isolated using monoclonal antibody as a probe. Northern blotting analysis revealed that transcripts with an estimate size of 2.0 kb are expressed in hemocyte, Ovary and testis, and not in fat body. Furthermore, the same transcripts are expressed maternally in the early embryo. STRUCTURE AND EXPRESSION OF VITELLOGENIN GENE OF BOMBYX MORI K.Yano, M.Toriyama, S.Izumi and S.Tomino Dept. Biol. Tokyo Metropol. Univ., Tokyo Vitellogenin (Vg), a precursor of major yolk protein of Bombyx mori is a tetramer composed of each two molecules of non- identical subunits. We have cloned the mRNA and gene sequences for Vg and analyzed their structures. The Vg gene is composed of 7 exons interspersed by 6 introns, which encodes the 5.7 kb Vg mRNA. The primary structure of peptide deduced from the mRNA sequence comprised 1782 amino acid residues with molecular weight of 203.0k. Since amino-terminal primary structures of each Vg subunit are mapped on the deduced amino acid sequence, it is highly probable that two subunits of the B. mori Vg are derived from a single precursor peptide by cleavage. In microsomal fraction prepared from the female fat body, a putative precursor peptide with molecular weight of about 200k was detected by immunoblotting. Northern hybridization of the fat body RNA during development proved that the level of Vg mRNA sharply rises in female fat body at about a period of larval-pupal ecdysis. Developmental profile of the amount of Vg mRNA was consistent with that of primary transcript of the Vg gene. These results conclude that the Vg synthesis in the B. mori fat body is regulated in a sex- and stage-dependent Manner at the level of transcription. CONSTRUCTION OF cDNA LIBRARY FROM CRAYFISH PROCAMBARUS CLARKII. W.-K. Kang and Y. Naya. Suntory Institute for Bioorganic Research (SUNBOR), Osaka. Using oligo-dT column, the polyA-mRNAs were purified from different organs and tissues of several tens of crayfish. Three to five wg of mRNAs from four kinds of materials (muscle, liver, eyestalk and Y-organ) were used to synthesize cDNA by reverse transcriptase. Then, each cDNA was ligated to AZAPII vector and used to be packaged. The average length of inserts from each library was found to be about 1 kb after PCR (Polymerase Chain Reaction) that was carried out with SK and T7 primers (of pBluescript vector) and phage stocks of plaques as templates. By nucleotide sequencing, it was found to contain ribosomal protein S14 equivalent and actin-homolog in the library of muscle. Both clones show polyA signal (AATAAA) and polyA tail in their C-terminal nontranslated region. The clone 25-3, ribosomal protein S14 equivalent, contains an ORF (151 amino acids) and shows 88% identity in amino acid level and 77.3% identity in DNA level to the gene for ribosomal protein S14 of Drosophila. Actin-homolog clone, 25- 12, is lacking of N-terminal 150 bp, however, shows 87% and 78.2% identity to actin gene of shrimp in amino acid level and DNA level, respectively. 1142 Biochemistry PHOSPHORYLATION OF HISTONE H1 DURING DNA SYNTHESIS IN RAT HEPATOCYTES K.Asami, .Miyashita, N.Sawada and T.Kishimoto“. Dept. Biol. and Dept. Pathol., Sapporo Medical College, Sapporo 060, Lab. Cell and Develop. Biolyn, Tokyo Inst. Technol., Yokohama 227. Phosphorylation of histone Hl occurs at the time of DNA synthesis in primary cultured rat hepatocytes. Effects of the compounds which inhibited DNA synthesis in different ways were examined on phosphorylation of histone H1 in the hepatocytes. Sodium butyrate inhibiting progression of the cell cycle at Gl phase inhibited both DNA synthesis and histone Hil phosphorylation. Hydroxyurea and aphidicolin inhibit DNA synthesis at the S phase ina different way. They inhibited both DNA synthesis and H1 phosphorylation at a similar concentration. But removal of these compounds from the culture medium restored DNA synthesis in a little different way. Removal of hydroxyurea partially restored both DNA synthesis and the phosphorylation, while recovery of DNA synthesis after removal of aphidicolin was slow and phosphorylation of histone H1 was very low. Thus, phosphorylation of histone Hl seemed to proceed as DNA _ synthesis proceeds. To investigate the protein kinase responsible for histone Hl phosphorylation, histone H1 kinase activity of the cell extract was examined. The kinase activity increased at the start of DNA synthesis, and a _ part of the activity was bound to p13suel beads. COLD ADAPTATION IN DROSOPHILA QUALITAIVE CHANGES OF TRIACYLGLYCEROLS C. Katagiri’, and T. Ohtsu’. 1Biochem. Lab., Inst. of Low Temp. Sci., and “Dept. Zool., Fac. Sci., Hokkaido Univ., Sapporo. Triacylglycerols are the major fuel for basal metabolism during the winter in temperate species of the Drosophila melanogaster species group. Differential scanning calorimetry analysis revealed tuniantey thie staal nis) 14 aso neetemplenacdl tUaEeTS a Ont triacylglycerol were lower in species or strains adapted to cooler climate than those adapted to warmer climates. This phenomenon was correlated to the fatty acid compositions of the triacylglycerols; the proportion of unsaturated fatty acids in triacylglycerols was higher in the species or strains adapted to cooler climates. Furthermore, in the temperate species of the montium species group, the amount of saturated triacylglycerols was smaller than the value expected on the assumption that fatty acids are randomly distributed in the triacylglycerols, suggesting that non-random distribution of unsaturated fatty acids among triacylglycerols. This may facilitate the lowering of the transition temperature of triacylglycerols and hence may be related to the ability of Drosophila to cope with temperate climates. COLD RESISTANCE OF CALCIUM TRANSPORT ACTIVITY IN SARCOPLASMIC RETICULUM OF SCALLOP STRIATED ADDUCTOR MUSCLE ----- --- CALCIUM-TRANSPORTING PROTEIN OR THE MEMBRANE LIPIDS ? D. Sato and J. Nakamura. Biol. Inst., Fac. of Sci., Tohoku Univ. Different from the sarcoplasmic reticulum (SR) of rabbit skeletal muscle, calcium-transporting ATPase of scallop (Patinopecten yessoensis) SR has been found to be able to operate at low temperature (0-20 °C) and to be irreversibly inactivated at high temperature (37 °C). Here , we. preliminarily studied the role of lipid of scallop SR in the cold resistant property. Temperature profile of the scallop ATPase activity was compared with that of the rabbit ATPase activity in the presence and absence of excess amount of nonionic detergent (C]2E8). Thier temperature profiles were not significantly affected by the detergent. The cold resistance of scallop SR may be based on the ATPase protein itself. ATP ENHANCES CALCIUM-DEPENDENT CALCIUM OCCLUSION IN CALCIUM- TRANSPORTING ATPase OF SARCOPLASMIC RETICULUM FROM RABBIT SKELETAL MUSCLE J. Nakamura, Biol. Inst., Fac. of Sci., Tohoku Univ. In the previous meeting, we reported that 45Ca2+-40Ca2+ exchangeability of 45Ca bound to the calcium transport sites of Ca2+-ATPase of the sarcoplasmic reticulum is heterogeneous in the absence of ATP: Half of the bound calcium is [Ca2+]- dependent in a slowly exchageable (k <0.3 en). "occluded" state in the Ca2+-ATPase, and the other calcium is [Ca2+]-independent in a rapidly exchageable (k=0.3 s~!), "unoccluded" state. Here, the excangeability of the bound calcium was studied in the presence of ATP at 0°C. By the addition of ATP,the degree of the occlusion became higher (k<0.003 s-1). The unoccluded calcium was, however, not significantly affected. These results suggest that ATP more highly occludes calcium, already occluded in the absence of ATP. Biochemistry 1143 MOLECULAR CLONING OF Na*,K*-ATPASE a-SUBUNIT GENE IN THE SEA _ URCHIN, Hemicentrotus pulcherrimus. K. Yamazaki’, H.Kawashita’, C.Okamura’, kK. Mitsunaga-Nakatsubo’, K.Yamada?, K.Akasaka?, H.Shimada*® and I.Yasumasu’. ‘Dept. of Biol., Sch. of Educ., Waseda Univ., Tokyo, “Dept. of Radiation Res., The Tokyo Metropol. Inst. of Med. Sci., Tokyo, *Zool. Inst., Fac. of Sci., Hiroshima Univ., Hiroshima. We have isolated the cDNA of Na’*,K*- ATPase a-subunit from embryos of the sea urchin, Hemicentrotus pulcherrimus, and determined its DNA sequence. Northern blot analysis with the cDNA probe indicated that this mRNA was expressed maximally at the messenchyme blastula stage preceding the increase of the activity. And its mRNA was predominantly expressed in ectoderm cells. To analyse the regulatory mechanism of the expression of Na‘,K*’-ATPase in the stage and cell-lineage, we have tried to isolate and characterize the gene. Since the gene encoding Na*,K*-ATPase a- subunit seemes to be so long, two probes were prepared, one of which encodes N- terminal reagion and the other C-terminal reagion. Ten positive signal clones were isolated by screening 2.5 x 10° of sea urchin genomic library with these probes. And now we are analysing these clones. MITOSIS -SPECIFIC PHOSPHORYLATION OF DYNAMIN S. KOMATSU!? , HHOSOYA!S, T. SHIMIDZU4 ,M.INAGAKI° , M.|KEGAMI2, K.YAZAKI! 1Tokyo Metro. Inst. Med. Sci., 2Tokyo Agri. Univ ., 3 Hiroshima Univ is 4Re ser. Inst. Polym. and Tex. Tokyo Metro. Inst. Geront . Cytosol preparations form mitotically selected Hela cells exhibited much higher kinase activities to phosphorylate a 100kD microtubule cross-linking protein, dynamin, than the preparations from non- mitotic cells. Based on substrate specificity and lack of sensitivities to characteristic activators or inhibitors, this dynamin kinase activity appears to be distinct from A-kinase, cGMP dependent protein kinase, C- kinase or Ca2+-calmodulin dependent protein kinase Il. Purified cdc2 kinase (p34°9°?-cyclin B complex) phosphorylated dynamin. Microtubule-binding activity of phosphoyrlated dynamin was reduced. FURTHER CHARACTERIZATION OF DYNAMIN. T. Shimizu", T. Kokubu', S. Ohashi’, Y.Y. Toyoshima?, and T. Miki-Noumura?. ‘Res. Inst. Polym. Text., Tsukuba, Ibaraki, and 2Ochanomizu Univ., Dept. Biol., Bunkyo, Tokyo, Japan. Dynamin (100k protein) isolated from rat brain high speed supernatant exhibited GTPase activity higher than other NTPase activity as reported (Shpetner & Vallee, (1992) Nature 355, 733). The GTPase activity depended upon pH, being high at an acidic pH (5.5 to 6) and low at pH 7 or higher. It decreased upon increase in ionic strength and was quite low at 0.2 M NaCl or higher. At PH 6.7 in the absence of NaCl, the apparent Km for GTP was 44M. Dynamin did not exhibit an initial phosphate burst with 304M GTP or ATP. Its GTPase activity was enhanced by microtubules (MTs) as reported by Shpetner and Vallee. Our previous dynamin preparation was shown to contain up to 0.03% kinesin by immunoblotting with poly- clonal kinesin antibodies. When kinesin contamination was eliminated by the use of DEAE-Sephacel chromatog- raphy, the resultant dynamin poorly translocated MTs. Adaition of the original contaminating level of kinesin was sufficient to restore MT movement. The previous obser- vation of MT translocation by dynamin preparation, there- fore, was likely to be due to kinesin. Thus, dynamin does not interfere with the kinesin motility, but even supports low density kinesin motility like casein or cytochrome c. IMMUNO-ELECTORON MICROSCOPIC STUDIES ON BETA- AND GAMMA- DYNEIN HEAVY CHAINS FROM TETRAHYMENA CILIA. D 1 E.Masuyama', M,Ishida“, K.Nakamura’, and Y.Shigenaka?. 1 Dep, gf Living Sci.,Univ. of Hiroshima Women's, Fac. Integr. Arts & Sci., Univ. of Hiroshima, Hiroshima, When Tetrahymena Ciliary 22S dynein was digested wi ermolysin, two protease- resistant_fragments which were designated TH-1 and TH-2 were obtained.The fragments were characterized by high ATPase acti- vities and consisted of two principal polypeptids (TH-1: 173K and 80K, TH-2: 173K and 120K). Polyclonal antibodies against these dynein fragments were prepared and used to investigate the Origin of the fragments and the Structural relationships between the polypeptides of the fragments. By immuno- Dlotting analysis, anti-TH-1 and anti-TH- 2 crossreacted strongly with the gamma- and beta-heavy chain of 22S dynein, respectively. The two antibodies recognized only the 173K polypeptide of each fragment. We also used these purified antibodies to further investigate the location of beta- and gamma-dynein heavy chains within the axoneme. In immuno-electron micrographs we observed that these antibodies labelled with Protein A-gold particles attached to outer arm region. Furthermore the two gold particles located in Slightly differing positions of the outer arm. 1144 Biochemistry MOLECULAR CLONING OF CYTOPLASMIC DYNEIN cDNA K. Ogawal and T. Shimizu2. 1Natl. Inst. for Basic Biol., Okazaki and ?Res. Inst. of Polym. Text., Tsukuba. Bovine brain cDNA library was screened by affinity-purified anti-MAPI1C (cytoplasmic form of dynein) antibody. One clone among 14 positive candidates gave a signal corresponding to 15 kb poly(A)+RNA in Norther blot. The signal was detected in human brain, heart, muscle and liver poly(A)+RNA. Sequencing showed that this clone (AJ073) encodes for the carboxy terminus of a protein. Homology search by MAILBLAST program picked up the carboxy terminal sequence of dynein B heavy chain of sea urchin. Therefore, we consider that this clone corresponds to the 3'- terminal part of cytoplasmic dynein heavy chain cDNA. CENTRAL-PAIR MICROTUBULES OF CHLAMYDOMONAS FLAGELLAR AXONEMES HAVE DYNEIN ATPase OR NOT. N.Ishii and T.Miki-Noumura. Department of Biology, Ochanomizu Univ., Ohtsuka, Tokyo. Little is known about the function of central-pair microtubules(central-pair MTs) in flagellar movement. We isolated central- pair MTs from Chlamydomonas flagellar axonemes (Hosokawa and Miki-Noumura,1987) and examined the polypeptide composition, attempting to make clear existence of dynein ATPase in them. We examined here the polypeptide composition in 7.5% SDS-polyacrylamide gel electropholesis. The central-pair MTs consisted of about 25 polypeptides, of which major one was tubulin. Using 4% and 3-5% gradient gel, we examined the bands in high=-molecular-weight region. Any bands corresponded to dynein was not found in the polypeptides of central-pair MTs. Next, we carried out immunological analysis of dynein bands, using antibodies against Tetrahymena and Chlamydomonas dynein heavy chain. The band in the high- molecular-weight region of the central-pair MTs did not react with these antibodies. Measurement of ATPase activity also showed no activity in central-pair MTs. The results, analysis of polypeptide bands, Western blot, and ATPase measurement, suggest that the central-pair MTs may not have dynein ATPase. KINESIN-DEPENDENT MOTILITY AND MICRO- TUBULE STRUCTURES. S. Kamimura! and E. Mandelkow~. Dep. Biol., Coll. Arts & Sci., Univ. Tokyo, Tokyo and *Max-Planck-Unit, Struct. Mol. Biol., c/o DESY, Hamburg, Germany Microtubules are build of tubulin subunits assembled into hollow cylinders which consists of parallel protofila- ments. Thus, kinesin interacting with a microtubule could do so either with one or several tubulin units. This makes it difficult to determine the structural requirements for the interaction One way to approach the problem is to alter the surface lattice. This can be done in several ways. Protofilaments can be exposed on their inside (C-tubules), they can be made antiparallel (zinc sheets), or they can be rolled up (duplex tubules). We have exploited this polymor- phism to study how kinesin attached to a glass surface interacts and moves the various tubulin assemblies. About 40% of C-tubules glided similarly to microtubules. Zinc Sheets showed motility but the motion was dis-— continuous. Duplex tubules did not move. This indicates (a) that a full cylindri- cal closure of microtubule is not neces- sary for movement, and (b) that the inside surface of microtubules does not support motility. The data can be ex- plained by assuming that the outside of one protofilament represents the minimal track for kinesin. (ref. ) Kamimura, S. & E. Mandelkow (1992) J. Cell Biol., 118:865-875. STUDIES ON TUBULIN ISOFORMS INVOLVED IN AXONEMAL FUNCT ION OF TETRAHYMENA CILIA. K. Nakamura E. Masuyama’, S. Wada“, and M. Okuno*. ‘Dept. of living sciences, iroshima Women's Univ., Hiroshima, Dept. of Biol., Col. of Arts and Sci., Tokyo Univ. Tokyo SS ea TOTS RONEM ET DT OTE TI consists of two subunits (alpha and beta-tubulins). Each tubulin subunit is a heterogeneous protein composed of a mix- ture of isotubulins. In order to investi- gate PHO CEHON La SSten SHS a SSDs lins on ciliary movements of [etra eM, it is essential to identify ese isoforms and to study their roles in axonemal movement. We have presented two novel methods which are useful for these analysis. One is the native gel electrophoresis with agarose and acryl- amide gel. Using this method and immunoblotting techniques with anti- tubulin antibodies, we have shown that tubulin molecules can interact with several proteins including dyneins. The other method is two-dimensional electro- phoresis designed for the separation of tubulin isoforms. This method uses 1so- electric phocusing at the second dimens- ion. At least 10 tubulin isoforms from Tetrahymena axonemes can be detected by this new two-dimensional electrophoresis. Using these electrophoretic systems, we have suggested that a beta-isotubulin, tentatively called as Ib, can interact with axonemal proteins including dyneins. Biochemistry 1145 PURIFICATION OF PROTFASOMES FROM SALMONID FISH SPERM AND THEIR LOCALIZATION ALONG SPERM FLAGELLA. K. Inaba, M. Morisawa and Y. Akazome. Misaki Marine Biological Station, Faculty of Science, University of Tokyo, Miura, Kanagawa. Motility of demembranated sperm in salmonid fish is inhibited by chymotrypsin inhibitors in an ATP-dependent manner. We purified chymotrypsin-like proteases from chum salmon sperm, with the molecular masses of 950 and 650 kD. Some enzymatic properties and molecular shape of the 650 kD protease showed that this protease was a multicatalytic proteinase (proteasome) as is well known to participate in ATP- dependent degradation of ubiquitinated proteins. We prepared polyclonal antibody against purified 650 kD proteasome. This antibody recognized mainly the 29 and 28 kD subunits of proteasome. Using immuno- fluorescent microscopy, we examined the subcellular localization of proteasomes in sperm. The result showed that proteasomes are located predominantly in sperm flagella. Interestingly, the anti-proteasome antibody did not stain overall portion of sperm tail but showed patches or somewhat periodical staining patterns along sperm flagellum. These results suggest that activation of proteasomes at regular intervals on sperm Elagellum causes ATP-dependent conversion of microtubule sliding to flagellar bending, resulting in the regulation of sperm motility. MITOSIS-SPECIFIC PHOSPHORYLATION OF MICROTUBULE ASSOCIATAED PROTEINS F. TAKAHASHI!» , H. HOSOYA!:3 , K. KOBAYASHI‘ , M. INAGAKI? , T. ARAF. , K. YAZAKI! 'Tokyo Metro. Inst. Med. Sci 42 Tokyo Sci. Univ ., 3 Hiroshima Univ a 4 Jikei Univ 3 “Tokyo Metro. Inst. Geront . Mitotic Hela cell extracts exhibited much higher kinase activities to phosphorylate brain microtubule associated proteins (MAP2 and MAPtc) than the extracts from non-mitotic cells. This MAP2/MAP1c kinase activity appears to be distinct from A-kinase, cGMP-dependent protein kinase, C-kinase or Ca¢t- calmodulin dependent protein kinase Il. Purified cdc? kinase (p34°9°?_cyclin B complex) phosphorylated MAP2 and MAP‘1c. PROTEIN PHOSPHORYLATION IN POLYMORPHO- NUCLEAR LEUCOCYTES RELATED TO THE SIGNAL TRANSDUCTION OF CHEMOATTRACTANT STIMULATION M.Shibata!, T.Ohoka!, Y.Yamakawa2, S.Mizuno2 and K.Suzuki2. !Dept.Biol., Fac.of Sci., Tokyo Metropol. Univ. and 2Nat. Inst. of Health. Human polumorphonuclear leucocytes (PMN) are known to be activated by various kind of chemoattractants, LUCT/IL-8, leucotriene B4 or FMLP, lead to migration, phagocytosis or O27 production. To elucidate the molecular mechanism of signal transduction, phosphorylated proteins are analyzed by two- dimensional electrophoresis and autoradiography, after the 32P- labelling of intact PMN and stimulation with FMLP . Protein subunits having molecular weight of 82, 66, 64, 58, 55 and 50 kDa were able to be detected, the marked phosphorylation was observed with 64 kDa proteins. One of the 64 kDa proteins are revealed to be phosphoglucomutase, this phosphorylation was stimulated in presence of a micromolar level of glucose, by a mechanism including hexokinase and substitution of 32P- phosphate from glucose-6-phosphate to active site seine. The other phosphorylatable 64kD protein (p64) was detected after FMLP stimulation, having a isoelectric point (pl=5.3) different from phosphoglucomutase. The pl shifted after phosphorylation from 5.3 to more acidic side forming pp64. The FMLP- stimulated phosphorylation was time-dependent and saturated within 5 min., the maximum stimulation was achieved with 10 nM FMLP. Phosphoamino acid analysis of the pp64 revealed the phosphorylation of the serine residue. Staurosporine (100 nM) and W-7 (100 mM) significantly inhibited the phosphorylation, H-7 slightly inhibited, H-8 and herbimycin did not affect. These data suggested that protein kinase C and calmodulin like protein(s) are concerned. From the purification studies of p64 and by amino acid analysis, p64 was identified as ]-plastin, one of the leucocyte specific proteins. Phosphorylation of this may play a role in cytoskeletal reorganization of PMN. INDUCTION OF AMOEBOID MOVEMENTS IN THE SEA URCHIN EGG BY PROTEIN PHOSPHORYLATION. -Mabuchi-, A.Morimatsu and H.Tosuji~. Dept. of Biol., Coll. of Arts and Sci., Winsk\7o Ore YON WO, Dept. Biophys. and Biochenm., ac. of Sci.,Univ. of Tokyo, Tokyo and ~Dept. of Biol., Fac. of Sci., Kagoshima Univ., Kagoshima. Calyculin A, an inhibitor of protein phosphatases type 1 and type 2A, induces cleavage-like changes in the unfertilized sea urchin egg (Tosuji et al., Proc. Nat. Acad. Sci. USA, in press). It induces formation of protrusion or amoeboid movements in the fertilized eggs. We recently found that calyculin A induces the amoeboid movement also in the unfertilized eggs at appropriate concentrations. These eggs usually did not cleave, but cleavaed by the addition of IBMX or TPA. TPA alone could induce the movements in the fertilized eggs. Tautomycin, which has been known to be another inhibitor of protein phosphatases type 1 and type 2A (Hori, M. et al., FEBS Lett., 285: 145-148, 1991) also induced the movements in the fertilized sea urchin eggs. These results strongly suggest that protein phosphorylation is involved in the cytoskeletal organization in the sea urchin egg. 1146 Biochemistry EFFECTS OF WORTMANNIN, AN INHIBITOR OF MYOSIN LIGHT CHAIN KINASE, ON THE CELL DIVISION OF CULTURED CELLS OR SEA URCHIN EGGS H.HOSOYA |: 2, ETAKAHASHI 2° ©, M.INAGAKI 3, Y.MATS UDA 4, Y.NONOMURA ®, TARAI ©, E.NAKAMURA 2: 7 H.SAKAI ”, K.YAZAKI 2 THiroshima Univ., 2 Tokyo Metro. Inst. Med.Sci., STokyo Metro. Inst. Geront., 4kyowa Hakko Inc., Suniv. Tokyo, °Tokyo Sci. Univ.,/Japan Women's Univ. We investigated roles of myosin light chain kinase (MLCK) on cell division by using a specific inhibitor of MLCK, wortmannin. Wortmannin inhibited cytokinesis with a half maximal inhibition at 1-2 pM. The nuclear division was accomplished normally at 10u.M where the cytokinesis was completely blocked. Fluorescent staining of actin filaments with rhodamine- labeled phalloidin revealed that the contractile ring was not formed in the cleavage-inhibited eggs. Wortmannin did not inhibit cdc2 kinase activity. ELECTRON MICROSCOPY OF 1200 kDa FRAGMENT OF a -CONNECTIN FROM RABBIT SKELETAL MUSCLE J. Suzuki, S. Kimura and K. Maruyama. Dept. Biol., Fac. Sci., Chiba Univ., Chiba. a -Connectin, an elastic protein, of striated muscle is hydrolyzed into £ -connectin and 1200 kDa peptide in situ, when exercised muscle is for about 15 h kept at AT. In the present work, rotaty shadowed images of the isolated 1200 kDa peptide were examined under an electron microscope. There were a main peak at 350 nm and a smaller peak at 170 nm in the length distribution. The width of the former was 3 nm and that of the latter was 4.5 nm suggesting that the latter was folded form of the former. It is concluded that the 1200 kDa peptide was a filament 350 nm long and 3 nm wide. Under the same conditions, £-connectin was observed as a filament 750 nm long. The molecular mass of £-connectin has been estimated to be approximately 2700 kDa. The ratio of molecular mass (1200 : 2700 = 1 : 2.3) was similar to that of molecular length (350 : 750 = 1 : 2.1). EPTOPE MAP OF ANTIBODIES TO CONNECTIN CHICKEN STRIATED MUSCLE. Y. Kawamura, Y. Ohtani, S. Kimura and K. Maruyama. Dept. Biol., Fac. Sci., Chiba Univ., Chiba. Connectin molecule,sMW,~3000,000,links the myosin filament to the Z line a sarcomere of vertebrate striated muscle.Immunoelectron microscopy reveals striation in a sarcomere due to the binding of an antibody to a specific epitope in connectin, because connectin filaments run parallel with the mainaxis of a sarcomere. We have examined the specific epitope sites of 11 kinds of monoclonal and 1 kind of polyclonal antibodies to connectin in chicken breast muscle. The results obtained were compared with those reported by K.Weber's and J.Trinick's groups. We have also compared the epitope locations in chicken breast muscle with those in chicken leg muscle. There were the cases where the epitope region was different between the two types of chicken skeletal muscle . SECONDARY STRUCTURE OF MUSCLE ELASTIC PROTEIN CONNECTIN AS REVEALED BY RAMAN SPECTROSCOPY. Y. Nakauchi', K. Maruyama’, S. Toyama?, and I. Harada’. ‘Dept. of Biol., Fac. of Sci., Chiba Univ., Chiba, *Fac. of Pharmac., Tohoku Univ., Sendai. It has been reported that muscle elastic protein connectin consists of 60% B-sheet and 30% f-turn based on circular dichroism measurements (Maruyama er a/., 1986). The present work using Raman spectroscopy extends the previous work. Ultraviolet resonance Raman spectra of B-connectin in solution indicated the presence of B-sheets and hydrogen-bonded irregular structures. Infrared spectra showed an abundance of B- sheets in B-connectin fiber, some of which were aligned with their main chain axes parallel to the fiber axes. Thus, the B-spiral structure proposed for elastin is improbable in connectin. We have also obtained infrared Raman spectra of a 1200 kDa peptide fiber. The 1200 kDa peptide which constitute elastic portion of connectin in situ was also rich in B-sheet structure. We propose a model of connectin structure based on the repetitive amino acid sequence of connectin (Labeit er a/., 1990, 1992) and also our finding. Biochemistry 1147 CONNECTIN FILAMENTS OF GTANT SARCOMERES OF CRAYFISH CLAW MUSCLE T. Manabe, H. Higuchi?, Y. Kawamura’, S. Kimura’, and K. Maruyama’. 'Dept. of Biol., Fac. Sci., Chiba Univ., Chiba, "Dept. of Physiol., Jikei Univ. Sch. Med., Tokyo In giant sarcomeres (10 jum at rest of crayfish claw muscle), there is 3000 kDa protein in addition to 1200 kDa projectin. The rotary shadowed image of the isolated 3000 kDa protein was very thin filament, maximumly 0.9 jum long. When skinned opener fibers were stretched up to 14 um sarcomere length, resting tension developed, but on mild treatment with trypsin resulted in the decrease in tension development accompanied by splittin g of the 3000 kDa protein. Immunofluorescence microscopy using anti-3000 kDa protein antibodies revealed that the protein linked the Z line to the edge of the A band and it was movable devending on the sarcomere length. From the present study, it is concluded that the 3000 kDa protein in the giant sarcomeres of crayfish craw muscle corresponds to connectin, elastic protein of vertebrate skeletal muscle. LOCALIZATION OF TWO MYOSIN ISOFORMS IN PERITONEAL NEUTROPHILS FROM GUINEA PIG. H.Takano-Ohmuro, M.Endo and K.Kohama* Dept. of Pharmacol.Fac. of Med., Univ.of Tokyo, Tokyo, and “Dept. of Pharmacol. Fac. of Med., Gunma Univ. Maebashi We previously reported that peritoneal neutrophils of guinea pig has two isoforms of myosin Il differed in the heavy chain (HC) (Zool. Sci., 1991) To localize the isoforms, we subjected the cell lysate of the neutrophils to the centrifugation at 120,000 g for 70 min. The supernatant was used as cytosolic fraction (Cyt). The precipitate (ppt) was suspended in an isotonic solution and the suspension was centrifuged at 10,000 g for 30 min. The ppt after 10,000 g centrifugation was used as 10K fraction (10K) and the suspension was further subjected to the centrifugation at 120,000 g for 70 min. The ppt was used as 120K fraction(120K). When Cyt, 10K and 120K were subjected to native pyrophosphate gel electrophoresis, they showed only one band. The mobility of myosin in cytosol did not coincide with that in 10K. From peptide Mapping and immunoreactivity, the difference could be explained by the difference in HC of both myosin. Myosin of Cyt comigrated with that of 120K. Our similar analyses showed that myosin of Cyt was identical in HC and light chains with myosin of 120K. The reason to explain the difference in the localization between Cyt and 120K remained to be examined. MYOSIN FROM THE SEA SPONGE, Halicondria okadai N. Kanzawa and K. Maruyama. Dept. Biol., Fac. Sci., Chiba Univ., Chiba. We have attempted to purify myosin from the sea sponge, Halicondria okadai. There has been no report on sponge myosin. Sea sponge myosin consisted of 220 kDa heavy chain and two species of light chains, 18 and 21 kDa. Two headed structure was observed under electron microscope. The K-EDTA activated ATPase activity was as high as 0.5 imole/mg/min, but the Mg?*-ATPase activity was low and only slightly enhanced by rabbit F-actin. Solubility of sea sponge myosin was lower than rabbit skeletal myosin: the former was only soluble by 40% at 0.3 M KCl as compared to the latter (by 80%). Sea sponge myosin formed thick filaments, 0.5-1 [1m long, at 0.3 M KCl, where rabbit skeletal myosin formed much smaller oligomers. ON THE TISSUE-SPECIFIC DISTRIBUTION OF TROPOMYOSIN ISOFORMS IN CRUSTACEAN MUSCLES T.Ishimoda-Takagi, S.Nakano, K.Hino and M. Itoh. Dept. of Biol., Tokyo Gakugei Univ., Tokyo. We have previously shown that several species of tropomyosin (TM) isoforms were included in the spiny lobster and American lobster. Distribution of the TM isoforms was tissue specific, and tissue-specifici- ty of the TM isoforms correlated consider- ably with the types of muscle fiber. In order to investigate generality of the tissue-specificity of TM isoforms in crus- tacean decapod, we examined tissue speci- ficity of TM isoforms in the prawn, Panaeus japonicus, and the crab, Erimacrus isenbeckii. Four TM isoforms were present in the prawn muscles. Distribution pat- tern of TM isoforms was similar to that of spiny lobster. The TM component a was a main component of the cephalo-thoracic and abdominal muscles. The component b waS mainly observed in the leg muscle. The component c was mostly involved in the dorsal muscles. Heart muscle contained a heart-specific TM isoform. The TM com- ponent corresponding to b was also observ- ed in the crab leg muscle. Furthermore, crab heart muscle contained heart-specific TM isoforms. However, the TM isoforms corresponding to the components a and c, which were involved in the muscles to move abdomen, could not be identified in the crab of which abdomen is greatly reduced. 1148 Biochemistry DNASE I-BINDING PROPERTY OF CHIMERIC ACTINS CONSISTING OF TETRAHYMENA ACTIN AND DICTYOSTELIUM ACTIN M. Hirono!, K. Sutoh?2, Y. Watanabe? and Tadao Ohno!. !RIKEN Cell Bank, Institute of Physical and Chemical Research, Tsukuba, 2Department of Pure and Applied Sciences, College of Arts and Sciences, University of Tokyo, Tokyo, “Institute of Biological Sciences, University of Tsukuba, Tsukuba. Tetrahymena actin has a unique primary structure and some unusual properties such as lacking a DNase I-binding activity. To know which sequence of Tetrahymena actin corresponds to those unusual properties, we constructed two chimeric actin genes from Tetrahymena actin gene and Dicty- ostelium actin gene, and those genes were expressed in Dictyostelium cells. One chimeric actin (designat- ed as Tet84Dic) was contracted from N-terminal portion (1-83 residues) of Tetrahymena actin and C- terminal portion of Dictyostelium actin (84-375 residues). The other chimeric actin (designated as Dic84Tet) was constracted from N-terminal portion of Dictyostelium actin C-terminal portion of Tet- rahymena actin connecting at the same site as Tet84Dic. Using DNase I-affinity column, we re- vealed that one of the chimeric actin gene product, Tet84Dic, was not retained in the column whereas intrinsic actin was retained. On the other hand, we also found that Dic84Tet was retained in the DNase I column. In conjunction with our previous data that Tetrahymena actin does not interact with DNase I, we suggest that the binding site of DNase lina ubiquitous actin is located in N-terminal region (residues 1-83). STRUCTURAL CHANGES OF ACTIN FILAMENTS INDUCED BY GONIODOMIN A S. Watanabe’, K. Maruyama!, K. Furukawa’, and Y. Ohizumi?. Dept. Biol., Fac. Sci., Chiba Univ., Chiba, 7Fac. Pharm. Sci., Tohoku Univ., Sendai. Goniodomin A purified from Goniodoma pseudogoniaulax increases the actin—activated Mg?*-ATPase activity of myosin at 107M and inhibits it at 10° M [ Ohizumi,Y., Folia pharmacol. japon. 100, 259-269 (1992)]. In the present study, the effect of goniodomin A on actin was investigated. Goniodomin A, 10° M, hardly affected fluorescence intensity of pyrenyl actin (Ex 365nm; Em 407nm), but at 3 x 10° M the intensity was remarkably dropped ( to 8% of the control). In the presence of 3 x 10°M goniodomin A, actin filaments were associated with each other to form a gel. Therefore, the aggregates were completely sedimented by centrifugation for 15 min at 12,000 x g. The present results sugggest that goniodomin A stoichiometrically bound to actin monomers in the actin filament and conformational changes of actin induced by goniodomin A may affect its interaction with myosin. A HIGH MOLECULAR WEIGHT PROTEIN FROM CHICKEN GIZZARD SMOOTH MUSCLE. A. Terasaki and K. Ohashi. Dept. of Biol., Fac. of ‘Sci., Chiba Univ., Chiba. Myofibrils of chicken gizzard smooth muscle were extracted with a high salt solution. Successively the residue was extracted with a low salt alkaline solu- tion (pH 9.0). The high molecular weight protein, whose molecular weight was more than 400,000, was not extracted from the residue at 4 °C but 37 °C. The extract was fractionated by ammonium sulfate. The precipitate of 20% saturation was suspend- ed in a solution containing 20 mM NaCl (pH 8.0) and dialyzed against the same buffer. A small part of the high molecular weight protein could be redissolved. The solu- tion was applied on a hydroxylapatite column. The high molecular weight pro- tein was eluted at the phosphate concen- tration of 0.15 M. The eluant was chro- matographed on a DEAE-Cellulofine column. The high molecular weight protein was eluted at the NaCl concentration of 0.25 M with a small amount of actin. This frac- tion was rechromatographed on a DEAE- Cellulofine column in the presence of 4 M urea to remove actin, The yield was 0.05 mg from 100 g of smooth muscle, The antibody against the high molecular weight protein stained the adhesion plaques of a cultured cell. Immunoblot analysis showed that the high molecular weight protein were present in smooth (gizzard), skeletal (breast), and cardiac muscles. 33KD PEPTIDES FROM CHICKEN GIZZARD SMOOTH MUSCLE. H.Nakagawa, M.Ishihara and K.Ohashi. Dept. of Biol., Fac. of Sci., Chiba Univ., Chiba. We prepared a pair of peptides, which were associated with actin and desmin, from chicken gizzard smooth muscle. Their apparent molecular weight on SDS-PAGE was approximately 33,000. Myofibrils of chick- en gizzard smooth muscle were extracted with Hasselbach-Schenider’s solution. Solid ammonium sulfate was added into the extract to 70% saturation. The supernatant was dialyzed against 0.1 M KCl. A small amount of precipitation was coilected by centrifugation and solubilized in 0.6 M KCl. The main components of this solution were’ the 33kD peptides. The yield of the 33kKD peptides was 0O.5mg from 100g of smooth muscle. The two peptide bands on SDS-PAGE were stained purple with CBB-R250 and easily distinguishable from the other blue peptide bands. The electron micro- scopic observation showed that the 33kD peptides bundled F-actin and formed dense aggregates of desmin filaments. The 33kD peptides were coprecipitated with F-actin and desmin filaments by centrifugation at 15,000 rpm. When G-actin and desmin proto- filaments were assembled in a _ solution containing the 33kD peptides at the same time, F-actin bundles decorated with many dense aggregates of desmin filaments were observed under an electron microscope. Biochemistry 1149 LOCALIZATION OF A 52KD PEPTIDE FROM CHICK- EN GIZZARD SMOOTH MUSCLE IN VARIOUS TYPES OF CELLS. H. Nakagawa, M. Nishimura, and kK. Ohashi. Dept. of Biol., Fac. of Sci., Chiba Univ., Chiba. A 52kD peptide, purified from chicken gizzard smooth muscle, had a weak interac— tion with actin fragments and localized at the leading edges of a moving cultured smooth muscle cell. Immunoblot analysis showed that the 52kD peptide was present in various types of tissues and cells of chicken, i.e. skeletal (breast muscle), cardiac, and smooth (gizzard) muscles, and nonmuscle tissues or cells (brain, small intestinal epithelial cells, and lympho- cytes). By immunofluorescence method, the antibody against 52kD peptide was strongly stained the junctional complex of an isolated small intestinal epithelial cell and also seemed to stain the costameres of a skeletal muscle fiber. In these cells, immunofluorescence of anti-52kD peptide antibody was observed at the vicinity of the plasma membranes. In cultured smooth muscle cells, the distribution of fluores- cence partially coincided with the stress fibers and cell edges. In addition, the distribution had a granular and a vesicu- lar component which are seen in both the perinuclear and peripheral regions of the cell. In cultured cardiac muscle cells, the vesicular components were rich in the contact regions of neighboring cells. CLONING AND SEQUENCING OF cDNA ENCODING CHICKEN PROFILIN. R. Mochizuki, N. Minami, H. Abe and T. Obinata. Dept of Biol., Fac. of Sci., Chiba Univ., Chiba. In the previous studies, we isolated and characterized an actin-binding protein of 16 kDa (profilin) from embryonic chicken skeletal muscle, which sequesters monomeric actin in the cytoplasm, and have demonstrated that this protein regulates differentially the assembly of cytoskeletal (8,y ) and sarcomeric (a ) actins during myofibrillogenesis. In the present study, we cloned cDNA encoding the profilin from the cDNA library prepared with poly(A ) RNA from embryonic chicken skeletal muscle by using specific antibodies as probes, and determined the sequence. The deduced amino acid sequence was compared with previously described non-muscle profilin sequences. The chicken sequence exhibited about 77 % identity with those of profilins from human and bovine non-muscle tissues. The similarity of the chicken profilin with those from echinoderm, amoeba, and Physulm was very low; 19 Z% identity with Clypeaster profilin and 17 % identity with Physulm profilin. Nevertheless, fifteen residues conserved beyond the species were observed. Northern RNA blot analysis showed that the expression of profilin is down-regulated in parallel with that of cytoskeletal (8 , Y ) actins during deve- lopment of chicken skeletal muscle. PURIFICATION OF DOMAIN-SPECIFIC ANTIBODIES USING AFFINITY CHROMATOGRAPHY AND SDS- PAGE. Y. Yoshihara and M. Kuroda. Dept. of Biol., Fac.of Sci., Shimane Univ., Matsue. By a combination of SDS-PAGE and affinity chromato: graphy, we have developed a new method to isolate domain- specific antibodies from polyclonal antiserum. Chymotryptic- digests of @—actinin was separated by SDS-PAGE. The 32K- head and 55K-rod bands were cut out from CBB-stained gels respectively. Gel pieces containing 32K or 5SK domain were placed on CNBr-activated Sepharose packed in a short glass tube. The 32K or 55K polypeptide was electro-phoretically eluted from gel pieces onto the CNBr-activated Sepharose. Then the Sepharose gel was used as an immuno-absorbant. Undigested 104K subunit of a-actinin was also treated in the similar way. Goat anti-chicken skeletal @-actinin antiserum was applied to these three affinity columns. The anti-55K IgG that had eluted from the 55K-rod conjugated affinity column reacted with all the rod-containing subfragments in chy- motryptic digests but did not cross-reacted with 32K-head fragments at all. Using 1D-peptide map of the 55K-rod domain, we assayed the epitope of the anti 55K IgG between Pro360-Arg502 of a@—actinin. Meanwhile, anti-32K IgG reacted slightly with some of the rod containing subfragments beside 32K-head. We assume that the anti-32K IgG recognized head-rod junction of @-actinin. ACTIN CYTOSKELETON IN CULTURED MYOGENIC CELLS TRASNFECTED WITH COFILIN cDNA. S. Ono, H. Abe and T. Obinata. Dept. of Biol., Fac. of Sci., Chiba Univ., Chiba. Cofilin is an actin-binding protein which modulates actin assembly in both muscle and non-muscle cells. To better understand the role of cofilin in myogenic cells, cofilin cDNA cloned into an expression vector was transfected into mouse C2 myogenic cells. We examined the effects of cofilin over-expression on the cellular morphology and actin assembly in C2 cells. Under standard culture conditions, alteration in actin filaments was not clear as examined by phalloidin- staining, although increased cofilin expression was confirmed by staining the cells with anti-cofilin antibody (MAB-22), but the cellular shape was slightly changed to become flat. When the transfected cells were incubated in the medium containing 10 % dimethylsulfoxide (DMSO), actin filaments were remarkably decreased and cofilin/actin rods were formed in the nuclei. More drastic results were obtained by heat shock, 1 hr at 43 C; actin filaments were almost completely disrupted and huge actin/cofilin rods were generated in the cytoplasm and the nuclear rods as well. A calmodulin inhibitor, W/, also caused disassembly of actin filaments. These results suggest that cofilin become active in the cells treated by heat shock or DMSO. 1150 Biochemistry PHOSPHORYLATION AND DEPHOSPHORYLATION OF COFILIN IN CULTURED FIBROBLASTIC CELLS. H. Abe, K. Okada, and T. Obinata. Dept. of Biol., Fac. of Sci., Chiba Univ., Chiba. Cofilin, a 20 kDa actin-regulatory protein, seems to be profoundly involved in the assembly and disassembly of actin filaments in cells. When cultured cells are exposed to heat or incubated in a medium containing 10 % dimethylsulfoxide (DMSO), intranuclear actin-cofilin rods are formed. Recently, it was demonstrated that dephosphorylation of cofilin accompanies its nuclear localization in the cells treated with heat or DMSO. In this study, we examined the phosphorylation state of cofilin and its cellular localization in Balb/e 3T3 and rat 3Y1 cells in a synchronous’ culture. When cells were arrested at GO phase by serum starvation, the amount of phosphorylated cofilin was significantly decreased, although about 50 % of cofilin was phosphorylated in an asynchronous culture. Most of cofilin was present in the cytoplasm of GO-arrested cells, as examined by immunofluorescence microscopy. The intranuclear actin-cofilin rods were induced within 15 min by treating the cells at the GO state with 10 % DMSO, but dephosphorylation of cofilin was not detected in this case. Therefore, we conclude that dephosphorylation of cofilin and its nuclear localization are independent phenomena. A STRUCTURAL STUDY OF A COFILIN ACTIVE SITE ANALOG PEPTIDE. Susumu Kotanil, Yutaka Ito’, Yutaka Muto2, Eisuke Nishida@, and Shigeyuki Yokoyama“. IDept. of Biochem. Eng. Sci., Kyushu Inst. Tech., lizuka, and Dept. of Biophys. Biochem., Fac. Sci., Univ. of Tokyo, Tokyo. Cofilin is a low molecular mass actin-— regulating protein. A cofilin active site analog dodecapeptide was analyzed by a series of two-dimensional 1H-NMR methods to determine the conformation. The pep- tide proton resonances were unequivocally assigned. The peptide alone in solution contained no detectable secondary struc- ture. However it takes a specific confor- mation when mixed with actin, as revealed by the transferred nuclear Overhauser effect (TRNOE) measurements. The intra- residue TRNOE’s were observed for all the 12 amino acid residues, suggesting that the dodecapeptide interacts with actin and PIPo along its entire length. The confor- mation of the cofilin active site was calculated using inter-residue and intra- residue NOE data. The dodecapeptide N- terminal five residues are folded into a turn structure, while the C-termical port- ion is extended. DESMIN NET SURROUNDS MYOFIBRILS: DETECTION BY IMMUNOELECTRON MICROSCOPY. M. Kuroda !, Y Jinguhji 2 and K.Fujiwara 2. 1 Dept. of Biol. Fac. of Sci., Shimane Uniy., Matsue, 2 Dept.of Strut Anal., Natl. Card. Cent. Res. Inst., Osaka. In the previous meeting, we showed that the distribution of desmin in chicken skeletal muscle was not restricted to Z-disks. We showed following two fluorescence patterns of desmin Staining that were significant especially when myofibrils were bundled side by side; (i) longitudinal staining between adjacent myofibrils in the bundle and (ii) diffuse staining on an entire myofibril or over a bundle of myofibrils. These fluorescence patterns suggested that desmin was not confined around Z- disks but surrounded the surface of myofibrils. This time we confirmed these observations by immunoelectron microscopy. As desmin was expected to distribute on the surface of myofib- rils, semithin unstained sections were examined using protein A colloidal gold probes. In cross-sectional images of muscle, Au-particles were detected around the periphery of each myofibril and in the inter-myofibrillar space. No decoration was Seen inside the cross-sectioned myofibrils. Meanwhile, in longitudinally sectioned images, continuous arrays of Au- particles distributed on myofibril besides Z-disk regions. Some decorated filaments obliquely crossed over myofibrils on an A- or I-band region. Branching of desmin filaments was a common figure. Consequently, desmin filaments intercon- nected each other to form networks around myofibrils. PURIFICATION METHOD FOR Fg, FROM SOLUBLE VITELLIN BINDING PROTEIN FROM LOCUSTA MIGRATORIA K. Yamasaki, Dept. Biol. Tokyo Metropol. Univ. Tokyo. Two types of vitellin binding activity have been reported for the ovary of Locusta migratoria. One of vitellin binding protein (VBP)responsible for the activity is local- ized in inner portion in oocyte and soluble in aqueous solution(s-VBP). The role of s- VBP was supposed to be vitellin condenser holding high concentration of vitellin in mature oocytes. Further purification of s-VBP was carried out and could be purified over 200 times compared with starting material. A component of the purified s-VBP, Fp was estimated to be an active binding portion with vitellin. This fraction was prepared by PAGE and electro-ellution. FITC-Fs produced complex with Vn . Specific binding of Fs with vitellin was clearly shown by native PAGE analysis. The purific- ation method for Fs involved complicated procedure. However, the more simple prepar- ation method for Fp, could be established by the results of characterization,especially behavior of the Fs for organic solvents. It is estimated Fx, contains several compo- nets active for Vn binding and have not suitable category for the characteristics as chemical substance. Biochemistry 1151 IDENTIFICATION OF TETRAHYMENA 14-NM FILA- MENT-ASSOCIATED PROTEIN AS ELONGATION FACTOR la Y.Kurasawa, O.Numata, M.Katoh, H.Hirano! and Y.Watanabe. Inst. of Biol. Sci., Univ. of Tsukuba, Tsukuba and !National Inst. of Agrobiol. Resources, Tsukuba Tetrahymena 14-nm filament-forming protein has dual functions as a citrate synthase in mitochondria, and as a cyto- skeletal protein involved in oral morpho- genesis and in pronuclear behavior during conjugation. The 14-nm filament protein fraction prepared by assembly and disas-— sembly contained two 49-kDa proteins whose isoelectric points were 8.0 and 9.0. Monoclonal antibodies against the 14-nm filament protein fraction were clearly separated into two groups reacting to pI 8.0 or pI 9.0 protein specifically. From the N-terminal amino acid sequence, the pI 8.0 protein was identified as the 14-nm filament-forming protein. The pI 9.0 protein is considered to be a 14-nm fila- ment-—associated protein since the pI 9.0 protein copurifies with the pI 8.0 protein during two cycles of an assembly and disassembly purification protocol. Cloning and sequencing the pI 9.0 protein gene from a T. pyriformis cDNA library, we identified the pI 9.0 protein as elonga-— tion factor la (EF-la) based on it sharing 73-76% sequence identity with EF-la from several species. PRIMARY STRUCTURE AND PROPOSED TERTIARY STRUCTURE MODEL OF THE SQUID SODIUM CHANNEL. Chikara Sato, Kiyonori Hirota and Gen Matsumoto. Electrotechnical Laboratory, Supermolecular Science Divison, Tsukuba. The complete amino acid sequence of a sodium channel from squid Loligo bleekeri has been deduced by cloning and sequence analysis of the complementary DNA. The deduced sequence revealed an organization virtually identical to the vertebrate sodium channel proteins; four homologous domains containing all six membrane- spanning structures are repeated in tandem with connecting linkers of various sizes. A unique feature of the squid Na channel is the 1,522 residue sequence, approximately three fourths of those of the rat sodium channels I, II and III. On the basis of the result, we have proposed a tertiary structure model of the sodium channel where the transmembrane segments are octagonally aligned and the four linkers of S5-6 between segments S5 and S6 play a crucial role in the activation gate, voltage sensor and ion selective pore, which can slide, depending on membrane potentials, along inner walls consisting of segments S2 and S4 alternately. The proposed model is contrasted with that of Noda et al.. The proposed model could explain various unsolved phenomena, for example, effect of TTX (tetrodotoxin) on the gating current of the sodium channnel. THE BINDING SITE FOR COLLAGEN FIBRILS ON A CELLULAR FIBRONECTIN (cFN) MOLECULE M. TSukahara, K. Yoshizato Moi. (Celli) Scr. (Zab. ,, Zool Inst. , Face of Sci., Hiroshima Univ., Higashihiroshima The collagen gel culture is a useful experimental system in which the mode of binding between fibroblasts (Fbs) and collagen fibrils (CL) can be analyzed. With this culture Fbs recognize CL through cFN but not plasma FN (pFN). We have obtained monoclonal antibody (A3A5) which inhibits human Fbs-mediated collagen gel contraction and recognizes human FN. Quantitative assays of the extent of Fbs spreading on CL also suggested the involvement of cFN in binding of Fbs to CL. The region in FN recognized by A3A5 was analyzed. Human pFN was digested with trypsin and was subjected to immunoblot analyses with A3A5. The shortest fragment with molecular weight 19.5 kDa which reacts with A3A5 was analyzed for the to amino acid sequence and was found to have a sequence of TAGPDQT at the N-terminal region. From this we suppose that the binding site of CL on cFN is localized between typeIII region on the N- terminal side of ED-A and the middle of Heparin domein. For more precise analyses on the recognition site of A3A5 were carried out using human FN cDNAs encoding cFN and pFN. FN cDNAs fragments contained resion of 19.5 kDa trypsin fragment were constructed in vector and expressed as a fusion protein. This protein is now under investigation for the reactivity with A3A5. BREAKDOWN OF LARVAL TISSUES OF AMPHIBIAN TADPOLE BY ASPARTIC PROTEINASE. M. Mukai, K. Yoshizato, Mol. Cell Sci. iets), “Aol AGNCigs., ICs Weise Whehhirg = Cpr Hiroshima, Higashi-hiroshima. Amphibian tadpoles undergo metamorphosis and convert themselves into frogs by destroying their larval tissues and developing adult ones. The activity of acidic proteinases increases markedly in metamorphosing tail tissue. They are one of key enzymes in this histolysis. This study examined the activity of aspartic proteinase in tissues of tail, limbs and dorsal body muscle of metamorphosing bullfrog tadpole ( Rana catesbaiana) in relation to changes in DNA contents. The results suggested that the process of tissue degradation is divided into two phases. In the first phase activity of aspartic proteinase increased which was observed in both tail and dorsal body muscle at Taylor and Kollros stage XXI. This phase did not show any changes in the DNA contents per tissue weights. In the second phase which was found only in tail tissue (especially strong in distal part of tail) at T-K stage XXII, the activity further increased with a concomitant rise of DNA content which is most probable due to tissue condensation. The first phase may reflect autolysis, while the second is heterolysis probably caused by invading Macrophages. The enzyme activity in limbs remained quite low during metamorphosis. 1152 Biochemistry BILIVERDIN-ASSOCIATED CYANOPROTEIN OF RIP- TORTUS CLAVATUS MOLECULAR CLONING, SE- QUENCE ANALYSIS AND REGULATION BY JUVENILE HORMONE. 1K. Miura, 2T. Shinoda, 1M. Yura, 1S. Nomura and ly. Chinzei. ‘Dep. Med. Zool., Mie Univ., Tsu, and 2Natl. Inst. for Tea, Mie Pref. Biliverdin-associated cyanoproteins of the bean bug, Riptortus clavatus (CP-1 to 4) are hexameric proteins composed of two different subunit, CPa@ and CPZ. The cDNA libraries were constructed from the fat body polyA(+) RNA of the reproduc- tive and diapause females. cDNA clones encoding CPa and CP8 were screened from the cDNA li- braries by using anti-CP-1 and anti-CP-4 sera. The nucleotide sequences of the CPa and CPB cDNAs predicted that CPa and CP8 contain 693 and 686 amino acid residues, respectively, and the identity at the level of amino acid sequence be- tween the two subunit calculated to be 68%. Homology search revealed that the cyanoprotein falls into ‘hexamerin’ superfamily which includes insect storage hexamers and arthropod hemocya- nins. The expression of the cyanoproteins were examined by northern blotting of the fat body total RNA using the CPa and CP8 cDNA clones as radioactive probes. Juvenile hormone (JH) treat- ment to the diapause bugs resulted in complete diapause termination. During the course of the diapause termination the shift of CP expression from CP8 to CPa@ was observed in the females while the JH treatment suppressed completely the expression of both CPa and CP in the diapause males. STRUCTURAL ANALYSIS OF INTERNAL TRANSCRIBED SPACERS(ITS) AND 28SrDNA OF APHID. D.Amako, O-Y.Kwon and H.Ishikawa. Zool.Inst., Fac.Sci., Univ. of Tokyo, Tokyo. In the process of forming mature 18SrRNA, 5.8SrRNA and 28SrRNA in eukaryotes, two regions of the pre-rRNA are cut off through post-transcriptional processing. One is ITS1 which is between 18SrRNA and 5.8SrRNA; the other, ITS2 between 5.8SrRNA and 28SrRNA. To characterize these regions of the pea aphid, Acythosiphon pisum, we determined their primary sequences and constructed their presumable secondary structures. ITS1 and ITS2 of aphid consisted of 229 bases and 280 bases, whose G/C contents were 70% and 74% respectively. These G/C contents were surprisingly high compared with other lower invertebrates. The aphid ITSs were rather comparable to those of vertebrates. In addition, it was shown that despite extensive divergence in nucleotide sequence and G/C content, the secondary structures of ITSs are conserved throughout species. We also determined the partial sequence of the aphid 28SrDNA which is exceptional in that its molecular weight is larger than other organisms by 200KDa, and that it does not contain "hidden break". So far about 3,000 bases have been sequenced and the followings were suggested: 1) G/C content was approximate 60%, 2) Not so similar to those of other organisms, 3) Highly conserved regions were separated from each other by long non-conserved regions. STUDIES ON THE MECHANISM OF FLIGHT MUSCLE BREAKDOWN IN APHIDS. M. Kobayashi and H. Ishikawa. Zool. Inst., Fac. of Sci., University of Tokyo, Tokyo. We studied flight muscle breakdown of the pea aphids, Acyrthosiphon pisum (Harris), in relation to their flight, feeding and reproductive behavior. Using the acetone-freeze dry microdissection technique, which enables us to isolate the indirect flight muscle (IFM) free from other tissues without proteolysis, we monitored its development and breakdown in terms of its protein content. IFM protein content reached the maximum on the 2nd day after the final ecdysis, and then turned to decrease. Histological studies indicat- ed this decline is due to the breakdown of IFM. Intriguingly, body weight of alatae, unlike that of apterae, decreased during the first 2 days after the final ecdysis in inverse proportion to IFM development, thus providing the suitable conditions for flight. In addition, body weight turned to increase coincidentally with the onset of IFM breakdown, followed by larviposition, suggesting that fecoing not only provides raw materials for producing progeny but also serves as certain stimuli to bring about muscle breakdown. In fact, in the starved aphids, neither the IFM breakdown nor reproduction took place until these insects were fed later. The fact that the administration of cycloheximide prevented IFM breakdown indicated feeding stimulates de novo syntheses of certain proteins essential to this process. AMINO ACID EXCRETION OF ENDOSYMBIOTIC SYSTEM OF APHIDS. T. Sasaki and H. Ishikawa. Zool. Inst., Fac. Sci., Univ. of Tokyo, Tokyo. Bacterial endosymbionts of aphids have been suggested to play important roles in the nitrogen metabolism of the host insects. We maintained symbiotic and aposymbiotic pea aphids, Acyrthosiphon pisum on a chemically defined syn- thetic diet, and analyzed nitrogen compounds in the honeydew excreted by the aphids. Unlike most insects, aphids did not ex- crete uric acid as a nitrogenous waste but instead amino acids. The principal amino acid in the honeydew of aposymbiotic aphids was glutamine, while symbiotic aphids scarcely ex- creted it. The amino acids in the honeydew are a mixture of metabolic products and those passed through the gut without being absorbed or subjected to metabolic manipulation. To tell one from the other, aposymbiotic aphids were kept on a diet from which glutamine was omitted. Even on such a diet, they excreted appreciable amount of glutamine, confirming that they excreted glutamine as a nitrogenous waste. For a further study, aposymbiotic aphids were kept on a diet containing [e- 1SN]glutamine. In their honeydew, asparagine was found labeled. We also found an unknown ninhydnn-positive spot on TLC which was highly labeled with 15N. Analyses by NMR and GC-MAS revealed that this spot is due to a mixture of y-glutamylglutamine and y-glutamylasparagine. It was sug- gested that aposymbiotic aphids excrete amides and y- glutamylamides, while these amides are reutilized by the symbiotic aphids, possibly with the aid of their endosym- bionts. Biochemistry 1153 PHOSPHORYLATION OF SYMBIONIN. M. Morioka and H. Ishikawa. JZool.Inst., Fac.Sci., Univ.of Tokyo, Tokyo. Symbionin, a GroEL-homologous heat shock protein of aphid endosymbiont, functions as molecular chaperone in folding and assem- bling polypeptide in vitro. Also, symbionin is autocatalytically phosphorylated in vitro in response to the temperature shift- up. In this paper, we report the biochemical significance of phosphorylation of symbionin as a molecular chaperone. When the chemically unfolded dihydro- forate reductase (u-DHFR) was diluted in refolding buffer, the spontaneous refolding of u-DHFR was strongly inhibited by GroEL but not by symbionin. By contrast, the refolding of u-DHFR was inhibited by the phosphorylated symbionin, suggesting that the latent domain contributing for the chaperonin activity of symbionin is activated by the phosphorylation. The latent chaperonin activity of symbionin was also activated with several kinds of salt. In addition, when the 1M NaCl-treated symbionin was subjected to native PAGE or gel-filtration, 70kDa-protein was separated from symbionin, suggesting that symbionin exists as a complex with 70kDa-symbionin binding protein, which plays an important role as a regulatory factor of molecular chaperone in the endosymbiont. PHOSPHOTRANSFERASE ACTIVITY SPECIFIC TO SYMBIONIN, A GROEL PROTEIN HOMOLOGUE. H.Muraoka, M.Morioka and H.Ishikawa. Zool.Inst.,Fac.Sci.,Univ.of Tokyo, Tokyo. EE ee ee eee Endosymbionts, when habored by the aphid bacteriocyte, selectively synthesize symbionin, a structual homologue of the GroEL protein, a heat shock protein of E.coli. Isolated endosymbionts under a temparature shift-up synthesize the 63KDa heat shock protein which is a phosphorylated form of symbionin. In this study, we showed that symbionin has auto-kinase activity, and that the phophorylated symbionin is able to transfer its phosphate to ADP. It was also shown that symbionin transfers phosphate group from ATP to GDP, while that the GroEL protein has ATPase activity but is the apparently without transferase activity. These results suggested that the phosphorylated symbionin has a high energy bond and serves as a high-energy phosphate donor. Therefore, it is likely that symbionin has other functions in addition to chaperonin activity common to the GroEL protein. 1154 Developmental Biology GRAFTS OF CENTER CELLS INTO EMBRYOS AT DIFFERENT STAGES IN HORSESHOE CRABS. T. ITOW. Dept.of Biol., Fac.of Edu., Shizuoka Univ., Shizuoka. The center cells under the blastopore of horseshoe crab embryos at early gastula stage induce secondary embryos after the grafting of the center cells into embryos at the same stage. Fot the understanding of characteristics of embryonic induction by center cells, the center cells of horse shoe crab embryos. at early gastrula stage were grafted into the embryos at the stages earlier than _ early gastrula. “Then the center cells of early gastrula were grafted into the embryos at the stages later than early gastrula. As the results of both grafts, the secondary embryos were not formed, and the treated embryos developed normally. Those facts mean that the center cells at early gastrula can not induce secondary embryos at the stages different from early gastrula. Besides, the center cells of embryos at the stage later than early gastrula were grafted into the embryos at early gastula stage. As the results of those grafts, secondary embryos were induced. The fact means that the center cells of embryos after early gastrula stage remain the ability of embryonic induction. ANALYSIS OF AMINO ACID - LIKE CHROMATIN COMPONENTS OF EARLY NEWT EMBRYOS T. Asao, Biol. Lab., Sch. Med., St.Marianna Univ., Kawasaki Amino acid-like components in the nuclear extract of newt embryos were analyzed by gel, ion exchange and reverse phase chroma-— tography. The results from chromatographic profiles showed, firstly, that these were a group of substances of similar molecular weight below several hundred daltons and were released from the nucleus most effi- ciently with around a 50 to 100 mM NaCl solution. Secondly, they were composed of three kinds of group, i.e.,hydrophilic, in- termediate and less hydrophilic substances showing amino acid properties, the last group of which increased as development proceeded from blastula to tail bud stage. Thirdly, the regional differences in the chromatographic profiles were noted among the anterior and posterior neural plates and the epidermis in neurula. Reverse phase chromatography showed that medium rearing gastrula tissue fragments for a day con- tained several substances similar to nucle- ar extract components. An infra red spec- trum of the specimens recovered from gel chromatography, however, was similar to that of fructose. Furthermore, the physio- logical activity in vivo of the specimens above was hardly positive. These unfavor- able results are thought to be due to the incomplete purification of the substances. DORSAL CYTOPLASMIC TRANSFER INTO XENOPUS EARLY EMBRYOS DEVELOPED FROM UV-IRRADIATED-FERTILIZED EGGS OR -OOCYTES. N. Mise and M. Wakahara, Zool. Inst., Fac. Sci. Hokkaido Univ., Sapporo. Previous works have shown that ultraviolet (UV) irradiation to vegetal region of fertilized eggs (UV-F) and oocytes (UV-O) of Xenopus laevis yields embryos that lack dorsal axial structures (dorsal axial structure deficiency) in the same way, but UV targets are thought to be different respectively. On the other hand, it has been shown that cytoplasm of dorsa! vegetal blastomeres of early embryo (dorsal cytoplasm) contains "dorsal determinants”. UV-F embryos had circular blastopores, and they did not have dorso-ventral polarity in appearance. UV—O embryos had normal blastopores with clear dorso-ventral polarity in early gastrula stage. Injecting dorsal cytoplasm from 8-16 cell stage embryos could rescue the dorsal axial structure deficiency of UV-F embryos. But we could not rescue UV—-O embryos by cytoplasmic transfer. These results suggest that the establishment of dorso-ventral polarity and the development of dorsal axial structures should be analyzed separately. EXPRESSION OF THE EMBRYONIC EPIDERMAL DIFFERENTIATION-SPECIFIC TRANSCRIPT IN ADULT TISSUES OF THE JAPANESE NEWT, CYNOPS PYRRHOGASTER. T.C.Takahashi and K.Takeshima, Radioisotope Research Center, Nagoya University, Nagoya 464-01 JAPAN Embryonic epidermal differentiation- specific cDNA isolated from Cynops neurula library (Develop.Growth&Differ., 34, 277- 283, 1992) was used for studying its gene expression both in embryos and in adult tissues. The HindIII-SphI fragment of the cDNA, pG3m11N138-48, was prepared for the probe for Northern hybridization analyses. When RNAs were isolated from the whole embryos of various developmental stages, the Message was first detected at stage 15, the stage characterized by the disappearance of the yolk plug prior to the neural plate formation. The message was then increased and was very abundant throughout the larval stages. When RNAs were purified from adult tissues, the message was not abundant and detected by less intensity. Interesting result was the appearance of different size of the transcript in adult tissues when compared to the size from larval and embryonic preparation. These results suggests that the expression of this gene is regulated in time and in space not only during the early development but also in post- metamorphotic growth and differentiation. Developmental Biology 1155 CON A-LIKE MOLECULES SPECIFIC TO THE ORGA- NIZER AND THE DORSALIZED VENTRAL MESODERM MRM, Wehey_p Mss Takata and S. Takesue-. 1 Nagoya University, College of Medical Technology, Higashi-ku, Nagoya 461 Doho University, General Education, Nakamura-ku, Nagoya. - Con A-treated ventral mesoderm was found to devel— op notochord and muscle tissues typical of the orga- nizer and was found to attain peak neural-inducing activity 24h after treatment (Diazetal., 1991). This time, at the protein level, wewere able to showthat a couple of bands were specific to both the organizer and the 24h-old dorsalized ventral mesoderm. Organizer samples were analyzed by SDS polyac- rylamide gel electrophoresis followed by immuno- blotting using anti-Con A as probe. A Several anti-Con A reactive proteins were de- tected. Compared with immunoblots of the endoderm and the ventral mesoderm, a few of those bands appeared to be specific to the organizer. Compared with the banding pattern of the 24h-old dorsalized ventral mesoderm, a couple of bands appeared to be common with the Organizer, but not with the untreated ventral mesoderm. These results offer additional evidence to the possi- bility that the organizer is aCon A-like substance. More detailed research onthe nature of the organizer at the molecular level may be approached using Con A system as the working model. INDUCING ABILITIES OF THE XENOPUS AND CYNOPS PRESUMPTIVE ECTODERM TREATED WITH ACTIVIN A. T.Ariizumi, Z.Hasegawa and M.Asashima. Dept. of Biol., Yokohama City Univ., Seto, Kanazawa, Yokohama 236. We examined the inducing abilities of the presumptive ectoderm of early amphibian embryos after treatment with activin A. We treated the ectoderm of late Xenopus blastula with 10 - 100 ng/ml of activin A for 10 minutes and "precultured" it for 0 - 7 hours. The treated ectoderm was sandwiched between two sheets of ectoderm from another late blastula embryos. Short- precultured ectoderm induced trunk-tail Structures, whereas long-precultured ectoderm induced head structures. These changes of inducing abilities, dependent upon the time of preculture, occurred more rapidly when the concentration of activin A was higher. Similar phenomenon was observed in the experiments using Cynops embryos. In addition, beating heart was also induced in the Cynops explant. These results suggest that the activin A-treated ectoderm behaves like the Spemann's organizer and functions as “head organizer" or "“trunk-tail organizer", depending upon two factors: the concentration of activin A, and the time of preculture. INDUCTION OF THE ANIMAL BLASTOMERES OF THE 8-CELL EMBRYO OF XENOPUS LAEVIS BY ACTIVIN A. T. Bessho, A. Fukui, H. Uchiyama and M. Asashima. Dept. of Biol., Yokohama City Univ., Yokohama 236. Xenopus blastula ectoderm is induced to mesoderm by activin A. However, maternal activin is presence in the unfertilized egg, and the real mesoderm induction is considered to occur earlier. We separated a single animal blastomere from 8-cell embryo, and at once treated it with 50 ng/ml of activin A for 1 h. The dorsal blastomere mainly formed notochord and neural tissues, in contrast to the ventral one which formed mainly mesenchyme and coelomic epithelium or atypical epidermis. Next, we precultured the blastomere in a saline for 1 or 2 h before treatment with activin. We had same results as the above experiment. When the blastomere is treated first with activin for 1 h, followed by follistatin at 500 ng/ml for 4 days, the mesodermal induction was observed. Respon- siveness of dorsal and ventral blastomeres to activin A are different. These results suggest that activin receptor exist in the animal blastomeres of the 8-cell embryo. MESODERM-INDUCING ACTIVITY OF INHIBINS IN XENOPUS EMBRYOS. H.Nakano', H.Sugino2, and M.Asashima'. ‘Dept. of Biol., Yokohama City Univ., Yokohama, @Inst. for Enzyme Res., Tokushima Univ., Tokushima. We previously reported about high mesoderm-inducing activity of activin A, and lower activity of inhibin than that of activin A. An activin A molecule is a homodimeric protein composed of two inhibin B,—-subunit chains. An inhibin molecule, heterodimeric protein consisted of a- and B-subunits, has two isoform types, such as inhibin A (a + B,) and inhibin B (a + Bg). Mesoderm—inducing activity of those inhibin A and B was tested by animal cap assay method using Xenopus laevis embryo. In the cases of both inhibin A and B, mesoderm induction accompanying with mesenchyme and coelomic epitheli- um was recognized at a concentration of 100 ng/ml, but not at of 10 ng/ml. The concentration of inhibins needed for the mesoderm induction is higher than that of activin A. To test the combination effect of inhibin A or B with activin A, activin A with inhibin A or B were co-incubated before bioassay. Then assay was carried out. As a re- sult, mesoderm-induction was reduced a bit, and per— centages of induced tissue types were slightly changed, respectively. Inducing activity and effect on induction of a-subunit monomers (26 and 46 forms) were also tested by the same bioassay method. The relationships be- tween composition forms of these proteins and meso- derm-inducing activity are discussed. 1156 Developmental Biology PURIFICATION OF XENOPUS ACTIVINS A, AB AND B, FROM CONDITIONED MEDIUM OF XTC CELLS. A. Fukuil, re Nakamura2, H. Uchiyamat, Be Sugino2 and M. Asashimat. 1pept. of Biology, Yokohama City Univ., Yokohama 236, Inst. for Enzyme Res., Tokushima Univ., Tokushima 770. Activin is composed of two inhibin 6 chain subunits. Activin A, AB and B isoforms are composed of fafa homodimer, Babe heterodimer and phy homodimer, respectively. Follistatin, an activin- binding protein, suppresses physiological activities of activins. We have purified Xenopus follistatin, and activins A, AB and B, from conditioned medium of XTC cells using four steps of column chromatography. The activins bounded with follistatin in the conditioned medium. The Activin- follistatin complex was separated by the last reverse phase HPLC, into follistatin and Activins, A, AB and B. The identities of these molecules were revealed by SDS- PAGE and immunoblotting. Xenopus activins were determined by erythroid differentia- tion activity (EDF assay) using porcine activins as standard. Xenopus activins induced mesodermal tissues from ventral to dorsal ones, in dose-dependently. OVER EXPRESSION OF FOLLISTATIN mRNA IN THE EARLY EMBRYO OF XENOPUS LAEVIS . M.Iwao, S.Shimazaki*, S.Nishimatsu**, N.Ueno**, A.Fukui, H.Uchiyama and M.Asashima. Dept.of Biol., Yokohama City Univ., yokohama; *Whittier Inst., San Diego, USA.; **Inst. of Appl. Biochem., Tsukuba Univ., Tsukuba. Activin, a Xenopus oocyte's maternal protein, has the mesodermal inducinhg activity on isolated early Xenopus animal caps. This inducing activity is inhibited in vitro by follistatin, an activin-specific binding protein. To investigate the activin function in vivo, we injected follistatin mRNA into blastomeres at St.3. The injected embryos delayed the closure of the blastopore, and had abnormal eyes. Many of them were small or lensless. On the other hand, some embryos had the third cement gland and the eye on their forehead. The ratio of the abnormal embryo was increased dose-dependently with the amounts of injected mRNA. When follistatin mRNA was injected with activin mRNA, they counteracted each other. This suggests that injected mRNA was translated, and that the products had the activity in vivo. Therefore, the effect of injected follistatin mRNA seems to be inhibit native activn activty. Translation of injected follistatin mRNA began at early gastrula, and decreesed from early neurula stage. Therefore, the mRNA of this follistatin was not translated during mesodermal induction (St.6=7) . MESODERM INDUCTION BY ACTIVIN A ON THE XENOPUS DIVIDING STAGE ANIMAL HALF CELLS. K.Kinoshita!, T.Kinoshita’?, M.Asashima?. 1Biol. Lab., Nippon Med. School, Kawasaki, 2Z001. Inst., Hiroshima Univ., *Dept. of Biology., Yokohama City Univ. Activins act on the stage 8 animal cap cells of Xenopus embryo to induce mesoder- mal tissues. However, we know little about when animal half cells become acceptable for activins. We isolated animal half blastomeres at early dividing stage (after st.4), then treated with 50 ng/ml activin A. RNAase protection assay showed that muscle a-actin mRNA was present in the explants derived from the blastomeres treated with activin at the onset of st.5. The level of mRNA remarkably increased following the stage of treatment. It was synthesized mostly in the future dorsal cells. When activin was treated on the cells of st.6 embryos, no evident differ- ence was observed between the cells of animal pole and dorsal marginal zone. The responsiveness to activin was also estimated by goosecoid gene, an organizer specific homeobox gene. The goosecoid mRNA was transcribed soon after activin treat- ment in st.8 animal caps. The timing of goosecoid expression was not influenced by the activin treatment at st.5. The results suggested that dorsal animal half cells of stage 5 possess some activin receptors and the timing of goosecoid gene expression seems to be regulated indepen- dently of the stage of mesoderm induction. AXIS FORMATION AND THE EXPRESSION OF A HOMEOBOX GENE IN EARLY XENOPUS EMBRYOS. Koichiro Shiokawal, Misaki Asanol, Yasuko Sugase~, Yasufumi Emori*, Kaoru Saigo4, Lab. Mol. Embryol., Zool. Inst., “Dent. Biophys. Biochem., Univ. Tokyo, Tokyo. In animal embryogenesis, various homeo- box genes and growth factors have been implicated as important factors responsi- ble for embryo axis formation. We have recently isolated a novel homeo- box gene of Xenopus laevis from the ovary cDNA library (Asano et al.: J. Biol. Chem., 267, 5044-5047, 1992). The homeodomain of the gene was homologous to that of Droso- hila Distal-less, and the gene was named Tl. The mRNA exists in the unfertilized egg’as maternal mRNA, and the level of the mRNA was constant during the cleavage to the early blastula, but increases from the blastula to gastrula stage. Embryo dissection experiments reveal- ed that the Xdll mRNA is most abundant in the head part, especially in the cement gland, and its amount is much smaller in the trunk and tail regions, The relative abundance of the mRNA coincides the antero- posterior axis, and the direction of the invagination of the mesodermal cells during the gastrula stage. These results suggest strongly that the Xdll mRNA may play an important role in determining the head to tail or antero- posterior axis in Xenopus embryogenesis. Developmental Biology 1157 SEA URCHIN HATCHING ENZYME: THE SUGGESTED "SECOND ENZYME" IS POSSIBLY A SMALL FORM AUTOLYTICALLY DERIVED FROM ENVELYSIN K. Nomura!, and N. Suzuki2, !Dept. of Biochenm., okyo Metropol. Inst. of Gerontol., Tokyo, and Noto Marine Lab., Kanazawa Univ., Uchiura The sea urchin hatching enzyme (HEz) belongs to the mammalian matrix metalloproteinase (MMP) family!,2 We have isolated from Hemicentrotus pulcherrimus a 37 kDa HEz, designated "envelysin", which alone is able to dissolve the fertilization envelope (FE). A possibility has been suggested that another enzyme is involved in the hatching process. Indeed, we found during the Red-agarose chromatography a fraction with caseinolytic activity but without hatching enzyme activity . We made a hypothesis that the enzyme is an auto- catalytic derivative of envelysin by truncation of its C-terminal domain having a role of binding to FE, the specific substrate. Supporting evidences are shown below. Envelysin, left at 27 °C for 13 h, autodegrades into 32 kDa form with drastically reduced hatching activity. However it retained almost full caseinolytic activity as well as full activity on (Xj j-antitrypsin. The caseinolytic fraction without hatching activity, that did not bind to the Red-agarose column, was inhibited by EDTA and the cysteine-switch peptide, typical inhibitors of envelysin. This enzyme seems to be distinct from the 64 kDa protease homologous to the human BMP-15. Experiments from protein chemical and immunological aspects are now in HOR RESS to further prove the above hypothesis. EMBO J. 9 3003 (1990). 2Biochem. 30 6115 (1991). SDevelopment 114 147 (1992). Analysis of binding action of HCE, a con- stituent of the hatching enzyme of medaka, toward chorion Lee K., S.Yasumasu, I.Iuchi, K.Yamagami Life Sci. Inst., Sophia Univ., Tokyo The hatching enzyme of medaka, Oryzias latipes, consists of two enzymes, HCE and LCE. HCE swells the inner layer of chorion by hydrolyzing it partially and LCE hydrolyzes the swollen part of the inner layer. HCE tends to bind tightly to the chorion when it causes the chorion swell- ing. Present study examined by SDS-PAGE whether the enzyme binds to chorion EUSMElYy DieiOGrm CO CloOri@lwSiss A monoclonal anti-HCE antibody(E72) inhibits the choriolytic activity of HCE, but it does not affect the proteolytic activity of the enzyme. But how E72 inhibits the choriolysis of HCE is not well known. When a definite amount of HCE was prein- cubated with varied amounts of E72, the binding of the HCE to the chorion decreased depending on the amount of the E72. This result strongly suggests an in- hibition of HCE binding to the chorion by aU Z < Wows le LS COMPAiPMGC wWAAwB wae choriolysis of HCE premises binding of the enzyme toward the chorion. HCE bound to the egg envelope of rainbow trout but not to insoluble collagen. The binding to the former was also inhibited by MAB B2e The Gene Structure of HCE, a Constituent of the Hatching Enzyme of Medaka, Oryzias latipes S.Yasumasu7, K.Inohaya* K.Yamada?, I.Iuchit+, H.Shimada? and K.Yamagamit 1: Life Sci. Inst., Sophia Univ. 2: Zool Inst., Hiroshima Univ. The hatching enzyme consists of two types of proteases (HCE, LCE). In cloning of the cDNA for HCE, two distinct cDNAs (HCE21, HCE23) having nucleotide sequences with 92.8% similarity were obtained. The genes encoding HCE, corresponding to HCE23 (heel) and HCE21 (hce2), were iso- lated from the genomic libraries con- structed from the drR strain of medaka. The structure analysis indicated that both heel and hce2 contained the entire coding sequences (approx 900bp), but lacked in- trons. At least thee or four of hcel and hce2 were tandemly repeated. The putative TATA box consensus sequences were located at about 50 bp upstreams from protein coding regions. Their 5'flanking regions locating in the upper stream of the protein coding regions (200-400bp long) highly resembled each other. Comparison of gene structures of HCE and LCE indicated that HCE gene was quite dif- ferent from LCE gene, which contained 8 exons and 7 introns. HCE gene (hcel) was located in about 8kb down stream of LCE gene. SOME PROPERTIES OF ERYTHROCYTE TRANSITION DURING POST-HATCHING DEVELOPMENT OF RAINBOW TROUT, Oncorhynchus mykiss. I. Iuchi (Life Science Institute, Sophia University, Tokyo) It is well known that larval type eryth- rocytes containing larval type hemoglobins develop first on 7-8th day after fertiliza- tion (15°C) and their replacement by adult type erythrocytes containing adult type hemoglobins occurs after hatching (Iuchi, Zool., Sci., 2, 11-23, 1985). Treatment of phenylhydrazine (10° °M) for 6-12 hr from 8th to 9th day after fertilization resulted in degradation of larval type erythrocytes. Embryos lacking almost all the erythrocytes thus prepeared developed normally and hatched out. They recovered neither larval type erythrocytes nor adult type eryth- rocytes, while they developed adult type erythrocytes after hatching. This recovery is ascribable to production of adult type erythrocytes in the way similar to that in non-treated alevins. Lack of larval type erythrocytes did not affect the production or differentiation of adult type eryth- rocytes. On the other hand, X-ray irradia- tion (2,000R) suppressed the production of adult type erythrocytes. These results sug- gest that the erythrocyte transition is not easily influenced by extrinsic factors but determined by some intrinsic constraints. 1158 Developmental Biology LOCALIZATION OF M PHASE CELLS IN SEA URCHIN BLASTULAE. H.Mizoguchi, Lab. of Biol., Jun. Col. of Rissho Univ., Saitama. Propidium iodide was known to stain nuclear DNA. We can infer M phase cells from the profile of propidium iodide stained nuclei. Hence, in the propidium iodide stained cells of sea urchin embryo at the blastula stage, cell proliferation study was performed to clarify the cellular mechanism of archenteron formation. Staining of the embryos by propidium iodide(0.5 uz g/ml) was performed at the swimming blastula stage( 11 hrs and 14hrs after fertilization at 18°C ) and mesenchyme blastula stage(17 hrs and 20 hrs after fertilization at 18°C ). Observations were performed by fluorescence microscope. M phase cells were found in the embryo uniformly at the swimming blastula stage. Most of the M phase cells were localized at vegetalized hemisphere at the mesenchyme blastula stage. Percentage of M phase cells to the total cell number of the embryo at the mesenchyme blastula stage is greater than that of the embryo at the swimming blastula stage. Changes in frequency of M phase cells in the embryo at the mesenchyme blastula stage may relate to archenteron formation. ARCHENTERON-FORMING ABILITY OF VEGETAL REGION OF STARFISH EGG (3). M.Kiyomoto and H.Shirai. Ushimado Marine Laboratory, Okayama Univ. Okayama. Archenteron-forming ability is localized in vegetal region in immature or mature oocytes of the starfish, Asterina pectinifera. Animal egg fragments, which are known to develop into only so-called permanent blastulae, fused with vegetal fragments can form archentera. If vegetal fragments have been labeled with RITC, only these archentera are composed of labeled cells. Fertilized eggs, whose fertilization membranes were removed, were fused with RITC-labeled vegetal fragments at the animal pole. Many of these eggs developed into gastrulae with two archentera; one was RITC-labeled and the other was non- labeled. The former was considered to be derived from fused vegetal fragment and the latter from the original vegetal region. This result showed that the determinant as well as other cytoplasm of the vegetal fragment remained through the cleavage stage in the original place at which the egg-fragment fusion had occurred. Thus, whole eggs (non-excised eggs with intact animal and vegetal region) can be used as recipients of the assay for the determinant, EXPRESSION OF ALKALINE PHOSPHATASE IN STARFISH LARVAE. Ritsu Kuraishi and Kenzi Osanai. Mar. Biol. Stn. Asamushi, Tohoku Univ., Aomori, In larvae of Asterina pectinifera, the presumptive area of esophagus first invaginates at the start of gastrulation. Then those of the stomach and intestine are added to the archeneron gradually after the early gastrula stage. To check whether these areas differentiate into endoderm simultaneously or separately, we examined expression of alkaline phosphatase (AP), a reported endoderm marker, histochemically from the start of gastrulation to the bipinnaria stage. As a result, the presumptive area of the esophagus and anterior part of stomach (area I) became AP-positive several hours after its involution (27 h), while that of the rest of the digestive tract (posterior part of stomach and intestine; area Il) remained AP-negative until the mouth- formation stage (42 h). The presumptive area of mesoderm tissues (that of the anterior coeloms and mesenchymes at the tip of the archenteron and that of the posterior coelom in the dorsal wall of the archenteron), on the other hand, was AP-negative throughout the stages examined. We have reported that complete removal of the area |! from immature oocytes forbids the larvae to gastrulate, though at least a part of the area Il is preserved. In those larvae, expression of AP was also inhibited. Iductive effect from the area | seems to be necessary for both involution and the following differentiation of the area Il. MORPHOGENESIS IN SPICULE-REMOVED EMBRYOS OF THE SEA_URCHIN. K. Shimizul2, y. Nakajima? and M. Ikeda! Dept. of Biol., Keio Univ.,Yokohama and 2Biofouling Project, ERATO, JRDC, Yokoha- ma. In order to investigate the role of spicules in morphogenesis of pluteus larvae, embryos of the sea urchin, Cle- peaster japonicus, were treated with sea water containing 10 mM hydrochloride for 15 seconds at various stages. Spicules were completely removed from embryos in the acidic sea water. Although the treat- ment delayed development, tissue differen- tiation of the treated embryos were not affected. More than 60 % of embryos treat- ed at the early gastrula stage normally developed into pluteus larvae. Almost embryos processed at the prism stage, however, became pluteus larvae with abnor- mal morphology; round shape with lobe-like short arms containing extraordinary spi- cules. The time required for beginning to regenerate spicules increased as the stages treated were advanced. Immunohisto- chemical study using monoclonal antibody P4 which recognizes the antigen in the spicule forming cells revealed that pat- terning of the spicule forming cells were disordered in the treated embryos. These results suggest that spicule formation is essential for morphogenesis of pluteus larvae and at the same time formation of proper pattern of spicules requires inter- action with other tissues in the embryos. Developmental Biology 1159 CHANGE iN MOLECULAR FORM AND ACTIVITY OF ACETYLCHOLINESTERASE DURING SPICULE FORMATION IN SEA URCHIN EMBRYO. K. Ohta, M. Sato, T. Nakazawa, Department of Biology, Faculty of Science, Toho University, Funabashi. In this study, the enzyme activity and molecular change of acetylcholinesterase were examined during embryonic development cf sea urchin, Hemicentrotus pulcherrimus. The acetylcholinesterase activity was detected originally in unfertilized egg, remaining in the low level before gastrula stage. At gastrula stage, two molecular forms of acetylcholinesterase were detected by polyacrylamide gel electrophoresis. The molecular masses were estimated as 140 kD and 300 kD, respectively. The appearance of higher molecular enzyme was consistent with the occurrence of a remarkable increase in the enzyme activity. This increase was sensitive to emetine or puromycin but not to actinomycin D. No activity of cholinesterase (butyryl) was detected throughout the development. The spicule elongation was suppressed by cholinesterase inhibitor, eserine. The suppression of spicule elongation was rescued by incubation in normal sea water. A remarkable increase in the activity of acetylcholinesterase and differentiation in the enzyme pattern wouid involve in the spicule formation. SPICULE FORMATION-INDUCING SUBSTANCE IN BLASTOCOELIC FLUID OF SEA URCHIN BLASTULA M.Kiyomoto!’, M.Maruoka? and J.Tsukahara?. 1Ushimado Marine Laboratory, Okayama Univ., Okayama, *Dept. of Biol., Fac. of Sci., Kagoshima Univ., Kagoshima. We reported spicule formation-inducing substance (SFIS) in blastocoelic fluid (BCF) of sea urchin blastula. The presence of SFIS in BCF can be assessed by spicule formation in decendants of isolated micromeres under the influence of BCF. SFIS is effective interspecificaly and is inactivated by trypsin. BCF of Toxopneustes pileolus and Hemicentrotus pulcherrimus was fractionated by gel filtration (HPLC, Waters Protein Pack 300). Activity of SFIS of IT. pileolus was eluted as a broad peak (M. W. ca. 1x10*), and that of H. pulcherrimus, as two peaks (M. W. ca. 3x105 and 1x10*). In ion exchange chromatography (HPLC, DEAE-Toyopearl 650), SFIS activity of T. pileolus was obtained in gradient elution at NaCl concentrations of ca. 0.5 M and 0.8 M, although total activity was markedly reduced. That of H. pulcherrimus, at ca. 0.3 M and 0.5 M NaCl in step-wise elution. ROLES OF TGF-B SUPERFAMILY GENES IN DIFFERENTIATION OF MICROMERES OF SEA URCHIN EMBRYOS. T. Kinoshita! and K. Kinoshita”. 1Zool. Inst., Fac. of Sci., Hiroshima Univ., Hiroshima and “Biol. Lab., Nippon Med. School, Kawasaki. The micromeres of 16 cell stage sea urchin embryos can be isolated and cultured in vitro. However, some serum factors or blastocoelic factors should be included in the culture media for the differentiation of spicule-forming cells. In order to examine the ability of several defined peptides to induce the spicule formation, micromeres of Hemicentrotus pulcherrimus were cultured in the sterile sea water supplemented with EGF, bFGF, TGF-B or activin A. Among the four peptides tested, only activin A possessed the inducing activity, although the percentage of cells induced was very low. Using cDNA fragments of Xenopus activin and BMPs as probes, homologues of these genes were screened from the cDNA library of gastrula stage sea urchin embryos. Seven clones were selected through the second screening and were subcloned. Northern hybridization analysis showed that mRNA of at least one clone existed in oocytes and cleavage stage embryos. The mRNA gradually decreases in amount after gastrulation. SPICULE FORMATION OF PRIMARY MESENCHYME CELLS IN THE ISOLATED BAGS FROM SEA URCHIN EMBRYOS T. Iwase and T. Miki-Noumura, Department of Biology, Ochanomizu Univ., Ohtsuka, Tokyo. The formation of spicular rudiments and the growth of spicules in the bags were examined in the culture media. Based on the method of Whiteley et al.(1980), we able to isolate the bags from sea urchin embryos (Clypeaster japonicus) before formation of spicular rudiments. In the bags, the mesenchyme cells were enclosed with basal lamina lining blastocoel. The number of mesenchyme cells in it was changed with the embryo stage. After adding epitherial cells to the bags, they were cultured in the culture media with or without 3% horse serum (HS). The bags were cultured without the epitherial cells showed no spicule growth; in contrast, the bags surrounded by reaggregated epitherial cells were assembled into a swimming gastrula-like structure having remarkable spicule growth. The culture medium containing HS induced higher rates in the formation of spicular rudiments and the growth of spicules than that containing no HS. So it can be said that the presence of epitherial cells are essential to induce spicule formation in the bags of the sea urchin embryos (Clypeaster japonicus). 1160 Developmental Biology METAMORPHIC POTENCY OF THE AGGREGATES DERIVED FROM MESOMERES ISOLATED FROM 16-CELL-STAGE EMBRYOS OF A SAND DOLLAR, PERONELLA JAPONICA. S. Amemiya, Misaki Marine Biological Station, University of Tokyo, Kanagawa. The developmental potency of the aggregates derived from mesomeres isolated from 16-cell-stage embryos of a sand dollar, Peronella japonica, was examined. The eggs whose fertilization membrane was removed chemically by treating with trypsin were cultured to 16-cell-stage in Cat*t-free sea water. Sixteen mesomeres isolated from the 16-cell-stage embryos were placed into 10-ml conical glass tubes containing 0.5 ml of culture medium (100 ug/ml streptomycin and 100 u penicillin/ml in Jamarin sea water= JSW), followed by gentle centrifugation in order to group them together on the tube bottom. The mesomeres formed an aggregate and developed into an embryoid. Two days later, the embryoids were removed into larger glass tubes containing 2 ml JSW, followed to culture for about 30 days more. The embryoids developed into larvae having most larval structures such as arm rods, archenteron and pigment cells within several days, then formed some adult structures such as spines and test plates. Finally, they formed lantern and tube feet and metamorphosed into complete juvenile sea urchins. FORMATION OF THE ADULT RUDIMENT AND METAMO- RPHOSIS OF SEA URCHIN BY THYROID HORMONES. Y.Chino, M.Kanda, K.Yamasu, T.Suyemitsu and K.Ishihara. Dept. of Regul. Biol., Fac. of Sci., Saitama Univ., Urawa, Saitama. During development of the sea urchin,the adult rudiment develops within the larval body and renders the larva competent for metamorphosis. Exploring the molecular mechanisms of larval development that lead to metamorphosis in the sea urchin, we found that thyroid hormone (TH) induces both formation of the adult rudiment and metamorphosis in the sea urchins. TH is actually present in the larval body, and we found that TH accumulates to a remark- ably high level in 8-armed larvae before metamorphosis, when larvae are reared in the presence of food, namely, algae, that contains TH. Removal of algae inhibits the accumulation of TH and arrests the forma- tion of adult rudiments, suggesting that TH is obtained from algae. Metamorphosis of competent larva can be induced by TH, but differences from natural metamorphosis are apparent. We like to propose for metamorphosis of sea urchin that it is better to understand as a consecutive process that can be divided into two stages; a preceding pro- cess of gradual formation of adult rudiment and absorption of the larval body and a ensuing rapid collapse of the residual larval body and appearance of adult struc- tures. EFFECTS OF TEMPERATURE AND Ca2* CHELATOR ON ATTACHMENT OF HEAD AND TAIL RESORPTION OF ASCIDIAN LARVA. Y. Sato and M. Morisawa Misaki Marine Biological Station, Fac. of Sci., Univ. of Tokyo. Kanagawa. EEE After hatching, tadpole larva of ascidian, attaches to the bottom and then starts metamorphosis ; the transition from the larval to the adult stage Stimulation of the attachment has been considered to be the signal for the later morphogenetic process, such as the resorption of the tail, loss of the outer cuticular layer of the tunic, the rotation of the visceral organs. As the first step to know the relationship between head attachment and tail resorption, we first studied the effect of temperature on both events in the larva of the ascidian Ciona intestinalis. When the larvae were incuvated at 4%, attachment and complete resorption did not occur for more than 24 hours though both events occurred at room temperature (230) within 5 hours after hatching. When temperature was brought back to the original condition, tail was resorbed rapidly without attachment in all larvae. Rotation of visceral organs and the formation of the stron were completed after tail was resorbed and then attachment of larvae began. These results suggest that larval attachment is not the trigger for the tail resorption. Then we investigated the role of Ca** on the process of metamorphosis. When hatched larvae were incuvated in the artificial sea water containing 10°°M BAPTA-AM to chelate intracellular (Ca**, attachment of larvae did not occur and the adhesive papillae remained intact at the tip of tadpole head. However, tail was resorbed completely suggesting that intracellular Ca** takes part in the attachment, but not play a key role in the tail resorption. . The experiments using temperature shock and (Ca** chelator suggest that attachment and tail resorption of ascidian larva may be controlled by different mechanism CHANGES IN ELECTROPHORETIC PATTERNS OF EGG PROTEINS DURING FERTILIZATION IN Oryzias latipes. T. Kani-e, T. Iwamatsu and T. Hirabayashi Dept. Biol., Aichi Univ. of Educ., Kariya 448 and Inst. of Biol. Sci., Univ. of Tsukuba, Ibaraki 305. Changes in the two-dimentional SDS elec- trophoretic patterns of extracts of fertil- izing Oryzias latipes eggs were surveyed. A major protein stainable with a coomassie brilliant blue R-250 in unfertilized eggs began to dissappear a few minutes and be- came undetectable 85 min after insemina- tion, followed by appearance of three dif- ferent proteins. within ten min (23°C). On the other hand, unfertilized eggs which were immersed .in 25% acetone-saline for 30 sec (26 C) and failed to exhibit exocyto- sis by insemination, were homogenized in to 100 min from the acetone-treatment, or insemination immediately after the treat- ment. The electrophoretic patterns in these eggs were compared with those in normally fertilized eggs. In acetone treated eggs. only a small spot of slightly alkaline pro- tein among three proteins described above became also detectable a few minutes after acetone-treatment regardless of insemina- tion. These observations suggest that in the medaka egg, the alkaline protein be- comes detectable by acetone-treatment, ir- respective of exocytosis, while other three change in a close relation with exocytosis. Developmental Biology 1161 1-METHYLADENINE PRODUCTION BY OVARIAN FOLLICLE CELLS RESPONSIBLE FOR OOCYTE MATURATION IN STARFISH M. Mita. Dept. of Biochem., Teikyo Univ. Sch. of Med., Tokyo. Resumption of meiosis in starfish oocytes is induced by l-methyladenine (1- MA) produced by ovarian follicle cells under the influence of a gonad-stimulating substance (GSS). This study was under- taken to estimate the concentration of 1-MA within the ovary of Asterina pectinifera after stimulation with GSS. After incubation of an ovarian fragment with GSS at concentrations of more than 0.1lmg/ml for about 20 min, spawning started simultaneously with germinal vesicle breakdown (GVBD) and follicular envelop breakdown (FEBD). About 5 min of treatment with either 0.1 mg/ml GSS or 0.1 pM 1-MA was sufficient for induction of oocyte release. The amount of 1-MA produced by the ovary increased as the GSS concentration was raised, and the longer the time treatment with GSS, the greater was the amount of 1-MA produced. Also, 1- MA production was markedly dependent on the content of ovary. About 130 oocytes were contained per mg wet weight of ovary and each oocyte was surrounded by a follicular envelop consisting of about 50 cells. Therefore, the content of 1-MA was calculated to reach 0.18 pM at 5 min after 0.1 mg/ml GSS treatment. These results strongly suggest that follicle cells are enough to produce 1-MA for oocyte Maturation in starfish. STARFISH OOCYTE P34C4C2 KINASE ASSOCIATED WITH DETERGENT-RESISTANT CYTOSKELETONS. K.Kishimoto, S.Hisanaga and T.Kishimoto. Lab. of Cell and Dev. Biol., Fac. of Bio- sciences, Tokyo Inst. of Tech. Yokohama. Onset of mitosis or meiosis is induced by activation of a serine/threonine kinase mamed maturation Eaanoiag factor (MPF), consisting of p34°9°* and cyclin B. Al- though MPF was assumed to be a soluble fac- tor, our and other's recent results suggest its, at least, four distinct subcellular states: freely soluble, microtubule-associ- ated, detergent resistant cytoskeleton- associated and chromosome-associated. To resolve how MPF displays different subcel- lular states, we studied and compared properties of detergent soluble _(DS)- and detergent insoluble (DI )-p34e¢e kinase. When starfish oocytes were treated with a buffer containing 0.5% Nonidet P-40, about half of histone Hl kinase activity was extracted and the remaining half was recov- ered in.the Di-fraction. Although DS- and DI-p34°9°* showed similar properties in the binding to p135¥°* and anti-cyclin B, and the behavior on gel filtration, vimentin kinase activity was different. The ratio of VME GE oy BASSE H1 phosphorylation by DI-p34°°°* was about 3-5 times lower than that of DS-p34°9°*, Sucl-affinity column purification of DI-p34°°°* increased _vimen- tin kinase to the level of DS-p34C9°¢. These results suggest the association of a Beoeas which Amina Lies) Heetsinteatin kinase of p34cdC2, with D1-p34Cde2 | BINDING OF CYCLIN B AND MAP MEDIATES THE ASSOCIATION OF MPF WITH MICROTUBULE. K.Ookata, Saris an Aga, H.Murofushi~, T.Itoh“, H.Hotani*, E.Okumura, K.Tachibana and T.Kishimoto. Lab. Cell Dev. Biol., Tokyo Inst. Technol., Yokohama, lDept. ENCIISIS 6 Biochem., Univ. Tokyo, Tokyo, and Dept. Biosci., Teikyo Univ., Utsunomiya. The onset of M-phase is controlled by MPF (Mepbgse promoting factor), a complex of p34°%C* and cyclin B. We demonstrated previously that a portion of the complex associates with microtubules (MTs) and accumulates on meiotic spindle of starfish oocytes. To address the mechanism and the role of this association in vitro, in the present study we used MAP4, which is a major and well characterized microtubule- associated protein in mitotic HeLa cells. The cdc2-cyclin B complex, which is purified from starfish oocytes with sucl- affinity column, coprecipitated with MTs in the presence of MAP4. GST-cyclin B- Sepharose 4B bound to whole MAP4, and preferentially to bacterially-produced "Pro-rich fragment" of MAP4. The dark field microscopic observation revealed that the phosphorylation of MAP4 by the cede2-cyclin B complex increased the instability of an individual MT. These results indicate that the association of the cdc2-cyclin B complex with MT is mediated by the binding of cyclin B and the "Pro-rich region" in the C-terminal half of MAP4 and the associated complex affects the MT dynamics. DISTRIBUTION AND CHANGES OF cdc2 HOMOLOGUES DURING CELL CYCLE IN THE NEWT EGGS. K. Takahara, Y. Iwao, M. Yamashita* and M. Nagahama Inst. Biol., Fac. Sci., Yamaguchi Univ., Yamaguchi. XLab. of Reprod. Biol., Natl. Inst. for Basic Biology, Okazaki. The accessory sperm nuclei in the vegetal hemisphere degenerate in the physiologically polyspermic newt, Cynops pyrrhogaster eggs, probably due to exposure to a low level of M phase- promoting factor (MPF) activity. We investigated the distribution of cdc2 kinase (a catalytic subunit of MPF) and its relative molecules with anti-PSTAIR- sequence antibody. A PSTAIR-containing protein (32kDa) was observed both in eytoplasm and in germinal vesicles of full grown oocytes. Three proteins, major 32kDa, minor 31kDa and 30kDa, were detected in the unfertilized eggs, but 81kDa protein appeared only in high MPF condition. The 30kDa protein may be cdk2 protein by cross-reaction with anti- goldfish-edk2 antibody. The animal hemisphere was abundant about 2 times in 32kDa protein, and 4 times in 31kDa and 30kDa proteins, compared with that in the vegetal hemisphere. These results suggest higher activity of MPF in the animal hemisphere than in the vegetal hemisphere. 1162 Developmental Biology CYCLIN B IN FISH OOCYTES: ITS APPEARANCE DURING OOCYTE MATURATION AND IMPLICATIONS FOR THE MECHANISM OF MPF ACTIVATION. Y. Katsu, M. Yamashita, H. Kajiura and Y. Nagahama Lab. of Reprod. Biol., Natl. Inst. for Basic Biology, Okazaki. Changes in the two components of maturation-promoting factor (MPF), p34°42 and cyclin B, during 17 @,208-DP-in- duced oocyte maturation in goldfish were examined using newly developed monoclonal antibodies against the C- terminal of goldfish p34°4? and E. coli-produced goldfish cyclin B. The C-terminal p34°4? antibody recognized a 35kDa protein in immature oocyte extracts and 35kDa and 34kDa proteins in extracts from oocytes undergoing germinal vesicle breakdown (GVBD). Cyclin B was absent in imma- ture oocytes and appeared at GVBD, coinciding exactly with the appearance of the 34kDa protein. Furthermore, immuno- precipitate experiments revealed that cyclin B formed a complex with the preexisting p34‘? as soon as it appeared. Introduction of E. coli-produced goldfish cyclin B into immature oocytes induced MPF activation both in vivo and in vitro. It is concluded that (1) 35kDa and 34kDa proteins are an inactive and active form of p34°4*, respectively, and (2) MPF activation in fish oocytes occurs when preexisting p34ed2 forms a complex with newly synthesized cyclin B, a situation differing from that in Xenopus and starfish, in which the p34¢d:2-cyclin B complex is already present in immature oocytes. MOLECULAR MECHANISMS OF ACTIVATION OF MPF. M. Yamashita, H. Kajiura, T. Hirai* and Y. Nagahama. Lab. of Reprod. Biol., Natl. Inst. for Basic Biology, Okazaki and “Dept. of Biosci., Nishi-Tokyo Univ., Yamanashi. Maturation-promoting factor (MPF) consists of catalytic p34°4? and regulatory cyclin B. p34°4 is activated after binding to cyclin B, and forms active MPF. Using a cell-free system derived from immature goldfish oocytes, we are investigating the molecular mechanisms of activation of MPF (p34°**), Activation of MPF was induced by introduction of cyclin B into immature oocyte extracts, in which p34°%? was present but cyclin B was absent. The activation was associated with phosphorylation of p34°%? on threonine and of cyclin B on serine. MPF activation was not induced when phosphorylation was inhibited by protein kinase inhibitors. To examine whether serine phosphorylation of cyclin B is required for MPF activation, we pro- duced a mutant cyclin B which lacks serine residues (Ser? and Ser”*) phosphorylated by p34°¢?. The mutant cyclin B was not phosphorylated during MPF activation, but it was still able to induce MPF activa- tion. These results indicate that serine phosphorylation of cyclin B is catalyzed by activated p34‘ and is not required for MPF activation. Using a mutant p34°¢, we are currently investigating whether threonine phosphorylation of p34°%? is a prerequisite for MPF activation. PROTEASOME DURING OOCYTE MATURATION AND EGG ACTIVATION IN GOLDFISH, CARASSIUS AURATUS T.Tokumoto, M. Yamashita and Y.Nagahama, Lab. of Reprod. Biol., Natl. Inst. for Basic Biol., Okazaki 444 Changes in protein levels and enzyme activity of proteasome were examined during 17a,208-DP-induced oocyte maturation and egg activation in goldfish. Protein levels were assessed using three kinds of monoclonal antibodies ( GC4/S, 3a and 3B ) against proteasome purified from goldfish oocytes, each of which is specific for one of the 10 proteasome subunits separated by SDS-PAGE. Enzyme activity was determined using a fluorogenic peptide ( Suc- Leu-Leu-Val-Tyr-MCA ) as a substrate. During oocyte maturation, there were two parallel peaks in the protein levels and enzyme activity of proteasome, one prior to migration of the germinal vesicle ( GV ) and the other just after the completion of GV breakdown. Immediately after egg activation, a two-fold increase in enzyme activity was observed, followed by a sharp drop after cyclin degradation. This decrease in enzyme activity was accompanied by a decrease in protein levels, except for one minor anti-GC4/S- positive component which remained unchanged during egg activation. These results suggest that proteasome is involved in oocyte maturation and egg activation, and plays a role in cyclin destruction that occurs soon after egg activation. GONADOTROPIN INDUCES AN INCREASE IN 17a,20B- DP RECEPTOR CONCENTRATION AND _ THE DEVELOPMENT OF OOCYTE MATURATIONAL COMPETENCE IN A_ TELEOST, THE HIRAME, Paralichthys olivaceus. M. Yoshikuni!, Y. Nagahama!, S. Adachi?, S. Ijin2 and K. Yamauchi?. \Lab. of Reprod. Biol., Natl. Inst. for Basic Biol., Okazaki, 2Dep. of Biol., Fac. of Fisheries, Hokkaido Univ., Hakodate. During the breeding season, hirame spawn daily at a specified time. Oocytes in various stages of development can be obtained by sacrifying fishes at various times before ovulation. Oocytes collected at 30 hr before ovulation undergo final maturation in vitro in response to human chorionic gonadotropin (HCG) or 170,20B-dihydroxy-4- pregnen-3-one (170,20B-DP, a presumptive maturation- inducing hormone in this species). In contrast, oocytes collected at 36 hr before ovulation mature with HCG, but not with 170,20B-DP; however, these oocytes become sensitive to 17a,20B-DP stimulated if exposed to HCG in vitro for 10 hrs. (3H]17a,20B-DP-binding activity was examined using oocyte cortices prepared from defolliculated oocytes. Cortices isolated from 17a@,20B-DP-sensitive oocytes (30 hr before ovulation) specifically bound [3H]17a,20B-DP, but those from 17a,20B-DP-insensitive oocytes (36 hr) did not. Treatment of the latter oocytes (36 hr) with HCG in vitro resulted in the appearance of 17@,20B-DP binding activity. These results strongly suggest that gonadotropin-induced maturational competence in hirame oocytes in mediated by an increase in 17a,20B-DP receptor concentrations on the oocyte surface. Developmental Biology 1163 ARTIFICIAL INDUCTION OF MEIOSIS-RESUMPTION WITH EMETINE IN PELECYPOD OOCYTES. K. OSanai and R. Deguchi. Marine Biological Station, Tohoku Univ., Asamushi, Aomori. A protein synthesis inhibitor, emetine is known to induce parthenogenetically meiosis in metaphase I-arrested pelecypod oocytes (Mytilus edulis, Dubé and Defresne 1990). We examined whether protein synthe- sis inhibitors induce meiosis-~resumption also in prophase arrested oocytes of pele- eypods (Crassostrea gigas and Limaria ha- kodatensis). In sea water containing 0. 1- 0.5 mM emetine, the oocytes underwent ger- minal vesicle breakdown and extruded the first polar body. After the first meiotic division, chromosomes’ remained in the proper oocytes duplicated in number and aggregated to several clusters, or dispers-— ed in cytoplasm. The aggregated chromosome elusters decondensed to swollen nuclei. The first cleavage often proceeded without nuclear division. Puromycin (0. 1-0.5 mM) induced also germinal vesicle breakdown, but the oocytes were arrested at the first metaphase of meiosis. These findings suggest that the mechanism of meiosis resumption in prophase-arrested oocytes differs from that in metaphase-arrested oocytes. OVARY EXTRACT OF THE ASCIDIAN, HALOCYNTHIA RORETZI1, THAT INDUCES THE OOCYTE MATURATION AND THE SELF- STERILITY. 11. Numakunai, and 7H. Yokosawa, Mar. Biol. Stat., Fac. of Sci., Tohoku Univ., Asamushi, 2Dept. of Biochem., Fac. of Pharmaceutical Sci., Hokkaido Univ., Sapporo. Ovarian egg mass was taken out of the gonad of H. roretzi by pipetting and suspended in sea water (sea water:egg mass=1:1). After stirring briefly, the suspension was centrifuged at 250xg for 15'. The resulting supernatant was boiled and centri- fuged at 25,00xg for 30'(ovary extract). The ex- tract was subjected to a gel filtration Sephadex G- 75 column, and the inhibiting or inducing activity of the oocyte maturation and the expansion of the peri-vitelline space(self-sterility) of the ovarian egg was assayed. When the extract was treated with 0.01% trypsin or allowed to keep O°C for several days, the inducing activity was detected as two peaks: the two activities showed different suscep- tibility to DFP, a serine protease inhibitor. At the beginning of the spawning season the strong in- hibitory activity was detected, while near the end of the spawning season the inhibitory activity was diminished and inversely the inducing activity be- came detectable. The extract of the gonad of Ciona savignyi was prepared and treated with trypsin. After applying the Sephadex G-75 column, each fraction was assayed for the inducing activity using the egg of H. rore- tzi. The same results were obtained as those in the extract of H. roretzi prepared near the end of the Spawning season. FUSION OF STARFISH OOCYTES WITH DIFFERENT MATURATION PHASES. Mitsuki Yoneda. Dept of Zoology, Kyoto Univ. Kyoto 606 Immature oocytes of Asterina pectinifera were fused by treating them with poly- ethylene glycol following the procedure of Vassetzky et al (1986). Electric pulses were occasionally applied. 1-methyl- adenine caused the fused pair to extrude pairs of polar bodies (PB1's and PB2's) on schedule of unfused controls, and the pair eventually formed two pronuclei. Two populations of oocytes with different maturation phases were then fused at random. I observed those fused pairs that extruded PB2 from one meiotic nucleus within + 8 min of the PB1 extrusion from the other nucleus. In these hetero- geneous pairs, the PB2 was not formed from the latter nucleus, and both nuclei went through to form pronuclei. Presence of a cytoplasmic factor to stop cell division in maturing starfish oocytes is suggested. Vassetzky et al (1986) Methods in Cell Biology, 27:359-378. REGULATORY REGION OF SEA URCHIN ARYL- SULFATASE GENE RESPONSIBLE FOR SPATIAL EXPRESSION. K.Akasaka, K.Yamada, S.Tanaka*, Y.Iuchi, K, Takata and H. Shimada. Dept. of Biol., Fac. of Sci., Hiroshima Univ., Higashi- Hiroshima, *Inst. of Life Sci., Mitsubishi-Kasei, Machida. The expression of arylsulfatase(Ars) gene begins at blastula stage and is restricted in aboral ectoderm cells in sea urchin embryos (H. pulcherrimus). We have demonstrated that the cis-regulatory elements responsible tor temporal expression locate in the fragment spanning from -3kb to -2b of Ars gene by in vivo transcription assay. In the present study, we have monitored spatial expression of CAT(chloramphenicol acetyltransferase) reporter gene in the microinjection mediated Ars(-3kb to -2b)- CAT fusion gene transfered embryos by using anti-CAT antibody. The expression of CAT was restricted in the aboral ectoderm cells as that of endogenous Ars gene and no ectopic expression was detected. It is suggested that the cis-regulatory elements responsible for aboral ectoderm specific expression also locate in the fragment spanning from -3kb to -2b of Ars gene. 1164 Developmental Biology QUANTATIVE ANALYSIS OF THE CIS-ACTING ELEMENTS REGULATING THE EXPRESSION OF ARYLSULFATASE (ARS) GENE IN SEA URCHIN EMBRYO. K. Yamada, S.Eguchi, K.Akasaka and H. Shima- da, Zool, Inst, Fac, Sci, Hiroshima Universi- ty, Higashihiroshima 724, Japan. We have previously reported that a pArs-CAT fusion construct, containing the 5’ flanking region of sea urchin (Hemicen- trotus pulcherrimus) Ars gene spanning from -2 to -3160, was expressed under a proper temporal control following injec- tion into unfertilized eggs. A number of cis-acting elements regulating the expres-— sion of the Ars gene was also found by deletion analysis. As a preliminary step for quantative analysis of the cis-acting sequences of the Ars gene, in this report we describe the factors that affect the level of Ars gene expression. The results of DNA titration showed that it is essen- tial to normalize the CAT reporter activ- ity to the plasmid contents per embryo is required for significant quantitation of the expression level of injected con- structs. Comparison of the normalized CAT activity among egg batches suggests that an experiment using the eggs from a single batch is required for precise evaluation of the transcriptional activity of inject- ed constructs. Quantative analysis of the upstream enhancer of sea urchin Ars gene indicates that at least three positive elements exist in this region. NEWLY DETECTED CIS-ACTING ELEMENTS REQUIRED FOR INCREASED EXPRESSION OF THE ARYLSULFATASE(Ars) GENE THE SEA URCHIN (H. pulcherrimus) EMBRYO Y.Iuchi, K.Yamada, K.Akasaka, and He ShilmadakyeZool einisit-1,.eh aCe SCllan Hiroshima Univ., Higashi-Hiroshima 724. When the Ars-CAT fusion gene containing the Ars sequence spanning from -3000bp to -2bp was constructed and CAT assayed, the expression of the reporter gene was fairly low as compared with that of the endogenous Ars gene, suggesting the presence of other cis-acting elements beyond this Ars sequence. In the present study, We screened for new cis-acting elements of the Ars gene using a microinjection-mediated gene transfer system, and we found two Ars sequences which activate the transcription of the reporter gene. One is in the region spanning from a part of the lst intron to the beginning of the 2nd exon, and the other spanning from the 3rd exon to the 3rd intron. Two distinct DNA binding proteins that sequence-specifically interact with these cis-element were detected by gel mobility shift assays. SINGLE AND TRIPLE STRAND STRUCTURES (H-DNA) IN REGULATORY REGION OF ARYLSULFATASE GENE OF SEA URCHIN EMBRYO. N.Sakamoto, T. Yamamoto, K. Yamada, K.Akasaka and H.Shimada. Zool. Inst., Fac. Sci., Hiroshima Univ., Higashi- Hiroshima 724 We have reported that the upstream region from —3000bp to -2bp of arylsulfatase(Ars) gene of sea urchin, H. pulcherrimus, is responsible for temporal and spatial regulation of expression of this gene. This region contains a long polypyrimidine = polypurine (pyr-pur) sequence. On the basis of Sil nuclease sensitivity, we have demonstrated that the pyr-pur sequence can adopt H-DNA structure under a moderately acidic condition. In the present study we have shown that the pyr-pur region can adopts the H-DNA configuration even under physiological pH using DEPC and Os04 as chemical probes. Cleavage sites of the plasmid containing the pyr-pur region by the chemical probes were similar to Sl nuclease reactive sites under the level of negative supercoiling higher than those of plasmids isolated from E. coli. Since chromosomal DNA is believed to be under high torsional stresses, it is likely that the pyr-pur region often form H-DNA configurations in chromosomal structures. It is also conceivable that such an unorthodox DNA structure may have an important role for regulation of gene expression. THE EGIP-BINDING PROTEIN IN EMBRYOS OF SEA URCHIN, ANTHOCIDARIS CRASSISPINA. Y.Fujita, K.Yamasu, T.Suyemitsu, and K.Ishihara. Dept. of Reg. Biol., Fac. of Sci., Saitama Univ., Urawa. We previously reported that sea urchin embryos contain exogastrula-inducing pep- tides (EGIPs) throughout the early devel- opment. In the present study, we investi- gated the location of EGIP-binding protein in the embryos. EGIP-D was incubated with homogenates of embryos at various stages, crosslinked to the binding protein using disuccinimidyl suberate (DSS), and analysed by western blot using anti-EGIP-D-antibody. As a re- sult, 30KD protein was detected in both eggs and embryos. To examine the localization of this pro- tein in the surface of embryos, EGIP-D was added to intact embryos, crosslinked by DSS, and analysed by western blot. The EGIP binding protein was detected in, 3nz tact embryos, but not detected in Ca, Mg- free sea water (CMF-SW)-treated embryos. It suggests that this protein exists in outer surface of embryos and can be extracted by CMF-SW. Actually, the effect of EGIP was markedly decreased on induction of exogastrula for CMF-SW-treated embryos. The role of EGIP and its binding protein in surface of embryos during normal developement are now under investigation. Developmental Biology 1165 STRUCTURAL ANALYSIS OF EXOGASTRULA- INDUCING PEPTIDE GENE Y.Haruguchi, K.Yamasu, T.Suyemitsu and K.Ishihara. Dept. of Regulation Biol., Fac. of Sci., Saitama Univ.,Urawa. We previously isolated the cDNA clone for exogastrula-inducing peptide(EGIP) precursor of sea _ urchin, Anthocidaris crassispina and showed that the EGIP gene is expressed in a stage-specific manner. In order to elucidate the regulatory mechanism of EGIP gene expression, we have tried to isolate genomic clones. Bulk of the EGIP gene has already been cloned, but the clones for the transcrip- tional initiation site and the upstream region have not been obtained. To com- plete the cloning of EGIP gene, we plated the genomic library of sea urchin on E.coli CES201 strain as a host, and screened it using 5'-terminal fragment of EGIP cDNA as a probe. Positive clones were subcloned into pUC19 vector and analyzed by Southern hybridization and sequencing. EGIP gene is at least 7kb in size and composed of 7 exons and 6 introns. Primer extension revealed that the capping site is about 100 nucleotides upstream of 5'-end of cDNA, indicating that the full-length mRNA is about 1750b. This result and the sequence of the genomic DNA upstream of cDNA sequence al- lowed us to determine the tentative transcriptional initiation site and the promotor region. ANALYSIS OF cDNA FRAGMENTS FOR PROTEIN TYROSINE KINASES OBTAINED BY RT-PCR METHOD. K.Yamasu, M.Sakuma, T.Suyemitsu and K. Ishihara. Dept.of Regulation Biol., Fac.of Sci., Saitama Univ., Urawa. Phosphorylation of tyrosine residues in various proteins by protein tyrosine kinases (PTKs) is now considered the es- sential aspect in signal transduction from outer environment into cells. It seems to regulate various cell functions such as proliferation, differentiation, and cell motility. This prompted us to examine the roles of PTKs in the development of sea urchin embryos. As a first step to this aim, we tried to clone partial cDNA frag- ments of PTKs by RT-PCR method. Using to- tal RNA from the embryos of sea urchin An- thocidaris crassispina as templates and primers designed to amplify the cDNA frag- ment corresponding to the highly conserved PTK catalytic domain, we obtained PCR fragments for different types of PTKs in- cluding cytoplasmic PTKs and receptor-type PTKs. The PCR fragments were obtained using RNA of all developmental stages ex- amined ranging from undifferentiated eggs to pluteus larvae. Northern analysis showed multiple bands with different length of 3-6 kb. mRNA of most of the PTK genes are present in unfertilized eggs and plutei. These suggest that PTKs are necessary in oogenesis stage or cleavage stage and then after gastrulation when tissue differentiation is initiated. IN SITU PROTEIN PHOSPHORYLATION IN A SEA URCHIN EMBRYO LOADED WITH RADIOACTIVE PHOSPHATE: ANALYSIS BY 2D-PAGE S.Takeda and H.Hayash Sugashima Marine Biological Laboratory, School of Science, Nagoya University, Toba. We have studied protein phosphorylation in detail during early developmental stage of sea urchin embryos. Proteins were labeled in situ by culturing embryos in artificial sea water containing [$2P]PO4. The TCA insoluble fractions of cell lysate were separated by two-dimensional polyacrylamide gel electrophoresis and phosphoproteins were detected by fluorography at -80°C. During the cleavage stage, the pattern of labeled proteins did not change substantially; i,e. most spots were found constantly throughout this period. However, one protein whose apparent molecular weight was 43 kilodaltons specifically phosphorylated in pre-hatching blastura. In the case of Clypeaster japonicus, labeling of this protein was first appeared at 6.5 hours after fertilization, almost coincident with 128 cell stage, and intensity of labeling of this protein was increased until beginning of hatching. In other species, Anthocidars crassispina Peronella_japonica_and Mespilia gulobulus, the similar proteins identically positioned on autoradiograms and phosphorylated in the similar manner were found. We have also noticed that the treatment of embryo with LiCl which is known as vegetalizing agent caused reduction of the labeling of this protein. These results suggest that phosphorylation of 43K protein in pre-hatching blastura might play an important role in embryogenesis of sea urchin embryo. PROTEINS TO BE PHOSPHORYLATED IN THE REACTIONS CATALYZED BY CAM KINASE, C KINASE, A KINASE AND G KINASE IN SEA URCHIN EMBRYOS. M.Okuyama, Y.Kamata, S.Furuya and I.Yasumasu. Dept. of Biol., Sch. of Educ., Waseda Univ., Tokyo. SDS-PAGE of proteins in whole homogenate of sea urchin embryos was performed after the exposure of whole homogenate to (f{-*?P)- ATP in the presence of protein kinase activators(CAMP, cGMP, Ca**-calmodulin, or the mixture of Case, TPA and phosphatizylserine), and protein kinase inhibitors(HA1004, H-7, H-8, W-5, W-7). On autoradiograph of SDS-PAGE, protein band with molecular weight of 63kD was found to become dense in whole homogenate exposed to ({-*7P)-ATP in the presence of Ca’*-calmodulin and was faint, when W-7 was supplimented. In the presence of W-5 at concentrations higher by about 10 holds then that of W-7, the band of 63kD protein, to be made dense by Ca**- calmodulin became quite faint. Dense band of 63 kD protein obtained in the presence of Ca**-calmodulin was not made faint by H-7 and H-8. This 63kD protein is probably main one to be phosphorylated by CaM kinase. Protein band with molecular weight of 53kD was made faint by H-7 and H-8. The density of 53kD protein band was not altered by W-5, W-7 or Ca**-calmodulin. These proteins were found in the whole homogenate of morulae, blastulae and gastrulae. 1166 Developmental Biology REGULATION OF EARLY EMBRYONIC HISTONE GENE EXPRESSION BY Ca** SIGNALS IN EMBRYOS OF THE SEA URCHIN, Hemicentrotus pulcherrimus. M.Kettoku’, M.Kanda’, K.Mitsunaga-Nakatsubo’, A.Fujiwara*’ and I.Yasumasu’ *Dept. of Biol., sch. of Educ., Waseda Univ., Tokyo, Dept. of Radiation Res., The Tokyo Metropol. Inst. of Med. Sci., Tokyo. It has been reported that pulse treat- ment with A23187 inhibits hatching enzyme synthesis but augments *“C-leucine incorpo- ration into proteins in sea urchin embryos. The pulse treatment with A23187 for lhr in pre-hatching period enhanced the rate of [U-**C]lysine incorporation into H,SO,- extractable proteins. The enhanced rate of [U-“*C]lysine incorporation was made low by tetracaine,ruthenium red and W7, but was hardly altered by H7 and H8. mRNAs encoding early embryonic histones were investigated by Northern blot analysis using genomic DNA clones of histones Hl and H3 as the probes. Pulse treatment with A23187 enhanced the levels of mRNAs encoding histones. The in- crease in the levels of these mRNAS was also blocked by tetracaine, ruthenium red and W7 but was not affected by H7 and H8. Probably, Ca** calmodulin-dependent reactions, enhanced by A23187-induced artificial Ca’ signals, enhance expression of several genes such as early embryonic histone genes in pre- hatching embryos of sea urchin, though expression of other genes, such as hatching enzyme gene, may be turned off by the signals. [ADP-RIBOSYL]ATION OF HISTONES IN NUCLEI ISOLATED FROM SEA URCHIN EMBRYOS S.Furuya, Y.Kamata, M.Okuyama and I.Yasumasu. Dept. of Biol., Sch. of Educ., Waseda Univ., Tokyo. Isolated nuclei were exposed to **P-NAD and histones were extracted by 0O.4N HCl. They were separated using two-dimensional electrophoresis of acid/urea/Triton and SDS gel. Dried gels were analysed for radioactive proteins by fluorography. These fluorographs established some differences in the modification pattern among histones in morulae and gastrulae. In morulae and gastrulae, histone Hl is present in mono modified form and histone H4, at least 4 [ADP-ribosyl]Jated forms. In gastrulae, histone 13 A= established 11[ADP- ribosylJated forms and in morulae 10 [ADP- ribosyljated forms. Histone H3.2 was not present in [ADP-ribosyl]ated form in gastrulae but it displayed up to 4 [ADP- ribosyljated bands in morulae. we AS expected that mono and poly [ADP- ribosyl]ated forms of histones have different functions in nuclei respectively. Changes in the [ADP-ribosyl]aion pattern among varients of histones in nuclei of sea urchin embryo during early development probably causes alternation of their functions. DOES CADP-RIBOSYLJATION OF PROTEINS IN NUCLEI CONTRIBUTE TO ECTODERMAL CELL DIFFERENTIATION IN SEA URCHIN EMBRYOS ? Y.Kamata, A.Fujiwara, S.Furuya and I.Yasumasu. Dept. of Biol., Sch. of Educ., Waseda Univ., Tokyo. The increase in the ADP- ribosyltransferase activity followed by its decrease occurred in pre- and post-hatching period of early development, with peaks of the activity at the morula and the gastrula stage. In embryo wall cells (ectoderm cells) isolated from sea urchin embryos at the late gastrula stage, the ADP- ribosyltransferase activity was markedly higher than in endoderm and mesenchyme cells. The increase in the enzyme activity between mesenchyme blastula and the gastrula stage, which was blocked by actinomycin D and cycloheximide, probably results from the increase in this enzyme activity in ectoderm cells. Expression of this enzyme gene is probably augmented in ectoderm cells. 3-Aminobenzamide (3-ABA), Evil iy EOS — eye ADP-ribosy!transferase, inhibited formation of ectoderm structure more strongly than that of archenterons (endoderm structure). In animalized embryos, which exhibited higher activity of this enzyme than in normal ones, formation of ectodermal structure was also blocked by 3-ABA. ADP-ribosyltransferase, enhanced in its activity due to expression of this enzyme gene in ectoderm cells during gastrulation, probably contributes to differentiation of ectoderm cells. PARTIAL PURIFICATION AND CHARACTERIZATION OF A CASEIN-KINASE 2-TYPE PROTEIN KINASE FROM SEA URCHIN M.Kawamoto?, K.Miyagawa?, T.Natsume? and I.Yasumasu*. +*Dept. of Biol.,) “Schueeod Educ., Waseda Univ., Tokyo., *Teikokuzouki co., LTD., Kanagawa. It has been reported that the activity of cyclic nucleotides independent protein Kinase, is inhibited by heparin in nucleus fraction of sea urchin embryos. The activity localized in nuclei is high in morulae and gastrulae. Extract with 0.4M NaCl of nucleus fraction isolated from gastrula was used for the purification of this enzyme, by sequential chromatography on Casein-Phosvitin-Sepharose, Heparin- TOYOPEARL, Sephacry! S-300HR. The finally obteined enzyme sample exhibited not only several bands on SDS-PAGE, which were judged to be subunits of this enzyme, but also faint bands of other proteins. The enzyme was only partially purified, though the specific activity increased markedly following sequential chromatography. Partially purified enzyme was’ inhibited by heparin. The enzyme was relatively insensitive to spermine, protamine and poly-:t-lysine and appreciably activated by poly-,_-arginine. Developmental Biology 1167 NEW HISTONE MOLECULES PRODUCED AT THE BLASTULA STAGE OF STARFISH EMBRYOS T.Shimizu, W.Teramura, T.Tsuruta, Y.Matsumoto, S.Ikegami. Dept. of Applied Biochem., Hiroshima Univ., Higashi- hiroshima, Hiroshima. We found that the application of trichostatin A (0.01-100mg/1) to fertilized eggs of starfish Asterina pectinifera prevents formation of msenchymal cells but not cleavages, blastulation nor gastrulation to occur. The period sensitive to trichostatin A was restricted to the midblastula stage (12-14 Iie eto] a8 SNe eal Patra heal(eyey) 4 By using a monoclonal antibody raised against p29, a chromatin protein produced during the blastula stage, we analyzed trichostatin A-treated chromatins of midblastulae. It was found that the appearance of p28, a chromatin protein reacted with anti-p29 antibody, was selectively suppressed by trichostatin A whereas p29 was present in the chromatin. Peptide mapping and partial sequencing experiments revealed that both p29 and p28 contained an undecapeptide and an octapeptide. The latter is a part of the sequence of histone H2B. On the other hand, the former LS MONE related to histones, suggesting that p29 and p28 are new histone H2B anchoring hither-to- unknown peptide. PROBABLE PARTICIPATION OF DNA METHYLATION IN ECTODERM CELL DIFFERENTIATION IN SEA URCHIN EMBRYOS. M.Nakasone, Y.Kamata, A.Fujiwara and Y.Yasumasu. Dept. of Biol., Sch. of Educ., Waseda Univ., Tokyo. In post-hatching period in early development, evident increase in the rate of DNA methylation occurred in sea urchin embryos between the mesenchyme blastula and the gastrula stage. The rate of DNA methylation was estimated by the radioactivity in DNA in nucleus fraction exposed to [{methy1-14c}s-adenosy1-L- methionine or in embryos kept with [methyl- 4c¢]methionine. In embryo wall cell fraction, the rate of DNA methylation was higher than in archenteron cells and mesenchyme cells. In animalized embryos, the rate of DNA methylation was evidently higher than in the control ones. Exposure of animalized and normal embryos to SIBA, which inhibited DNA methylation, strongly blocked formation of ectoderm structure. Augmentation of DNA methylation by exposure to methionine at concentration above im, which augmented the rate of methylation, produced abnormal embryos which were alike to animalized embryos. SIBA canceled the effect of methionine on embryonic development. Probably, DNA methylation contributes to differentiation of ectoderm cells. ABNORMAL EMBRYOS DERIVED FROM EGGS TREATED WITH SCN-. A. FUJIWARA, T. NAKAGAWA AND I. YASUMASU Dept. of Biol. School of Education Waseda Univ., Tokyo. Sea urchin eggs, kept for 2 hr in arti- ficial sea water (ASW) containing 0.1-0.3M SCN- in place of Cis; developed to animalized embryos. Embryos washed 4 times with SCN--containing ASW during 2 hr culture with this ASW developed to several types of abnormal embryos such as animalized ones and those alike to vegeta- lized ones. Non-dialyzable compounds extracted from eggs with SCN- were found to bind with embryos and exerted animaliz- ing effect on sea urchin development. Without washing eggs with SCN--containing ASW, these compounds, which are extracted during SCN- treatment, seem to bind again with embryos upon dilution of SCN- concentration at the end of the treatment. Translocation of these compounds is assumed to result in production of animalized embryos. Animalizing effect of these non-dialyzable compounds was canceled by tetracaine, ruthenium red and W7. Treatment of embryos with W7, as well as tetracaine, produced vegetalized embryos. Ca’, calmodulin dependent reactions seems to be activated by these SCN--extractable compounds resulting in production of animalized embryos. EFFECTS OF NON-DIALYZABLE COMPOUNDS EXTRACTED BY SCN- FROM SEA URCHIN EGGS ON MORPHOGENESIS IN EMBRYOS. T. NAKAGAWA, A. FUJIWARA AND I. YASUMASU Dept. of Biol. School of Education, Waseda Univ., Tokyo Non-dialyzable compounds extracted with 0.1-0.3M SOM iieroin sea urchin eggs produced abnormal embryos which were alike in their morphological characteristics to animalized embryos. SCN--extract hardly contained DNA, RNA and substances to be dissolved with ethanol-ether. This SCN— extract did not exhibit any activities of cytochrome c¢ oxidase, rotenone insensitive NADH cytochrome c reductase, phosphorylase and glucose-6-phosphate dehydrogenase. SCN--extract from plasma membrane fraction isolated from sea urchin eggs exhibited almost the same activity to cause abnormal embryo formation to the activity in the extract from egg homogenate. These compounds were bound again with embryos. Thus, we assumed that these compounds is localized to egg surface. Chromatography of SCN--extractable matter on Sephadex G- 75 or G-100 column was performed. The fractions thus obtained, exhibiting a dense band at the molecular weight of about 40K as well as other faint bands on SDS-PAGE, showed the activity to induce abnormal embryos. 1168 Developmental Biology INSULIN RECEPTOR OF THE CULTURED CELLS DERIVED FROM MICROMERES OF SEA URCHIN EMBRYOS S.Kuno, T.Nagura and I.Yasumasu. Dept. of Biol., Sch. of Educ., Waseda University., Tokyo. In a previous study, we found that the micromere-derived cells cultured with insulin undergo outgrowth of pseudopodial cables without formation of spicule rods. Hence, it is assumed that receptors for insulin or insulin-like compounds exist on the surface of the micromere-derivrd cultured cells. In the present study, SDS- PAGE of proteins in micromere-derived cells in culture cross-linked with 257-1abeled insulin was performed to find out receptors for insulin ( or insulin-like protein ). Radioautograghs of SDS-PAGE show a band at the molecular weight of 44k in non-reduced condition and 41k in reduced condition. Insulin receptor is known to have protein tyrosin kinase domein in mammalian cells, probably, these cultured cells have protein tyrosin kinase domein. We carried out immunoblotting with anti-phosphotyrosine antibody, PY20, to find out the phosphorylated protein by the protein tyrosin kinase domein. Immunoblotting shows bands at the molecular weight of 35 and 55k. DIFFERENTIATION OF SMOOTH MUSCLE CELLS DURING HISTOGENESIS OF VESSEL WALL IN THORACIC AORTA OF MOUSE EMBRYO. Y. Takahashi. Vessel Research Laboratory, Fac. of Pharm. Sci., Teikyo Univ., Sagami- ko, Kanagawa. As a basis for studying mechanism of histogenesis of arterial wall, differenti- ation of smooth muscle cells in thoracic dorsal aorta of mouse embryo was investi- gated immunohistochemically using mono- clonal antibodies against two molecular markers, a-actin and smooth muscle- specific myosin heavy chain. At 9 day of gestation, the aorta con- sists of endothelial monolayer. Alpha- actin positive cells appear at 10 day, when they surround the endothelium as a single layer. In 11 day embryos, two or three layers of cells around the endothe- lium express both a-actin and smooth muscle myosin heavy chain. At 13 day, 4-5 layers of cells with both markers are histologically distinguishable from sur- rounding mesenchyme. The number of smooth muscle cell layers hardly increases until 15 day of gestation and neonatal period. In neonatal aortas, there are 1-2 layers of a-actin negative cells outside the smooth muscle layers, showing formation of adventitia. These results suggest that the initial phase of possible smooth muscle induction in mouse aorta occurs before 10 day of gestation. CHARACTERIZATION OF MUSCLE-CELL-SPECIFIC NUCLEAR PROTEINS, P32 AND P30 D. Takai, M. Kirinoki and T. Hirabayashi. Inst. Biol. Sci., Univ. Tsukuba, Tsukuba. Taking advantage of the autoimmunity of some nuclear proteins, we detected and characterized muscle-cell-specific nuclear proteins. We injected the protein components of the nucleus isolated from chicken muscles into chicken breast muscle. Three proteins were detected on a 2D-PAGE pattern of isolated nuclei with the antiserum. Two of them were not detected in the nuclei of liver, brain, cardiac muscle and slow skeletal muscle, but detected in the nuclei of smooth muscle(gizzard) and fast skeletal muscle at all stages examined. They were detected only in a DNase treated sample prepared in the fractionation of muscle cell nuclei, suggesting that they are proteins which weakly bind to chromatin. ANALYSIS OF PROTEINS IN MUSCLE NUCLEAR FRACTION M. Kirinoki, D. Takai, and T. Hirabayashi, Inst. Of (Biiely (Sci. sUnsse of Tsukuba, Tsukuba. In order to examine the change of nuclear protein components during cell differentiation, we compared protein constituents of nuclei between several tissues by 2-dimensional gel electrophoresis. Among proteins which seemed to have tissue specificity, we picked up and analyzed proteins that formed several spots with a molecular weight of 108 kDa in a range of isoelectricpoint from pH 7.2 to pH 7.4 on a 2-dimensional pattern of chicken breast muscle nuclear fraction. The antiserum against 108 kDa proteins showec by immunoblotting tests that i08 kDa proteins were in nuclear fractions of gizzard and heart as well as of breast muscle, but not of brain nor of liver from 1-day-old chicks. The 108 kDa proteins were first detected in the breast muscle of the 15-day-old embryo. Their amount increased until hatching and was maintained for 1 week, then decreased gradually. In sequential extraction experiments, the 108 kDa proteins were found to be soluble ina solution of high salt concentration after NP40 treatment and DNase I digestion. Developmental Biology 1169 TROPONIN T ISOFORM EXPRESSION AND CHIMERIC FIBERS IN REGENERATING MUSCLE Y. Yao and T. Hirabayashi, Inst. of Biol. Sci., Univ. of Tsukuba, Tsukuba Using antibodies against anti-fast troponin T and anti-slow troponin T, we examined the regenerating muscle cells obtained by transplantation of anterior latissimus dorsi (slow muscle) into pectoralis major (fast muscle) and found by 2-D SDS-PAGE and immunoblotting that they expressed slow type troponin T isoforms. Thus, the pattern of skeletal muscle troponin T isoform expression is fixed in cell lineage. Futhermore, the chimeric muscle fibers composed partly of fast and partly of slow fibers were found in regenerating muscle blocks, suggesting that the fast and slow myoblasts fused during regeneration. EXPRESSION OF MYOGENIC FACTORS IN DENERVATED CHICKEN BREAST MUSCLE: ISOLATION OF THE CHICKEN MYF5 GENE. 2 0.Saitoh!, A.Fujisawa-Sehara“, Y.Nabeshima , and M.Periasamy>. lDept. of Mol. and Cel. Neurobiol., Tokyo Metropol. Inst. for Neurosci., Fuchu, 2 Div. of Mol. Genet., Natl. Inst. of Neurosci., Natl. cent. of Neurol. and Psychia., Kodaira, 3 Dept. of Physiol. and Biophys., Univ. of Vermont, Vermot, USA. 2 We have isolated chicken Myf5 gene in addition to cDNAs for MyoD1 and myogenin, and analyzed their mRNA levels during chicken breast muscle development. We found that chicken Myf5 and MRF4 genes are tandemly located on single genomic DNA fragment, and that chicken Myf5 gene is organized into at least three exons. The analyses of expression revealed that myogenin expression is restricted to in ovo stages in breast muscle, and is not detectable in neonatal and adult stages. On the other hand, Myf5 expression is detectable until day 7 post-hatching and is not found in adult muscle, whereas high levels of MyoD1 and MRF4 are detectable at all stages. To further understand the roles of innervation during muscle Maturation, we analyzed the expression of the myogenic factors in denervated adult breast muscle. We found that MyoD1, myogenin, and MRF4 are induced at high levels in denervated muscle, whereas no change occurs in the level of Myf5. CAN COLLAGEN SYNTHESIS TRIGGER THE EXPRESSION OF MYOGENIN IN CULTURED MUSCLE CELLS? R. Matsuda. Dept. of Biol., Col. of Arts & Sci., Univ. of Tokyo, Tokyo. It has been known that the myogenic regulatory factor (MyoD family) triggers muscle cell differentiation. In cultured skeletal muscle cells, the decrement of serum or fibroblast growth factor concentration is crucial to activate MyoD family genes. On the other hand, the extracellular matrix (ECM) is known to be an important environmental factor to support muscle cell differentiation. To study the effect of ECM formation on the expression of MyoD family gene in cultured BC3H1 cells, we added 200M of ascorbic acid 2-phosphate (a co-factor for collagen synthesis) into growth medium and cultured for 4 days. The cells continued to proliferate and formed well developed ECMs and multiple cell layers. Immunofluorescent study revealed that only the cells on the bottom of the culture dish expressed myogenin. The result suggests that the enhanced formation of ECM is one of the important factors to up-regulate the expression of MyoD family gene during muscle development. Involvement of Protein Tyrosine Kinase in the Regulation of Myogenin Expression N. Hashimoto, M. Ogashiwa and S. Iwashita. Mitsubishi Kasei Inst. Life Sci., Machida, Tokyo 194. Myogenin is one of the muscle-specific transcription factors that regulate myogenic differentiation accompanying growth arrest. We are focusing on the role of tyrosine phosphorylation in the process of myogenic differentiation of cultured myoblast C2C12 cells. Using an affinity-purified anti-myogenin antibody, we revealed the expression pattern of myogenin during the course of differentiation of C2C12 cells. Myogenin'was first detected by immunoblot analysis as a pair of bands prior to myotube-formation and expression levels remain high in myotubes. To examine the role of tyrosine phosphorylation in myogenesis, the effect of genistein, an inhibitor of protein tyrosine kinase, was examined. When C2C12 cells were cultured for 2 days in differentiation medium containing various concentrations of genistein, myogenin expression was suppressed in a dose- dependent manner. Furthermore, myotube-formation was also completely inhibited in the presence of 50 uM genistein. Taken together, these results indicate that genistein blocks myogenesis through the suppression of myogenin expression. The present study suggests that tyrosine phosphorylation plays a key role in the regulation of myogenin expression and myogenesis. 1170 Developmental Biology IMMUNOCYTOCHEMICAL EVIDENCE OF BINDING OF ANTI- BIOTICS TO THE VITELLINE AND FERTILIZATION ENVELO-— PES OF CHERRY SALMON BGGS AND THEIR BINDING GLYCOPROTEINS S. Kudo! and S. Yazawa*. ‘Depts. of Anat. and ?Tegal Med., Gunma Univ. Sch. of Med., Maebashi The binding of three antibiotics (gentamicin, Oleandomycin and chloramphenicol) to the vitelline (VE) and fertilization envelopes (FE) and their binding glycoproteins were investigated using cherry salmon eggs. Purified VEs and FEs were incubated in isotonic solution (pH 7.0) containing each of the antibiotics (1 mg/10 ml) for 2 hr at room temperature. After a thorough wash in the isotonic solution, immunostaining was performed using antiserum against each of the antibiotics, followed by photomicrography and immunoelectron microscopy. Purified VEs or FES were treated with enzymes (neuraminidase, B-galactosidase, a-manno- sidase,N-acetyl-8-D-glucosaminidase, and a-L- fucosidase) , followed by incubation in the anti- biotic solution and subsequent immunostaining and immunocytochemistry as described above. The main binding sites of the antibiotics were demonstrated to be the VE or FE outermost layer and cortical alveolus exudates. Enzyme treatment suggested that the binding of gentamicin and chloramphenicol to the VE or FE might be related to sialic acid residues and that of oleandomycin to fucosyl residues. An extract from the FEs was analyzed by SDS-PAGE separation, and proteins in SDS slab gels were transferred electrophoretically to nitrocel- lulose membranes for immunoblotting analysis. The binding of the antibiotics was revealed using their antisera as several bands showing differences and common features in both position and reactivity. MEDAKA OOCYTES ROTATE WITHIN THE OVARIAN FOLLICLE UNDER in vitro CONDITIONS. T. Iwamatsu. Dept. Biol., Aichi Univ. of Educ., Igaya-cho, Kariya 448 In a previous study (Zool. Sci., 9 (3): 589-600, 1992), it has been inferred that in medaka follicles, oocytes surrounded by a granulosa cell layer may begin to rotate within the basement membrane before or when the oocyte axis is established in the early stage of oogenesis. It is the pur- pose of this study to ascertain whether oo- cytes really rotate within follicle. Iso- lated medaka follicles were incubated in L- 15 Medium supplemented with 10% fetal bo- vine serum and 40 ug/ml gentamicin sulfate for a few hours at about 26°C. During in- cubation, movement of oocytes under a mi- croscope was recorded on a video cassette recorder and analysed. Movement of the fol- licular surface was marked by carbon parti- cles which stuck to its surface, and the movement of intrafollicular oocytes was traced by dislocation of its attaching and non-attaching filaments on the chorion. Oo- cytes exhibited the rotation around the predetermined animal-vegetal axis, some- times at a slightly oblique angle to the axis. The velocity of oocyte rotation which might depend on movement of granulosa cells was about 30-50 wum/hr and deviated in oo- cytes of different stages in the early vi- tellogenic phase of oogenesis. The present result indicates that medaka oocytes virtu- ally rotate within follicle. FORMATION OF FILAMENTS ON THE CHORION OF OOCYTES IN THE MEDAKA, ORYZIAS LATIPES. S.Nakashima and T.Iwamatsu. Dept. of Biol., Aichi Univ. of Educ., Kariya. The formation of attaching and non-at- taching filaments on the chorion of oocytes of the medaka, Oryzias latipes and the re- lationship between the distribution of granulosa cells in the animal hemisphere (AH) and vegetal pole area (VPA) and that of attaching and non-attaching filaments were studied as a step in the clarification of the mechanism for the determination of egg polarity. The oocyte at stage II which is less than 100 pp in diameter has an almost smooth surface with a number of tufts of micropro- jections. Attaching and non-attaching fila- ments are first recognized as prominent structures from the oocyte surface at the end of stage II as reported by Tsukahara (1971). Most these structures are formed where adjacent granulosa cells joined by desmosomes meet, and electron dense materi- als are observed on the prominent struc- tures. After that very thin rudiments of chorion are formed. In stage IV attaching and non-attaching filaments become uni- laterally curved candle-like structures. Iwamatsu (1992) reported that the in- creasing rate of the distance between fila- ments is higher in that of AH. Granulosa cells of AH are larger and flatter than those of VPA. The difference in the distri- bution and the shape of granulosa cells may relate to the difference in the distribu- tion of filaments in the AH and VPA. IMMUNOELECTRON MICROSCOPIC LOCALIZATION OF VITELLOGENIN IN THE KOKANEE SALMON LIVER AND OVARY. H.Ueda! and A.Hara2. 1tToya Lake Stn. for Environ. Biol., Abuta-gun, and 2Nanae Fish Cult. Exp. Stn., Kameda-gun, Fac. of Fish., Hokkadio Univ. Fish vitellogenin (Vg) has been considered to split into lipovitellin (E1) and phosvitin-B component (E2) in oocytes. However, little is known about Vg production in liver cells and Vg uptake into oocytes. This study was carried out to observe immunoelectron microscopic localization of Vg, El and E2 in kokanee salmon (Oncorhynchus nerka) liver and Ovary using specific antisera against chum salmon (Q. keta) these three proteins by means of immunogold technique. In the liver cells, specific immunoreactions of anti-Vg, El and E2 sera were similarly detected in the Golgi apparatus and in granular inclusions of 100-1500 nm in size. The double labeling technique using gold particles of different size revealed that most of these inclusions contained both El and E2, but some of these inclusions possessed either El or E2. In the oocytes, immunoreactive gold particles to these three proteins were equally observed in endocytotic vesicles, yolk granules and yolk globules. These findings Suggest the possibility that Vg is produced in the forms of El and E2 in the liver cells, secreted into blood, and absorbed into oocytes. Developmental Biology 1171 Hustches Puninvcation of H=-Sh, Putative Precursors of Major Egg Envelope Con- stituents K. Murata, I.luchi, K.Yamagami Life Sci. Inst., Sophia univ. ,Tokyo Spawning female-specific (SF) sub- stances, putative precursors of major com- ponents of egg envelope glycoproteins (ZI- 1,-2,-3), can be classified into two groups in terms of their immunoreactivity and molecular weight. One is low molecular weight SF (L-SF), the precursor of ZI-3 and the other is high molecular weight SF (H- SF) which presumably includes those of ZI- Ip Se H-SF was partially purified from the as- cites of the Ez2-treated male fish. Ascites buffered with 20 mM phosphate buffer (pH 6.8) containing 40 mM EDTA and 0.4 mg/ml PMSF was fractionated with a Sephacryl S-200 HR column. The fractions containing H-SF substances were subjected to anti-Cl IgG affinity column chromatography (Cl:a purified protein fraction of the hatching enzyme digests of medaka egg envelope), followed by fractionation by HPLC with an AX-300 column. Determination of purity and the molecular weight of the obtained H- SF was carried out by immunoblotting analysis and SDS-PAGE. H-SF seemed to con- sist of 3 bands of proteins and they were eluted in the first peak of the AX-300 DEAE silica column. On 8%-gel SDS-PAGE, these proteins showed the same relative mobilities as those of the ZI-1,-2. IMMUNOLOGICAL CHARACTERIZATION OF THE VITELLINE ENVELOPE COMPONENTS IN THE NEWT, CYNOPS PHYRRHOGASTER. T. Suzuki, T. Adachi and K. Onitake. Dept. of IOI 6 5 Rac. of Seat. p Yamagata University, Yamagata. Using the antibody specific to the uterine egg envelope(UE), we have shown that in Cynops phyrrhogaster, vitelline envelope began to be formed in stage II according to Dumont (1972), and that the follicle cells surrounding oocytes and some of liver cells were stained with anti-UE antibody immunohistochemically. Erle eS present study, we examined to characterize the common antigen(s) recognized in the liver(L-Ag) . Immunoblotting analysis revealed that molecular weight(MW) of L-Ag WiclS ue D)- Indirect immunofluorescent histochemistry using the antibody against vitronectin(VN) of whose MW was same as L-Ag revealed that the most outer layer of UE was specifically stained, while coelom- ic egg envelope(CE) was never stained. These results suggests that vitronectin which is cell-adhesive glycoprotein may be one of the components of UE. Futhermore, it was demonstrated that UE of eggs pass- ing through UP1l corresponds to pars recta of anura did not react with anti-VN anti- body immunohistochemically, but the eggs Passing through UP2 which is posterior region of UP1 were stained of its outer most layers of UE. It appears that the addition of molecules to CE from oviduct is necessary for the completion of UE. IMMUNOHISTOCHEMICAL ANALYSIS OF POLYFUSOME IN EARLY OOGENESIS OF DROSOPHILA Y.lwai, F.Maruo and M.Okada. Inst. Biol. Sci., Univ. of Tsukuba, Ibaraki. We have isolated a monoclonal antibody (MAb), BN461, which reacts with a structure extended through the ring canals (RCs, cytoplasmic bridges) within the early germline cell-cluster. On the other hand, polyfusome (PF) is previously described as the structure extending through the RCs. To test the possibility BN461 MAb recognizing PF, we made precise observations of the structure reacted with BN461 MAb in whole mount ovaries using laser-scanning confocal fluorescent microscopy. 3-D images of the structure were reconstructed from serial fluorescent optical sections by using volume rendering system. In all stages of cystocyte divisions, the branching patterns of the structure corresponded to the previously described branching pattern of PF. Furthermore we found that there was asymmetrical distribution of BN461 Ag between two cells that were two equivalent candidates for the oocyte. Immunoblot analysis of the sample from ovaries resolved the Ag into an acidic protein of MW 220kD. Identification of protein component of the PF in this study will facilitate molecular and genetic analyses of PF function during early oogenesis. SEARCH FOR A DROSOPHILA PROTEIN HAVING THE SAME EPITOPE OF THE TETRAHYMENA 49K PROTEIN T. Arai, Y. Niki, J. Chiba!, H. Suzuki? and O. Numata“~. Dept. of Biol., PAE 5 Sci., Ibaraki Univ. Mito. Dept. of Bigil, SCi, Aime Weeing , Sea, Wray, of Tokyo, Noda. MAH Oi WilOil, Seite, Univ. of Tsukuba, Tsukuba. Tetrahymena 49K protein has dual function to form a 14nm-filament involved in activity of the pronuclei during conjugation and to be citrate synthase in mitochondria. We found that there presents a protein having the same epitope of the 49K protein in Drosophila Ovaries by the methods of Western blotting and of immuno-histochemistry. The Drosophila protein having the same epitope of the 49K protein is 40 kDa and distributes in the cytoplasm of developing egg chambers and of the epithelial and the peritoneal sheath, muscular tissues surrounding ovaries. Because of its molecular weight and characteristics of staining pattern, this protein is different from actin and tubulin, and also would not be one of other known muscular proteins in multicellular organisms. 1172 Developmental Biology EFFECTS OF THE BRL-CONDITIONED MEDIUM ON MOUSE PRIMORDIAL GERM CELLS IN CULTURE. E.Kawase~’ Y.Shirayoshi~, K.Hashimoto”, N.Nakatsuji+. ‘Mammal. Dev. Lab., Natl. Inst. of Genet., Mishima and “Div. of Dev. Biol., Meiji Inst. of Health Sci., Odawara. SF (steel factor), LIF (leukemia inhibi- tory factor) and bFGF promote proliferation and/or survival of mouse primordial germ cells (PGCs); The rate of proliferation in vitro, however, is still much lower than that in vivo. Recently, we found that the Buffalo rat liver cell-conditioned medium (BRL-CM) promoted survival of PGCs isolated from 12.5 dpe mouse embryos and cultured for 2 days. In this study, we isolated PGCs from 7.5 dpe embryos as allantois fragments or early 8 dpe (5 or 6-somite stage) embryos (allantois-hind gut fragments), and cul- tured them in media with BRL-CM. Our re- sults indicated that BRL-CM promoted the proliferation and survival of PGCs. It is already known that BRL-CM contains soluble type of SF (rSF) and LIF. Addition of purified samples of rSF and LIF, however, gave much smaller effects than BRL-CM. Moreover, addition of antibodies against growth factors (TGFB1, EGF or bFGF) did not inhibit this BRL-CM effect. Therefore, other unknown/known factor(s) effective on PGCs seem to present in BRL-CM. This work was supported by the Science and Technology Agency, using the special Coordinating Funds for Promoting Science and Technology. RECONSTITUTION OF SEMINIFEROUS TUBULES IN VITRO BY DISSOCIATED FETAL TESTICULAR CELLS. K. Hashimoto, T. Mitani, E. Kawase and N. Takahashi. Div. of Dev. Biol., Meiji Inst. of Hlth. Sci., Odawara Primordial germ cells and gonia could be promising target cells for genetic manipula tion. We are trying to develop new methods of production of transgenic mice by using these cells. Recently, we have succeeded in obtaining offspring derived from cultured oogonia (DGD,34,233,1992). However, we have no methods for obtaining offspring derived from manipulated spermatogonia. Fetal testicular cells dissociated with trypsin-EDTA were plated on partially gell- ed Matrigel (Hadley et al.,1990). They pene- trated into the gell and aggregated depend- ing on the cell density. At low density, cells were organized into many small spheres, whereas at high density they first formed a cell sheet, which was gradually transformed into a large irregular reaggre- gate by 12 hr. At about 5x10° cells/cm? cells were organized into tubules. After 2d culture, many cells positive for alkaline phosphatase could be detected in the recon- stituted tubules. Kuroda et al.(1989) re- ported that type A spermatogonia differenti- ated into spermatids when seminiferous tu- bules isolated from cryptorchid testes were transplanted into adult testes. Then, we are trying to transplant these tubules into adult testes. This study was supported by the Special Coordination Funds for Promot- ing Science and Technology. THE COOPERATIVE ROLES OF GERM CELLS AND SOMATIC CELLS IN TESTICULAR TERATOCARCINO- GENESIS IN RECONSTITUTED TESTES IN MICE M.Noguchi and T.Sugiyama. Dept. Biol., Fac. of Sci., Shizuoka Univ., Shizuoka Testicular teratomas can be induced from germ cells by grafting 12.5-day male genital ridges to the adult testes in 129/Sv-ter strain of mice, whereas no tera- toma can be induced by grafting 14.5-day fetal testes. In order to analyze the roles of germ cells and gonadal somatic cells in testicu- lar teratocarcinogenesis, we examined the susceptibility to teratocarcinogenesis of the testes reconstituted from germ cells and somatic cells in different stages. The 12.5- and 14.5-day testes and 9.5-day hindguts were dissociated, and germ cells were separated from somatic cells. Then, germ cells and somatic cells were reaggregated by hanging drop-gyratory culture. The resultant aggregates were grafted to the adult testes. It was found that the seminiferous tubules have been reconstituted in all combinations; 12.5-day germ cells-12.5-day somatic cells, 12.5- 14.5, 14.5-12.5, 14.5-14.5)5) 905-455 end that teratomas have been only induced in 12.5-12.5 aggregates, not in any other combinations. These results suggest that the cooperative interaction between germ cells and somatic cells in 12.5-day testes plays important roles in testicular teratocarcinogenesis in 129/Sv-ter strain. THE ter MUTATION, RESPONSIBLE FOR PRIMORD- IAL GERM CELL DEFICIENCY IS ALLELIC WITH NEITHER W._LOCUS NOR S1 LOCUS IN THE MOUSE. M.Noguchi~, T.Sakurai~* K.Moriwaki and H.Katoh*. 1bep.of Biol., hac ohescresona zuoka Univ.,Shizuoka, “Yokohama City Univ., Yokohama, ~Natl. Inst.of Genetics, Mishima, Central Inst.for Exp. Animals, Kawasaki A recessive mutation, ter(teratoma) cau- ses primordial germ cell(PGC) deficiency in ter/ter mice of both 129/Sv-ter strain, in which ter also causes a high incidence of congenital testicular teratomas(Noguchi and Noguchi.1985, Noguchi et ai.1985), and ter congenic strains, C57BL/6J-ter(Bé6- ter) and LTXBJ-ter (Noguchi et al. 1987, 1988, Noguchi and Kobayashi. 1991). The series of W(dominant white spotting, Chr S) and S$l(Steel, Chr 10) mutations also cause the PGC deficiency in their homozy- gotes. In order to examine the allelism of ter with W or Sl locus, the linkage tests betw- een ter and Si) or Pgm-1(phosphoglucomu- tase-1) which is mapped about 3 cM from WwW were Carried out, by using offspring produced by genetic test crosses between +/ter mice of Bé-ter and DBA/2J, C3H/HeJd or 129/Sv-S1CP. The results showed that ter was neither linked to nor allelic with Sl and WwW (the vicinity of Pgm-1), indicating genet- ically that ter is a new mutation which affects the nature of PGCs, differing from Wand Sl. Developmental Biology 1173 SEARCH FOR FACTORS RELATED TO SEX DIFFERENTIATION IN FISH Y. Fujiwara! , T. Hirabayashi? and J.-I. Miyazaki?. 'Inst. of Envi. Sci., ?Inst. of Biol. Sci., Univ. of Tsukuba, Tsukuba. A temperate wrasse (Halichoeres poecilopterus) is known to undergo sex reversal (protogyny). To detect factors related to sex differentiation which seem to appear during sex transition, we compared protein constituents between transitional and mature gonads by _ two- dimensional gel electrophoresis. By comparison between transitional gonads and Ovaries, six proteins were found to be specific to or significantly increased in the transitional gonads. Three of the six proteins were found in testes. We prepared an antiserum against whole ovarian proteins. After incubating the antiserum and whole testicular proteins and removing immuno-precipitates by centrifugation, the Supernatant was used to prepare an antiserum against testis-specific proteins. The antiserum recognized one of the proteins shared by transitional gonads and testes. The protein had an apparent molecular weight of 26 kDa and an isoelectric point at pH 5.3 and was not detected in the liver and muscle. The protein is a possible candidate for the factor which is related to sex differentiation and expressed continuously in mature testes. HETEROCHRONIC pPGCs IMPLANTED INTO HOST NEURULAE CAN MIGRATE, TOGETHER WITH HOST pPGCs, TO GENITAL RIDGES OF EXPERIMENTAL TADPOLES. T. S. Tanaka and K. Ikenishi. Dept. of Biol., Fac. of Sci., Osaka City Univ., Osaka. In normal development of Xenopus, presumptive primordial germ cells (pPGCs) which have acquired the migratory activity at around the mid-tailbud stage are thought to be able to migrate to the dorsal crest of endoderm by responding a signal from the dorsal axial mesoderm. Then, they emerge from the crest through the dorsal mesentery and migrate to genital ridges at the tadpole stage, responding to fibronectin. When the labelled pPGCs in explants at the same 'age' as host pPGCs were implanted into unlabelled host neurulae, they could migrate, together with host pPGCs, to the genital ridges of the experimental tadpoles (kKenishi et al., 1984). Employing essentially the same experimental system as in the previous study, we investigated whether implanted pPGCs of different ‘age' from host pPGCs can migrate, together with host pPGCs, to the genital ridges of the experimental tadpoles in response to the signal. There were no _ significant differences in Proportions of the experimental tadpoles with labelled PGCs, irrespective of the '‘age' of implanted pPGCs. This indicates the implanted, heterochronic pPGCs can migrate to the genital ridges of the host tadpoles, responding the signal properly. STRUCTURES OF CORTICAL ALVEOLAR-DERIVED GLY COPOLYPROTEINS (HYOSOPHORIN) FROM THE EGGS OF MEDAKA FISH SPECIES - ORYZIAS LATIPES (ORL) AND O.MELASTIGMA (ORM) T. Taguchi’, A. Scko’, K. Kitajima’, Y. Inoue’, S. Inoue’, and T. Iwamatsu? Dept. of Biophys. & Biochem., Fac. of Sci., Univ. of Tokyo, Tokyo, *School of Pharm. Sci., Showa Univ., Tokyo, *Dept. of Biol., Aichi Univ. Educ., Kariya. We determined the complete structure of L—hyosophorins isolated from two species of medaka fish. Orl and Orm. They were shown to have the identical core peptide sequence: Asp- Ala—Ala—Ser—Asn(CHO)-GIn-Thr-Val-Ser. The structures of their glycan chains (CHO) were found to have some characteristic features as summarized below: (i) The glycan units of Orl and Orm hyosophorin share commonality in having (a) a huge N-linked form of which apparent molecular weight is ~7K, (6) a novel galactose clusters (Galf1—+4GalB1—4Galf1—), and (c) uniquely branched galactose residues (4GIcNAch1—3(—4GalB.1—4)GalB1—); (ii) L-hyosophorin (Orm) has a tetraantennary glycan unit while Orl L—hyosophorin contains a pentaantennary unit; (iii) The sialic acid residues (NeuSAc) are a2,3-linked to the terminal galactose residues in Orm hyosophorin whereas they were a2,3—linked to the terminal and/or penultimate galactose residues in Orl hyosophorin: (iv) Or! hyosophorin contains fucose residues, but Orm hyosophorin is totally devoid of them. DETACHMENT OF THE GLYCAN CHAIN FROM HYOSOPHORIN DURING EARLY EMBRYOGENESIS OF MEDAKA ASekol, K.Kitajima!, Y Inouel, S.Inoue2, IDept. of Biophys. and Biochem., Fac. of Sci., Univ. of Tokyo, Tokvo. 2School of Pharm. Sci., Univ. of Showa, Tokyo. We found in early embryos of medaka a free glycan chain having the structure identical with that of the sugar chain of hyosophorin, a cortical alveolar glycopolyprotein. The free glycan, not present in unfertilized eggs, was shown to be liberated at the stage of gastrulation from L-hyosophorin which was formed from hyosophorin on fertilization. Either the apo- peptide or the free glycan derived from L-hyosophorin may possibly be relevant to biological function of L-hyosophorin. In this study, we identified, in early embryos of medaka, the presence of an_N-glycanase responsible for the detachment of the glycan chain, and characterized the partially purified enzyme. 1174 Developmental Biology ISOLATION AND CHARACTERIZATION OF HYOSOPHORIN FROM BUFO JAPONICUS Y. Shimoda’, K. Kitajima’, Y. Inoue’ and S. Inouc’. ‘Dept. of Biophys. and Biochem., Fac. of Sci., Univ. of Tokyo, Tokyo; *School of Pharmaceut. Sci.. Showa University, Tokyo With the aim to isolate cortical alveolar—derived carbohydrate rich glycopolyprotein (hyosophorin), we applied the isolation procedures established for fish eggs to Bufo japonicus eggs and carried out structural determination of a glycoprotein thus obtained. In brief, after removal of jelly from the fertilized eggs of B. japonicus, the homogenate was treated with phenol. Following dialysis of the aqueous phase, it was chromatographed on a DEAE-Sephadex A-25 column. When the fraction eluted at ~0.15 M NaCl was then subjected to Sephacryl S—200 chromatography, we obtained a glycoprotein which eluted at position slightly after breakthrough. Interestingly, this glycoprotein was found to have amino acid and carbohydrate compositions closely similar to those of hyosophorin of Oryzias latipes. Sequence analysis of the core protein of the Bufo glycoprotein revealed that it comprises tandem repeats of nonapeptide exactly identical with apo—H—-hyosophorin of O. latipes. Furthermore, methylation analysis of the huge carbohydrate chains showed the presence of a number of partially methylated alditol acctates which mostly share with those formed upon methylation analysis of the O. latipes hyosophorin, strongly indicating that the glycan units of the Bufo hyosophorin has a N-linked bulky pentaantennary structure. The possible physiological significance of the present findings of closely similar hyosophorin molecules in toad and medaka is considered in the light of their unique structural features. IMMUNOELECTRON MICROSCOPE OBSERVATIONS ON THE FORMATION AND DEVELOPMENT OF CORTICAL GRANULES IN XENOPUS LAEVIS OOCYTES. N. Yoshizaki, Dept. of Biol., Fac. of Gen. Educ., Gifu Univ., Gifu. The origin and development of cortical granules were observed by treating sections of Xenopus laevis oocytes with a rabbit antiserum against cortical granule lectins and with a gold-conjugated goat antiserum against rabbit IgG. In stage I oocytes, gold particles were present on small numbers of cortical granules of 200-600 nm size. In stage II and III oocytes, they appeared on granules in the Golgi complexes as well as on large numbers of cortical granules, ranging in size from 200 nm to 1.4 um, in the cortical cytoplasm. Some of these cortical granules showed an irregular shape, indicating fusion of small granules into a large one. Gold-labeled granules disappeared from the Golgi complexes at stage IV. Cortical granules in stage V oocytes consisted exclusively of large granules and they were alined beneath the oolemma at stage VI. These results suggest cortical granules form in the Golgi complexes of stage I to III oocytes and coalesce in the cortical cytoplasm of stage i to" LV oocyvelss. VITELLIN COAT LYSINS FROM Mytilus edulis SPERM. T.Takagil, A. Nakamura?, R. Deguchi? and K. Kyozuka? 1lBiol. Inst., Fac. Sci., Tohoku Univ., Sendai, Dept. Pharmacol., Gunma Univ., Sch. Med., Maebashi, 3Marine Biol. Station, Fac. Sci., Tohoku Univ., Asamushi The acrosomal proteins obtained from Mytilus edulis were separated by a reverse phase HPLC. They were separated into 11 peaks and 3 of them (M3, M6, M7) showed strong vitellin coat lysin activity. The amino acid sequences of these proteins were determined by peptide sequence analyses. The sequence of M7 was confirmed by cDNA sequence analysis and revealed to have a signal peptide of 38 residues. M6 and M7 were composed of 180 amino acid residues and sequences were 76 % identical. On the other hand, M3 was composed of 148 residues and the sequence was different from M6 or M7. No sequence homology with lysins of abalone and Tegula was observed. Although the sequence of M3 was different from those of M6 and M7, all three proteins have a typical C-type lectin structure. No lectin activity was observed, but they were coprecipitate with isolated egg membrane. Mytilus lysins, M3, M6 and M7 are supposed to recognize the carbohydrate moieties of proteins involved in vitellin coat membrane and bind them and destroy the the structure of membrane. These processes can explain that stoichiometrical amount of lysin is necessary to destroy membrane. SPECIES-SPECIFIC SEQUENCES OF TEGULA VITELLINE COAT LYSJNS. K. Hanno SR ase! SONI: Sakai2, M. Tanaka & Y. Nagahama“. ~Tokyo Metropol. Univ., Tokyo, & “Natl. Inst. for BasvesRiaiis Okazaki. The vitelline coat lysin (VCL) of Tegula, a marine Mollusca genus, is released from acrosomal vesicles of the sperm during acrosome reaction and can lyze the vitel- line coats of only the same species. Thus, the lysin action is extremely species- specific. cDNA libraries were constructed from the testicular poly(A) RNAs of Tegula rustica (Owase) and T. lischkei with lambda gtl0 phage as a vector. A clone bearing the mRNA sequence for each VCL was isolated by screening the corresponding libraries with a 5’-terminal region; 400 bp (ORF-370) was obtained from the cDNA clone for VCI. of 7. pfeifferi. The cDNAs of T. rustica (Owase) and T. lischkei contained an open reading frame encoding 162 and 173 amino acid residues, respectively, and indicated the occurrence of 22 residues of signal se- quence at the amino terminal region of the nascent peplide. Both deduced amino- and carboxyl-proximal domains were virtually identical lo those determined for the VCIL of T. pfeifferi. However, the deduced sequences at the central domain (position 77-92) were different among the three species. This variable domain may account for the species specificity of the lysin. Developmental Biology 1175 A 70KD GLYCOPROTEIN ISOLATED FROM THE VITELLINE COAT IN THE ASCIDIAN,HALOCYN- THIA RORETZI S. Takizaw and M. Hoshil»2 1 Dept.of Eusewscie, -Gene Res. Cent, fokyo Inst. of Tech., Tokyo) Sugar chains of vitelline coat glycopro- teins are important for sperm-egg interac- tion in the ascidian, Halocynthia roretzi. Since no information is available on the chemical structure of the protein portion of the vitelline coat, we have isolated the major glycoprotein of 70kDa mainly due to the insolubility of the vitelline coat. We found that the major glycoprotein of 70kDa was selectively solubilized from the vitelline coat by DMSO. The solubilized glycoprotein was purified by SDS-PAGE and digested by lysylendopeptidase. Two major fragments purified by SDS-PAGE are under the process of sequencing. PROPERTIES OF A GLYCOSIDASE PURIFIED FROM EGGS OF THE ASCIDIAN, HALOCYNTHIA RORETZI. K. Matsuura, H. Sawada, and H. Yokosawa. Dept. of Biochem., Fac. of Pharmaceutical Sciences, Hokkaido University, Sapporo. In ascidians, Lambert has proposed that an egg glycosidase plays a key role in establishment of the polyspermy block through its binding to the sperm receptor on the vitelline coat. Fertilization of the ascidian, Halocynthia roretzi, was inhibited by GlcNAc-specific lectin (WGA) and the GleNAc'ase activity was highest among glycosidase activities released from the eggs by Ca2+ ionophore. Standing to the above proposal, these results imply the involvement of the egg GleNAc'ase in polyspermy block of H. roretzi. The GlcNAc'ase was purified from eggs to apparent homogeneity by chromatographies on DEAE- Toyopearl, SP-Toyopearl, Sepharose 6B, and Mono S. The molecular weight of the enzyme was estimated to be 520K by gel filtration and 66K by SDS-PAGE, suggesting the oligomeric nature of the enzyme. Isoelectric point was determined to be 7.0. The enzyme was activated with the isolated vitelline coat in artificial seawater. This result suggests that the GlcNAc'ase secreted from the eggs upon fertilization is activated in contact with the vitelline coat and probably functions in the polyspermy block. EXOCYTOSIS DURING FERTILIZATION OF A SEA URCHIN EGG DETECTED BY FLUORESCENCE DEQENNCHING METHOD Kazuhisa Takemoto’, Ken-ichi Hirano’, Tsuyoshi Hayakawa® and Hideyo Kuroda’. ‘Sugashima M.B.L., Fac. of Sci., Nagoya Univ., Toba 517, “Tsukuba Res. Lab., Hamamatsu Photonics, Tsukuba 300-26 and *Biol. Inst., Fac. of Sci., Toyama Univ., Toyama _ 930. In general, it is not easy to observe directly the exocytosis of living cells under a light microscope. We attempted to detect the exocytosis during in the fertilization of sea urchin eggs under a microscope using fluorescence dequenching technique with a membrane probe, 5-(N- octadecanoyl)aminofluorescein (AF18). The dye dissolved in the lipids of cell membranes was self-quenched at the concentration up to 5 mol%. When an AF18- stained egg was fertilized, the fluorescence increase was observed and propagated from the point of sperm entry to the opposite for 25 sec. The fluorescence increase is due to the dequenching caused by the diffusion of AF18 from plasma membrane to cortical vesicles during exocytosis. From the simultaneous measurement of the changes in fluorescence and light scattering which reflects exocytosis, the delay from exocytosis to dequenching was within 1 sec. These results show that the fluorescence dequenchching of AF18 is useful for studying exocytosis. SURFACE MEMBRANE RETRIEVAL VIA ENDOCYTOSIS IN THE DARK WAVE OF ACTIVATION WAVE FOLLOW- ING THE CORTICAL GRANULE EXOCYTOSIS IN THE EGG OF XENOPUS LAEVIS. T.Kageyama and H.Kubota. DYE, Cyt Iai < |, Kyoto Pref. Univ. of Medicine, Taishogun, Kee a kupmiyiOlCO MOOS manda) e pit mon ZOolin Fac. of Sci., Kyoto Univ., Kyoto 606. Unfertilized eggs were dejellied, and the yolk membranes were removed manually. Denuded eggs were artificially activated by pricking. Artificially inseminated eggs were used for following the later develop- ment. The internalization of surface membrane was examined in whole mount prepa- ration with Lucifer Yellow CH by fluores- cence microscopy, and ultrastructurely with horseradish peroxidase (HRP) as the marker of fluid phase endocytosis. Unfertilized eggs did not internalize the marker. The endocytosis began and was most prominent in the region where the dark wave (0.01-0.05; normalized time after the fertilization. 1.0; the first cleavage) of activation wave was passing through, immediately after the cortical granule exocytosis in the white wave (0-0.04)(Takeichi & Kubota, 1984). Elongated microvilli were reshortening to become globular in the dark wave. Endocyt- ic activity was detected in the region around the (presumptive) polar body in the early half till the first cleavage, and in the stress folds around the cleavage furrow during the cleavage stage. 1176 Developmental Biology BOTULINUM EXOENZYME C3 INDUCES ELEVATION OF THE VITELLINE COAT OF ASCIDIAN EGGS. S. Toratani and H. Yokosawa. Dept. of Biochem., Fac. of Pharmaceutical Sci., Hokkaido Univ., Sapporo. In the fertilization process of the ascidian, Halocynthia roretzi, expansion of the perivitelline space, called elevation of the vitelline coat, is observed within 30 minutes after insemination or addition of calcium ionophore. This phenomenon is thought as a result of exocytosis of intracellular granules likely as elevation of the fertilization membrane of sea urchin eggs. In this study, we examined the effects on elevation of the vitelline coat of H. roretzi eggs of botulinum exoenzyme C3, an enzyme which transfers ADP-ribosyl moiety from NAD to low molecular weight (LMW) GTP-binding proteins, and a monoclonal antibody which inhibits C3-catalyzed ADP- ribosylation, using microinjection technique. Elevation of the vitelline coat was induced by C3 and the elevation was inhibited by heparin that was co- injected with C3. The monoclonal antibody inhibited elevation induced by insemination, but its inhibition was canceled by the subsequent addition of calcium ionophore A23187. These results suggest that LMW GTP-binding protein(s) activated upon fertilization stimulates mobilization of intracellular calcium, which induces exocytosis. THE ABILITY OF AXIAL POLARITY EXPRESSION IN BODY FRAGMENTS OF POLYANDROCARPA MISAKIENSIS DURING CHIMERA FORMATION. T.Ishii and Y.Saito. Shimoda Mar. Res. Ctr., Univ. of Tsukuba, Shimoda, Shizuoka. In the compound ascidian, Polyandrocarpa misakiensis, when two body fragments from different adult zooids fuse with each other at their cut surfaces, a chimera zooid is formed. In the present study, to examine the ability of axial polarity expression of each body fragment during chimera formation, four kinds of fragments were used: normal anterior fragments (NAF) and normal posterior ones’ (NPF) from normal situs viscerum (NSV) zooids, and reversed anterior ones (RAF) and reversed posterior ones (RPF) from situs inversus viscerum (SIV) zooids. Analyzing two axial polarities, anteroposterior (A-P) one and heart-gut (H-G) one of obtained chimera zooids, it was found that there were differences in the ways of A-P axial polarity expression and H-G one. The results showed some differences of expression ability among the body fragments of NSV and SIV zooids; The NPF had the most stable ability in two axial polarity expressions, because it had expressed own axial polarities whomever it fused with. On the other hand, the ability of the RAF was the weakest in chimera formation. It could not express own axial polarities, when it fused with an NAF or an NPF. EFFECTS OF X RAY AND/OR MAGNETIC FIELDS ON SEA URCHIN DEVELOPMENT. N.Uto!, Y.Yamahama’, M.Takai’, T.Masui¥, and M.Kaneko*’ '‘Dept.of Biol.,?Dept.of Radiol., Hamamatsu Univ. School of Med., Hamamatsu. it has been reported the biological action of X ray or electromagnetic fields on development respectively. In the present study, we investi- Gated the effects, specially multiple effects, of X ray and magnetic fields on development of sea urchin embryo. Exposure of X ray (65 cGy/min) for 4-8 min on sea urchin (Hemicentrotus p.) caused a significant delay on early development. The influences of static magnetic field (2 Tesla) for 30 min on development appear negligible. Wheras changing magnetic field for 90 min caused a little number of short-armed plutei in some cases in spite of no developmental delay. Some of embryos exposed to both X ray and magnetic field tend to be in developmental abnormalities and delay. Amplified effects of magnetic field on X lay were uncertain. The present study will extend the information about potential effects on growing body cell. THE PARTICIPATION OF PHOSPHORYLATED PRO- TEINS IN MICROTUBULE NUCLEATING ACTIVITY OF MTOG ISOLATED FROM SEA URCHIN EGG MITO- TIC APPARATUS. E.Ueda! .E.Okumura? ,S.Hisanaga’” ,T.Kishimoto 2 and M.Toriyama!. 'Dept. of Biology, Fac. of Liberal Arts, Shizuoka University, Shizuoka and ?Lab. of Cell and Develop- mental Biology, Fac.of Biological Science, Tokyo Institute of Technology, Yokohama. A dramatic change in microtubule net- work occurs at the transition from inter- phase to M-phase. This change includes the increase in microtubule nucleating activity of centrosomes. To elucidate the activation mechanism of centrosome at M- phase, we isolated microtubule organizing granules (MTOGs) from sea urchin mitotic apparatus. The activity of MTOG decreased upon incubation with alkaline phosphatase. MPF but ATP restored the nucleation capa- city of dephosphorylated MTOGs. Treatment with 1M KCl inactivated MTOGs by solubili- zation of the component (s) responsible for microtubule nucleation. But sea urchin egg extract restored the microtubule nuc- leation. The extract contained proteins which were phosphorylated by MPF. In the SDS-PAGE these proteins had the same mobi- lity as the proteins that was existing in MTOG fraction and was phosphorylated by MPF. These results suggest the partici- pation of phosphoprotein(s) in the acti- vity of MTOG. Developmental Biology 1177 DEFORMATION OF THE NUCLEAR ENVELOPE CAUSED BY MICROTUBULES DURING CELL DIVISION IN SEA URCHIN EGGS. Y. Hamaguchi, M. S. Hamaguchi and S. Sato. Biol. Lab., Fac. of Sci., Tokyo Inst. of Technol., Tokyo. In order to investigate the process of nuclear disappearance, the image of the nucleus in the living cell was improved with the image processor, Image-1 (Universal Imaging Corp.). Clypeaster japonicus eggs were fertilized, deprived of the fertilization envelope and cultured in Ca-free sea water. The nucleus was clearly observed at prophase in the egg, centrosomes were positioned on the edges of the nucleus which became football- shape, and the nuclear envelope was dimpled near the centrosomes. Just before the disappearance, projections appeared at the dimpled areas in the nucleus and elongated deeply up to 5 um or more into the nucleus with time. Then the image of the nucleus became obscure, which means that the envelope broke down. Those projections were observed not only at the first cleavage but also at successive divisions. In the eggs treated with colcemid, there was no projection. By immunofluorescence microtubules were observed in the nucleus before the disappearance as well as around the nucleus. Therefore, the nuclear envelope mechanically pressed into the nucleus by microtubules from the centrosomes may be observed as the projections. RELATIONSHIP BETWEEN ASTER FORMATION AND CELL DIVISION THROUGH MATURATION TO CLEAVAGE REVEALED BY TRANSPLANTATION OF THE STARFISH CENTROSOME. T. Saiki and Y. Hamaguchi. Biol. Lab., Fac. Sci., Tokyo Inst. Tech., Tokyo. In order to investigate differences among the first and second maturation divisions, and the first cleavage division, we transplanted a centrosome at each division into maturating oocytes or zygotes of the starfish, A. pectinifera. In the zygote, the centrosome extracted at every divisions formed an aster, which induced cytokinesis, and eventually the recipient zygote divided into three blastomeres after the first cleavage. The centrosome extracted at any division also formed an aster in maturating oocytes. However, the polar body extrusion always eccurred normally; namely, an aster additionally formed in the recipient eocyte never induced cytokinesis. Stabilizing the mitotic aster with hexylene glycol treatment demonstrated clearly that the size of the aster in maturation divisions was smaller than that in the first cleavage. Asters reduced in size in the first cleavage with colcemid treatment failed in inducing cytokinesis. In conclusion, 1. the centrosome has no functional difference in aster formation among maturation and cleavage, and, however, 2. the size of the resultant aster determines whether or not cell division is induced. DETERMINATION OF THE MOTIVE FORCE GENERATED IN THE CELL USING A CENTRIFUGE- MICROSCOPE. Y.Hiramoto and M.Kikuyama. slob, IeWorS 5 Univ. of the Air, Chiba. We designed and constructed a new centrifuge-microscope with which microscopic objects can be observed through differential interference contrast optics with a 40x objective lens during application of a centrifugal force up to 1000x gravity. It consists of a Nikon inverted microscope (TMD), a custom-made centrifuge machine, a flash lamp with a very short flash duration (170ns), and a video-recording system. The cell ina centrifuge chamber being spun was illuminated with a flash whenever it passed through the front of the objective lens, and its microscopic image was recorded with the video-system. We tried to determine the motive force for chromosome movement during anaphase of mitosis by measuring the speeds of chromosomes in a centrifugal field applied in the direction of the spindle axis. The chromosome speeds should increase or decrease by the forces applied to the chromosomes as a result of the centrifugal acceleration and the density difference between the chromosome and the surrounding protoplasm. Experiments in sea urchin eggs and in grasshopper spermatcytes are in progress. It is expected that force- velocity relations in chromosome movement are determined. BEHAVIOR OF NUCLEI AND CHROMOSOMES FOR PARTHENOGENETIC STARFISH EGGS TO ESTAB- LISH TETRAPLOIDY Setsuko Washitani-Nemoto and Shin-ichi Nemoto Biol. Lab., Hitotsubashi Univ., Kunitachi, Tokyo and Tateyama Marine Lab., Ochanomizu Univ., Koh-yatsu Umi-no-Hoshi, Tateyama, Chiba, Japan. In starfish, artificial parthenogenesis Is associated with suppression of polar body (PB) extrusion. Both of par- thenogenetic eggs lacking PBs (OPB eggs) and eggs with 1st PB alone (1PB eggs) develop as tetraploids. To know how and when they become tetraploid, nuclear behavior of the parthenogenetically stimulated eggs of Asterina pectinifera (2n=44) was examined by stain- ing them with aceto-orcein. OPB eggs: After 22 tetrad chromosomes finally divided into 88 monads through two rounds of separations, they came together to form a single nucleus. This then disappeared and a diaster spindle was formed to divide 88 bivalents into two groups, followed by first cleavage. 1PB eggs: After meiotic 22 dyads divided into monads, they came together to form a single nucleus. When it disappeared, only a_half spindle was formed. Forty four bivalents separated into 88 univalents, but the eggs failed to cleave. In the next round of mitosis, a diaster spindle appeared to cleave the eggs into two blasto- meres, each of which contained 88 chromosomes. Thus, both types of the parthenogenetic eggs, OPB and 1PB eggs, become tetraploid before first cleavage. 1178 Developmental Biology DNA REPLICATION FOR PARTHENOGENETIC STARFISH EGGS TO ESTABLISH TETRAPLOIDY A. Nomura’ and S. Nemoto2. ‘Dept. of Zool., Fac. of Sci., Kyoto Univ., Kyoto, Japan, 2Tateyama Marine Lab., Ocha-no-mizu Univ., Umi-no-Hoshi, Tateyama, Japan. In starfish, suppression of polar body (PB) extrusion is prerequisite for production of parthenogenetic development. Eggs lacking PBs (OPB eggs ) have chromosomes equivalent to tetraploid , and eggs bearing 1st PB only (1PB eggs) have diploid . Despite of the difference of ploidy, both types of parthenogenetic eggs develop to tetraploid embryos. In the present study, we timed the DNA replication periods (S phase) up to the first cleavage in these parthenogenetic eggs of Asterina pectinifera. The eggs were labeled with BrdU and the incorporated BrdU was detected with anti-BrdU monoclonal antibody. In the both types of parthenogenetic eggs, each S phase was detected on the same schedule as would be observed in normally fertilized eggs. In the 1pb eggs, they did not cleave between first and second S phase, but did cleave between second and third S phase. This means that there was an extra round of S phase that was not followed by cytokinesis. The tetraploidy of these embryos is ascribed to the extra S phase. In the Opb eggs, on the other hand, each S phase and cleavage was observed alternatively, as in normally fertilized eggs. This maintains the tetraploidy of these types of eggs. METAPHASE IS PROLONGED IN SEA URCHIN EGGS TREATED WITH LOW PH SEA WATER CONTAINING SODIUM ACETATE. K. Watanabe, M. S. Hamaguchi, Y. Hamaguchi. Biol. Lab., Fac. of Sci., Tokyo Inst. of Tech., Tokyo. The relationship between mitosis and the intracellular pH was further investigated during the first cleavage of sea urchin eggs. When the eggs of Scaphechinus mirabilis were treated just before nuclear envelope breakdown with Ca*°-free sea water containing 10-20 mM CH3COONa and/or 10-20 mM NH, C1 pH-SW) whose pH was adjusted from 6.2 to 8.0, the intracellular pH was measured using a fluorescent indicator, pyranine. The values changed from 7.3 nearly to the pH values of pH-SWs within a few minutes and became constant. The volume of the spindle decreased linearly when the intracellular pH was increased from 6.8 to 8.0. Mitosis progressed normally in the eggs treated with pH-SWs at pH7.3 or more, but it didn't progress in those treated with pH-SWs at pH6.5 or less. When the pH was lowered by treatment with pH-SWs at pH6.8 or 7.0, metaphase was prolonged. In the cases of S. mirabilis and Clypeaster japonicus this period at pH6.8 was longer than that at pH7.0. In the case of Hemicentrotus pulcherrimus, as reported in MAO, Kyeshs th Wi V7 (1991), the eggs did not enter anaphase but remained at metaphase more than 30 min. These results suggest that the transition from metaphase to anaphase depends on the intracellular pH. PSEUDOPODIA FORMING ACTIVITY DURING EARLY CLEAVAGE STAGES OF SEA URCHIN EMBRYOS. T.Kominami. Dept. of Biol., Fac. of Sci., Ehime Univ. Matsuyama. After the 6-7th cleavage, blastomeres of sea urchin embryos become closely attached one another and change into columnar configuration. The purpose of this study is to find out the causal forces that operate during such a process of blastulation. The dissociated blastomeres of mid-to-late stage embryos were found to form several blebs at the cell surface. These blebs were retracted in a few minutes if they failed to attach the the substratum, and new blebs were formed at different sites. Blebs which succeeded in making contact with the substratum became typical lobo- or filopodia. Detailed observation revealed that such pseudopodia forming activity (PFA) appeared after the 7th, 6th and 4th cleavage in macro-, meso- and micromere derived cells, respectively. To know the factors which correlate such a cleavage-dependent phenomenon, size of blastomeres, nuclei and nucleo-cytoplasmic volume ratio was measured after each cleavage. Among these factors, the nucleo- cytoplasmic ratio was found to closely correlate the appearance of PFA. The ratio initially increased logarithmically during several cleavages, and the ratio was Maintained at a constant level once the maximum level had been reached. In any lineage of blastomeres, the ratio was over 0.1 when PFA appeared. IMMUNOLOGICAL STUDIES ON EXTRACELLULAR MATRIX PROTEINS OF SEA URCHINS. Y.Yokota', V. Matranga*, F. Zito?, M. Cervello* and E. Nakano”. !Biol. Lab., Aichi Pref. Univ., Nagoya. 2Ist. Biol. Sviluppo, Palermo, Italy. Two extracellular matrix (ECM) proteins of mole- cular weight 105 kD and 180 kD were isolated from the sea urchins, Temnopleurus hardwicki, Para- centrotus lividus and Pseudocentrotus depressus. A polyclonal antibody against the 105 kD protein of T. hardwicki was raised and employed in immuno-— blotting and immunofluorescent microscopy. The 105 kD protein of P. lividus and "echinonectin"” were cross-reacted with this antibody, whereas the 105 kD protein of P. depressus was cross-reacted weak- ly. ‘The cross-reactivity of the 105 kD protein with the antibody seems to have relevance to the behavior of the protein in the affinity chromato- graphy on gelatin-Sepharose. Immunof luorescent microscopy indicated that this antigen is distri- buted uniformly in the unfertilized eggs but is localized mainly in the surface area of embryos. The embryos of T. hardwicki were cultured in the presence of Fab of the antibody. No significant effect of Fab on development was observed unti the gastrula stage but the elongation of skeleton was inhibited in plutei. These results suggest that the 105 kD ECM protein is secreted from the cytoplasm to the outer surface of embryo and plays some roles in development. Developmental Biology 1179 PATE OF AN EXTRACELLULER MATRIX COMPONENT OF FERTILIZED SEA URCHIN (HEMICENTROTUS PURCHERRIMUS) EGGS Y.Myotoishil N.Kitai, T.Fukada, S.Ikegami, K.H.Kato.? IDept. of Applied Biochem. Hiroshima Wimelavaenr Higashi-hiroshima, Hiroshima, and 2Bi0l.Lab., Coll. General. Educ. Nagoya City Univ., Nagoya. The peptides with the apparent molecular Masses (Mr) of 99, 52, 49, 39 and 32kDa are released from the sea urchin blastulae at hatcing. Components reacting with antisera raised against the peptide with the Mr 99 (p99) are concentrated in the cortical granules of an untertilized egg. Antigen components translocated into the fertilization envelope and on the surface of the plasma membrane. The antigen components were not removed upon treatment with Ca?*-,Mg**-free sea water, suggesting that the components are not hyaline. The results of the present study demonstrate the occurrence of a new extracelluler Matrix component surrounding the outer surface of the embryo, which is removed at hatcing. COLLAGENOUS FIBERS IN THE SEA URCHIN PLUTEI BLASTOCOEL. Hee Nakajimal, and Katsuhiko Shimizu! 2. Dept. of Biol., Keio Univ., Yokohama 223, 2Pusetani Biofouling Project, Yokohama 235. The existence of collagenous fibers in the blastocoelic space of the sea urchin, Hemicentrotus pulcherrimus, Scaphechinus mirabilis, and Clypeaster japonicus plutei, was demonstrated by the transmission elec- tron microscopy. A considerable amount of fibrils were retained in the blastocoelic Space of pluteus which were prepared by quick freeze and freeze substituted tech- nique. Some of them show cross striation (60 - 70 nm periodicity) and had a width of 20 nm. They were morphologically resemble to the type I collagen. Fibroblastic mesenchymal cell in the blastocoel of pluteus arm spun out fibers from the cyto- plasmic pit. Two types of collagen disrupt agents, B-aminopropionitrile and aa'-dipyridyl, dose dependently inhibited further exten- sion of the arms of C. japoinus prism and pluteus larvae. Collagen fibers in the blastocoelic space of pluteus may contrib- ute to the morphogenesis of pluteus. It might be still remain to be further verified whether the blastocoelic meshwork of rapidly frozen and freeze substitution of the Srtongylocentrotus purpuratus blas- tula reported by Cherr et al.(1992) were due to ice crystals with the surrounding eutectic phase of blastocoelic substances. ULTRASTRUCTURE OF THE AXIAL ROD COMPOSED OF A BUNDLE OF ACTIN FILAMENTS IN ABALONE SPERM REVEALED BY QUICK FREEZE TECHNIQUE. Yoo sieOiyiai mance Nee leers akan, By OM | IhENS 5 Wayo Women's Univ., Chiba. eee An axial rod in abalone (Haliotis discus) sperm is a motile structure com- posed of a bundle of actin filaments. The axial rod elongates anteriorly to form an acrosomal process during the acrosome reaction. The ultrastructure of the axial rod was examined using quick freeze tech- nique. Thin sections of quick freeze and freeze-substituted sperm revealed that the actin filaments within the axial rod are hexagonally packed in a paracrystalline array almost through its entire length With an average center-to-center spacing of 12nm. Periodic transverse bands were also observed across the actin filament bundle. Quick-freeze deep-etch analysis provided the three-dimensional view of the axial rod. Actin filaments exhibiting 5.5- 6nm spaced striations were observed to run in parallel with each other. Cross- bridging materials were displayed between adjacent filaments. These results suggest that the actin filaments in the axial rod are probably held together by regular cross-bridges to form paracrystalline ar- ray, and also cross-linked by 3-4nm fila- ments to the lateral membrane which closely surrounds the anterior half of the actin filament bundle. ACROSOME REACTION AND SPERM PENETRATION IN THE BIVALVE. LATERNULA LIMICOLA. K. Hosokawa and Y.D.Noda“. Biol. Lab., Tokyo Dent. Coll., Chiba. ?Biol. Inst., Fac. Sci., Ehime Univ., Matsuyama. Acrosome of the mature sperm of lLaternula limicola are presented at the posterior end of the mitochondria in the mid-piece. To ascertain the role of the posterior acrosome, the process of acrosome reaction and sperm penetration were observed by an electron microscopy. The first step of the acrosome reaction is a pit formation at the posterior tip of the acrosome vesicle. The second step takes place at the ab- dominal part of the acrosoinmal vesicle, namely, the sperm plasm membrane and the outer membrane of the vesicle burst open at the abdominal part and fuse with one another. In the third step, membrane re- gression occurs immediately follow in the membrane fusion. As a result, the interior membrane of the vesicle replaces the exterior membrane of the sub- acrosomal region and obtains the capacity to fuse with the oolema. In the fourth step, the replaced membrane of the subacrosomal region makes contact with the tip of one of the microvilli remaining in continuity with the oolema and rapidly fuses with microvilli membrene. A fertilization cone is formed at the point of the membran contact. Finally, sperm penetration begins from the posterior ventral region of the sperm. The sperm organelles incorporated into the fertilization cone preceed the microfilament, follow by five mitochondria and the sperm nucleus is the last to the enter the ooplasm. This form of sperm penetration is a new mode of fertilization. 1180 Developmental Biology MORPHOLOGICAL CHANGES OF HAMSTER ISOLATED SPERMATOGENIC CELLS AFTER ELECTROFUSION WITH HOMOLOGOUS OVA. N. Usuil, a. Ogura? and R. Yanagimachi3. lpept. of Anat., Teikyo Univ. Sch. of Med., 2Dept. of Vet. Sci., Natl. Inst. of Health, Tokyo and 3Dept. of Anat. & Reprod. Biol., Univ. of Hawaii Sch. of Med., Hawaii. When an electrofusion pulse is applied, hamster mature ova fuse with isolated round spermatids and most of them develop to the 2-cell stage (Ogura et al, '93, Zygotes, in press). To elucidate the effect of ooplasm on the nuclei of spermatogenic cells (SCs), ova fused with isolated SCs were examined by means of thin sectioning. At 3-5 h after fusion, the SC nuclei re- mained almost unchanged in their shapes and sizes; the acrosomal vesicle was found at- tached to the spermatid nucleus. Detailed observations, however, revealed structural modifications in the spherical nuclei of early spermatids (the Golgi and cap phases) as well as spermatocytes. As female pro- nucleus developed, these nuclei gained the characteristics of the pronucleus: (1) prom- inent spherical nucleoli, (2) the so-called blebbing of the nuclear envelopes and (3) annulate lamellae in their vicinity. Perhaps, MPF did not affect the SCnuclei because it had disappeared before intermin- gling of the SC cytoplasm with the ooplasm, whereas cytoplasmic factors responsible for pronuclear development remained and induced modifications in the spherical SC nuclei comprising dispersed chromatin. BINDING OF BUFO JAPONICUS SPERM TO THE VITELLINE COAT AND ITS RELATION TO FERTILIZATION. S. Omata and Ch. Katagiri, Zoological Institute, Faculty of Science, Hokkaido University, Sapporo Dejellied uterine eggs (UE) of Bufo japonicus are not fertilized in 1/20 De Boer's solution (1/20DB), but are fertilized in the reconstituted salt solution (RSS) which mimics the ionic conditions in jelly envelopes. Concomitant with this difference in fertilizability, the Vitelline coat (VC) of the eggs inseminated in RSS had more than 20 times as many sperm bound to those in 1/20DB. Determination of the number of sperm bound to a unit area (0.2mm*) VC indicated that the sperm binding to UEVC (a) is dependent on the concentrations of both Ca** and Mg** in RSS, (b) is lost after activation of eggs in 1/20DB but not in DB, and (c) is competitively inhibited by the VC materials solubilized by SDS. The rate of binding was extremely low to the VC of coelomic eggs (CEVC), but increased to a level equivalent to that of UEVC after the treatment of coelomic eags with the extract of pars recta (PR), the upper 1/20 portion of oviduct, or with trypsin. This effect of PR was ascribable to the trypsin-like enzyme which caused a limited proteolysis of CEVC. Observations on transmission electron microscopy revealed that the sperm bound to VC were not acrosome-reacted. These results suggest that the proteolysis by oviducal PR protease enables VC bound to fertilizing sperm before the occurrence of acrosome reaction. SCANNING ELECTRON MICROSCOPIC STUDIES ON THE ACROSOME REACTION INDUCED BY JELLY LAYERS AND JELLY EXTRACTS IN THE NEWT. K. Onitake, I. Matsumoto and N. Nishiyama. Dept. of Biol., Fac. of Sci., Yamagata University, Yamagata. When the oviducal eggs were inseminated, all sperm remaining in the capsular cham- ber caused the acrosome reaction (AR) and the acrosomal caps disappeared following exposure of their perforatoria. Further- more, sperm incubated with jelly extracts (JE) caused acrosome reaction in very high rate. In the present study, we examined a process of acrosome reaction of the newt, Cynops pyrrhogaster, by JE in comparison with sperm passing through jelly layers by SEM. When spermatozoa were incubated with JE, acrosome reaction was caused within 60 sec. Process of acrosome re- action was as follows: (1) the disruption of acrosomal cap began within 30 sec, and (2) disrupted acrosomal cap was stripped and the perforatorium was exposed within 60 sec. On the other hand, the disruption of acrosomal caps of sperm passing through the jelly laysers occured within five min- utes. Some of sperm observed in Jl to J4 caused acrosomal reaction completely,and the other was on the way of AR. Sperm just after passed through Jl(inner most) showed complete AR or incomplete disruption of acrosomal cap. These results may suggest important role of jelly on the fertiliz-— ation and gentle polyspermy block in the newt which is physiological polyspermy. ON THE ROLE OF ACROSOME REACTION INDUCING FACTORS FROM EGG JELLY IN THE NEWT FERTILIZATION S. Nishida and K. Onitake. Dept. of Biol., Fac. of Souls Yamagata University, Yamagata. We have reported that the extracts from the newt, Cynops byrrhogaster, jelly layers(jelly extracts:JE) had the acrosome reaction(AR) inducing factors, and they were synthesized at the specific region of the oviduct in the HCG-injected ovulating female. In the present studies, we investigated the role... jo& AR-inducing factors on the fertilization using the antibodies against JE. When JE were incubated with anti-JE antibodied or papain digested univalent antibodies, AR inducing activities were neutralized. AR could not be induced completely by JE incubated with univalent antibodies. Moreover, the fertilization ratio of uterine jelly eggs was reduced strongly when they were treated with univalent antibodies and inseminated artificially. In addition, the oviducal tissues obtained from ovariectomized newt for 6 months were not stained with anti-JE antibodies by the indirect immunofluorescent histochemistry. These results suggests that AR inducing factors in the jelly layers were closely related with fertilization in vivo, and the synthesis of them was controlled by the hormone produced in the ovary. Developmental Biology 1181 POLYSPERMY BLOCK IN THE LAMPREY, LAMPETRA JAPONICA EGGS W. Kobayashi’, Y. Baba?, and T. Shimozawa? Zoological Institute, Faculty of Science, Hokkaido University, Sapporo. 21 ab. of Neuro—Cybernetics , Res. Inst. for Electronic Science, Hokkaido University, Sapporo. Sperm entry in the lamprey eggs is restricted toa region near the animal pole (sperm entry site, SES). Upon insemination of the eggs, the membrane potential rapidly sifted from -12 mV to +36 mV (fertilization potential). Sperm were unable to fuse with the eggs voltage clamped at more than +10 mV, indicating that the fertilization potential participates in the fast block against polyspermy. Duration of the effective fast block was about 160 seconds. Previous studies showed that sperm-—egg fusion has been established at the tip of the acrosomal filament of sperm although its nucleus remains outside the egg envelope, and that sperm-egg fusion is prerequisite to the passage of sperm through the envelope. In this study, transmission electron microscopic observations revealed that the acrosomal filaments were unable to touch the ooplasmic surface after separation of the egg envelope from the ooplasmic surface. This fact indicates that formation of the perivitelline space at the SES is responsible for a permanent block against polyspermy. Establishment of the permanent block was about 160 seconds after initiation of the fertilization potential. These results suggest that the permanent block is not quite fast enough to replace the electrical block against polyspermy, and do not rule out the occurrence of another intermediate block. ACTIVATION OF STARFISH EGGS BY CAFFEINE: Ca2*- RELEASE, CORTICAL GRANULE EXOCYTOSIS AND OXYGEN CONSUMPTION. S. Nemoto!, K. Kato*, A. Hino, T. Mohri, Y. Hamaguchi4 and H. Nakagima>. !Tateyama Marine Lab.,Ochanomizu Univ., *Nagoya City Univ., 7Kanagawa Univ., “Tokyo Univ. of Technol. and >Aichi Univ. of Edu., Japan. As we previously reported, caffeine activates both immature (GV stage) and maturing (after GVBD) oocytes. In the present study, we determined Ca2* release on caffeine activation of both immature and maturing oocytes of Asterina pecunifera using Fura-2 in relation to the formation of the fertilization envelop (FE), cortical granule (CG) exocytosis and consumption of oxygen. There was no difference between maturing and immature oocytes in caffeine concentrations required for inducing Ca2* release comparable to that in normal fertilization (10 mM or higher). Immature oocytes, however, formed only a partial FE, in contrast with maturing oocytes that did fully elevated the FE and a significant number of CGs were observed to remain unbroken in immature oocytes. A transient increase in Oxyge consumption (burst) was small in immature oocytes as compared with that in maturing oocytes. These differences between both oocytes were the same in A 23187 activation. The present results suggest that contact of CGs with egg cell membrane is established by the time of GVBD after the Teinitiation of meiosis and that Ca**-release Ca2+ machinery may participate in Ca** increase on fertilization. EFFECTS OF H-7 AND W-7 ON CHANGES IN THE RATE OF OXYGEN CONSUMPTION AND THE CONTENT OF ARGININE PHOSPHATE FOLLOWING TREATMENTS WITH ACTIVATING REAGENTS IN SEA URCHIN EGGS M.K. Kojima, N.Iishizawa, H.Hirai and S. Nakamura. Dept. of Biol., Fac. of Sci., Toyama Univ., Toyama We reported that various kinds of activat- ing reagents, such as A23187, procaine, ammonia and TPA, can induce both an en- hancement of oxygen consumption and an in- crease of the content of arginine phosphate (ArP) in unfertilized sea urchin eggs. And we suggested a possibility that metabolic stimulation by these reagents have a close relation with a release of cytoplasmic Ca?* and a rise of cytoplasmic pH. Therefore, in the present study, it was determined whether W-7, a calmodulin inhibitor, and H-7, an inhibitor of protein kinase C, can suppress such a rise of 0, consumption or of the ArP content following treatments with above-mentioned activating reagents. Thus, it was revealed that an enhancement of O21 consumption is cancelled only by com- bining treatments of TPA with H-7, and by treatments combined A23187 with W-7, but not by combination of H-7 or W-7 and pro- Caine or ammonia. On the other hand, H-7 has no effect on increased ArP content after treatments with the above-mentioned four activating reagents. Similar results are obtained by treatments combining W-7 with those activating reagents. POLARITY OF UNEQUAL CELL DIVISION IN GRASSHOPPER NEUROBLASTS. K. Kawamura. Lab. of Biol., Rakuno Gakuen Uviv., Ebetsu, Hokkaido. ¢ Grasshopper neuroblasts repeat unequal cytokinesis along the dorso-ventral axis of the embryo. They produce a small ganglion cell to the dorsal side. Each neuroblast is accompanied with one or more cap cells on the ventral side surface and a column of ganglion cells extending to the dorsal side. When the spindle body at metaphase was rotated approximately 90° by a microneedle, the rotated spindle returned to the original direction by autonomous turning. Therefore, the polarity of neuroblasts have been already determined by metaphase. In the present study, various microdissections such as the spindle rotation, the spindle shifting, the bending of half spindle, the destruction of surrounding cap cells or ganglion cells, were performed in dividing neuroblasts. The subsequent cell divisions in these microdissected neuroblasts were traced in order to analyze the polarity of unequal cell division. The results obtained here suggests that the preceding division axes participate in determination of the division axis in subsequent division. The cortical differentiation concerning the polarity seems to be commenced at late anaphase of the preceding division, and to be established only in the condition that cytokinesis is completed. 1182 Developmental Biology BEHAVIOR OF CENTRIOLE PAIRS IN YOUNG NEUROBLASTS OF CHORTOPHAGA. N. Yamashiki. Biology, Rakuno Gakuen Univ., Ebetsu, Hokkaido. Neuroblasts in the grasshopper embryos repeat unequal cell division that produces a small daughter cell (a ganglion cell (GC)) and a large daughter neuroblast (NB). In the previous electron microscopic studies, centriole behavior was observed by uSing 16-17 day old embryos in which mitotic activity of NBs is quite high. The centriole pair in the centrosome were close together at both spindle poles by metaphase. In late anaphase when the spindle took an eccentric position close to GC-side, the centriole pairs at the two spindle poles behaved differently. The pair at NB-side pole were separated, while the pair at GC-side pole remained close together. In this study, centriole behavior was observed in young NBs from 8- 10 day old embryos. The young NBs have experienced one or two mitosis after their banotchis In metaphase, the both centriole pairs were close together, as observed in 16-17 day old embryos. However, centriole behavior in late anaphase was different in the young NBs. The centriole pairs at the both spindle poles were slightly separated, and no differential behavior was detected. The differential behavior of centriole Pairs between the two spindle poles seems to be acquired with ageing of the NBs. FLUORESCENE MICROSCOPIC OBSERVATIONS OF CONE MOSAIC OF THE RETINA OF MEDAKA. Y.Nishiwakit, T. Oishi, s.Horiuchi2, T.Morita and F.Tokunaga”. IDept. of Biol., ac. of Sci., Nara Women's Univ., Nara, ME Ne, CRE iil, (@WwlS oir (en. JaolbKeE , eyeVol “dKeyoe > ee Uet@l,, jee Cr Sel. - Osaka Univ., Toyonaka. The distribution of cones in the retina of teleost has been observed as two organizational patterns, a square mosaic and a row pattern. In the medaka, three kinds of cones form a square mosaic pattern which consists of two rows, rows of zig-zagging double cones and rows of alternating short and long single cones. In the section of the retina of adult medaka, some kinds of cells could be detected by its autofluorescence without staining, using a fluorescence micro- scope, equipped with blue excitation filter. We discriminated each type of cones by their morphological differences. While this mosaic pattern was hardly observed in the pigment layer with conventional light microscope, these cones were clearly identified by their emission of autofluorescence against the pigment layer as a background. The same cone mosaic pattern could be detected in growing retinae after hatching. This autofluorescence method is useful to analyze the pattern of cone mosaic in the retina of the medaka. FORMATION AND DISTRIBUTION OF VISUAL CELLS IN CHICKEN RETINA S. Horiuchi’, H. Kobayashi'?, F. Tokunaga?, Y. Fukada‘, Y. Shichida*, T. Yoshizawa’, T. Morita! and Y. Koshida!. 1Dept. of Biol., Coll. of Gen. Educ., Osaka Univ., Toyo- naka, *Dept. of Biol., Fac. of Sci., Osaka Univ., Toyonaka, ‘Dept. of Biophys., Fac. of Sci., Kyoto Univ., Kyoto, *Dept. of Appl. Phys. and Chem., Univ. Electro-Communications, Tokyo. Present adress: Dept. of Biophys. Engineer., Fac. of Engineer. Sci., Osaka Univ., Toyonaka. To investigate the histogenesis of chicken retina, espe- cially the formation of the distribution pattern of rod and cone cells, we applied monoclonal antibodies (mAb) raised against cattle rhodopsin (Tokunaga et al., Zool. Sci. 6:167- 171, 1989) and raised against chicken iodopsin and R-photop- sin (Shichida et al, Exp. Eye Res., 48:281-293, 1989) for im- munohistochemical studies to the developing retinae of chick embryos. Using FITC indirect immunofluorescence method, FITC positive visual cells could be detected at first in 16-day embryo by anti-cone visual pigment mAb and 3 days later rod cells could be detected by anti-cattle rhodopsin mAb. No particular regularity of distribution pattern of rod and cone cells was recognized in developing chick embryo retinae. These mAbs are useful marker to examine which of the visual cells differentiate formerly in the juvenile retinae. MONOCLONAL ANTIBODIES SPECIFIC FOR PHOTO- RECEPTOR AND BIPOLAR CELLS IN THE NEWT RETINA. F.Maruo, Y.Sakaki, Y.Kaneko and T.Saito. Inst. Biol. Sci.«,. Univ” Of eetsuncmbay Tsukuba. Regeneration of the adult newt retina follow- ing its destruction provides us with a useful system for understanding the genesis of neural circuitry in the vertebrate central nervous system. In this study, we prepared monoclonal antibodies (MAbs) against neural retinas of adult newts, Cynops pyrrhogaster, to obtain markers of cell types for analysing retinal development and regeneration. MAbs were screened with immunofluorescence staining using frozen sections of retina. RB-1 MAb stained a subtype of bipolar cells and the cone-type photoreceptor cells. During normal development, RB-1 MAb first reacted with photoreceptors in the retina before segregation into distinct synaptic layers at stage 35, and then reacted with both photoreceptors and a bipolar cell subtypes in the retina with segregation into the synaptic layers at later stages. RB-1 MAb did not stain other type of cells during retinal development. RB-2 MAb stained both cone and rod photoreceptor cells and a subtype of bipolar cells. RB-2 MAb bound to a single band of 26 kDa in the immunoblot analysis. This band was also reacted with antiserum against recoverin, which is a Ca*’- binding protein found in photoreceptors of bovine retina. RB-1 and RB-2 MAbs could be used as differentiation markers for specific retinal cells. Developmental Biology 1183 EFFECTS OF SAPOSINS ON THE DEVELOPMENT OF ORGANOTYPIC CULTURE OF NEWBORN MOUSE CEREBELLUM. D.Satomil, Y.Kishimoto2. 1Dept. of Biol., Coll. of Arts and Sci., Univ. of Tokyo, Tokyo, 2Ctr for Molec. Genet., Univ. of Calif., San Diego, La Jolla, USA. Saposins(A,B,C and D),sphingolipid act- ivator proteins,are all small heat-stable glycoproteins derived from a common precu- sor, prosaposin. Recently, it was found that saposins bind strongly gangliosides and are present at high concentrations in mervous tissues. To investigate the roles of saposins on neuronal development, we used cerebellar culture system. Saposin A or B was added to feeding medium from 2 days in vitro(2 DIV). In saposin A treated explants, ependymal formation was observed at 8 DIV. Myelination did not occur during incubation. A number of large flat cells with few long processes were present char- acteristically in the circumference of 15 and 22 DIV explants. The levels of 2',3'- eyclic nucleotide 3'-phosphodiesterase (CNP) and glutamate decarboxylase(GAD) were low during incubation. In saposin B treated explants, morphologically ependy-— mal formation and myelin formation were observed. The activities of CNP and GAD increased nearly same manner as those of control.These observations indicate that biological function of saposin A and B is Significantly different despite their sim- ilarity in structure. ANALYSIS OF CYTOSKELETON OF GnRH NEURONS MIGRATING ALONG OLfACTORY NERVE IN CHICK EMBRYO. M.Miyakawa, K.Wakabayashi_ and Y.Arai. Dept; Anat - Juntendo Univ.Sch.Med., Tokyo and ~Inst.Endocrinol.Gunma Univ.,Maebashi. In recent years a considerable body of evidence has been accumulated suggest- ing the migration of GnRH neurons from the olfactory placode to the brain along the olfactory nerve in chick embryos. To know the possible mechanism of neuronal migra- tion, cytoskeletal elements of these neu- ronal substrates were studied. The locali- zation of actin filaments was observed by rhodamine-labelled phalloidin. Monoclonal antibodies to GnRH and d&-tubulin were used to detect the presence of GnRH and microtubules, respectively by immunohisto- chemical methods. By double staining of GnRH and 4-tubulin, GnRH-immunoreactive (IR) neurons were observed to be closely associated with the olfactory nerve axons from the olfactory epithelium to the brain. Double staining of actin and GnRH or tubulin revealed that the nerve associ- ated with GnRH-IR neurons showed plenty of phalloidin-labelled actin compareing to the other nerve bundles. The coexistance of GnRH-IR cells and abundant actin fila- ments was marked at the sites of the emer- gence from olfactory epithelium and the entrance into the brain. The actin-rich processes of GnRH-IR neurons may play an important role in the neuronal migration scaffolded by the olfactory nerve. EXPRESSION OF SC2 ANTIGEN ON FASCICULATING PRIMARY AFFERENT FIBERS IN THE DORSAL FUNICULUS OF CHICK EMBRYO SPINAL CORD. T.Shiga’. Te Shire "and Mi, Tamale. "Dept of Anat., Yamagata Univ., Yamagata and Dept. of Neurosci. & Immunol., Kumamoto univ., Kumamoto. We have been investigating the mechanisms of pathway formation of spinal primary afferent axons (PAAs) in chick embryos. In the present study, we examined the expression of SC2 antigen (specific cell surface molecule for dorsal root ganglion (DRG) cells and PAAs, Tanaka and Obata ‘84), using immunocytochemi cal method. Surfaces of DRG cells and PAAs became SC2 positive on embryonic day 3 (stage 19-20 of Hamburger and Hamilton, monly) when the earliest growing PAAs entered the spinal cord from DRG. Thereafter PAAs ran rostrocaudally in the dorsal funiculus in a fasciculated manner These PAAs expressed strongly SC2. On day 6 when the first PAAs entered the dorsal horn through the dorsal funiculus without fasciculation, PAAs in the dorsal funiculus and dorsal horn became weakly SC2 positive or negative, respectively. In contrast, PAAs expressed neuron-glia cel | adhesion moleculue (Ng-CAM), irrespective of the state of their fasciculation. Therefore, these results suggest that SC2 may be involved in the fasciculation of PAAs. NEURITIC PROCESS OUTGROWTH FROM CALCITONIN- PRODUCING CELLS IN VITRO. I.Nishiyama!, T.Seki?, T.Oota3, M.Ohta* and M.Ogiso‘. Dept. of Pharmacol., Teikyo Univ. Sch. of Med., 2Dept. of Anat., Juntendo Univ. Sch. of Med., 2Iso- tope Cent., Tokyo Univ. of Agri., and *Dept. of Physiol., Toho Univ. Sch. of Med., Tokyo. A calcitonin-producing cell (C-cell) is one of the endocrine derivatives of the neural crest, and it shares several properties with the neuron. The phenotypic plasticity of the C-cells was examined in culture systems. The ultimobranchial bodies, the embryonic organs composed mainly of C-cells, were isolated from 16-day rat fetuses and cultured in serum-supplemented conventional medium. At Day 4 in vitro, the cells were subjected to double immuno- staining using an anti-calcitonin antiserum and a monoclonal antibody (MAb) 12E3 which reacts specif- ically with polysialic acid portion of embryonic neural cell adhesion molecule. Most of the C-cells were polygonal or ovoid in shape, and few of them were positive to MAb 12E3. When a protein kinase inhibitor H-7 (30 to 70 M) was added to the cul- ture medium at Day 3 in vitro, many C-cells were found to extrude long neuritic processes and to exhibit intense immunoreactivity to MAb 12E3 within 24 h. The longest process reached 300 pm in length. Some processes had growth cones at their terminals. The effect of H-7 was apparently antago- nized by 40 nM okadaic acid, a potent inhibitor of protein phosphatases. Practically the same results described above were observed in rMTC 6-23 cells, a C-cell line derived from rat medullary thyroid carcinoma. These results suggest the involvement of some protein kinase(s) in morphoregulatory sys- tem in the C-cells. 1184 Developmental SNAIL-LIKE MOVEMENT OF THE CHICK PRE- STREAK STAGE EPIBLASTIC CELLS IN VITRO Ryuji Toyoizumi and Shigeo Takeuchi. Dept. of Biol. Sci., Fac. of Sci., Kanagawa Univ.. Hiratsuka. To understand the mechanism how the epiblastic cells (EC) of amniotes move to form the primitive streak, we dissected the cells from chick pre-streak stage embryo, dispersed into single cells, cultured with our conditioned medium, and observed their behavior in vitro . On the substratum coated with laminin (LN) gel, EC attached in a spherical form, and protruded one or a few very long stick-like lobopodia extending up to 20 um, whose figures looked like snails. But they hardly locomote on it. After 24 hr in culture. each cell dissolved the LN gel beneath it to make a hole and settled in it. On the substratum lined in parallel with fibrous LN gel, EC extended the long lobopodia along the lines. But, also in this case, they hardly locomoted. Single epiblastic cells poorly attached to the substratum coated with fibronectin (FN). However, on the substratum lined in parallel with LN gel supplemented with a small amount of FN, they protruded the long lobopodia along the lines from their spherical cell bodies, and locomoted unidirectionally along it. Substratum containing both LN and FN, somehow align in the blastoderm might support the migration of EC to form the pnmitve streak in situ. HOW DOES REPLICATION TIMING OF SPECIFIC DNA ESTABLISH IN DEVELOPMENT. - A STUDY OF IMAGE ANALYSIS. S. Tanaka, T.Tsuji, Y. Kawasaki and T. Saitoh Mitsubishi Kasei Institute of Life Sciences. 11 Minamiooya, Machida-shi, Tokyo. Replication timing of genes and subchromosomal segments in differentiated cells is fixed throughout successive cell cycles and closely related with their states of activities. Although replication timing of respective replication domains (replicons) is presumed to be established during embryonic development, thorough studies are lacking and _ its developmental significance is uncertain. Present study is aimed to exploit a method of image analysis for measurements of replication timing of specific DNA of avian scale dermal cells during their initial morphogenesis. Experimental conditions and relevant methods were examined in various points such as follows: doses and time intervals of double labeling of DNA with bromodeoxyuridine (BrdU) and iododeoxyuridine (IdU) during S phase in jn vivo system, immunocytochemical detection of BrdU and IdU, multiparametric microfluorometry of DNA content, amounts of BrdU and IdU of individual cells, image capturing of double stained replicons by confocal laser scanning microscope and image processing for identification and characterization of replicons classified by kinds of combinations of the labeling. Biology ROLES OF HEMOPOIETIC CELLS IN LYMPHOID FOLLICLE FORMATION IN THE BURSA OF FABRI- CIUS. Wer SiloaiGyyprirent Dept. Biol., Fac. Sci., Shizuoka Univ., Shizuoka. Lymphoid follicles develop from the epithelial part which hemopoietic cells invade in the bursa of Fabricius of the avian embryo. The present study was under- taken to reveal what roles hemopoietic cells play in the lymphoid follicle forma- tion of the bursa. Bursas of 6-day quail and 7-day chick embryos which were not colonized by hemo- poietic cells were transplanted onto the chorioallantoic membrane (CAM) of chick or quail embryos. The chick bursas were also co-transplanted with the axial mesenchyme containing hemopoietic cells onto the CAM of quail embryos. The transplants was examined histologically. Quail bursas developed abundant lymphoid follicles containing host hemopoietic cells when transplanted on the CAM of either chick or quail embryos. In contrast, chick bursas hardly developed lymphoid follicles when transplanted on the CAM of quail embryos. Co-tranplantation of chick bursas with the axial mesenchyme produced lymphoid follecles in the transplants. These results suggest that chick hemopoietic cells are necessary for chick bursal follicle forma- tion. Invasion of hemopoietic cells in the bursal epithelium may be indispensable for the lymphoid follicle formation in addition to the bursal epithelium and mesenchyme. AN ELECTRON-MICROSCOPIC STUDY ON THE EARLY DEVELOPMENT OF THE FRESHWATER PLANARIAN BDELLOCEPHALA BRUNNEA, WITH SPECIAL REFERENCE TO THE FORMATIONS OF GROUP AND SYMCYTIUM OF YOLK CELLS. W. Teshirogi, H. fujino, S. Sugimori and S. Ishida. Dept. of Biol., Fac. of Sci., Hirosaki Univ., Hirosaki. The egg of freshwater planarians is a composite egg, composed of several egg cells and numerous yolk celis. The yolk cells around the egg cell surrounded the egg cell radiately, shortly after egg-laying. These yolk cells then formed the syncytium. Seilern-Aspang (1958) and Koscielski (1966) have assumed that the grouping substance released from the egg cell ' at 1-cell stage, and syncytial substance released from blastomeres. This time, we have examined these in embryo at 1-to 32-cell stage by electron microscopy. The cytoplasm of egg cell is filled with ER of vacuole type and free ribosome, and the ER vacuole are released outside of the egg. This is regarded to be closely related with grouping substance. After that, not only ER vacuoles but also plenty of vacuoles originated from mitochondria are released into the syncytium. We believe that these vacuoles are related with syncytial substance. The activities of these releases are high at 4-to 8-cell stages and it falls remarkably at the 16- ceil stage. The lysosomes and autophagosomes that appear in the yolk cells probably bring about autodigestion of the cytoplasm. Developmental Biology 1185 CHANGES OF CELL DISTRIBUTION AND STRUCTURE IN DIFFERENT BLASTEMAL REGIONS OF THE PLANARIANS. I.Hori. Dept.of Biol.,Kanazawa Med.Univ., Uchinada-machi, Ishikawa. It is a well-established fact that a head regenerates at different rate from various levels along the longitudinal axis of the planarian body. This study was designed to examine this phenomenon cytologically. Two species, Dugesia and Phagocata, were used. The animals were transected respectively at four levels; just behind eyes(region A), pre-pharyngeal(B), post-pharyngeal(C) and tail(D) regions. The 3rd and 5th-day blastemata were obtained from each piece. In region A, cell density in the blastema decreased significantly between 3rd and 5th day while it did not change in regions C and D. This was in accordance with the differentiation rate of regenerative cells. One of the morphological features of well- differentiated cells was numerous elongated cell processes with microfilaments. When cytochalasin B was administered in the late phase of regeneration, these processes were inhibited indicating that a scaffold of differentiating cells within the blastema is produced by such processes which contain microfilaments. Staining with ruthenium red revealed a large quantity of the reticular filaments surrounding these processes. In regions, C and D, the less differentiated cells occupied the blastema but they had few prominent cell processes. The extra- cellular matrix was organized poorly. THE OBSERVATION OF GASTRODERM-FORMATION BY IMMUNO ELECTRON MICROSCOPY IN THE FRESHWATER PLANARIAN PHAGOCATA VIVIDA- T. Kuwahara, S. Ishida and W. Teshirogi. Dept. of Biol., Fac. of Sci., Hirosaki Univ., Hirosaki. Monoclonal antibody (mAb) 6@12B-D reacted specifically to gastrodermal phagocytic cells in Phagocata vivida. We reported with light microscope that this antibody have reacted to differentiating cells associated for the repair of gastroderm (1991). This time, we attempted to detect the specific antigen of mAb §12B-D by immuno electron microscopy using ABC and PAG methods in phagocytic cells. And we also observed undifferentiated cells (neoblasts) and differentiating cells into gastrodermal cells. The specific binds of colloidal gold were recognized lipid droplets in phagocytic cells. In differentiating cells, the specific binds were also recognized the newly-formed lipid droplets. PHARYNX-FORMATION IN REGENERATION OF THE FRESHWATER PLANARIAN PHAGOCATA VIVIDA. K. Onodera, S. Kobayashi, S. Ishida and W. Teshirogi. Dept. of Biol., Fac. of Sci., Hirosaki Univ., Hirosaki. Some histilogical investigations have revealed that regenerated pharynx including its inner epithelium are derived from gastrodermal cells (Teshirogi (96)Asai 1991 and others), This time, pharynx-formation was observed using monoclonal antibody (mAb) 612B-D which react specifically to gastrodermal phagocytic cells of Phagocata vivida,immuno histochemically. The antigen of mAb was recognized in the pharyngeal inner epithelium of the basal region of differentiating pharynx. As a conclusion, it was suggested that the new pharyngeal inner epithelial cells originate from old gastrodermal cells. We have also obtained a mAb which recognize the materials existing abundantly in the pharynx. PRESENCE OF RHODOPSIN-LIKE PROTEINS IN THE PLANARIAN (Dugesia japonica Japonica). Ss Nakamura! , T. Shinozawa“, S. Ishida! 1:Dept. of Biol., Fac. of Sci., Hirosaki Univ., Hirosaki. 2:Dept. of Biol. and Chem. Eng., Fac. of Eng., Gunma Univ., Kiryu. Planarian has two sensory organs. One is visual system which consisted of pigment-cup ocells and the other is auricles as tactile system and chemore- ceptor. In Western blot analysis of the planarian head, anti-frog-rhodopsin rabbit IgG recognized the presence of rhodopsin-like proteins with apparent molucular weight of 62,000 and 65,000(Fujita et al. 1991). In this report, we examined the localization of rhodopsin-like proteins by fluorescent antibody technique and Protein-A gold method. Rhodopsin-like proteins were locarized in the membrane of microvilli of the eyes. We considered that the auricles were associated with not phototaxis but a different role to the light stimulation. 1186 Developmental Biology INTERACTION BETWEEN EYES AND BRAIN OF PLANARIAN (Dugesia Japonica), ANALYZED BY THE ANTI-VISUAL CELL SPECIFIC ANTIBODY. --FORMATION OF SUPPLEMENTARY EYES AND THEIR PROJECTION ONTO VISUAL CENTERS. F.Sakai,K.Agata,K.Watanabe, Lab.of Regen- eration Biology, Fac.of Sci., Himeji Inst. of Technology. Ako, Hyogo, 678-12 Japan. Supplementary eyes of planarian occur spontaneously and experimentally. Their visual system were examined to analyze mechanism of eye formation and interrela- tion between eyes and brain. A monoclonal antibody was used to visualize a whole image of planarian visual cells by fluores- cent antibody technique. With a binocular, supplementary eyes were classified into two types: separate eyes and partially joined eyes(two pigment eye cups with a single mass of visual cells). However, microscopic observation showed similarity between two types. Optic nerves of supplementary and original eyes run along separate pathways towards brain. Projection from the fore eye was always located more medially than that of the hind eye. Two optic nerves (on the one side) shared same projection cen- ters in some cases, and did not in others. Some regenerants had several eyes joined with pigment cells. Their eyes had been arranged into two ,three or four eyes. Supplementary eyes may come from defect in regeneration process. Stability of eye structure may demand functional interrela- tion between eyes (optic nerves) and brain. MYOSIN HEAVY CHAIN IN PLANARIAN Kobayashi,S.,A.Yamada,H.Orii,K.Agata and K.Watanabe. Lab.Regeneration Biol.,Fac.Sci Himeji Inst. Tech., Harima Sci. Park City, Akou, Hyougo, Dept.Biol., Fac.Sci., Hirosaki Univ., Hirosaki, Aomori. Identification of molecules specific for different types of cells is a key point on understanding of planarian regeneration. Although we have made many trials, it is very difficult to prepare cell type speci- fic monoclonal antibodies(MAs) by standard method. Almost all MAs recognized sugar and were not suitable for gene cloning. To avoid this,we prepared polyclonal antiserum against planarian tissue and screened an expression library. Positive clones can be used for analyzing the genes and also for epitope selection,preparing the monospeci- fic antibodies good for Western blot and immunostaining. We report here structure of a myosin heavy chain gene isolated using this method. The myosin gene is similar to that of mammalian muscle and exp- ressed.in pharynx of planarian. HOMEOBOX GENES IN PLANARIAN. Orii,H., K.Agata and K.Watanabe Lab.of Regeneration Biol., Fac.,of Sci., Himeji Inst. Tech., Harima Sci. Park City, Akou-gun, Hyougo. How does planarian regeneration occur? Our knowledge of it is very poor, especial- ly at the molecular level. Homeobox genes have been isolated or identi tified in many organisms belonging to di- fferent phylogenetic groups and they maybe play key roles in cellular differ- rentiation and development. In order to understand planarian regeneration, we focused on planarian homeobox genes. Using PCR technology and sequence analysis we have identified 19 types of homeobox genes in thts organism. This suggests that planarian has a large number of homeobox genes like nematode. We found Antp-like homeobox gene in them, suggest- ing that HOM-C gene cluster may be con- served in planarian. STUDY ON THE EYE-INDUCTIVE SUBSTANCE IN FRESHWATER PLANARIANS. S.Ishida and S.Matsuguti Dept. of Biol., Fac. of Sci., Hirosaki Univ., Hirosaki. Though Lender called the eye-inductive substance "organishine"(1952,1956), the chemical nature has not been well known yet. This time the next was clarified. The substance is distributing over the whole body, but it is activated only in the brain. The inductive ability is appeared beyond the concentration of 25 ug protein/worm in head extract of Polycelis auriculata. It is supposed that the substance is protein or peptide. As the eyes are inducted by the ammonium chloride treatment, it is suggested that ammonium ion is one of the materiales which active eye-inductive substance. An inhibitory substance against eye induction is existing in the region which can not regenerate their head in Bdellocephala brunnea. There is no difference between normal eyes and inducted eyes in ultrastructure. it is possible that ammonia excreted inside worm body is one of the factor which act on the increase of eye-number in Genus Polycelis. Developmental Biology 1187 DETECTION OF THE PLANARIAN (DUGESTA JAPONICA) REGENERATION REGULATOR S. Shiozaki, H. Fujino, T. Tanaka, M. Ezaki, T. Saheki and T. Shinozawa. Dept. of Biol. and Chem. Engineering, Faculty of Engineering, Gunma Univ., Gunma Lender described that crude homogenate of planarian heads inhibited brain regen- eration of decapitated planarians. In this study, planarian head extracts were fractionated by DEAE-Sephadex, CM-Sepha- dex, Con A-Sepharose, Ammonium sulfate precipitation, and Phenyl-Sepharose. Each of the fractions were examined for their effect on the growth of Neuro2a or PC-12 cells. The 40% ammonium sulphate precipitated fraction of the planarian- head extracts inhibited the growth of Neuro2a cells. More over, the same frac- tion inhibited the brain regeneration of decapitated planarians. These results suggest that planarian head extracts contain growth inhibitor of neuronal cells and the utility of Neuro2a growth assay in the purification of planarian brain regeneration inhibitor. References: 1. TH. Lender, J. Embryol. exp. Morph., 8(3) 291-301, 1960 2. H. Fujino, T. Tanaka and T. Shinozawa, Zool. Sci., 8(6) 1072, 1991 ENDOGENOUS ION CURRENTS AND REGENERATION OF TAILS IN THE JAPANESE NEWT, CYNOPS PYRRHOGASTER. T.Nawata, College of Medical Sciences, Tohoku Univ., Sendai. Endogenous ion currents near amputated newt tails were measured with a vibrating electrode technique. The currents around an intact tail before amputation were less than 0.4yA.cm~ in density and entered the surface of the tail. After amputation of the tail, steady intens currents with a density of 50-450yA.cm~ left from the end of the stump. The stump currents continued to flow during the first 2-3 days and declined to about 10A.cm~2 by 4 days after amputation. A sign of regeneration of amputated tails could be observed about 7 days after amputation. To maintain the intense stump currents measured after amputation, an electric current with an intensity of 0O.1yA was artificially imposed on the end of the stump over 14 days. Under this experi- mental condition, a sign of regeneration of amputated tails could be observed about 3 days after amputation. In con- trast, when such a current was imposed in the reverse direction, the obvious regen- eration was undetectable within 14 days. These findings suggest that the en- dogenous stump currents play some causal role in initiating regeneration. SUBCELLULAR LOCALIZATION AND MOLECULAR NA- TURE OF THE no. 48 ANTIGEN FOUND IN XENOPUS GERM PLASM BY FLUORESGENCE MICROSCOPY. Ikenishi , K. Itoh” and M. Furusawa‘’. Dept. of Biol., Fac. of Sci., Osaka City Univ., Osaka and ~Furusawa MorphoGene Pro- ject, ERATO, JRDC, Tsukuba. — Subcellular localization and molecular nature of the corresponding antigen to the no. 48 antibody, which had strongly reacted with the germ plasm (GP) or cytoplasm of the germ line cells by fluorescence micro- scopy in the previous study, were investi- gated in Xenopus embryos. The antigen was demonstrated to be pre- sent on the GP-specific germinal granules or their derivatives, mitochondria not only in the GP or the cytoplasm of the germ line cells but also in cytoplasm of every soma- tic cells, and myofibrils of muscle cells by immuno-gold labelling. In order to know the molecular nature of the antigens on those organelles, immunoblottings for the subcellular fractions of the cleaving em- bryos and the myofibrillar fraction of the tadpoles were carried out. The Qo. 48 antibody recognized a Mr 53 x 10° band in the precipitate of 7,000 g §"mitochondrial fraction") and a Mr 40 x 10> band in the supernatant of 20,000 g, but it did not react with any band in the precipitate of 20,000 g (a "post-mitochondrial fraction" in which the germinal granules are to be contained). It also detected a Mr 43 x 10 band, probably actin, of the myofibrillar fraction. 3 LIPID COMPOSITION IN VEGETAL, ANIMAL, VENTRAL AND DORSAL HALF EMBRYOS DURING EARLY DEVELOPMENT OF RANA JAPONICA. M. Ryuzaki! and Y. Sone?. Dept. of 'Biol. and ?Biochem, Kitasato Univ. School of Medicine, Sagamihara : The authors conducted quantitative and qualitative analysis of triglyceride (TG), phospholipid (PL) and total cholesterol (TC) from vegetal(VE) and animal half embryos(AE) at stages], 2 and 10, from vegetal 4-cells and animal 4-cells at stage 5, and ventral (VnE) and dorsal half embryos (DE) that divided near primitive gut at stages 17 and 20 of Rana japonica. Weight percentages of TG, PLand TC to total lipid content in these specimens and their whole embryos were approx. 48-60% 15-27% and 4-6% respectively. TG (52-53%, PL(21% and TC6% in AE and TG(4485-50%, PL(27% and TCS-6% in VE were the same for stage 1 and 2. TG and PL in VE and VnE remaind unchanged following fertilization during embryogenesis and throught stage 20, hatching, at ca 46- 50 %and 20-27% and in AEand DEat ca 53-60% and 15-21% respectively. Except for minor quantitatve changes, the gross patterns of TG.PL and TC in the spcimens showed no _ significant change during embryogenesis. An anthron sulfuric acid positive lipid was detected in a fertilized egg for the first time as a single band and was also found in embryos at stagesS and 10 during early development on TLC. But this band was not detected in embryos at stage 17 or 20. 1188 Developmental Biology SPATIAL DISTRIBUTION OF MITOCHONDRIAL IrRNA IN DROSOPHILA EARLY EMBRYOS S. Kobayashi, R. Amikura and M. Okada. Inst. of Biological Sciences, Univ. of Tsukuba, Tsukuba, Ibaraki Polar plasm localized in the posterior polar region of early Drosophila embryos contains factors required for germ-line establishment. We have reported that mitochondrial large ribosomal RNA (mtIrRNA) is a cytoplasmic factor that rescues embryos from u.v.-caused inability to form pole cells, or germ-line cells. In this study, we analysed spatial distribution of mtIrRNA in early embryos using in situ hybridization technique. MilrRNA was enriched in polar plasm of cleavage embryos, but was no longer enriched in pole cells. U.v.-irradiation to the posterior polar region apparently lessened the mtIrRNA signal in polar plasm. Furthermore, Bic-D mutation, which mislocalizes the genetic information for abdomen formation, but not one for pole cell formation, in the anterior pole region, never affected the localization of mtIrRNA. These results suggest the possibility that the enrichment of mtIrRNA in polar plasm represents an indispensable step for pole cell formation in normal development. CELL DETERMINANT, MITOCHONDRIAL LARGE RIBOSOMAL RNA, IN DROSOPHILA CYTOPLASMIC DISTRIBUTION OF A POLE EMBRYOS. R. Amikura, S. Kobayashi and M, Okada. Inst. Biol. Sci., Univ. of Tsukuba, Tsukuba. Mitochondrial large ribosomal RNA (mtIrRNA) is a candidate for the germ cell determinants in the Drosophila embryo. We have presented the idea that mtIrRNA is transported out of mitochondria into the cytosol and takes part in pole cell formation (Kobayashi and Okada, 1989). In this study, we visualize this phenomenon using the jn situ hybridization method in electron microscopy. We could detect mtIrRNA outside mitochondria but not inside with this method. In the early cleavage stage, mtIrRNA signal was frequently found to attach to mitochondria. Some of the signal was located on polar granules. On the contrary, the control RNA signal (ND-1 RNA, Mitochondrial small rRNA) was not detected from cytoplasm nor from mitochondria. In the pole bud stage, most of polar granules were free from the signal. In pole cells, neither polar granules nor nuclear bodies exhibited signal. Considering those polar granules probably contain pole cell determinants, the localization of mtIrRNA in polar granules suggests the importance of mtirRNA in pole cell formation. CELL SHAPE CHANGES DURING DROSOPHILA VENTRAL FURROW FORMATION. M. Matsuda! and M. Yasutomi2. lDept. of Embryo., Inst. for Dev. Res., Kasugai, Aichi, “Lab. of Biol., Aichi Med. College, Nagakute, Aichi. Cell shape changes during ventral furrow formation were measured by using NIH Image 1.41 in cross sections of Drosophila embryos. To ascertain which changes are responsible for the process, models of ventral furrow formation were made. The results indicated that changes of the apical and basal cell surface width in the presumptive mesoderm cells, which were subdivided into the central and peripheral cells, generated ventral furrow formation. Apical cell surface elongation and basal cell surface contraction in the peripheral cells and the reverse phenomenon in the central cells resulted in invagination of the presumptive mesoderm cells. The furrow deepened by the events that cells located in the center of the central population elongated their basal cell surface and cells located in the side of the central population elongated their apical cell surface and contracted their basal cell surface. The results also indicated that the distribution of microfilaments and the position of nuclei May Correlate to changes of the apical and basal cell surface width. STUDIES ON A NEW MUTANT OF DROSOPHILA THAT CHANGES DEVELOPMENT OF IMAGINAL DISCS. M. Sawa’, M. Onuma?, M. Hatsumi*, K. I. Wakahama* and Y. Oguma*. ' Dept. Biol. Aichi Univ. Educ., Kariya, ? Dept. Biol. Fac. Sci., Shimane Univ., Matsue, * Inst. Biol. Sci., Univ. Tsukuba, Tsukuba. We got a P[lwB) insertion strain, 416, of Drosophila melanogaster that shows aber- rations in adult morphology, degenerated eyes, wings, legs, halteres, genitalia, and testes in varing degree. Femurs, tibiae and tarsi are degenerated or fused together. Wings and halteres are severely reduced. Testes are not elongated but spermatogesis did not appear abnormal. The imaginal discs of legs, wings and halteres from third instar larvae were observed. The discs derived from 416 lacked central regions which correspond with distal part of each organ in its fate map. The location of the P element insertion of this strain was mapped at 66B on the third chromosome by in situ hybridization. Thirty eight excision strains were constructed and twenty two of them produced flies of normal phenotype, which confirmed the aberrations in 416 should be resulted by the P[JwBJ insertion. Ten strains showed mild defects and one strain showed defects as severe as 416. These may have deletions of DNA segments which removed by the P element. Futhermore, five excision strains were lethal, suggest— ing that the product of the locus may be indispensable for normal development. Developmental Biology 1189 Sequential Appearance of Sensory Mother Cells (SMCs) of Microchaetes on the Drosophila Notum: Spacing Pattern of SMCs. K. Usui, K.-I. Kimura. Laboratory of Biology, Hokkaido University of Education, Iwamizawa Campus, Iwamizawa, Hokkaido. We have examined the spatio-temporal appearance of SMCs of microchaetes during pupal period using an enhancer trap line Al01 where lacZ is expressed in SMCs. In the dorsocentral (DC) region, microchaetes occur in evenly-spaced manner in five longitudinal lines (L1-5 medio-laterally). At 8 hr after puparium formation (APF), SMCs were first detectable in a DC line (L5) which runs anteriorly through anterior and posterior DC macrochaetes. At 10 hr APF, two more lines of SMCs (L1&3) were added in an even-spaced manner, in addition to LS. Two hours later, moreover, two lines (L2&4) were intercalated among these three pre-existing lines. In each line, SMCs did not appear synchronously but in a skipped pattern. In the case of the well-fed flies, additional SMCs appeared in the largest space among the five pre-existing lines of SMCs. scute (sc) gene was ubiquitously expressed by heat-shocking an hsp-sc chimeric gene in achaete/sc mutant flies which lack all microchaetes. LS was preferentially rescued in a stage-independent manner during sensitive period, showing that the enhanced sensitivity for SMC development existed in L5. ROLE OF HEMOLYMPH 350kDa LECTIN ON PROGRA- MMED CELL DEATH OF THE ANTERIOR SILK GLANDS OF THE SILKWORM, BOMBYX MORI. oie Soma »8: Sakurai-, K. Amanai* and T. Ohtaki-. ept. Biolgy, Kanazawa Univ., Kanazawa, ,“Naatl. Inst. for Basic Biol., Okazaki, 3Nishogakusha Univ., Kashiwa, Japan. Silk gland, a larval specific tissue, initiates to degenerate immediately before pupation in the silkworm, Bombyx mori. At the same time, the contents of 350 kDa lectin is found abundantly in hemolymph. Since hemolymph lectin was indicated to be involved in non-self recognition in Sarcophaga larvae, it was possible that the Bombyx hemolymph lectin is involved in the cell death of the silk gland, especially in the removal of the gland by phagocytes. Degeneration of the silk gland is induced in vitro when the glands are cultured with 20-hydroxyecdysone and hemocytes. We examined the above possibility by addition of gulucuronic or galacturonic acid, both of which inhibits hemagglutinating activity of the lectin, or by adding an anti-lectin monoclonal antibody to the culture to inhibit the function of lectin. Under these conditions, the gland degenerated, indicating that the hemolymph 350 kDa lectin may not be involved in the degeneration of anterior silk gland. CELL DEATH IN WING MORPHOGENESIS OF LEPIDOPTERAN INSECTS 2 A.Yoshidg 1 R-Kodama , G.Eguchi™ and T.Mitsui~. Project Office, Biohistry Res. Hall, Tokyo, Div. of Morph.,, Natl. Inst. Basic Biol., Okazaki, and ~Lab. of Insect Toxicol. and Physiol., Inst. of Phys. and Chem. Res. (Riken), Wako. Degeneration of the marginal region in the pupal wing epidermis is involved in wing morphogenesis in the lepidopteran insects (SUffert, 1929). First, we exam- ined the pupal wing epidermis of the small white cabbage butterfly, Pieris rapae, with a light and an electron microscope. Second, we injected BrdU into early pupae, fixed them after 2h., and then detected BrdU incorporated into the nuclei with enzyme antibody technique. Until 3rd day after pupation, morpho- logical change do not occur in the degen- eration region. The epidermis bulges out dorsoventrally at 3.5th day, dramatically contracts at 4th day, and finally disap- pears at 4.5th day. At 3.5th and 4th day, degenerating cells are visible along the basal surface of the wing epidermis. During 1 day after pupation, BrdU- incorporating nuclei are visible in the degeneration region, but almost absent after 1 day. No mitosis is observed in the degeneration region after pupation. This suggests that cell death occurs after DNA synthesis as in the digit formation of the chicken embryo (Tone et al., 1988). SPUR PATTERNS ON THE TIBIA AND FEMUR OF THE GERMAN COCKROACH. A. Tanaka and S. Kitamura. Dept. of Biol., Fac. of Sci., Nara Women's Univ., Nara. Spur patterns were observed on the tibias and femurs of all the legs throughout postembryonic development of the German cockroach, Blattella germanica (L.). All the spurs were named by a row-and-level method. Six rows of spurs were recogniz- able on the tibia in the adult stage. One of the rows on the posterolateral surface was designated as row 1. Five remaining rowS were named consecutively as rows 2-6, clockwise or anticlockwise over the distal face of the right or left tibia, respectively. Spurs belonging to the same row were named alphabetically from the distal level. The number of spurs per row differed with rows and on the three pairs of legs. A clear gradient was found with respect to the size of spur: the more distal ones in the same row tended to be larger than the more proximal ones. Three rowS were recognizable on the femur and designated as rows I-III. Levels of spurs were also named alphabetically from the distal position. Spurs on the femur were not always larger more distally but rather showed double gradients with respect to size. In the larval stage, both on the tibia and on the femur, spinelike hairs replaced spurs at some positions of the rows and levels. The number of spurs steadily increased instar by instar until the last instar. 1190 Developmental Biology ON THE OOGENESIS OF FRESH WATER HYDRA AND SOME SPECIES OF MARINE HYDROZOA K. Noda!, C. Kanai!, S. Kubota® and H.Namikawa% 1Ultrast.Res. Tokyo Metropol. Inst. of Gerontol. Tokyo, @Seto Marine Biol. Lab., Kyoto Univ. Shirahama. “Dept. of Zool.,Natl.Sci.Museum. Tokyo Many oocyte-groups appeared among numerous oo- gonia in an egg-forming area of fresh water polyp, Pelmatohydra robusta. But, only one cell of the oocytes situated at the central part of an egg-forming area was always preferred as pre- sumptive egg by ectodermal epithelial cells After one oocyte was selected, the base of ecto- dermal epithelial cells of egg-forming area were broken. We also examind a course of oogenesis during morphogenesis of gonophore/medusa of 7 species of marine hydrozoa in special refer- ences to 1) the stage of germ cells migrating into inner ectoderm, 2) migrating route of germ cells, 3) the number of oocytes in an egg- forming area and 4) structures of gonophore/ medusa when germ cells developed to oocytes (in inner ectoderm). Marine materials used in the study were anthomedusa, Eudendrium, 3 species of Stylactaria, Halocordyle disticha, Sarsia japonica, and leptomedusa, Eugymnathea japonica Final structure of gonophore/medusa seemed to be closely depend on the pattern of oogenesis exhibited by each species. REGURATIVE DEVELOPMENT OF HYDRA EMBRYO T. Oda Dept. of Biol., Fac. of General Edu., Yamagata Univ., Yamagata. Hydra embryos in various stages were divided with razors edge and the subsequent development of their isolated blastomeres or halves were ob- served. When the blastomeres from 2- and 4-cell embryos were separated, some of the cells develop- ed into a complete and well-proportioned adult. Thus, the early cleavage stage blastomeres could regulate their development so as to produce a com- plete organism. When embryos at the hollow blas- tula and gastrula stages were cut in half meridi- onally through animal and vegetal poles, both halves produced entire organisms. Moreover, when embryos at the same stages were split equatorially separating animal and vegetal poles, both halves also developed into normal organisms. These may happen because the cells of early embryo are equi- potential in the sense that there is no difinite relation between the position of any of the blas- tula cells and the specific tissue it will form in the developing embryo. This idea may be supported by the following observation. Gastrulation in hydra was caused by the multipolar ingrowth, in which endodermal cells migrated into blastocoel from not only vegetal pole but also other posi~ tions of ectodermal layer. From the results, it became clear that hydra embryo from 2-cell to gas- trula stage had a great ability to regulate devel- opment. BASAL DISK FORMATION IN HYDRA ;PROSPERITY AND DECAY OF RF-AMIDE+ NEURONS DURING THE FOOT REGENERATION ¥..KOBAYAKAWA® A.MAKI2 & O.KOIZUMI? Ol. ibe) 5 Coll. gf Genl. Educ., Kyushu Univ., Fukuoka, ‘Physiol. Lab., Dept. Sci., Fukuoka Women’s Univ., Fukuoka RF-amide+ neurons are located in the tentacles, hypostome and peduncle, but not in the body column or basal disk in Hydra magnipapillata. Ectodermal epithelial cells also show position specific charac— ters. In the basal disk, epithelial cells are special mucous secreting cells (BDGCs ;Basal Disk Gland Cells). When a hydra was amputated its foot at the middle part of the body column, as the first step of foot regeneration, the epithelial cells at the proximal end differentiated BDGCs. Then, the RF-amide+ neurons reappeared in the new peduncle region, but not in the basal disk region. When a hydra was amputated its basal disk at the lowest part of its peduncle, RF-amide+ neurons remained at its proximal end. Then, the epithelial cells at the proximal end began to differentiate BDGCs. The RF-amide+ neurons, however, did not begin to decay from the proximal end before the basal disk regeneration pro- ceeded to a certain extent. It is suggested that during the foot regeneration in hydra, the epithelial cell differentiation precede the change of neuropeptide phenotype in nerve net. NERVE NET FORMATION BY NERVE PRECURSOR (INTERSTITIAL) CELLS INTRODUCED INTO NERVE- FREE HYDRA S.Minobe and O.Koizumi. Physiol.Lab.,Fukuoka Women’s Univ., Fukuoka Hydra has a simple nervous system consisting of a nerve net that extends through the body. The nerve cells are continuously produced by the differentiation from interstitial multipotent stem cells even in adult hydra. Hydra consists of two independent cell lineages, epithelial and interstitial cell lineages. In this study, epithelial hydra that contains no nerve cells was produced. Hydra was treated with colchicine to eliminate interstitial cells. The resultant hydra could be maintained by force-feeding. Nerve net formation was examined after the interstitial cells were introduced into the epithelial hydra. The nerve net of both ganglion and sensory cells had formed from the base of tentacles toward the tip. The rate of the nerve net formation corresponded with that of the displacement of the tentacle epithelial cells. Unlike neurons, nematocystes had recovered quickly through the tentacles. Thus the mechanisms for the repopulation are different between nerve cells and nematocytes. In conclusion, the nerve net formation occurs at the base of the tentacles and then it is displaced along the tentacles with the epithelial tissue. Developmental Biology 1191 REGENERATION PROCESS IN HYDRA CELL AGGRE- GATES ANALYZED BY THEIR DEVELOPMENT OF ORGANIZED MOTION. Sato-Maeda,M.*,and Tashiro,H.,Photodynamics Res Ctr, Inst Phys/Chem Res (RIKEN) ,Sendai. i Hydra can regenerate even from a disso- ciated cell aggregate. To know how and when an aggregated medley of dissociated cells initiate self-organization to form the body axis which may guide the direction of the subsequent growth, we analyzed motion of ag- gregates in early stages of regeneration. Aggregates made of cells which are selec-— tively gathered from the gastric region of intact animals were prepared in the size in which only one head was expected to form. After the final dilution of medium at t= 22hr, the motion of an aggregate was con- tinuously recorded by a timelapse VIR. Both the widths of x- and y-axis directions of the aggregate during the period were mea- sured with an image processor. Until 30hr, aggregates stayed spherical. They showed slow expansion or slow asymmetric motion. Aggregates started to show distinct anisotropic motion with a single axis after a few times of big contractions. The ampli- tude and frequency of the asymmetric motion increased. Taking account of observations for tissue-piece or aggregates of low nerve-— density, these results are understood that (1)the determination of the body axis was correlated to the appearance of the an- isotropic movement and (2)the nerve system that is completed later relates twitching of spike-like contraction-recover motion. A NERVE DERIVED FACTOR REQUIRED FOR SUR- VIVAL AND PROLIFERATION OF INTERSTITIAL STEM CELLS IN HYDRA T. Fujisawa. Natl. Inst. of Genetics, Mishima Interstitial stem cells (i-cells) in Hydra proliferate continuously in asexual- ly budding animals, but their density is always maintained at a constant level in the tissue. In the course of the study how this i-cell homeostasis is maintained, I have found that some factor(s) derived from nerve cells is required for survival and proliferation of i-cells. Partial purifi- Cation and characterization of the factor Suggest that it is a neuropeptide which resembles head activator (Schaller and Bodenmueller, 1981). Synthesized head activator in a dimerized form (a gift from Dr. Schaller) also exhibited an activity to support survival and proliferation of i-cells. These results suggest that head activator is at least one of the paracrine growth factors of i-cells in Hydra. A SUBPOPULATION OF INTERSTITIAL CELLS IN HYDRA MAGNIPAPILLATA THAT IS RESTRICTED TO DIFFERENTIATE INTO GERM CELLS. C. Fujisawa! and T. Sugiyama?. !Dept. of Genetics, School of Life Science, Grad. Univ. for Advanced Studies, Mishima and 2Natl. Inst. of Genetics, Mishima Interstitial cells (i-cells) in hydra are undifferentiated stem cells that proliferate continuously. They also differentiate into nerve cells, nematocytes (stinging cells), gland cells and germ cells (eggs and sperm). To examine the differentiation capacity of individual i-cells, we used hydroxyurea treatment to obtain i-cell clones from a male strain of Hydra magnipapillata. We have previously cloned i-cells which differentiated only into sperm (1991 Meeting of Japan Soc. of Devl. Biologists). Using the same strain (male), we have also isolated, to our surprise, i-cell clones which differentiate only into eggs. These results suggest that male polyps contain a subpopulation of i-cells restricted to egg differentiation, whose differentiation is Suppressed in male tissue. THE DIGESTIVE TRACT OF THE ASCIDIAN POLYANDROCARPA MISAKIENSIS. |. ESOPHAGUS. H.Koyama, Dept. Anat., Sch. Med., Yokohama City University, Yokohama. The organization of the esophagus and its associated structures was studied ina styelid ascidian, Polyandrocarpa misakiensis. The simple squamous branchial epithelium gradually becomes thick and possesses apical metachromatic granules at the entrance to the esophagus. The esophagus is surrounded by connective tissue, in which many blood sinuses and terminal branches of the pyloric gland are embedded. The peribranchial epithelium Surrounds the esophagus as a rough periesophageal epithelium containing many coelomic cells. Many circular and longitudinal smooth muscles are present between the pene soplegeas. epithelium and esophagus. The lumen of the relaxed esophagus has three (left, right, and posterior) main folds. Pseudostratified regions occur at the base of each fold. The remaining part of the esophagus is a simple columnar epithelium, which mainly consists of ciliated mucous cells. The anterior basal side of the left fold has a group of cells different from typical mucous cells. The mucous cells have some Cilia, microvilli, granules, much rER, Golgi apparatuses, and mitochondria. There are two types of granules, one large and electron-translucent, and the other small and electron-dense. These granules lack a limiting membrane. There are also paraneuron-like cells with apical microvilli and dense cored vesicles mostly in their subnuclear regions. 1192 Developmental Biology ON THE EGG CYTOPLASMIC FACTORS RESPONSIBLE FOR THE MUSCLE CELL DIFFERENTIATION OF ASCIDIAN CIONA SAVIGNYI: EGG FRAGMENTS PRODUCED BY CENTRIFUGAL FORCE. Y. Marikawa and N. Satoh. Dept. of Zoology, Fac. of Sci., Kyoto Univ., Kyoto. Dechorionated unfertilized eggs of C. Savignyi were incubated for 10 min in the sea water that contains 10 ug/ml cytochalasin B and then centrifuged for 20 min at 1500xg. ‘These treatments separated eggs into several types of fragments, large red fragments (-~60% egg volume), small gray ones (~10% egg volume), and small transparent ones. Red fragments contained egg chromosomes. If the fragments were inseminated, they developed into the so-called permanent blastulae, in which epidermal differentiation was evident but muscle cell differentiation was not detected. Gray and transparent fragments never cleaved even if they were inseminated. However, if red fragments were inseminated after the fusion with gray fragments they developed into the embryos in which muscle cell differentiation was conspicuous. The embryos derived from these fused fragments sometimes appeared as normal tadpole larvae. These results suggest that muscle determinant in the eggs was mainly Partitioned into gray fragments and not into red fragments. AN ATTEMPT TO ISOLATE cDNA CLONES SPECIFIC FOR B4.1 BLASTOMERES OF THE ASCIDIAN EMBRYO T. Miya!, K. W. Makabe, 2 and N. Satoh! 1Dept. of Zool. , Fac. of Sci. , Kyoto Univ. , Kyoto , 2Div. of Biol. , Califommia Inst. of Tech. , Pasadena , USA During ascidian embryo genesis, differentiation of primary lineage muscle cells, which are derived from B4.1 blastomeres of the 8-cell embryo, seems to be controlled by factors localized in the egg cytoplasm. To analyze mRNAs specific for B4.1 blastomeres, we collected B4.1 blastomeres and blastomeres of the animal hemisphere (a4.2+b4.2) from 8-cell stage embryos of the ascidian Halocynthia roretzi, and we made each cDNA library. Then we made a subtracted cDNA library, and by differential screening we isolated some cDNA clones which are present in the B4.1-cDNA library but not in the animal hemisphere-cDNA library. Now, we are continuing farther analysis of these clones. LOCALIZATION PATTERNS OF CYTOSKELETAL COMPONENTS AND MYOPLASMIN-C1 DURING EARLY DEVELOPMENT OF THE ASCIDIAN EMBRYO. Miki Y., Ashida K., Tanaka R., Yokoyama K. and Nishikata T. Dept. of Biology, Fac. of Science, Konan Univ., Kobe. The myoplasm of the ascidian egg is believed to contain muscle determinants, and is distributed into muscle precursor blastomeres through the ooplasmic segregation. The segregation consists of two phases, each mediated by different systems, the first by microfilaments and the second by microtubles (Sawada & Schatten, 1989). Myoplasmin-Cl is one of the myoplasmic components which are thought to play an important role in the muscle cell differentiation. In order to examine the interaction of myoplasmin-Cl and other cytoskeletal proteins, we immunologically detected tubulin, actin and p58 (resembles to the porcine neurofilament 160; Swalla et al, 1991) with myoplasmin-Cl on the same sections. During the first phase of the segregation, the distribution of myoplasmin-Cl was closely related to that of the actin rather than the tubulin. On the second phase, myoplasmin-Cl migrated posteriorly together with the sperm aster. As far as we examined, myoplasmin-Cl and p58 segregated to the same area of the egg during ooplasmic segregation. These results implicate the image of the myoplasm which is composed of the intricate complex of the cytoskeletal components; these components dynamically move, rise and fall, associate and disassociate with each other. ANALYSIS OF THE EXPRESSION OF THE MYOGENIC bHLH PROTEIN GENE IN THE ASCIDIAN 4alocynthia roretz2. I.Araki!, N.Satoh!, K.W.Makabe?, H.Saiga®>. Dept. of Zool., Fac. of Sci., Kyoto Unaiv., Kyoto, *California Institute of Technology, Pasadena, *Dept. of Biol., Fac. of Sei., Tokyo Metropolitan Univ., Hachi-oji. To address the question whether MyoDi-like factors play pivotal roles in muscle differentiation in #4.roretzz embryo, we isolated by PCR method two cDNA clones and one genomic clone encoding a protein which belongs to the myogenic bHLH protein family. The difference hetween cDNA clones occur in the poly(A) sites. The gene consists of four exons. In the 5’ upstream region four’CANNTG motifs (or E-boxes) are clusterd around -550. They may be target sites of its autoregulation. This protein consists of 435 amino acid residues and its bHLH domain is well conserved. RT-PCR analysis has demonstrated that the expression of this gene begins at 64-cell stage and continues until swimming larva stage. In adult, body wall muscle and heart express this gene, while liver, gill and intestine don't, although none of the members of the myogenic bHLH protein gene was expressed in the heart of mammals. These results suggest that a MyoDl-like factor may be involved in ascidian muscle differentiation Developmental ACTIVITY OF THE UPSTREAM REGION OF HALOCYNTHIA RORETZI MUSCLE ACTIN GENE IN CIONA SAVIGNYI EMBRYOS. A. Hikosaka, T. Kusakabe, K. W. Makabe, and N. Satoh. Dept. of Zoology, Fac. of Sci., Kyoto Univ., Kyoto. HrMA4a, one of the muscle actin genes of the ascidian H. roretzi, is expressed specifically in larval muscle cells during embryogenesis. We have already shown that about 1.4-kb 5' upstream region of this gene has the function to control muscle- specific activation of this gene. In this study we examined whether the upstream regulatory region of HrMA4a works in the other ascidian Ciona savignyi. We constructed pHrMA4a-Z that contains the upstream regulatory region of HrMA4a and coding region of bacterial €-galacto- sidase gene (lacZ). We injected this fusion gene into C. savignyi eggs, cultured them until hatching, and assayed for B-galactosidase activity by histochemical staining. It was observed that the gene was expressed predominantly in larval muscle cells. However, in some cases the gene expression was detected in unidentified cells located at the ventral side of larval trunk region and in some other cases it was also activated in mesenchyme cells although its expression was weak. pHrMA4a-Z deletion mutant that has only 216 bp upstream region was also expressed in basically same pattern, although in some specimens it was expressed in larval brain cells. AN ATTEMPT TO ISOLATE MSH GENE OF THE ASCIDIAN HAROCYNTHIA RORETZI K.Tagawa and N.Satoh. Dept.Zool.,Fac.Sci.,Kyoto Univ., Kyoto. msh (muscle segment homeobox) is a gene that has been isolated from Drosophila, and known to be expressed in the mesoderm, developing CNS and the muscle of the fly. In order to study whether msh gene homolog of H.rorerzi plays an important role in the ascidian development, we tried to isolate msh gene homolog of H.roretzi. We constructed a genomic library of H.roretzi and screened the library using Ciona intestinalis msh as a probe. So far, we obtained several canditate clones, of which sequences are now investigated. Biology 1193 AN ATTEMPT TO ISOLATE A NOTOCHORD SPECIFIC GENE OF THE ASCIDIAN HALOCYNTHIA RORETZI H. Yasuo and N. Satoh. Dept. Zool. , Fac. Sci. , Kyoto Univ. , Kyoto. The ascidian is primitive chordate, which develops a notochord only during larval stage. We want to know the origin of notochord in the animal phylogeny, by studying of the molecular mechanism of the notochord cell differentiation in the ascidian. The Brachyury (T) gene is a genc that has been isolated in mouse and Xenopus , and the gene is specifically expressed in their notochords. The amino acid sequences of the putative Brachyury proteins of mouse and Xenopus are highly conserved in the N-terminal half. By using the Xenopus Brachyury cDNA fragment encoding the conserved region as a probe, we isolated a genomic clone of an ascidian homolog of Brachyury . We used this clone to probe Northern blots of RNA prepared from H. roretzi embryos at different stages. Maternal transcript was not detected. A transcript, about 2.3kb in length, was first detected in gastrula,and the expression was maximal at gastrulation. The amount of the transcripts, however, decreased following neurula stage. The size and the expression pattern of the ascidian gene closely resemble those of mouse and Xenopus Brachyury genes. Now we are screening a gastrula cDNA library with a probe prepared from the genomic clone. THE MOLECULAR STRUCTURE OF cDNA CLONES SPECIFIC FOR THE EPIDERMIS OF THE ASCIDIAN EMBRYO. 1T.Ueki, 7K.W.Makabe and!N.Satoh. Dept. of Zool., Fac. of Sci., Kyoto Univ., Kyoto. 2Div. of Biol., California Inst. Tech., Pasadena. USA. To investigate the molecular mechanism involved in the differentiation of epidermal cells of the ascidian embryo, we have isolated eight different cDNAs for mRNAs that are specifically expressed in the epidermal cells of Halocynthia roretzi embryo. The temporal expression patterns of the mRNAs have been revealed to be classified into four types. We choosed four genes, HrEpiA,B,C and E, that represent each of the four types. We isolated and analysed longer cDNA clones of them. The sequences of the cDNAs were determined by dideoxy method. The longest cDNA of HrEpiA was about 1.2kb, nearly full-length of its mRNA. It encodes a 241amino- acid peptide started from the first methyonine and the peptide sequence was revealed to be similar to epimerases. The longest cDNAs for other genes,HrEpiB and C, were several hundred bases shorter than the predicted size of their mRNAs. The amino acid sequences predicted by the longest open reading frame were used for the database search, but the search revealed no significant similarity between the amino acid sequences and any previously reported protein sequences. The longest cDNAs for the HrEpiE gene so far obtained was about 1 kb shorter than its 3.0 kb mRNA. This cDNA was thought to cover length of the trotein not long enough for database search. 1194 Developmental Biology Tissue compartments in Drosophila gut epithelium: minimum tissue units of gene expression. R. Murakami, A. Shigenaga!, A, Matsumoto’, I. Yamaoka and T. Tanimura’. Biol. Inst., Yamaguchi Univ., Yamaguchi. Biol. Lab., Kyushu Univ. Ropponmatsu, Fukuoka. Spatial expression patterns of lacZ reporter gene were analyzed in the gut of Drosophila larvae transformed with P elFement-lacZ vector (P-lacZ). lacZ expression by this method has been shown to reflect the expression of a gene neighbouring the insertion of P-lacZ vector. lacZ-positive cells formed distinct domains with transverse and longitudinal boundaries along the gut tube. Boundaries of lacZ-positive domains were included in consistent demarcation lines which subdivided the gut epithelium into about 40 partitions regardless of the strain. We called the partitions "tissue compartments". lacZ- positive domains of the strains appeared as a single tissue compartment or as a combination of several compartments, Overlapping with each other. All the cells in a tissue compartments were equivalent as to lacZ expressions, suggesting that the tissue compartment is a minimum tissue unit of gene expression comprising the cells with a common gene expression pattern. Analysis of the proctodeum gene which causes malformation of the hindgut of Drosophila embryo. A. Shigenaga, I. Yamaoka and R. Murakami. Biol. Inst., Yamaguchi Univ., Yamaguchi. A recessive lethal mutant with malformation of the hindgut was isolated from P-lacZ enhancer trap strains. We called the mutant proctodeum (proc) since the proctodeum (hindgut blastema) degenerated during stage 12-14 in proc embryo. Target tissue of this mutation corresponded to JacZ-positive domain in the proc embryo, suggesting that the lacZ expression of this strain represented the expression of the proc gene. lacZ expression was first detected shortly after the beginning of cellularization at stage 5. lacZ-positive domain formed a ring covering approx. 10-20 % egg length. This domain included future ectodermal proctodeum and visceral mesoderm of the hindgut. lacZ activity continued to be detected in the developing hindgut in embryos of later stages. lacZ activity was still positive in hindgut epithelium of larva and adult. Since the lacZ of the Proc was not expressed in the trunk and torso embryos in which anteroposterior axis was disorganized, expression of the proc was thought to be under the control of these maternal genes. PRODUCTION OF A MONOCLONAL ANTIBODY AGAINST KERATIN EXPRESSED IN THE CHICKEN EMBRYONIC DIGESTIVE TRACT EPITHELIUM K.Sato, K.Urase and S.Yasugi. Dept. Biol., Fac., of Sci., Tokyo Metropolitan Univ., Tokyo We found that the expression of certain keratin molecules was related to the differentiation of chicken embryonic digestive tract epithelium. To investigate more precisely the nature and changes of keratin molecules during development, we tried to get monoclonal antibodies against chicken keratins with using s-carboxymethylated extract of day 13 chicken gizzard (GZ) as antigen. We obtained a monoclonal antibody (IgM) which recognized 72 and 78.5 kDa polypeptides on immunoblotting after SDS- PAGE of the extract. We examined the staining patterns by this antibody in the whole embryos in early stages (day 1- 4) and the digestive organs (from day 5) . In early stage embryos and in digestive organs, the endoderm gave consistently positive signals. In the GZ and proventriculus (PV), the positive structures appeared mainly as filaments running parallel to the apico-basal axis of the epithelial cells. In the GZ epithelium from day 14 afterwards, the staining pattem changed from filamentous to granular. In the PV, from day 8 afterwards when the deep glands begin to be formed, the positive structures in the cells of deep part of the glands became granular and were more faintly stained. The outermost layers of epithelium of ectodermal derivatives such as skin and scale also positively reacted to the antibody. SUPPRESSION OF DNA SYNTHESIS IN THE CHICKEN EMBRYONIC DIGESTIVE ORGANS BY AN INHIBITOR OF PROTEIN KINASE C. Sadao Yasugi! and Etsuko Yasugi?. 1Dept. Biol., Fac. Sci., Tokyo Metropol. Univ., Tokyo, and 2Div. Clinic. Biochem., Clinic. Inst., Natl. Med. Center, Tokyo, Japan. We reported the suppressive effect of H7, a potent and specific inhibitor of protein kinase C (PKC), on the differentiation of digestive organs of chicken embryos in vitro. To analyze the _ relationship between suppressive action of H7 and proliferation of epithelial and mesenchymal cells of organs, we cultivated 6-day proventriculus and intestine and assayed the incorporation of BudR into DNA of cells of these organs on the 3rd day of cultivation when the suppression of epithelial differentiation was already observed. In the case of proventriculus, the control explants showed the labeling of 6.2% of mesenchymal cells and 14.9% of epithelial cells. When 100 uM of H7 was added to the culture medium, the labeling index in mesenchyme lowered to 2.3%, whereas that of epithelium remained at the level comparable to that of control explants. The coexistence of H7 and SC9, an activator of PKC, in the medium restored the labeling index of mesen- chyme to 4.7%. The similar tendency was observed as to the proliferation of intestinal explants. These data suggest that H7 affects primarily the prolifer- ation rate of mesenchymal cells and brings about a reduced growth, and this in turn results in the suppression of normal epithelial differentiation. Developmental Biology 1195 DIFFERENTIATION OF CHICK EMBRYONIC OESO- PHAGEAL EPITHELIUM CULTURED IN A BASEMENT MEMBRANE-LIKE MATRIX. S.Matsushita. Dept.of Biol., Tokyo Women’s Medical College, Tokyo. The oesophageal epithelium of 6-day-old chick embryos isolated by collagenase- treatment was embedded in a small volume of a basement membrane-like matrix (Matrigel), placed at the interface of air containing 5% co, and a liquid medium con- sisting of 75% Medium 199, 20% 12-day chick embryo extract and 5% foetal bovine serum, and cultured at 38°C. The oesophageal epithelium, which had been pseudo-stratified columnar-type and had a tubular shape, came to form a small vesicle within one day in culture, and be- came stratified-type within 2 days. After 4 days, part of the epithelium became mucus-secretory type, while the rest of the epithelium increased in cell layer and developed cilia on the luminal surface. After 6 days, mucous epithelium and strat- ified squamous epithelium containing many tonofilaments were found, which resembled the glandular and luminal epithelia of the mature oesophagus of hatched chicks, res- pectively. Thus, the present culture meth- od could support the full differentiation of the oesophageal epithelium of the 6-day chick embryo in the absence of mesenchyme. THE INFLUENCE OF A NEW ANESTHETIC AGENT ‘ISOFLURANE ' ON THE HATCHED CHICKEN. -Nonoyama , H.Tanaka and H.Haseggwa O Aichi Univ. of Educ., Kariya and ~“Chukyo Womens Univ., Ohbu. The authors reported on the anesthetic agents such as halothane, enflurane and sevoflurane caused the inhibition of grow- th,liver damage and malfolmation of chick embryos previously. In this studies, we examined on the influence of a new anesth- etic agent, isoflurane to the hatched chi- cken according to the previous reports.The ratios of the body weights of 2% treated chickens were suppressed 16.4% and 18.2% on 20 and 25 days after respectively, as compared with the control. The values of serum GOT, GPT and r-GTP of the control were 340, 7, 27; on the contrary the val- ues of the treated chickens were 391, 7, 25 (after 20 days),and 439, 8, 21 (after 25) respectively. The variation of serum LDH patterns ofthe control was as follows: Ds YW = /Oo8, wo As 1760, wos 3S 2 5a, LDH 4 = 2.1 and LDH 5 = 3.3. And that of the 20 days' treated hatched chicken was 51.6, 15.8, 7.8, 8.2, and 16.6; and the a5 Casyys Was @Soilp Wolh Vas, BoB, ~ evacl 18.8 respectively. Many stretched micro- villis and lymphatic cells on the surface of disse cavity of the treated chicken liver were obserbed. However, not so sig- nificant difference was recognized bet- ween the treated chicken liver and the control, from the scanning electron mic- roscopic observation. POSSIBLE INVOLVEMENT OF GP-100 GLYCOPROTEIN IN THE PROCESS OF BUDDING AND REGENERATION OF THE POLYSTYELID ASCIDIAN, POLYANDROCARPA MISAKIENSIS. Y.Katsuyama , S. Fujiwara and K.Kawamura . Dept. of Biol. , Fac. of Sci. , Kochi Univ., Kochi GP-100 is a glycoprotein that binds to the galactose-specific lectin, T'C-14 extracted from the polystyelid ascidian Polyandrocarpa misakiensis. Purified GP-100 was separated into two subunits (20kDa and 26kDa) on SDS-PAGE under reduced condition. In the asexual life span, it increased remarkably in amount during budding, and decreased to a minimum level in functional animals of pre-budding stage. GP-100 also increased in amount in the process of regeneration, although the amount was not so high as that of developing buds. We prepared anti- GP-100 monoclonal antibody that recognized specifically the 20kDa subunit of the glycoprotein. Immunostaining showed that in adult animals GP-100 was localized in the mesenchymal space between the epidermis and atrial epithelium, indicating that this glycoprotein is a major coelomic component. GP-100 was also located in the vacuoles of a certain kind of blood cell, probably leucocyte. Thus, this kind of cell is considered as GP-100-producing cell. Interestingly, GP-100 disappeared from the morphogenetic region of both developing buds and regenerating adult animals.GP-100 was also extractable from other budding polystyelid ascidians such as Metandrocarpa, but not from solitary ascidians and budding entrogona such as Aplidium. The results of this study suggest that GP-100 might be involved in budding and regeneration of polystyelid ascidians, but that it might act as a negative regulator of morphogenesis rather than a factor that promotes morphogenesis. Retinoic acid can induce the ectopic morphogenesis via mesenchymal cells in the palleal bud of Polyandrocarpa misakiensits. K.Hara, S. Fujiwara, K. Kawamura, Dept. of Bio., Fac. of Sci., Kochi Univ. Kochi. We have already shown that retinoic acid (RA) can induce the secondary antero-posterior axis in the palleal bud of Polyandrocarpa misakiensis In this study, we examined whether mes en- chymal cells could mediate the effect of RA on the induction of the ectopic morphogenesis. The mesen- chymal cells were isolated from adult animals and treated with 10°>M RA, or DMS Oas a control. They were implanted into the bud to assess their morpho- genesis inducing-activity. RA treated mesenchymal cells could induce the ectopic morphogenesis in 45% in the most efficient cases, but DMSO treated ones could not do so. This activity of mesenchymal cells depended on the time length of exposure to RA, during which RA had no apparent effect of growth of the mesenchymal cells. These results indicated that RA could induce the ectopic morphogenesis via the mesenchymal cells. They also suggested that the mo rphogenesis-inducing activity of mesenchymal cells was acquired without any cell growth. 1196 Developmental Biology DEVELOPMENTAL ROLE OF ALDEHYDE DEHYDROGENASE IN THE PROCESS OF BUDDING AND REGENERATION OF THE TUNICATE, POLYANDROCARPA MISAKIENSIS. K. Kawamura and S. Fujiwara. Dept. of Biol., Fac. of Sci., Kochi Univ., Kochi. We have previously shown that in the budding tunicate Polyandrocarpa misakiensis retinoic acid can induce an ectopic morphallactic field of developing buds. From this animal. we extracted endogenous retinoids and, using HPLC, identified some major peaks as cis-retinal, all-trans-retinal and all-trans- retinoic acid, of which cis-retinal was most abundant. During both budding and regeneration, cis-retinal decreased remarkably in amount. Concomitantly, aldehyde dehydrogenase that could metabolize retinal in vitro was induced in epithelial cells and then in mesenchymal cells. Both kinds of cells were major sources of endogenous retinoids. In developing buds, the enzyme always appeared at the proximal end. that is, the future morphogenesis domain, whereas in regenerating animals it was observable at the whole area of the zooid piece. When exogenous retinoic acid was applied to buds, the enzyme was induced in the secondary morphallactic field, as in the primary field. The results of this work show that Polyandrocarpa aldehyde dehydrogenase is a potent retinoic acid synthase induced specifically in the process of budding and regeneration. They also suggest that the enzyme plays a role in morphallactic development of Polyandrocarpa buds and regenerates via positive auto-regulation of retinoic acid synthesis. THE INHIBITION OF SPERMATOGENESIS IN THE MEDAKA, ORYZIAS LATIPES, EXPOSED TO HIGH WATER TEMPERATURE. S. Haniaeuicnet ; M. Takakuwa2 Yamazaki Dept. Biol., Coll. Gen. Educ., “Dept. Biol., Fac. Sci., Niigata Univ., Niigata and It is well known that spermatogenesis is inhibited when mammalian testes are exposed to high temperature. We reared the medaka, Oryzias latipes, at 27, 30, 33 and 36°C of water temperature, and found that the inhibition of spermatogenesis occur in 33 and 36°C groups. From the analysis of the areas occupied by germ cells at each stage of spermatogenesis, conspicuous decrease in the type B sperma- togonia were noted in 33 and 36°C groups. The number and labeling index of type A spermatogonia were not affected, which indicates that the proliferative activity of type A spermatogonia is normal at high temperature. The number of cysts of labeled type B spermatogonia was extremely decreased. The fish which had an injec- tion of BrdU were kept at 27 and 33°C, and examined the most advanced stage of spermatogenesis of the labeled germ cells. The results showed that labeled spermato- zoa appeared 5-9 days after the injection at 27°C and 3-5 days at 33°C. These results indicated that the dif- ferentiation of type B spermatogonia is specifically inhibited at high tempera- ture. ON THE SPERMATOGENESIS OF THE FRESHWATER EARTHWORM, BRANCHIURA SOWERBYI. I. THE FINE STRUCTURE OF THE CENTRAL MASS. M.Shirasawa and N.Makino. Dept. of Biol., Tokyo Med. Coll., Tokyo. The spermatogenesis of the freshwater earthworm, Branchiura sowerbyi was stud- ied by light and electron microscopy. Male reproductive cells are released from the testes as spermatogonia, and maturation takes place in coelomic pouches called se- minal vesicles. Male cells in every stage of maturation make a morula-like cluster, connected together by nucleus-less proto- plasm, called central mass. Central mass, which is thought to serve as a supporting or nutritive struture for the male cell, contains many small mitochondria, free ri- bosomes, and granular or agranular endo- plasmic reticulum. Mitochondria of the central mass, which originate in spermato- gonia, propagate during early stages of maturation and seem to be stored as sup- plies of mitochonria for the formation of the chondriosome body in spermatids. An- other principal organella of the central mass is membrane-less, pyronin-positive, large dense bodies. They seem to originate from chromatoid bodies of spermatogonia and are thought to concern with the pro- duction of free ribosomes of the central mass. Golgi apparatus was observed in the central mass, though secretion was not yet obvious. LECTIN BINDING TO TESTIS OF CRICKET GRYLLUS BIMACULATUS. A.Suzuki, K.Nishimura and M.Obika. Dept. of Biol., Keio Univ., Yokohama. The sugar chain expression in germ cells of cricket Gryllus bimaculatus during the spermatogenesis was investigated using fluorescent lectins or peroxidase conjugated lectins. When spermatozoa and spermiogenic cells smeared on a slide glass were treated with fluorescein isothiocyanate-labelled concanavalin A or rhodamine-labelled wheat- germ agglutinin, acrosomal area was labelled intensely but other part also reacted weakly, as described in Drosophila and two species of Orthoptera. Soy bean agglutinin (SBA) bound very specifically to spermatogonia and spermatocyte among peroxidase conjugated six lectins (Con A, WGA, PNA, UEA-1, SBA and DBA) tested for their affinity to paraffin section of testis. In young spermatids beginning the tail elongation, a round or cup-shaped organelle was the only structure that bound SBA. The SBA binding figure disappeared during the process of maturation of spermatid to sperm. The organelle corresponded to the structure of dictyosome and acroblast by electron-microscopical analysis. Developmental Biology 1197 SPERM SURFACE AUTOANTIGEN INVOLVED IN FERTILIZATION IN THE NILE TILAPIA (Oreochromis niloticus). K. Mochida, S. Adachi, K. Yamauchi and H. Takahashi. Dept. of Biol., Fac. of Fish., Hokkaido Univ., Hakodate. In the testis of fishes, several specific proteins are expressed on the sperm plasma membrane during spermiogenesis. These proteins heve been known as autoantigens which are isolated from fishes' own immune system by the blood-testis barrier, and may have specific functions during and after spermiogenesis. In order to investigate whether the sperm surface autoantigens are involved in fertilization, fertility of sperm coated with anti-sperm autoantibody was examined in the Nile tilapia. Moreover, partial purification of sperm autoantigens was attempted in the present study. Anti-sperm autoantibodies were isolated from serum of male Nile tilapia immunized with allogeneic sperm emulsified with Freund's complete adjuvant. Freshly spawned eggs were inseminated with sperm reacted with autoantibody in artificial seminal plasma. The fertlity of sperm coated with autoantibody was prominently lower than that of sperm coated with non- specific antibody. The result suggests that at least one of the autoantigens plays an important role in fertilization in the Nile tilapia. Membrane proteins of sperm were solubilized with non-ionic detergent, octyithioglucoside. The extract was loaded onto the affinity column using anti-sperm autoantiboy-Sepharose 4B, and autoantigens were eluted with 8 M urea. The eluted fraction was analyzed by SDS-polyacrylamide gel electrophoresis. Under reducing condition, the fraction showed major polypeptide bands centered at 80 kDa as well as some minor bands. By immunoblotting analysis by autoantibodies, four main bands containg the 80 kDa band were identified as autoantigens. LOCALIZATION OF SPECIFIC RECEPTOR FOR ARIS ON SPERMATOZOA OF STARFISH, Asterias amurensis A. Ushiyama', K. Chiba’ and M. Hoshi! ‘Dept. of Life Science and Gene Reserch Center, Tokyo Institute of Technology, Yokohama. Starfish egg jelly coat contains a highly sulfated glycoprotein, which is called acrosome reaction-inducing substance (ARIS). ARIS is essential for inducing the acrosome reaction. We previously demonstrated that FITC labeled ARIS bind specifically to homologous sperm, indicating the presence of ARIS-receptors on the sperm. By using two methods, we studied the distribution of ARIS-receptors on the sperm surface. Competition of FITC-ARIS binding was assayed with a head fraction and a tail fraction of spermatozoa. Head fraction bound FITC-ARIS specifically almost to the same extent as whole sperm. Tail fraction did not bind FITC-ARIS appreciably. ARI|S-binding was also assayed by microscopical observation of intact spermatozoa after incubation with fluorescent polystylene beads (Fluosphere) coupled with ARIS. The binding of fluosphere coupled with ARIS was specific and restricted to the anterior part of sperm heads. Weconclude fromthese datathat ARIS-receptors localize on the anterior surface of sperm heads. THE ER PROTEIN, CALRETICULIN, IS PRESENT IN THE ACROSOME OF POST-MEIOTIC CELLS IN RAT TESTIS. M.Nakamura! ,M.Moriya’ ,Y.Michikawa’ ,T.Baba‘’, S.Okinaga’,T.Kobayashi’ and K.Arai'’. ‘Dept. of OB/GYN,Sch.of Med.,Teikyo Univ.,Tokyo, ?Zool.Inst.,Fac.of Sci.,Hokkaido Univ., Sapporo, and *Inst.of Appl.Biochem.,Univ.of Tsukuba, Tsukuba. In this study, this protein has been identified as a calreticulin. Immuno-stains for the localization of this protein in rat testis revealed that the protein is present in the ER, the Golgi body and the acrosomal vesicle of round spermatids, and in the acrosome of sperm. The complete amino acid sequence of calreticulin deduced from the cDNA nucleotide sequence appeared to be Similar to that of mouse calreticulin. The cDNA encoded a protein of 416 amino acids, but a 17-residue NH,-terminal signal sequence was removed.The mature 399 residue protein of Mr=46347 ends in Lys-Asp-Glu-Leu. There were 7 amino acids replaced. When Northern blot analysis, using RNAs prepared from purified populations of rat spermato- genic cells, was performed,the calreticulin mRNA was found in pre- and post-meiotic cells. Furthermore, the purified protein has two Ca?* binding sites. These results suggest that calreticulin, of which biosynthesis is already started before meiosis, plays a role in Ca’?* storage in spermatogenic cells of rats, and that this ER protein incorporates into the acrosomal vesicle via the Golgi body without glycosylation during spermiogenesis. EFFECTS OF HORMONES ON THE TESTIS FRAGMENT CULTURED IN VITRO IN THE MEDAKA. M. Matsumoto and K. Onitake. Dept. of Biol., Fac. of Sci., Yamagata University, Yamagata. We have reported that the spermatogenesis progressed under the conditions of cell culture of spermatocytes and organ culture of testis fragments. In the present study, effects of several hormones, human chorionic gonadotropin:HCG, follicle stim- ulating hormone: FSH, testosterone and 17- Sestradiol, on the differentiation of spermatogonia in the testis fragaments cultured in L-15 medium supplemented with fetal bovin serum. To identify the differ- entiation process more cleary, BrdU was added in the culture medium and BrdU in- corporating spermatogonia were pursued immunohistochemically using the anti-BrdU monoclonal antibody. When FSH and 17-£8 estradiol were added, the number of sper- magonial cysts increased and the differ- entiation process from spermatogonia to spermatocytes was observed. Addition of HCG was also effective as same as FSH. Testosterone was neither effective on in- crease of the number of spermatogonial cysts nor the differentiation of spermato- gonia to spermatocytes. Furthermore, after 5days of culture, spermatids containing BrdU appeared. These results suggests that spermatogenesis in the testsis fragments cultured in vitro was controlled by the hormones and progressed as same as in vivo. 1198 Developmental Biology IN VITRO SPERMATOGENESIS FROM THE PRIMARY SPERMATOCYTES UNDER THE BROMODEOXYURIDINE CONTAINING MEDIUM IN THE MEDAKA. M. Suzuki and K. Onitake. Dept. of Biol., Fac.of Sci., Yamagata University, Yamagata. We have revealed that the isolated pri- Mary spermatocytes of the medaka completed first and second meiotic divisions and differentiated into functional sperm in vitro. In the present study, in order to get more direct evidence of the production of functional sperm in vitro, primary spermatocytes were precultured in L-15 medium supplemented with 10% fetal calf serum containg BrdU of l1pM/ml for 3 hr. After preculture, they were cultured in L- 15 medium without BrdU for 5 days. Process of spermatogenesis was pursued immuno-— histochemically using anti-BrdU monoclonal antibody. After 2 days of culture, sperma-— tocytes in l1yM BrdU/ml completed first and second meiotic divisions and differenti- ated into flagellated spermatids. Time course of differentiation was almost same as in the normal culture medium, while the spermatocytes cultured in the medium containing BrdU of 50y4M /ml required 4 days for completion of the first meiotic divisions,but second meiotic divisions never occured. These results indicate that DNA syntheses at primary spermatocytes were necessary for the completion of second meiosis follwing further differ-— entiation into spermatozoa. DIFFERENTIATION OF SECONDARY SPERMATOGONIA TO PRIMARY SPERMATOCYTES IN ORGAN CULTURE OF TESTES FRAGMENTS FROM CYNOPS PYRRHOGASTER: ANALYSIS OF PROTEIN SYNTHESIS. , K. Maekawa and S. Abe. Dept. of Biol. Sao MACS C1 SStlo A UMEMOLO Wrrsiys , Kumamoto. We have previously shown that secondary spermatogonia differentiate into primary spermatocytes by FSH in organ culture of testes fragments and that FSH induces proliferation of spermatogonia in testes fragments which were incubated in a control medium for a week(Ann. Meeting of Dev. Biol., 1992). In this study, we examined if the synthesis of new proteins is induced by the addition of FSH in the testes fragments which were cultuped for a week in the absence of FSH. [~~S]methionine was incorporated into the testes fragments, the cell extracts were electrophoresed in two dimensions, the gels were exposed to an imaging plate for 3-4 days. Analysis by an image analyzer showed that at least two spots (pI6.9, MW1.8K and pI5.5, MW1.3K) were detected to appear within 1 day after the addition of FSH. These results indicate that FSH triggers the synthesis of some proteins in Sertoli cells and/or secondary spermatogonia, which leads to the proliferation of spermatogonia. DIFFERENTIATION OF PRIMARY SPERMATOCYTES TO ELONGATING SPERMATIDS BY FSH IN ORGAN CULTURE OF TESTES FRAGMENTS FROM CYNOPS. Z. S. Ji and S. Abe. Dept. of Biol.Sci., Fac. of Sci., Kumamoto Univ., Kumamoto. To study if the processes in spermatogenesis beyond primary spermatocytes (PC) depend on hormones and Sertoli cells, testes fragments rich in PC were cultured in a chemically defined L-15 medium supplemented with various hormones and vitamins. In a control medium a few percent of cysts contained round spermatids but no elongating spermatids on the second week, while both of the diameter and viability of germ cells decreased to about 40%. On the other hand, ina medium supplemented with various hormones and vitamins more than 10% of the cysts differentiated to elongating spermatids and both the diameter of cysts and viability of cells were maintained at the initial level. Selective removal of hormones and vitamins revealed that FSH alone promoted the differentiation but neither androgens nor vitamins did. This is consistent with our previous results that FSH binds to the newt testes in PC- stage as well as in spermatogonia-stage. This result indicates that FSH and Sertoli cells play a major role in initiating nuclear elongation of spermatids. STABILITY OF MANCHETTE (MICROTUBULE BUNDLES) IN NEWT ELONGATE SPERMATIDS.IV. Y. Mazaki, K. Takamune and S. Abe. Dept. of Balol. Sci) RalceaOmepotneery Kumamoto Univ., Kumamoto. We have previously shown that Manchette structure in newt elongate spermatids(ET) is resistant to the treatment of Ca“* and low temp and that 1M NaCl sup from ET-rich testes contains MAPs which promote tubulin polymerization and confer the low temp- resistance to the microtubules(Ann. Meet. Zool. Soc., 1991). Copolymerization of porcine brain tubulin with the 1M NaCl sup and porcine brain MAPs(pbMAPs) showed that the amount of 76K and 59K of 1M NaCl sup bound was reciprocal to that of pbMAPs bound, suggesting that the binding sites On microtubules for 76K and 59K were common for pbMAPs. Western blotting of cell extracts of purified populations of primary spermatocytes(PC), round spermatids(RT), ET and mature sperm(MS) with anti-76K and anti-59K as probes showed that 76K was present through PC to ET but was absent in MS and that no 59K was present through all stages. These results indicate that 76K is responsible for promoting polymerization of tubulin and resisting disassembly and that it is somehow regulated so that 76K does not confer low-temp resistance to microtubules in PC and RT in vivo. Developmental Biology 1199 HOW IS THE FLAGELLAR LENGTH OF MATURE SPERM DETERMINED? VI. QUANTITATIVE ANALYSIS OF TUBULIN SYNTHESIS RATE IN SYNCHRONOUS CULTURE OF ROUND SPERMATIDS FROM CYNOPS AND XENOPUS. i A. Yoroisaka, K. Takamune and S. Abe. DAPEG O8 BWiOGl, SEilop WAGES O88 Sto, Kumamoto Univ., Kumamoto. We have shown that the difference in the period of flagellar growth between Cynops and Xenopus may be due to the difference in the period of tubulin synthesis between the two species(Ann. Meet. Zool. Soc. 1990). In this study, we measured tubulin Synthesis rate quantitatively in Synchronous culture of round spermatids from both species: [ 5s )}methionine was incorporated to round spermatids, the proteins extracted were electrophoresed, the gels were exposed to an imaging plate and the intensity of the tubulin band was analyzed by an image analyzer. In Cynops, tubulin synthesis rate rose up to about 150% by day 3 and fell down to about 40% of the initial value by day 6. Then, the value declined slightly by day 12 but about 20% remained on day 12. On the other hand, in Xenopus, the synthesis rate on day 3 was almost the same as the initial one but then it dropped to reach 5% on day 9. These results demonstrate and confirm the previous results that tubulin synthesis in Cynops round spermatids continued longer than in Xenopus. GENE ANALYSES OF THE SPERM-SPECIFIC BASIC PROTEIN IN, XENOPUS LAEVIS. N.Ariyoshi', K. akamune , S-Abé!, K.Mita2, and Ch.Katagiri~. Dept.Biol. Cop Meo Sci., Kumamoto Univ., Kumamoto, “Zool. Inst., Fac.Sci., Hokkaido Univ., Sapporo. The chromatin proteins of the mature sperm of Xenopus laevis comprise six sperm-specific nuclear basic proteins (SP1-6) in association with four types of core histones. Southern analyses of the genomic DNA from liver of the Xenopus J strain using SP4 cDNA as a probe revealed that four fragments (12Kbp, 7Kbp, 4Kbp and 2Kbp) of the EcoRI digests and two frag- ments (15Kbp and 10Kbp) of the HindIII digests contain SP4 genes. Mapping of both SP4 genes and restriction enzyme sites on the 12Kbp fragment of EcoRI digest indi- cated the existence of three loci for the SP4 genes and a HindIII site on one of the intermediate regions between the SP4 genes. These results suggested that two hybridizable fragments with SP4 cDNA of HindIII digest are linked together, so that plural SP4 genes are arranged over about 25Kbp area. S1 nuclease mapping and complete base sequences of one of these SP4 genes and its flanking region showed that TATA box and CCAAT homology (opposite strand) were 28bp and 84bp nucleotides upstream from the transcription start site, respectively. The SP4 gene had a single, short intervening sequence in contrast with the rooster and trout protamine genes which are intronless. ANALYSIS OF NUCLEAR BASIC PROTEINS AND CHROMATIN STRUCTURE OF RANA CATESBEIANA MATURE SPERM Meee Olle pan Kae Ohsumi2, K. Takamune?, and Ch. Katagiri. Zool. Inst., Hokkaido Univ., Lab. Cell Dev. Biol., Tokyo Inst. Technol., “Dept. Biol., Kumamoto Univ. Previous studies reported that the testis proteins in Rana catesbeiana contain testis-specific histone Hl and four core histones (H2A, H2B, H3, H4). Analyses on acetic acid/urea/Triton X-100 (AUT)-PAGE of acid extracts from mature sperm separated by Percoll density gradient centrifugation showed sperm-specific proteins besides core histones. Fractionation of these proteins using reversed-phase HPLC and SDS-PAGE and their amino acid analyses revealed three sperm-specific, very lysine-rich Hls and four somatic core histones. Incubation of sperm in nucleoplasmin (NP) resulted in pronounced nuclear decondensation and loss of sperm-specific H1ls but not core histones. Sperm chromatin was totally re- sistant to micrococcal nuclease digestion, but yielded 150- and 350 bp nuclease- protected DNA fragments after incubation with NP. Electron microscopy of NP-treated sperm nuclei showed beaded fibers possessing variable linker lengths. These results suggest that Rana sperm chromatin contains nucleosome-like structure with repeat lengths different from that of typical somatic chromatin. cDNA CLONING AND EXPRESSION OF ACTIVIN B IN THE TESTIS OF JAPANESE EEL, ANGUILLA JAPONICA T. Miural, M. Tanaka, K. Yamauchi2, M. Asashima3 and Y. Nagahama’. ‘Lab. of Reprod. Biol., Natl. Inst. for Basic Biol., Okazaki, 2Fac. of Fisheries, Hokkaido Univ., Hakodate and 3Dept. of Biol., Yokohama City Univ. Yokohama A cDNA insert encoding activin B was isolated from a Japanese eel testis cDNA library. The nucleotide sequence of eel activin B cDNA is 3.3 kb long. The insert contains an open reading frame predicted to encode a protein of 395 amino acid residues, of which the C-terminal 115 residues represent the activin B-subunit proper. The deduced protein of the activin B subunit has 89% homology with that of the human and chicken subunit, and 88% homology with that of the pig and Xenopus subunit. Northern blot analysis of testis RNAs using this cDNA insert demonstrated a single mRNA species approximately 3.3 kb in length. Testis expression of activin B mRNA was very low or undetectable prior to HCG treatment, was highest one day after HCG treatment and declined as spermatogenesis progressed. The tempora pattern of expression of activin B mRNA after HCG injection is strongly suggestive of a role for activin B in early spermatogenesis in the eel. 1200 Developmental Biology ANALYSES OF PROTEIN-DNA INTERACTIONS IN THE PROMOTER AND INTRON REGIONS OF THE MOUSE AND HUMAN HAPLOID-SPECIFIC Hsp70 HOMOLOGUE GENES H.Ando, Y.Saijoh, Y.Itol, J.Higuma and H.Fujimoto, Lab. of Cell Biol., Mitsubi- shi Kasei Inst. of Life Sci., Machida-shi, Tokyo (1univ. of Hiroshima, Hiroshima). Recent advances of molecular biological techniques revealed that some of genes are exclusively expressed in the post-meiotic cells. We cloned mouse and human haploid- specific Hsp70 homologue genes, hsc70t and hum70t, respectively. To investigate mechanisms of haploid-specific gene transcription, genomic structures of both genes were analysed. They had only one in- tron in the 5' untranslated region and their genomic organizations were con- served. After determination of a transcriptional starting point of the mouse hsc70t gene, nucleotide sequences of both genes were aligned in the promoter and intron regions. Several stretches of sequences were shared between mouse and human genes. The interaction of mouse nuclear proteins to these stretches was analysed by the gel shift assay. A DNA- protein complex caused by one of these stretches was shifted in nuclear proteins extracted from adult mouse germ cells, but not from somatic tissues examined nor younger mouse testis without spermatids. Functional significance of these regions remains to be determined by analyses of transgenic mice. EFFECTS OF ASCORBIC ACID PHOSPHATE ON THE AUTOCRINE GROWTH OF CHONDROGENIC CELLS OF THE CHICK EMBRYO T.Miyazaki, K.Watanabe, M.Amano Cell and Developmental Biology Laboratory, Faculty of Integrated Arts and Sciences, Hiroshima University, Hiroshima. Perichondrial cells, the scleral fibro- blasts of chick embryo, proliferated in protein-free culture. The cells died within a week after reaching stationary phase. Ascorbic acid phosphate(Asc-P; mixture of 2- and 3-phosphates) rescued this type of cell death. Four growth promoting factors(A-I~ A-IV) were detected by DEAE-Sepharose chromato- graphy in the conditioned medium obtained from Asc-P containing culture. These were different from previously described SAF-I and SAF-II separated from the conditioned medium without Asc-P. A-II and A-IV manifested growth-promoting activities to the scleral fibroblasts and the scleral chondrocytes, but A-I did only to the scleral fibroblasts. A-III showed the activity only to the scleral chondro- cytes. They promoted growth, but did not promote cartilage differentiation. These results suggested Asc-P altered secretion patterns of growth factors. The perichondrial cells were considered to produce multiple autocrine growth factors, which played roles on the own cells and ma- tured chondrocytes differently in autocrine and/or paracrine manner. VIMENTIN EXPRESSION DURING THE DEVELOPMENT OF SOMATOPLEURAL MESODERM OF THE CHICK EMBRYO. K.Hayashi, Y.Hagiwara and E.Ozawa. Div. of Cell Biology, Natl. Inst. of Neuroscience, NCNP, Tokyo. The limb fields are eStablished by an unknown inhibitory change on the limb- forming potential of the flank mesoderm. We obtained a monoclonal antibody that reacts to the flank mesoderm of the stage 22 chick embryo, but not to the mesoderm of neck, wing bud, leg bud and tail. Further investigation of this antibody revealed that the antigen iS vimentin. Vimentin expression during the development of the somatopleural mesoderm was studied. All the mesodermal cells expressed vimentin at stage 10. But the expression disappeared at the somatopleural mesoderm by stage 16. The flank somatopleural mesoderm re-expressed vimentin at stage 18 at the dorso-medial region, and the whole flank mesoderm expressed vimentin at stage 20. On the other hand, the limb bud mesenchymal cells did not re-express vimentin until stage 27 (about 2 days later). Considerably earlier onset of vimentin re-expression at the flank region May be a useful molecular marker for the study on the establishment of the limb fields. THE EXPRESSION PATTERN OF Chox—] GENES IN THE TRUNK OF CHICKEN EMBRYOS. Y.Yokouchi_ and A.Kuroiwa, Dept. of Cell BiCIoe ys Res. Inst. for Tuberculosis & Cancer. Tohoku Univ. Sendai. We have already reported that the expression pattern of homeobox genes belonging to Chox—I cluster are closely related to the segmental structure of the carti- lage along proximo-—distal axis in developing chick limb bud. ; We investigated the expression patterns of these genes in the trunk of embryos. In somite derived tis— sues, Chox—].8 was expressed between somite 26 and tail. On the other hand, Chox- J.9 was expressed between somite 29 and tail end. In contrust, Chox- 1.10 was not expressed in somite derived tissues. In neural tube, Chox—1.8 and Chox—1.9 were ex— pressed at the same position as somatic expression were found, and Chox—J.10 was expressed from the region correspondent to somite 33 to tail end. In the gut, we found that these genes were expressed along the anteroposterior axis correlated to the func— tional domains of the gut. Chox—1.8 was expressed in the mesoderm of the cecum, Chox-J.9 was expressed in the mesoderm of large intestine and Chox-J.10 was expressed in the mesoderm of the cloaca and the endoderm of the hind gut. These results suggested that these pene might be responsible for the determination of the positional specificities along the anteroposterior body axis. Developmental Biology 1201 BASIC FGF MAINTAINS THE CHARACTER OF THE PROGRESS ZONE CELLS OF ST.20 CHICK LIMB BUDS IN CULTURE. A.Watanabe,K.Ohsugi and H.Ide. Biol-.Inst., Fac.of Sci.,Univ. of Tohoku, Sendai. Distal region (progress zone,PZ) of chick limb bud is’ important for the cartilage pattern formation, in which mesodermal cells are Maintained in an undifferentiated and rapidly growing state by the interaction with the apical ectodermal ridge (AER). It is known that the AV-1 antigen expresses in distal- anterior region and Chox.7 gene expresses in distsal region of st.20 chick limb bud and these expressions depend on the AER. We have already reported that bFGF activated AV-1 expression in cell culture of st.20 chick limb buds by the combina- tion with the BRL3A conditioned medium (BRL3A-CM).Growth of the PZ cells is stimulated and cartilage differentiation is inhibited under this condition. In this study, we found that bFGF maintained Chox.7 expression in cell culture of st.22 limb buds, and affected growth and cartilage differentiation in the cell culture of st.20 and st.22 limb buds by the combination with the BRL3A-CM. Furthermore,it was indicated that after bFGF treatment, the grafted PZcells fitted well with limb cartilage pattern in vivo. These indicate that bFGF is effective for the maintenance of the PZ character at molecular and cellular level in vitro, and that bFGF may act as a PZ Maintenance factor in vivo. MRC-5 CELLS CAUSE A-P DUPLICATION IN CHICK LIMB BUD. S.Yonei-, K.Tamura!, E.Koyama“, S.Noji%, H.Ide~. Biol. Inst., Tohoku Univ., Sendai!, Dept. Biochem Okayama Univ. Dental School, Okayama and Dept. Biol. Scj. and Tech nol. Tokushima Univ. Tokushima~. Grafting of the polarizing region (ZPA) to the anterior margin of chick limb bud leads to mirror-image duplication of Cartilage pattern. In this study, we found that MRC-5 cells (normal human lung fibro- blast) showed the polarizing activity when grafted to the anterior region of chick leg field at pre-limb bud stages (st.14 - st.17). MRC-5 cells could induce addition- al digits and some duplications at tibia/fibula level. However, the activity attenuated in stage 18 leg buds and disap- peared in stages 19-20 leg buds. Chimera analysis showed that slow growth of MRC-5 cells occurred in the leg bud and the cells located proximal region. When the cells were implanted into the anterior region of wing field, the percentage of duplicate formation was very low. These results suggest that products of MRC-5 cells are involved in three possible processes of the duplicate formation that is, induction of polarizing activity, maintenance of the activity which is present weakly at pre-limb bud stages, and determination of antero-posterior axis as the ZPA factor(s). DIFFERENTIATED LIMB CARTILAGE CELLS CAN PARTICIPATE IN LIMB CARTILAGE PATTERN FORMATION ACCORDING TO THEIR POSITIONAL VALUES. M. Sato and H. Ide Biol. Inst., Tohoku Univ., Sendai. In developing limb bud, the cells emerging from progress zone participate in limb pattern formation according to their allocated positional values. To examine whether differentiated cartilage cells retain their positional values, we grafted tissue blocks of chick leg cartilage, femur and metatarsus, to the progress zone of stage 20 quail wing buds. Both cartilage tissues participated in the host wing cartilage pattern formation, and the grafts of metatarsus localized in more distal region than those of femur, suggesting that the limb cartilage cells may keep their original positional values along PD axis. When non-limb cartilage tissue, scapula, sternum, rib or sclera, was grafted to the wing buds, scapula and sclera participated in wing pattern formation but rib and sternum, which were of somite origin, did INOVIE c Mixed cultures with these differentiated chondrocytes and limb bud cells revealed that sternum cells and limb bud cells segregated one another although sclera and femur cells were intermingled with the limb bud cells, suggesting that the difference in cell adhesiveness may be a cause of difference in the participation in limb cartilage pattern formation. THE PROXIMALIZATION BY RETINOIC ACID IN DEVELOPING CHICK LIMB BUD. 2 K.Tamyra™, Y.Yokouchi“~, A.Kuroiwa H.Ide-. Biol. Inst.1 and Res. Inst. for TB and Cancer Tohoku Univ., Sendai. In regenerating urodele limbs, retinoic acid (RA) has been known to proximalize the positional value, resulting in the forma- tion of tandemly repeated limbs along the proximo-distal axis. However, there is no evidence which supports the proximalization by RA developing limb bud at present. When the beads pre-soaked in RA are implanted into the anterior margin of chick limb buds, they posteriorize limb bud tissues and induce ZPA. In order to estab- lish the proximalization by RA in the chick limb buds, 24 hr after implantation of the RA beads to stage 20 wing bud, the tissue blocks which contain new ZPA induced by RA and the linked distal cells were trans- planted into other wing buds at stages 16- 17. Following this operation, the tissues often developed a complete set of limb cartilage elements along proximo-distal axis from humerus to digits (control grafts adjacent to the DMSO-containing beads formed no cartilage structure). These ectopic structures were found to consist of graft tissue by chimera analysis, indicat- ing that the limb bud tissues under the influence of RA could form more proximal elements than the presumptive ones by the transplantation to early wing buds. The proximalization was also supported by the expression pattern of Chox-1 genes. and 1202 Developmental Biology FETAL WOUND HEALING. EXPRESSION OF SOME DIFFERENTIATION MARKERS AT THE WOUND SITE. S. Ihara and Y. Motobayashi-. Dey. Biol. Lab., Dept. of Plast. Surg., and ~Dept. of Biochem., Kitasato Univ. School of Medicine, Sagamihara. We have studied by in situ hybridiza- tion the expression of several differen- tiation markers at the wound site in day- 17 fetal rat skin 24 h after intrauterine wounding. Oligonucleotide probes (40-mers) were synthesized for the following mark- ers: Bactin; 60k and 67k type II kera- tins; a1l(1I) and al(III) procollagen; and C-terminus and EIIIA segments of fibro- nectin. Feasibility of the probes was con- firmed by Northern analysis with poly(A)- RNA prepared from day 16-20 fetal and newborn rat skin, although the expression of 60k keratin was exceptionally undetect- able at all stages. The expression of £8 - actin was ubiquitous and its signal inten- sity was unchanged during normal skin mor- phogenesis, but strong signals were seen at the upper half epidermal layer of the wound edge. Unlike widespread expression of 60k keratin, the expression of 60k keratin was restricted to the lower half epidermal layer close to the wound edge. No special induction of the two types of collagen was observed, whereas panniculus carnosus and epidermis expressed EIIIA* fibronectin predominantly at the wound edge. The present results suggest a delay of differentiation at the wound sites. PRODUCTION OF MONOCLONAL ANTIBODIES AGAINST MOUSE EMBRYONIC SUBMANDIBU- LAR GLANDS. Y. Mori’, Y. Hieda!, Y. Kano”, T. Morita! and Y. Nakanishi’. 1Dept. Biol., Coll. Gen. Educ., Coll. Bio-Medical Tech., Osaka Univ., *Dept. Chem., Fac. Sci., Nagoya Uniy. The morphogenesis of the mouse embryonic submandibular gland, lung and kidney has been proved to proceed under the influence of epithelial-mesenchymal interactions. We have tried to produce monoclonal antibodies against mouse or- gan rudiments to find novel extracellular matrix components, which would be involved in such interactions, and also to ob- tain valuable epithelial markers for the investigation of the morphogenetic movement. Embryonic submandibular glands or the mixture of submandibular glands, lung and kidneys were injected intraperitoneally into rats, and the splenocytes were fused with mouse myeloma cells. Hybridoma media were assayed by staining cryostat sections containing sub- mandibular gland, lung and kidney. More than 60 mono- clonal antibodies against components of basal lamina, mes- enchyme and epithelium were obtained. No obvious modula- tion with these antibodies of the epithelial morphogenesis of submandibular gland has been observed yet. Among those, mAb 65 showed a unique, fibrous staining at the basal lam- ina areas and the basal part of clefts. Concentrated mAb 65 stained clearly the extracellular matrix of the mésenchyme, suggesting that the antigen would be of mesenchymal nature. Interestingly, mAb 65 stained roofplate area of the neural tube in trunk region of the 13-day mouse embryo in addition to the extracellular matrix of bovine and mouse skin. Im- munoblot analysis showed that mAb 65 recognized a band of MW more than 400 kD. GLYCOSAMINOGLYCANS AND MORPHOGEN- ESIS OF MOUSE EMBRYONIC SUBMANDIBU- LAR EPITHELIUM. Y. Nakanishi!,Y. Mori?, K. Yoshida?, T. Morita?, and Y. Fukuda*. 'Coll. Bio-Medical Tech., 7Dept. Bicl., Coll. Gen. Educ., Osaka Univ., *Seikagaku Kogyo Ltd. and *Dept. Pa- thol., Nippon Medical Sch. The initial branching morphogenesis of the mouse embryonic submandibular epithelium has been suggested to be regulated by extracellular matrix components including glycosamino- glycans. Immunohistochemical studies with monoclonal an- tibodies against intact and chondroitinase-degraded chon- droitin sulfate chains showed that they were located at the extracellular spaces of the mesenchyme and epithelial cell mass in addition to basal lamina. Basement membrane type heparan sulfate proteoglycan (HSPG) was found in the basal lamina. Chondroitinase ABC in the medium did not inhibit the initial branching of the late 12-day gland though chon- droitin sulfate chains in the tissues were almost removed by the enzyme. In contrast, heparitinase I brought about a sig- nificant inhibition of the DNA synthesis and complete in- hibition of the initial branching of the late 12-day gland. Matrigel-clotted epithelium cultured under the influence of the mesenchyme did not undergo a specific branching mor- phogenesis in the presence of heparitinase I. Collagen IV and HSPG core protein in the heparitinase-treated glands were found both in the epithelial cell mass and in the basal lam- ina. Transmission electron microscopic study indicated that intimate contacts between the mesenchymal and epithelial cells were much fewer in the heparitinase-treated glands than in the controls. These results suggested that changes in the basal lamina architecture altered the growth and morphology of the epithelium and epithelial-mesenchymal interactions. ELASTIN GENE EXPRESSION PATERN ON FIBROBLASTS OF FASCIA DURING MOUSE ORAL DEVELOPMENT. T.Yamaai, Dept. Oral Anat., Okayama Univ. Dent. Sch., Okayama. Fascia is a term so vague in usage that it signifies envelopes of muscles, nervefibers, vessels, renal and splenic tissues. The arrangement of such fascia is highly valuable for sustainment and isolation of tissues. Elastin is the major component of elastic fibers and is found abundantly in fascia. Prior to the post fixation by a microwave processor for 5min at 37°C , C3H mice were fixed by perfusion with 4% paraformaldehyde. The gene expression of elastin in fascia of perinatal mice was investigated by a simplified in situ. hybridization method with 35S labeled riboprobes on 5um thick serial sections. After 10-day exposure, signals were observed on autoradiograms. Signals of the anti-sense probe were observed on fibroblasts of fascia from 16-day fetus to 2-week neonate. The signal intensity in fascia of buccal glands increased synchronously and became maximum at 1-week neonate. However the signal intensity in dermis, perimysia, periostea and perineuria increased asynchronously. The signal intensity of signal detectable fibroblasts in these tissues was not homogeneous. The intense, weak or no signal detected cells could be observed at same stage. The signal was more intense on the portion which had higher mobility in these tissues. It was suggested that the elastin gene activation was influenced by degree of intensity of occlusal stimulation and stimulation from outside. Developmental Biology 1203 MORPHOGENESIS OF THE TONGUE IN THE FROG LARVA ( Rana tagoi )- Masahiko Kumakura and Kan Kobayashi. Dept. Anat., School of Dentistry at Niigata, Nippon Dent. Univ., Niigata The process of lingual development in R. tagoi was observed by using the light and electron microscopies. At the stage of first appearance of hindlimb bud in larva, a pair of finger-like protrusion was appeared in the bottom of the oral cavity before the beginning of lingual development. Lingual development began at the time when the hindlimb bud was well developed. At this stage, epithelial cells surrounding the finger-like protrusions prolifelated, and a butterfly-like primitive tongue was appeared. A typical shape of tongue of the frog was formed at the stage of emergence of the forelimbs. In this time, numerous cone-like papillae were distributed densely on the lingual dorsal surface. During shortening of the tail, the fungiform papillae were begun to form. In the process of fungiform papillary differentiation, microvilli first appeared on the cell surface of the top of the cone-like papilla. Subsequently, they differentiated into microridges and, as a result, sensory disk was completed. At the final stage of metamorphosis, fungiform papillae showed fundamentally similar structure to that of adult form, while structures of filiform papilla and lingual gland _ were still remained as immatured compared to those of adult frog. ULTRACYTOCHEMICAL STUDIES OF PROTEOGLYCANS IN THE ANURAN LARVAL NOTOCHORD. F.Sasaki, J.Hirata, S.Nishikawa, *H.Iseki. Dept. of Biol., School of Dent. Med., Tsurumi Univ., Yokohama. *Tokyo Med. and Dent. Univ., Tokyo. During metamorphosis an anuran larval noto- chord degenerates both in body and tail. The immunohistochemical and ultrastructural localiza- tion of proteoglycans were studied in the noto- chord of Rana japonica and Bombina orientalis. The notochordal sheath and the peripheral region containing connective tissue were strongly la- beled with anti-chondroitin-4-sulfate. At the ultrastructural level, we examined the presence of sulfated proteoglycans in the extracellular matrix of notochord using the cationic dye poly- ethyleneimine (PEI) and cationic colloidal gold (CCG). After staining with PEI, the proteogly- Cans appeared as granules in the extracellular matrix of fibroblast, chondroblast, and chondro- cyte. PEI-positive material was removed after treatment of the tissue with chondroitinase ABC. The labeling with CCG at pH 1.0 was also observed on the chondroblast matrix and notochordal sheath. The present observations suggest that PEI-positive structures and CCG labelings repre- sent sulfated proteoglycans aggregates. OBSTRUCTION EFFECTS OF RETINOIC ACID ON METAMORPHIC RECONSTRUCTION OF LOWER JAW OF BROWN FROG, RANA ORNATIVENTRIS. S. Kurabuchi. Dept. of Histol., Sch. of Dentistry, Nippon Dental Univ., Tokyo. Rana ornativentris tadpoles were used in the current study. Retinoic acid (RA), 50, 5 or 0.5 uwg/weight g, dissolved in dimethyl sulfoxide, was injected into the individ- uals one time at the metamorphic crimax stages. When the metamorphosis completed, the gross-structural abnormality of the body was searched. Thus, the RA treatment resulted in the delay of metamophosis and the reduction defects of lower jow. Such malformations were frequently yielded in the group of the RA 50 ug/g injected at the stage XX (Taylor and Kallros, '46). The victria blue-stained whole-mounts for visu- alization of the morphology of cartilages (Bryant and Iten, '74) showed that the hyoid and the mandibla were undersized or partially lost in such malformed lower jaws. Furthermore, when the labeling of 5-bromodeoxyuridine (BrdU) was detected by immunohistochemistry(PAP method), the BrdU- immunoreactive cells remarkably decreased about 6-12 hours after RA-injection especially in the cartilages of the lower jaw. These results suggest that the RA may obstruct the reconstruction of the cartila- genous skeletons from the larval form to the adult form, therefore may cause abnormal morphogenesis. ANAYSIS OF METAMORPHIC CHANGES IN SMALL INTESTINE OF XENOPUS LAEVIS BY MONOCLONAL ANTIBODY T. Sano, A. Kawahara, and M. Amano Cell and Developmental Biology, Faculty of Integrated Arts and Sciences, Hiroshima University, Hiroshima Amphibian metamorphosis is regulated by thyroid hormones and prolactin (PRL). Amphibian small intestinal epithelium changes from larval type to adult one at metamorphic climax stage. To define the role of these hormones, we raised a monoclonal antibody specific to adult epithelium (A5D9 antibody). Fragmented small intestines of larva at st. 56-57 were cultured and treated with T3 and/or PRL. The results showed that A5D9 antibody recognized only an- terior intestinal epithelium which had been treated with T3 for five days. On the other hand, PRL inhibited this T3- dependent production of A5D9 antigen. Next, we examined whether A5D9 antigen could be also induced in vivo by T3 treatment for five days. A5D9 antigen was not able to be detected in this as- say, indicating that T3 activity was suppressed in vivo. The results suggested that the metamorphic transition of intestinal epithelial cells was directly promoted by thyroid hormones, but suppressed by PRL activity. 1204 Developmental Biology DIFFERENTIATION OF CONNECTIVE TISSUE CELLS OF THE XENOPUS SMALL INTESTINE CULTURED IN THE ABSENCE OF EPITHELIUM. A.Ishizuya-Oka and A.Shimozawa. Dept .of Anat.,Dokkyo Univ. Sch. of Med., Tochigi. We have previously reported that cells originating from the larval connective tissue (CT) are involved in metamorphic epithelial changes in the anuran small intestine; an aggregation of fibroblasts rich in rough endoplasmic reticulum (Fr) and macrophages (M@) participate in the adult epithelial development and the lar- val epithelial degeneration, respectively. In the present study, we investigated influences of the epithelium on differen— tiation of Fr and M@. The epithelium was removed by dispase treatment from tissue fragments isolated from the anterior part of the Xenopus small intestine at NF stage 57. Some of them were recombined with the epithelium (CT+E), and the others remained epithelium-free (CT-E). After cultivation in the presence of thyroid hormone, the number of Mgd@ that showed non-specific esterase activity was significantly larger in CT+E than in CT-E. The aggregation of Fr was observed just beneath the epithe- lium in CT+E, but was not observed in CT-E at alle These results indicate that the epithelium influences differentiation of CT cells, which in turn influences epithe- lial changes in the small intestine during anuran metamorphosis. cDNA CLONING OF COLLAGEN OF BULLFROG TADPOLE (Rana catesbeiana) S. Yomori, K. Oofusa and K. Yoshizato Molecular Cell Science Lab., Zool. Inst., Fac. of Sci., Hiroshima Univ., Higashi- hiroshima Collagens are major proteins of extracellular matrices and play critical roles in the process of formation of tissues and organs. The metabolism of collagen is markedly activated in the process of metamorphosis. In this study, the synthesis of collagen during metamorphosis of bullfrog tadpoles was examined by RNA blot analysis and cDNA cloning was performed. Using the human al(I) collagen cDNA and al(II) collagen gene as a probe, RNA blot analysis was performed for measurement of collagen mRNA levels in the tail of tadpoles. Both typel and typeII collagen mRNA levels were reduced by thyroid hormone. In contrast, they were up-regulated by prolactin that delays metamorphosis of frog tadpoles. A cDNA library was constructed from poly(A)* RNA obtained from tail of bullfrog tadpoles. Fifty four positive clones were obtained by plaque hybridization using the human al(1) collagen cDNA as a probe. The insert size of one of these clones named G6A-1 was largest and this clone was subjected to DNA sequencing. CLONING AND CHARACTERIZATION OF cDNA AND GENE FOR THE BULLFROG TADPOLE COLLAGENASE K. OOFUSA, S. YOMORI and K. YOSHIZATO Molecular Cell Science Laboratory, Zoological Institute, Faculty of Science, Hiroshima University, 1-3-1 Kagamiyama Higashi-hiroshima 724 A cDNA clone encoding the entire sequence of bullfrog (Rana catesbeiana) tadpole collagenase was fsolated from cDNA library constructed from the bullfrog tadpole back skin. This clone (Tscl) was similar to both porcine and human fibroblast collagenases; the similarities of deduced amino acid sequence were 78% and 65 %, respectively. RNA blot analyses revealed that the transcripts for collagenase increased in both back skin and tall tissues of thyrold hormone-treated tadpoles. In addition, mRNA for collagenase was decreased in tadpole talls by treatment of prolactin. Genome DNA was isolated from liver and whole blood of adult bullfrog (female) and were subjected to DNA blot analysis using pTSCl as_ probe. A genome DNA library was constructed from partially digested DNA and plaque hybridization was performed with pTSCl. Two positive clones were Isolated from this. library. Both of them carried about 20kbp Inserts and subjected to restriction enzyme mappings. CYCLIC NUCLEOTIDE-DEPENDENT PROTEIN DEPHOS- PHORYLATION IN SPERMATOZOA OF SEA URCHIN HEMICENTROTUS PULCHERRIMUS T.Harumi, K.Hoshino and N.Suzuki. Noto Marine Lab. Kanazawa Univ. Ishikawa. When CHAPS-solubilized sperm proteins were incubated with [7 -*?PJATP, a 48 kDa protein was phosphorylated within 1 min. Addition of cAMP or cGMP induced a rapid dephosphorylation of the **P-phosphorylated 48 kDa protein. Half-maximal effective con- centrations of cAMP and cGMP on the dephos- phorylation were 0.3 » M and 4 yw M, respec- tively. Protein phosphatase inhibitor, calyculin A or okadaic acid inhibited the dephosphorylation. TSKgel G3000SW chromato- graphy of the CHAPS-solubilized sperm proteins containing the *°*P-phosphorylated 48 kDa protein showed that the 48 kDa protein was eluted in the fraction contain- ing proteins whose molecular masses were from 250 kDa to 400 kDa. Cyclic nucleotide- dependent dephosphorylation activity for the *?P-phosphorylated 48 kDa protein was eluted in the fraction containing proteins with molecular masses of over 400 kDa. Photoactivatable cAMP analogue 8-Ns- [°?7P]cAMP was incorporated into the 48 kDa protein. These results suggest that H. pulcherrimus spermatozoa possess cyclic nucleotide-dependent dephosphorylating activity for a *?P-phosphorylated 48 kDa protein and the dephosphorylation occurs upon binding of cyclic nucleotides to the protein which probably exists as a hetero- oligomer. Developmental Biology 1205 CHARACTERIZATION OF TWO DIFFERENT PROTEIN PHOSPHATASES IN SPERM TAILS OF THE SEA UR- CHIN HEMICENTROTUS PULCHERRIMUS. K.Hoshino, T.Harumi and N.Suzuki. Noto Marine Lab. Kanazawa Univ. Ishikawa It is known that receptor for sperm- activating peptide-I (SAP-I) and guanylate cyclase, which is a phosphoprotein, local- ize in the sperm tails of the sea urchin Hemicentrotus pulcherrimus. Upon treatment of the spermatozoa with SAP-I, guanylate cyclase looses its phosphate groups. In the present study, using serial chromatography on DEAE-Sephacel, Sepharose 6B and histone- agarose columns we purified and charac- terized two different protein phosphatases (Type A and B ). Type A enzyme was isolated from the particulate fraction of the sperm tails with a solution containing 1% CHAPS. The molecular weight of the enzyme was es- timated to be 43,000 by gel filteration. An apparent Km for dephosphorylation of 32P- phosphorylated histone (%?P-histone) was 23.4 uw M and an apparent Vmax was 1.8 nmol Pi/min. Okadaic acid inhibited the enzyme activity with IDso of about 5x10-8M. Type B enzyme was isolated from the soluble frac- tion of the sperm tails. The molecular weight of the enzyme was estimated to be over 200,000 by gel filteration. An apppa- rent Km for dephosphorylation of 32P-hist- one was 23.0 uM and an apparent Vmax was 2.2 nmol Pi/min. The activity of type B en- zyme was more sensitive to okadaic acid.(IDso0; 1x10-°M). CLONING OF CDNAS ENCODING GUANYLATE CYCLASE AND SPERM ACTIVATING PEPTIDE-I RECEPTOR EXPRESSED IN HEMICENTROTUS PULCHERRIMUS SPERM T.Shimizu, Y.Sendai, N.Suzuki. Noto Marine Laboratory, Kanazawa University, Ishikawa The H. pulcherrimus testis cDNA library was constructed in Lambda gtl0, using poly(A)+RNA isolated from testes collected in November and December H. pulcherrimus and oligo(dT) primer. As a result the cDNA library containing 4.9x105 reconbinants was produced. For screening cDNAs encoding guanylate cyclase, oligo(dT)-primed cDNA library from H. pulcherrimus testis in Lam- bad gt10 were screened with two custom syn- thesized oligonucleotides (45mer; AACAT- GATCGCCATCATGGAGCGCTACACCAACAATCTGGAGGAG, 45mer ; GAGATTCTGCACGAGGAGTACTACGCGGGATTCGATC CATGGGAC). Six clones were isolated with two oligonucleotides. The longest clone had an insert of 4171bp containing a poly(A)+ tail and open reading frame encoding 1125 amino acids. For screening cDNAs encoding sperm activating peptide-I(SAP-I) receptor, oligo(dT)-primed cDNA library from H. pul- cherrimus testis in Lambda gt10 were screened with custom synthesized oligo- nuclotide (45mer;GGCATAGTCGTACGCACAGGG ATGTGGGTATTGACCCTGCAACCA). Forty clones were isolated with an oligonuclotide. The major clone had an insert of 2443bp con- taining a poly(A)+ tail and open reading frame encoding 532 amino acids. CHARACTERIZATION OF WHEAT GERM AGGLUTININ- BINDING PROTEIN IN SPERM TAILS OF THE SEA URCHIN HEMICENTROTUS PULCHERRIMUS Y.Sato, T.Shimizu, Y.Sendai and N.Suzuki, Noto Marine Lab. Kanazawa Univ. Ishikawa A 220 kDa wheat germ agglutinin (WGA) - binding protein was isolated from 0.1%Lubrol-solubilized fraction obtained from sperm tails of the sea urchin Hemicentrotus pulcherrimus by chromatog- raphy on a WGA-Sepharose column and preparative SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The protein was digested with protease V8. The resulting protein fragments were purified by SDS-PAGE and the partial sequence of a fragment (VD2, 29kDa) was determined to be -Val-Ser- Ser-Ile-Asp-Asn-Ile-Phe-Arg-Val-. The sequence which was designated by the under- line was identical to the conserved sequence of H*-ATPase beta chain from various sources including plants and animals. A mixed antisense oligonucleotide probe [5*’ GACACGGAAGATGTTGTCGATGCT (or AGA)GCT(or GGA)GAC 3’] which corresponds to the partial sequence was synthesized and used for screening of 6x104 clones of a cDNA library from H. pulcherrimus testes in 2 gt10. A cDNA (2.3kb) was isolated and the nucleotide sequence was determined to have 2264 bases. An open reading frame predicts a protein of 523 amino acids which is 85% identical to rat H*-ATPase beta chain. CLONING OF cDNAs ENCODING THE PUTATIVE PRECURSORS FOR SPERM-ACTIVATING PEPTIDE (SAPs). H.Kinoh!, H.Fujimoto? and N.Suzuki!. ‘!Noto Marine Lab. Kanazawa Univ. Ishikawa, ?Dept. Orr Hol, Celi, Oi ee ke Sn, Winsiyo Ese Tokyo, Tokyo. The egg jelly is composed mainly of two large acidic glycoproteins, a sialoglyco- protein and a fucose sulfate glycoconjugate (FSG). In the previous study, we demonstrated that the polycolnal antibody against FSG reacted with the accessary cells and surface region of the oocytes in the ovarian lumen. In the present study, to investigate the origin of SAP-I and its derivatives by in situ hybridization, we cloned a cDNA en- coding a putative precursor for SAP-I and its derivatives in the sea urchin Hemicentrotus pulcherrimus . About 3x104 clones of cDNA library from H. pulcher- rimus ovary in A gtll was screened with a 32P random primed cDNA encoding for a sperm-activating peptide precursor of Strongylocentrotus purpuratus. Fourteen clones were isolated, and the largest clone had a 2.0kb insert, the remaining 13 clones had a 1.3kb insert. Northern blotting analysis demonstrated that the mRNA for the 1.3kb precursor was detected during whole breeding season (from Nov. to Apri.). However, the mRNA for the 2.0kb precursor was detected only on January. 1206 Developmental Biology PARTIAL PURIFICATION OF SPERM-ACTIVATING AND SPERM-ATTRACTING SUBSTANCE IN THE ASCIDIAN, CIONA INTESTINALIS. M.Yoshida, K.Inaba, and M.Morisawa. Misaki Marine Biological Station, Fac. of Sci., Univ. of Tokyo, Kanagawa. Spermatozoa of the ascidian, Ciona intes- tinalis, exhibit chemotactic behavior to eggs prior to fertilization (Miller,1975; Yoshida and Morisawa, 1990). Here we tried to purify sperm-activating and sperm- attracting substance from unfertilized eggs. The eggs were suspended and incubated in artificial seawater for about 12 hr, and supernatant obtained by brief centrifuge (egg seawater) which exhibited strong sperm- activating and sperm-attracting activies was used as a starting material. The egg seawater was lyophilized, then extracted by absolute ethanol. After evaporation of ethanol, the extract was applied successively to reversed-phase (Sep-Pak C18), gel filtra- tion (Bio Gel P-4), and cation exchange (CM-Sephadex C-25) column chromatographies. During these purification process, specific activity of sperm activation and attraction increased and both activities are always co-migrated. These suggest that sperm-— activating and sperm-attracting substance is partially purified and both activities are derived from the same molecule. Nature of the substance is acidic, and molecular weight of this molecule was estimated by gel filtration as 2 ~ 2.2 kDa. PURIFICATION OF SPERM-ACTIVATING PROTEINS FROM UNFERTILIZED HERRING EGG. II. S. Odat, H. Ohtake, yY. Igarashi®, K. Sakai‘, Y. Shimizu? and M. Morisawa?. imMisaki Marine Biol. Stat., Fac. of S@ilo 5 Univ. of Tokyo. Kanagawa. Dept. of Physi- ol. and “Dept. of Biochem., Dokkyo Univ. Sch. Med., Tochigi. * Dept. of Mol.Biol., Keio Univ. Sch. Med., Tokyo. Unfertilized eggs of the pacific her- ring, Clupea palasii, release proteins which activate motility of herring sperma- tozoa. We have previously reported the purification of herring sperm activating proteins (HSAPs) and revealed that HSAPs are small acidic proteins on SDS-PAGE (Ohtake et al., 1990). To analyze the molecular natures of HSAPs, the HSAPs obtained from isoelectric focusing column were further analyzed by immobilized pH gradient gel electrophoresis. HSAPs are at least 5 proteins which pI values are 4.8, 4.9, 5.0, 5.1 and 5.4, respectively. The MW of all HSAPs purified on immobi- lized pH gradient gel electrophoresis are almost equal and are estimated less than 7 KD on SDS-PAGE. Gel filtration estimates the MW of HSAPs about 14 KD, which value is twice the MW obtained on SDS-PAGE. These suggest that HSAPs exist as dimers under the physilogical conditions. SDS- PAGE also revealed that HSAPs are composed of at least two peptides having different MW. Role of Ca2*-channel in activation of Xenopus egg by sperm extract. Y. Iwao, N. Se, and S. Jikumaru. Biol. Inst., Fae. Sci., Yamaguchi University, Yamaguchi. Potential changes as well as voltage- dependence on fertilization of amphibians are characteristics of sperm species, so that we attempted to obtain and characterize a sperm factor to induce egg activation. The extract obtained from Cynops pyrrhogaster sperm induced activation of dejellied, unfertilized Xenopus laevis eggs. The eggs treated with the extract elicited a short-lived, positive-going potential after appearance of a deep hyperpolarization. The pattern of potential changes induced by the sperm extract was quite similar to that by Cynops sperm, but not by homologous sperm. Immature oocytes or fertilized eggs show no potential changes upon treatment with the extract. When the unfertilized eggs were treated with the extract in 340 pM Ca“, the onset of cortical contraction and the positive-going potential was accelerated. The activation was inhibited by voltage=clanping at higher than -10 my in 34 pM Ca” , or -20 mV in 340 uM Mg , respectively. The activation) was not inhibited at +20 mV in 340 pM Ca These results indicate that the activation by the Spey extract is mediated by opening of Ca channels on egg plasma membrane. HEPARIN INHIBITS CALCIUM TRANSIENTS IN FERTILIZED SEA URCHIN EGGS INDUCED BY SPERM AND ITS SOLUBLE EXTRACTS. M.Osawa ,_H.Uchiyama~, H.kysudas, N.Kaneko and H.Kuroda“., Sugashima -B.L.,Sch of Sci., Nagoya Univ. Toba, Dept. of Biol., Fac of Scaly toyamd Univ., Toyama. Fertilization is known to initiate a transient increase of intracellular calci- um concentration (Ca;-transient) and an accompanying change of membrane potential in sea urchin eggs. Using aequorine, we previously showed that sperm and its soluble extract caused a Ca,-transient in fertilized eggs from which fertilization membranes and the hyaline layers were removed. In this report, we quantitatively meas- ured intracellular Ca concentration with Indo-1 microfluorometry and showed that the microinjection of heparin (final concentration of approx. 1mg/ml) into fertilized eggs inhibited the Ca;- and voltage- transients by sperm and sperm extract. The sperm extract which was diluted to 10-fold with artificial sea water (ASW) increased the intracellular Ca*+ concentration from 240nM to 760nM (in average values). The sperm extract which diluted to 1000-fold lost activity. The molecular weight of the active factor in the sperm extract was less than 5000. Developmental Biology 1207 TPA-SENSITIVE PROTEIN PHOSPHORYLATION INTERFERES WITH CORTICAL REACTION AND INDUCES CYTOSKELETAL CHANGES IN THE SEA URCHIN EGG. A. Morimatsu*, H. Murofushi* and I. Mabuchi*. *Dept.,of Biophys. and Biochem., Fac. of Sci., Univ. of Tokyo, Tokyo.,and Department of Biology, Fac. of Arts and Sciences, Univ. of Tokyo, Tokyo. When unfertilized sea urchin eggs were treated with TPA and then inseminated, no cortical reaction occurred and no fertilization membranes (FMs) appeared. Also calcium ionophore A23187 did not induce the formation of the FMs in the TPA treated eggs. These effects were completely abolished by a C-kinase inhibitor H7. Furthermore, calyculin A-treated unfertilized eggs did not form the FMs upon addition of A23187. Numerous bundles of actin filaments were observed in the TPA-treated eggs. Next, we added 200nM Ca** to cortices isolated from unfertilized eggs. The cortical reaction did not occur in those isolated from the TPA treated eggs, but it did in the control COrtices and the FMs were formed. When the TPA-treated eggs were inseminated, they became polyspermy. They began to divide irregularly at 120-150min after insemination to produce 4 to 17 cells. At this stage some eggs did not divide but began to move like amoebae (Mabuchi, Morimatsu & Tosuji, this meeting). These results indicate that some DG-dependent kinases are involved in the cortical reaction and cytoskeletal organization in the sea urchin eggs. CHANGES IN INTRACELLULAR FREE CALCIUM AT FERTILIZATION IN MACTRA CHINENSIS OOCYTES. R. Deguchi and K. Osanai. Mar. Biol. Stn., Asamushi, Tohoku Univ., Aomori. Oocytes of Mactra chinensis (Mollusca, Pelecypoda) are spawned at prophase-| and resume meiosis by fertilization. The prophase-| arrested oocytes (GV- oocytes) were injected with fluorescent Ca-indicator fura-2 to measure changes in intracellular free Ca™ (Ca?*i) at fertilization. Shortly after insemination, Cai increased rapidly and decreased within 2 min. After that, Ca*i was retained higher than the resting level (post- fertilization level) for a while and returned to the resting level about 10 min following fertilization. Germinal vesicle breakdown (GVBD) occurred 12-15 min after fertilization. External Ca* was required to maintain the post-fertilization level because Ca**i returned to the resting level immediately after replacing external medium with Ca-free artificial seawater containing 10 mM EGTA. In order to induce GVBD, Ca?*i had to be kept above the post- fertilization level for more than 4-5 min. Oocytes of another bivalve Limaria hakodatensis didn't require Ca™i increase to undergo GVBD. In Limaria oocytes, Ca*i level was higher than the post-fertilization level in Mactra oocytes. It is probable that Ca?*i level above a threshold is prerequisite for GVBD in bivalve oocytes. ULTRASTRUCTURAL STUDIES ON A TISSUE- SPECIFIC DIFFERENTIATION OF MELANOPHORES IN AN ORANGE-COLORED VARIANT OF MEDAKA FISH, ORYZIAS LATIPES. Eu. Hirose and J. Matsumoto. Department of Biology, Keio University, Yokohama In an orange-colored variant of the medaka fish Oryzias latipes which inherently devoid of the dominant gene B, melanophores exhibit a tissue-specific differentiation, as exemplified with failure of melanogenesis in the skin , an incomplete or deteriorated melanogenesis in the choroid and the peritoneum and formation of a mosaicism like melano-iridophores in the peritoneum. Ina wild strain of this species carrying B gene, all melano- phores are terminally differentiated irrespective of the tissues they are located. This indicates that the deficiency of B gene impairs the differentiation of melanophores in the medaka. Electron microscopy disclosed that the failure or deterioration of melanogenesis in fish deficient in B gene occur inside the melanosomes and that the manner by which deterioration give rise to is different between melanophores in the skin and the choroid or the peritoneum. The ubiquitous occurrence of reflecting platelet-laden melanophores in the peritoneum of this variant and the total absence of a mosaicism in pigment cells of the wild strain indicate that the deficiency of B gene imposes melanoblasts distributed in this tissue to an ambiguous state with regard to their differentiation. Little difference is observed with regard to melanosome maturation in pigment epithelial cells between orange- colored variant and the wild strain, indicating an silent role of the B gene in their differentiation. THE EXPRESSION OF CELL ADHESION MOLECULES IN PIGMENT CELLS. fe Fukuzawa and M. Obika, Dept. of Biology, Keio Univ., Yokohama, Japan To study the molecular mechanisms of pigment pattern formation, we examined the expression of cell adhesion molecules(CAMs) in pigment cells, since little is known about the cell-to-cell communication in pigment cells. Three types of pigment cells (melanophores, xanthophores, and iridophores) were cultured from the medaka, Oryzias latipes, and the expression of N-CAM and N-Cadherin was examined by immunocytochemistry. The results showed that both N-CAM and N- Cadherin were expressed in xanthophores, but not in melanophores and iridophores. N-CAM staining was observed in the tip or the base of the dendrite, or in the edge in-between the dendrites of xanthophores as a spot. If the shape of xanthophores is round in the presence of fibronectin and collagen type I, N-CAM was detected in the edge as a short line, or in the peripheral area as a half-ring whose center lay on the edge. The staining pattern of N- Cadherin in xanthophores was almost the same as that of N-CAM, however, the staining area of N-Cadherin was broader than that of N-CAM. Furthermore, the number of stained area of N-Cadherin was higher than that of N-CAM. In our knowledge, this is the first demonstration of CAMs in pigment cells. 1208 Developmental Biology DETERMINATION OF GENOTYPES IN QUAIL PLUM- AGE MUTANT (BLACK AT ATCH, Bh) MBRYOS . Y. Kubotg’, N; Shiojiri', T. Kaneko” and A. Nakamura“. Dept. Biol., Fac. Sci., Shi- zuoka Univ., Shizuoka. Dept. Biol., Hamamatsu Coll., Univ. Shizuoka, Hamamatsu. The Bh homozygotes die during early development (3-7 days), showing subcutane- ous haemorrhage, and degeneration of liver and neural tube tissues (Minezawa & Wakasu-— gi, 1977). No reports have been done on determination of genotypes of Bh embryos before feather germ development. We report the way of determining the genotypes of young Bh embryos with chorioallantoic membrane (CAM) grafting of the skin. Embryos obtained by the cross of Bh/+ x Bh/+ quails were used. The 7-day thigh skin fragments in which no feather germs developed yet were grafted onto the CAM of chick embryos, and cultured for 9 days. The grafts developed feather germs with black and yellow stripes, grey feather germs, and brown feather germs. The ratio of the grafts developing the striped, grey and brown feather germs was 1:2:1. The results suggest that the grafts of the striped, grey, and brown feather germs are derived from +/+, Bh/+ and Bh/Bh embryos, respectively. A few embryos with brown feather germs and subcutaneous haemorrhage which might be homozygous were observed at 10 days, supporting the above idea. In the 7-day Bh/ Bh embryos, subcutaneous haemorrhage occurred, but liver and neural tube degeneration was not always seen. IN VITRO CLONAL ANALYSIS OF MOUSE NEURAL CREST DEVELOPMENT. K.Ito!, T.Morita!, and M.Sieber-Blum?. Honea, one ihloit,, (ili, Cn’ (en, Jachoe. . Osaka Univ., Osaka, Dept. of Cell Biol. and Anat., Med. Coll. of Wisconsin, USA. We established a clonal culture system of mouse neural crest cells in order to analyze developmental potentials of these cells. Three morphologically distinct types of ‘clones were observed. (1) "Pig- mented clones" consisted of melanocytes only, suggesting that the clone-forming cells were committed to the melanogenic lineage. (2) "Mixed clones" consisted of pigmented and unpigmented cells. The clones contained up to four types of cells; melanocytes, S100-positive cells (Schwann cells or melanogenic precursor cells), serotonin (5-HT)-positive auto- nomic neuron-like cells, and substance P (SP)-immunoreactive sensory neuron-like cells. Thus, at least some mixed clone- forming cells are pluripotent. (3) Un pigmented clones" consisted of unpigmented cells only. The clones contained up to three types of cells; S100-positive cells, 5-HT-positive cells, and SP-immunoreactive cells, Suggesting that some precursor cells of these clones have partially restricted developmental potentials. These results indicate that the mouse trunk neural crest is a heterogeneous population of cells with different types of developmental potentials. MELANOCYTE DIFFERENTIATION AND C-KIT EXPRES-— SION IN CULTURED MQUSE NEURAL EST CELLS. H.Ono?, _ Y.Kawa*, M.Sato*, T1.Baba2, .Kubota2, M.MizoguchiY~ and T.Takeuchil. Biol. Inst., Fac. of Sci., Tohoku Univ. 2Dept. Dermatol., St. Marianna Univ. Sch. of Med. The signal transduction system by c-kit and SLF (Steel factor) affects development of melanocytes. In this study, we attempted to detect the c-kit expression in neural crest cells (NCCs) by monoclonal antibody, ACK2, and compare it with the pattern of melano- cyte differentiation in vitro. NCCSs were induced to differentiate into mature melano- cytes by addition of 10 ng/ml PMA (phorbol 12-myristate 13-acetate) into the culture medium. Further addition of 10 nM cholera toxin resulted in an increase in the number of melanocytes. Melanocyte differentiation seems to be correlated with the c-kit ex- pression in NCCs. The c-kit positive cells were more than 1000 cells/explant when treated with PMA and cholera toxin, and about 500 cells with PMA only by 15 days in culture. The number was less than 100 cells without PMA. (Total number of NCCs were not affected by the culture media.) We assume that PMA induces c-kit expression and that cholera toxin supports this action. Subse- quently, the c-kit/SLF signal transduction system accelerates the differentiation into melanocytes. The c-kit positive NCCs formed clusters in explant at 9 days in culture, this indicates that NCCs are heterogeneous and that some of NCCs have lost ability of c-kit expression by this stage. KERATINOCYTES REGULATE THE DEVELOPMENTAL CHANGES OF THE PROLIFERATIVE ACTIVITY OF MOUSE EPIDERMAL MELANOCYTES IN SERUM-FREE CULTURE. . T. Hirobe. Div. of Biol., Natl. ainsteusot Radwvols (Scie, Chibas The proliferative activity of mouse epidermal melanocytes is known to change during development. In order to make clear the control mechanism of melanocyte proli- feration, epidermal cell suspensions from mice of various ages were cultured with serum-free medium containing dibutyryl cyclic AMP and basic fibroblast growth factor. When the epidermal cell suspensions of dorsal skins of 0.5-, 2.5- and 4.5-day- old C57BL/10J mice were cultured on poly- styrene dish, melanoblasts proliferated well around keratinocyte colonies. In contrast, when the epidermal cell suspen- sions of 7.5-, 20.5- and 60.5-day-old mice were cultured similarly, keratinocytes failed to attachto the dish and melanoblasts did not proliferate. However, when the epidermal cell suspensions of 7.5-, 20.5- and 60.5-day-old mice were cultured on Type I collagen-coated dish, keratinocytes attached to the dish and melanoblasts pro- leferated around the keratinocyte colonies. These results suggest that keratinocytes are involved in regulating the prolifera- tion of mouse epidermal melanocytes and that the developmental changes in the pro- liferative activity of epidermal melano- cytes are caused by the changes in the cell adhesion property of keratinocytes. Developmental Biology 1209 CLEAVAGE ASYNCHRONY IN THE TUBIFEX EMBRYO: INVOLVEMENT OF CYTOPLASMIC AND NUCLEAR FACTORS fT. Shimizu, Zool. Inst., Fac. of Sci., Hokkaido University, Sapporo 060 AB cell of the 2-cell embryo of Tubifex divides 40 min later than CD cell. A previous study has shown that AB cell enters M-phase 5 min later, and spend in prometaphase portion of M-phase about 35 min longer than CD cell. In the present study, nuclear behavior was examined in embryos where first cleavage furrows were regressed or blastomeres were electrically fused. When AB and CD nuclei were brought into a common cytoplasm, they entered M- phase simultaneously. The durations of M- phase portions from prometaphase through telophase were not altered even in a common cytoplasm, as long as mitotic figures of AB and CD nuclei were separated from each other. Only when the mitotic spindles of both nuclei were united at poles, the length of prometaphase of AB nucleus was significantly shortened, and phases of the chromosome cycle became synchronous. Mitotic spindles are morphologically distinct between AB and CD cells of intact embryos; morphology of the spindle was not altered by changes in cytoplasmic environment. These results suggest that cleavage asynchrony in the Tubifex embryo is brought about not only by cytoplasmic components but also by nucleus-associated factors. PRODUCTION OF GERM LINE CHIMERAS IN MEDAKA. Y.Wakamatsu', M.Kinoshita?, H.Toyohara?, M.Sakaguchi?, T.Iwamatsu*, Y.Taguchi‘, H. Tomita®, K.Ozato'. 'Fac. of Lib. Arts and Sci., and ?Fac. of Agri., Kyoto Univ., Kyoto, *Dept. of Biol., Aichi Univ. of Edu., Kariya, ‘Inst. of Radiol. Sci., Chiba, ®*Fac.of Sci., Nagoya Univ., Nagoya. To produce germ line chimeras in medaka, cells of morula or blastula embryos of an inbred strain of wild type (HNI-I) were taken out and injected into about 200 embryos at the same stages of inbred (HO5) and outbred strains of an Orange-red variety and two strains of albinos. In some experiments, dechori- Oonated embryos were used. After injection, embryos were cultured and bred at 26°C. Black-pigmented cells developed in yolk sacks, pigment epithelia of eyes, skins, fins, and peritonea. Until now, 26 orange- red individuals have sexually matured. They were mated with the same strain. About 10% of F, progeny from a male chimera exhibited the wild type body color. Isozyme analyses showed that these wild type F, were hybrids of the donor and recipient strains. When the wild type F, were backcrossed to the recipient strain, phenotypes in the body color in BC, segregated into the wild type and orange-red at a 1:1 ratio. MORPHOGENESIS OF MEDAKA EMBRYO (FORMA- TION OF EMBRYONIC AXIS) Y. Ebina and S.Yokoya Div. Cell Sci. Fukushima Med. Col. Fukushima In the medaka embryo, shortly after gas- trulation begins, cell convergence occurs and the embryonic axis appears at one side of blastodisk (the dorsal side) where the embryonic shield is formed. The mechanisms involved in the formation of the embryonic axis in the teleost are not well under- stood. We examined the cell morphology in the prospective embryonic shield region in the medaka embryo during epiboly and gastrula- tion with scanning electron microscopy. SEM revealed that the marginal cells of the enveloping layer in the prospective embryo- nic shield region at late blastula are different in shape and in contact relation with inner yolk syncytial layer from the Marginal cells in the other region. These differences may cause cell conver- gence. 1210 Genetics KARYOTYPES AND BANDING PATTERNS IN EIGHT SPECIES OF THE SCORPLONFISH(SCORPAENIDAE). T. Yokoyama’, N.Ebitanivand T.Kubo!. Biol. Lab., Sophia Univ., Tokyo and *Biol. Lab., Shohoku Coll., Atsugi. Somatic chromosomes were prepared from the cultured cells derived from fins of fishes. The chromosome numbers in eight species of scorpionfishes(Helicolenus hilgendorfi, Scorpaena neglecta miostoma, S.izensis, S,neglecta neglecta, Sebastes inermis, S.matsubarae, Sebastiscus albo- fasciatus and S.marmoratus) were all 48. Karyotypes in these species, in the order of the species mentioned above, were as follows: 2M+2SM+44A, 8M+16SM+34ST, 8M+24ST +16A, 6M+2SM+30ST+10A, 2M+46A, 2M+46A, 4M +2ST+42A and 2M+46A. One pair of AgNORs were found on short arms of metacentrics in the three species of genus Scorpaena and on acrocentrics in S.matsubarae and S.albofasciatus. In the other three spe- cies, two pairs of silver-stained bands were revealed on two pairs of acrocentrics. C-bands were found on one pair of subtelo- centrics in the fishes of genus Scorpaena. In the other five species, they were found on more than eight pairs of acrocentrics. Based on these findings,relation between the characteristics of chromosomes and diversification of species in the scorpion- fishes was discussed. CELL CULTURE AND CHROMOSOME ANALYSES OF XENOPUS BOREALIS. K. Sekiya Dept. of Biol., Fac. of Sci., Niigata Univ., Niigata Cell nuclei of Xenopus leavis are stained homogeneously by fluorescent dye quinacrine, whereas those of X. borealis, which are able to get interspecies hybrids with xX. 1., are stained with about 15 bright fluorecent spots. As a result of staining cell nuclei of X.b. by quinacrine mustard, acridine orange and DAPI, bright spots could be observed in the various tissues. Especially DAPI and quinacrine mustard staining is clearly visible. X. b. whole embryos (st. 44) were used for setting up the culture, and in the primary culture, the surface of the bottle was covered with some different-type cells. The floating cells, selected by light pipetting, could be kept more than 3 months. In the cells attached to the surface, their fluorescent spots tend to become unclear and decrease, but the spots are kept even in the 15th subculture. The chromosome of both species is Similar, but their different type of sat- chromosome could be confirmed in silver staining. The results obtained in these cell culture will be very useful for interspecies cell fusion. ANALYSIS OF MALE CHROMOSOMES FROM THE HYBRID ANDROMEROGONES OBTAINED BY USING CRYOPRESERVED SPERM OF SEA URCHINS. K. Saotome!, R.Kamimura2, S.Kurokura? and R. Hirano. !Yokohama City Institute of Health, Yokohama, 2Kitazato Univ., Iwate and 3Univ. of Tokyo, Shizuoka. Analysis of male chromosomes is pos- sible by the use of hybrid andromerogones obtained by inseminating non-nucleate egg fragments of Hemicentrotus pulcherrimus with sperm of different species (Saotome, 1991). This system, however, was not applicable to the species having different breeding season from that of_H. pulcherrimus. Cryopreserved sperm was used to dissolve this problem. The freeze-thaw procedure of sperm was carried out as follows:1l)dry sperm was diluted with sea water containing 10% DMSO at the con- centration of 2-10%, 2) the suspension was pipetted into plastic insemination straws, heat sealed and frozen gradually to -196°C, 3) straws were stored in liquid nitrogen and 4) frozen samples were thawed rapidly by immersing the straws in sea water at room temperature. The sperm of five speceis was used;Strongylocentrotus nudus, Pseudo centrotus depressus, Strongylocentrotus intermedius, Glyptocidaris crenularis and H. pulcherrimus. Active swimming of sperma— tozoa was observed after thawing. The rate of cleavage varied from 6 to 84% according to species or batch of egg fragments and cryopreserved sperm. Chromosome numbers were haploid of those already reported in five species and karyotype of their chromosomes could be analyzed. CHROMOSOME MOVEMENT DURING FIRST POLAR BODY FORMATION IN THE ISOLATED MEIOTIC APPARATUS OF TUBIFEX HATTAI (OLIGOCHAETA, TUBIFICIDAE) M-. Matsumoto. pept. of RBiol., Fac. of sci., yamagata yniv., yamagata yolky eggs of the freshwater oligochaete j~. nattai were freed from their cocoons in Lehmannts solution. The vitelline mem- brane and egg cortex of the fresh eggs were ruptured and yolk granules were dis- persed in a petri dish containing 2y glycerol. The opaque core of the egg gradually emerging within 2-3 minutes of the dispersion was transferred to isolation medium. These meiotic apparatus were glued on a slide glass, and stained. puring anaphase J, formation of the cleavage furrow of tne polar body began during migration of the half-bivalent into the polar body when the temperature was changed. Furthermore, the separation of the first polar body from the egg was completed before the termination of chro- mosome segregation. That is, some cnhromo- somes that should have migrated into the polar body remained in the egg cell. These phenomena were found 34 of the cells with a temperature shift of 20¢—+26¢, but 294% with a shift of 26¢—23%. when meiotic apparatus were taken from eggs with shifts of 19¢—922% and 22¢-+17%, we found a complete polar body. Genetics EFFECTS OF MICROWAVE ON GERM CELLS OF DROSOPHILA. K.Kimura-. tokyo Hokkaido Y.Tonomura? and Womans ' Christ. Univ. and Inst. Tech. The Study aimed at finding the effects on germ cells. 1) Specimens used were 72-hour old D.melanogaster, late third-instar larvae, young pupae and late pupae with microwave appli- cation time of 5 seconds, 10 seconds and 30 seconds using microwave of 2.45 GHz at 200W, to see the mechanism of cell division and the protein synthesis of adult flies. 2)In this study, cytological specimens were made by air-dry method and the primary electro- phoresis method were used for the bio- logical analysis of protein synthesis of sibling cells. 3)We are currently conducting an observation using more than 690 microphotograph of germ cells of 20 females and 20 males taken from each group exposed to microwave at varied duration (excluding late pupae) to see whether or not cell division has taken place. An electrophoresis study disclosed no differences in the effects of microwave irradiation on larvae while some differences were observed on pupae, which may suggest the loss of protein bands in the molecular-weight region of 100,000. A GROUP OF CAFFEINE HYPER-SENSITIVE MUTANTS IS HIGHLY MUTABLE. T.Kusano!, Y.Wataya2, K.Okada3 and H, Shinkawa4. ‘Hiroshima women's Univ. 20kayama Univ. Hiroshima Univ. Hospital and 4Hiroshima Univ.of Economics. Caffeine is a clastogen but not likely mutagen to mammalian cells in culture. To catch any clues of much unknown effects of caffeine to cells,we isolated caffeine sensitive'cellfrom mouse FM3A cell line. So far, we could identified two groups. First group which Fcaf22B (C2)belongs to had not shown hyper-mutability. In second group Feaf(NQ@) 1 (NQA), which was selected in diluted caffeine showed hyper-mutability and hyper-sensitivity to bromodeoxuridine. FBrdUrd 1 (A2) was another caffeine sensi- tive mutant because this was selected first as a bromodeoxyuridine sensitive mutant. A2 mutant too found hyper mutable,but hyp- er sensitivity decreased in the presence of deoxycytidine. These results caused douts if caffeine could have potential mutagenic activity as bromodeoxyuridine has. Hyper mutability of both cell lines was seen frequently but mutation rate was not likely to differ from parent FM3A . dNTP pool size were mesured and in NQA cell an unbalnce was observed. Unbalnce of dNTP could occur frequently and spontaneou fre- quent mutation in NQA and A2 mutants might corelate to this phenomenon. Caffeine is still an atractive mutagen. 1211 POPULATION STRUCTURE OF ALLELES FOR THE PLASMODIUM FALCIPARUM MEROZOITE SURFACE PROTEIN. So Jongwutiwes’, f° Nakazawal, H. Kanbara and K. Tanabe~. Div. of Protozool., Inst. of Trop, Med., Nagasaki Univ., Nagasaki and “Lab. of Biol., Osaka Inst. of Technol., Osaka. The precursor to the P. falciparum major merozoite surface protein (MSP1) is one of the potential malaria vaccine can- didates. Our previous studies have demonstrated that the MSPl gene of laboratory strains of P. falciparum ex- hibits allelic variation. Here, we ex- amined the extent of the allelic variation in natural isolates obtained from an en- demic area of malaria by Southern blot hybridization. DNA fragment probes and oligonucleotide probes were derived from variable blocks of two allelic forms of the MSPl gene. Hybridizations against genomic DNA of 18 isolates obtained from Mae Sod district in Thailand revealed the existence of 7 MSPl alleles, 2 of which were newly identified. Coexistence of different alleles in individuals was not rare. It was also noted that the 7 al- leles did not occur at the same frequency, but rather several alleles predominated in the population of P. falciparum in the malaria field analysed. 1 DISCRIMINATION AMONG APHID CLONES BY ARBITRARILY PRIMED POLYMERASE CHAIN REACTION (AP-PCR). T. Fukatsu and H. Ishikawa. Zool. Inst., Fac. Sci., Tokyo Univ., Tokyo. When genomic DNA is amplified by PCR with a primer of an aibitrary sequence in low stringency and electrophoresed, ladder—pattern specific to the genome used will be obtained. This method, AP—PCR, seems to be superior to the conventional DNA fingerprinting in the following aspects; 1) universally applicable to any organism's genomic DNA, 2) only very small amount of template DNA is needed and 3) can be done without blotting or hybridization procedures. We applied this method to discrimination among aphid clones. We adopted a 15mer primer, S'atgcaggaptcgcat3', in this study. When DNAs from various organisms such as aphids, moth, fish and human, were amplified with this primer, species—specific ladder patterns were ob— tained in all of them. To establish optimal reaction condition and to confirm reproducibility, the genomic DNA from a laboratory—maintained clone of Acyrthosi— phon pisum was subjected to AP=PCR. Through a wide range of template DNA amount, from ae 800ng, identical ladder—patterns were obtained. he patterns were also identical irrespective of aphid's instar. The patterns did not change through five: parthenogenetic generations. 2 : p Ceratovacuna nekoashi is an aphid which forms banana-like galls on storax. A gall is composed of more than 10 subgalls, and all the insects in a gall are parthe— nogenetic offsprings of a single fundatrix. When AP— PCR was applied to this aphid species from several galls on the same storax twig, the epraules were, as expected, identical between subgalls of the single gall but distin— gushable between different galls. 1212 MACRONUCLEAR ANLAGEN INDUCES PROLONGED BACKWARD SWIMMING IN NONEXCITABLE MUTANTS OF PARAMECIUM, CNRS, JUST AFTER TRANSPLANTATION. N. Haga. Dept. of Biotech., Senshu Univ. of Ishinomaki, Ishinomaki. Paramecium has two types of nuclei, micro- and macronucleus, in the cell. These nuclei are same in developmental origin but different in size, DNA content and function. During macronuclear development, reorganization of chromosomal DNA including gene amplifications and rearrangements has been done. However the molecular mechanisms of nuclear differentiation is still unknown. | found that when macronuclear anlagen was transplanted into a vegetative cell of wild type in behavior, the recipient cell showed backward swimming for about 60 sec in the K-Dryl's solution. Physiological and electrophysiological studies have demonstrated that ciliary reversal is induced by the increase of intracellular calcium ion concentration. Since the control injection of vegetative macronucleus into a wild type cell induces short backward swimming for about 10 sec, nonexcitable mutants, CNRs, were used as recipients. Both cnrA and cnrB cells showed backward swimming for 50-60 sec when macronuclear anlagen was injected but did not swim to back when vegetative macronucleus was injected. These results suggested at least two possibilities, one is that Calcium ion is enriched in macronuclear anlagen and the other is anlagen induces the increase in intracellular Calcium ion. PHENOTIPIC CHANGE IN SYNGEN SPECIFICITY OF MATING TYPE BY INJECTION OF MACRONUCLEAR NUCLEOPLASM IN Paramecium caudatum. M. Hori and M. Takahashi. Inst. Biol. Sci., Univ. of Tsukuba, Ibaraki Paramecium caudatum has sixteen sub-groups called 'syngen' which consists of complementary mating types (E and O mating types). From cross breeding analysis, Tsukii(1985) showed that syngenic specificity are controlled by three loci: E type is controlled by codominant alleles at the Mt locus [e.g. E type of syngen 3 (E°) is controlled by Mt*] , O type by MA and MB. To get more information about the relationship between syngens, transplantation of nucleoplasm of macronucleus was performed between two syngens by microinjection using the cells immobilized by partial decilliation with 5% ethanol. In intra-syngen transplantation, recessive phenotypes in behavior and exocytosis (cnrB and tnd2 ) were rescued by the nucleoplasm of wild type and O mating type also was turned to E type by injection of E type nucleoplasm. On the other hand, in inter-syngen transplantation between syngen 3 and syngen 12, although both cnrBand tnd2 were rescued by dominant genes over syngen, the mating types, however, changed their syngen specificity. That is, when nucleoplasm of E'? was injected into macronucleus of O°, 11 out of 43 clones established expressed E” in spite of containing no Mt° allele. It was also true in the reciprocal combination, E® to O'*. This result suggests that syngenic specificity of Mt gene converted that of donor into recipient by inter-syngen transplantation. Genetics A MICRONUCLEUS-SPECIFIC SEQUENCE EXISTS IN 5’-UPSTREAM REGION OF CALMODULIN GENE IN TETRAHYMENA THERMOPHILA. M.Katoh, M.Hirono!, T.Takemasa, M.Kimura and Y.Watanabe. Inst. of Biol. Sci., Univ. of Tsukuba, Tsukuba and !Cell Bank, Inst. of Phys. and Chem. Res. (RIKEN), Tsukuba. To know relationships between DNA rearrangement and switching of gene ex- pression during macronuclear development in Tetrahymena thermophila, we analyzed micro- and macronuclear genomic DNAs for calmodulin gene by Southern hybridization and sequencing. Here we presented evidence that a 1.4 kb micronucleus (mic)- specific sequence existed about 3.5 kb upstream from the initiation codon of calmodulin gene, suggesting the occurrence of DNA rearrangement. The mic-specific sequence was highly AT-rich (85%) and had ATTA at both ends. The mic-specific sequence included a T-rich tract, Ti6CTs5, in the middle region and nearly complementary A- rich tract, As5TA10GAs, existed at 7 bp upstream of initiation codon. In addition, 20 bp repetitive sequences TAAT(TAAC) 4 were found about 100 bp upstream of the mic-specific sequence and in the promoter region of calmodulin gene (about 120 bp upstream of initiation codon). Although Significance of 1.4 kb mic-specific sequence remains unclear at the present, the TAAT(TAAC)4 and mic-— specific sequence including TisCTs might exert an influence upon the conformation of 5’-flanking region and the transcrip-— tion of calmodulin gene. IN VITRO ANALYSIS OF THE PROMOTER REGION OF TETRAHYMENA ACTIN GENE. M. Kimura’, M. Hirono“, T. Takemasa! and Y. Watanabe-. “Inst. of Biol. Sci., Univ. of Tsukuba, Tsukuba and “Cell Bank, RIKEN Inst. of Phys. and Chem. Res., Tsukuba OE EE ———————————————e In ciliates, little is known about the promoter elements and their protein factors in a certain mRNA transcription. Here we demonstrated fourteen protein binding sites (I-XIV) in the 5’- flanking region of Tetrahymena actin gene by using gel retardation assay and DNase I footprinting. Some of these sites are homologous to the conserved promoter elements, such as TATA box (I), GC box(II), GATA box (III and VI), homeobox protein binding site (IV), TRE (V), and CCAAT box (VII and IX). The other sites are little homologous to the previously known promoter elements. We further investigated the roles of such protein binding sites in Tetrahymena actin gene transcription by using in vitro transcription assay. The binding sites I, II, III, IV, V, X, and XI activated the transcription of Tetrahymena actin gene, whereas the binding sites VI, VII, VIII, and IX repressed the transcription under the conditions where nuclear extract from exponentially growing cells was used. The present results suggest that these elements (protein binding sites) regulate the transcription of Tetrahymena actin gene through interactions with nuclear protein factors. Genetics IDENTIFICATION OF HOMEOBOX-CONTAINING GENES IN THE MOST PRIMITIVE METAZOA, SPONGE. M.Seimiya+ , Y.Watanabe!, and Y. Kurosawa“, ‘4pDept. of Biol., Fac. of Sci., Ochanomizu Univ., Tokyo, 2Inst. for Comprehensive Med. Sci., Fujita Health Univ., Aichi. Porifera is the most primitive phylum of metazoa. Genomic DNA from freshwater sponge (Ephydatia fluviatilis ) was subjected to amplification by the polymerase chain reaction using two primers corresponding to helix 1 and helix 3 regions in the homeodomain. Sequence analysis of the amplified products revealed presence of two kinds of homeobox-containing genes, designated prox-1 and prox-2, in sponge. The amino acid sequences of the homeodomains of prox-1 and prox-2 showed 72 and 62% identities with those of NK-3 and om of Drosophila, respectively.These results indicated that when metazoa appeared during the course of evolution, multiple and distinct classes of homeobox- containing genes identified in higher organisms had already existed. CLONING OF 5'REGION OF TYROSINASE GENE IN ASCIDIANS AND LAMPLEYS. H.Masuya,H.Yamamoto,and T.Takeuchi Biol.Inst.,Tohoku Univ. Tyrosinase, the key enzyme of melanin bio synthesis, functions in various organ- isms. In vertebrates,tyrosinase gene ex- pression is restricted in pigment cells. In order to elucidate evolution of this tissue spesific expression, we have cloned and compared tyrosinase genes from various vertebrates including human, mouse, Japa- nese quail, and snapping turtle. In this study, we carried out cloning of tyrosinase gene in ascidian, Halocynthia roretzi, and agnathian, Entosphenus japon- ics. In the gene cloning in ascidian, 10kb of genomic fragment involving 590 base region that has homology to the first exon of mouse tyrosinase gene (60% DNA, 48% amino acid identities) was obtained by screening genomic library with mouse tyrosinase cDNA (Tyrs-J). In putative cupper binding reg- sion 1, 14/19 (73.6%) amino acid identities and two Hiss those are probabie cupper binding sites were conserved. In lampley, a partial library was con- structed of DNA extracted from a portion of gel where Southern blot signal was detect- ed. 4 clones were obtained by screening the library. 1213 PRODUCTION OF TRANSGENIC ORANGE- COLORED VARIANTS OF MEDAKA BEARING MOUSE TYROSINASE GENE BY MEANS OF ELECTROPORATION AND EXPRESSION OF WILD TYPE PIGMENTATION IN THEIR SKIN J.Matsumoto', E.Hirose’, K.Miyazaki’, H.Yamamoto? and T.Takeuchi? ‘Dept. of Biol., Keio Univ., Yokohama, *Biol. Inst., Fac. of Sci., Tohoku Univ., Sendai. We introduced reconstructed mouse tyrosinase gene, mg-Tyrs-d, into fertilized eggs of an orange-colored variant of medaka Oryzias latipes by means of electroporation. Of 589 eggs treated, 38 fish (6%) exhibited brownish skin pigmentation which was variable individually but discernible from the untreated. Piercing the chorion with a tungsten needle before electroporation markedly increased the yield of individuals with brown Pigmentation. Crossing between transgenic founders thus obtained yielded offsprings with heavily brown pigmentation. Light and electron microscopy disclosed that melanization was occurring restrictedly in particular dendritic or disc-shaped cells which were presumed to correspond to "amelanotic” melanophores present in an orange-colored variant of this species. The melanosomes found in such melanophores were peculiar with respect to their internal structure and different from those in melanophores of the wild medaka. Immunohistochemistry using the antibody raised against mouse tyrosinase disclosed that these melanized cells were specifically labelled, indicating the expression and transmission of the gene introduced in transgenic medaka. FUNCTION AND STRUCTURE OF THE AGOUTI LOCUS IN THE MOUSE y.Sato and T.Takeuchi .Biological Insti- tute, Tohoku University, Aoba-yama, Sendai 980 Japan The agouti locus( a_) on chromosome 2 encodes the gene product involved in hair pigmentation. Especially,it determines type of melanin synthesised in hair-bulb melanocytes. However,this gene expresses not in melanocytes but in cells surrunding melanicytes indicating that an intercellular communication gives rise to this phenomenon. In our previous studies,a polyclonal antibody against skin of the lethal yellow mouse(A_” /a) was prepared. Some lethal yellow mouse specific CDNA were cloned by screening lethal yellow mouse skin cDNA library with this antibody ( Sato et al., 1988). In order to verify the specificity of these clones, Southern blot analysis of genomic DNA obtained from several congenic mice concerning a-locus was performed ,using one of the clones as a probe. When genomic DNA was digested with BamHI and ECORI,Specific bands were detected. There- fore,it seemes possible that the clone rep- resents the A ” allele. 1214 RS-2 AND MO MUTANTS IN THE MEDAKA (ORY ZIAS LATIPES) . H. Tomita, Lab. of Freshwater Fish Stocks. Fac. of Sci., Nagoya Univ., Nagoya. The rs-2 mutant(small scales) were found twice. At the first case, the rs-2 mutant showed pleiotorpism, that is, (1) small scales, (2) deformed fin fold (membrane fin) at larval stages and (3) inhibition of melanin formation. They were weak and maintenance of them was difficult. At the second case, the rs-2 mutant showed small scales only. They were kept easily. The F, progeny of crosses between the first and second rs-2 mutants were small scales. In the first rs-2 mutant, (1), (2) and (3) char- acters might link with each other. The mo mutant caused pleiotorpic char- acters, that is, (1) dilution of black color of melanophores(dilute brown), (2) disappearance of leucophores at adult fish, and (3) reduced deposition of gua- nin on eye balls at larval stages. The gu-3 mutant resembled to (3) of mo mutant in a phenotype. The F. progeny of crosses between gu-3 and mo mutants caused reduced deposition of guanin on eye balls. The pleiotorpic characters of mo mutant consisted of (1) and (2) characters. The mo mutant might link with the gu-3 mutant. THE BASIC BODY WEIGHT OF JAPANESE QUAIL (Cuturnix cuturnix japonica) FOR BOTH SEXES A.Nakamura!, T.Kaneko! and K-.Nishimura? 1 Univ. of Shizuoka, Hamamatsu Col., Hamamatsu. 2? Dept.of Biol., Keio Univ., Yokohama. It is usually considered that’ the weight of the female quail body is heavier than that of males throughout their devel- opment and adult stage. However, as for the growth curve of pure strain quails,there was no difference between male and female. The growth curve difference is due to dimorphism,rapid de- velopment and slow gene development. Com- mercial quail lines are caused by contami- nation of both these genes. The hens during breeding season were temporarily heavier, but the body weight of adult hens and migrating wild quail hens was equal to that of males during the nonbreeding season. Domestic commercial hens were always in an artificial breeding season. It is considered that the difference in commercial quail body weight between male and female is caused by dimorphism of the sex-linked semi-dominant genes, moreo- ver,commercial quail hens are always in an artificial breeding season. The basic body weight of Japanese quails is the same _ for both sexes. Genetics ESTABLISHMENT AND SEVERAL PROPERTIES OF NEW INBRED LINES DERIVED FROM THE JAPANESE HOUSE MOUSE, Mus musculus molossinus. Takashi A. Nomaguchi and Youko Sakurai. Department of Cell Biology, Tokyo Metropol - itan Institute of Gerontology, Tokyo. a PT a SS Inbreedings of 8 lines in molossinus mouse were started from (Miura ? x Urawa ¢) F1(MU) in the stock captured at the Kanto district in Japan and performed by each primiparous littermate under a clean con- ventional condition. The respective number of inbreeding generations of MU/g, MU/a and MU/£ now exceeds 22, 21, 20, respectively. Inbreedings of MU/d and MU/e reached to F18 at present, but there were extinct at F11 for MU/h, F9 for MU/b and MU/c. Time required for inbreeding from F1 to F20 was BAST AO 3 NASoiW Cles7So Generation time expressed as days between birth and primiparous dates of the female in each successive generation was 117.7 + 50.5 days. It is characteristic for the mouse to be very small in body weight; the body weights linearly increased from neonatal period to 1 month and also further grew with age, but were constant after 7 months (19.9 + 1.6 g) for male and 9 months(19.3 + 0.7 g) for female. Maximum body weight was 21.5 g for male and 20.1 g for female. Genotypes of Es-1, Es-2, Es-3, Gpd-1, Hbb, Id-1 and Mod-1 in the 5 lines were determined and compared with strains of the laboratory mouse: MU/a for C3H/He type, MU/d and MU/f£ for C57BL/6, MU/e for C57BR/cd and MU/g for DBA/2, respectively. Physiology 1215 TWO KINDS OF FILAMENTS IN THE SMOOTH MUSCLE CELLS IN THE ADDUCTOR OF A PECTEN, CHLAMYS NOBILIS. A.Matsuno, H.Hori and O.Yamada, Dept. of Biol., Fac. of Sci., Shimane Uni., Matsue The opaque portion of the adductor of a pecten was investigated ultrastructurally. The portion contained smooth muscle cells which are composed of thick(myosin) and yhin(actin) filaments. It is well known that thick filaments in molluscan smooth muscle cells are composed of a paramyosin core and myosin molecules coarted the core. The core shows a regular periodicity. Thick filaments in the pecten adductor were classified into two kinds, thinner and thicker, according to the statistical anal- ysis of their diameter accumulated from cross sections. They were correspondingly classified into two kinds, shorter and longer, according to the statistical anal- ysis of their length from isolated native filaments. These thick filaments were con- sequently classified into two kinds; thinner and shorter filaments(about 26.5nm in diameter and 7.5\m in length), and thicker and longer ones(about 42.0nm in diameter and 13.0\m in length). A regular periodicity appeared on the surface of each kind of filaments, when coarted myosin was removed from the sur- face. Intervals of the periodicity were Similar in each kind of filament. It is still obscure whether paramyosin molecules are differnt each other in the two kinds of thick filaments, or not. RESIDUAL FORCE ENHANCEMENT AFTER STRETCH IN FROG SINGLE MUSCLE FIBRES. T. Tsuchiya. Department of Physiology, School of Medicine, Teikyo University, Itabashi-ku, Tokyo. Stretch of active muscle at a slow velocity causes force to increase above the isometric level and this force is composed of, at least, two component forces, high and quickly decaying one and low and long lasting one; the latter being called the residual force enhancement after stretch. More than one mechanisms are known to be involved in the former high force enhancement early after stretch (Sugi and Tsuchiya, 1988; Amemiya et al. 1988) but the mechanism of the latter is not well known and investigated in the present study. A living single skeletal muscle, tibialis anterior, of the frog was stretched during tetanus in the low temperature (2-3 C). The residual force above isometric force 4 or 5s after stretch was constant irrespective of stretch velocity and change of velocity during stretch if the stretch amplitude was constant. The residual force was higher at longer sarcomere length in the range between 2.0-3.0um. The ratio of residual force to isometric force was very much constant at low and high temperature. Hypertonicity by 98mM Sucrose had no effects on this ratio of the residual force. The possibility that the passive elastic element parallel to cross-bridges was involved in this phenomenon was discussed. EXCHANGEABILITY OF F-ACTIN-BOUND NUCLEO- TIDE IN THE SLIDING MOVEMENT. N. Oishi Radioisotope Research Center, Sch. of Med., Teikyo Univ., Tokyo. It has been shown that F-actins can change their conformations. A typical conformation is the rigid form, in which nucleotides are firmly bound, and the other is the flexible form that feasibly exchange the bound-nucleotides. To study of the structural change in actin during the sliding movement, the exchangeability of the actin-bound nucleotide was investi- gated in an in vitro system. F-actin containing 3H-ADP was prepared from rabbit muscle, and the released 3H- ADP was measured at 30°C. In a nitro- cellulose-coated microchamber with heavy meromyosin(HMM), the rate of exchange of F-actin-bound nucleotide was similar in the conditions of either sliding(with 1m™ ATP) or rigor(with 1lmM ADP). In the case of HMM treated with N,N'-p-phenylenedi- maleimide (pPDM), which showed no ATPase activity and weak affinity for F-actins, F-actins were non-motile, but the rate of exchange of the bound-3H-ADP was almost the same again. In the presence of both of the pPDM-treated HMM and the untreated- HMM,the sliding of the F-actins were hindered and slowed, and the rate of the nucleotide-exchange was greatly enhanced. These data suggest that the load or tension may transform the actin structure and facilitate the exchange of the bound nucleotide. The Properties of Scallop and Rabbit Striated Muscle Thin Filaments. Y. Yazawa and M.Kamidochi. Dept. of Nutritional Physiol., Hokkaido Univ. of Education at Asahikawa. Asahikawa. eraction is regulated by troponin-tropomyosin system de- pendent system dependent on the Ca?' concentration. In contrast, (a** regulation of molluscan actomyosin ATPase is known to be associated with the myosin molecule and myosin-linked system had been regarded as essential. Recently, we have detected troponin-like proteims in the striated muscle of scallop In the present study, we prepared thin filaments from scallop striated muscle and rabbit skeletal muscle. When scallop thin filaments were added to scallop myosin, the Mg**-APTase activity was more deeply inhibited in the presence of 10°’M Ca** and further activated in the pr- esence of 10 ‘M Ca** as compared that of actomyosin re- constituted of scallop and actin. When rabbit skeletal thin filaments were added to scallop myosin. the Mg?* - ATPase activity was slightly inhibited in the presence of 10°’MCa?* as compared with that of scallop thin fil- aments. we concluded the properties of scallop thin fil- aments were similar to that of vertebrate skeletal trop- oninI. 1216 Physiology Purifications and Properties of Two Isoforms of Lamprey Skeletal Creatine Kinase. Y. Yazawa, Y. Nakajima and J. Soga. Dept. of Nutritional Physiol., Hokkaido Univ. of Education at Asahikawa. Asahikawa. Two isoforms of creatine kinase, CK] and CKII, were isolated from lamprey skeletal muscle, respectively. CK ] was purified with ammonium sulfate fractionation, DEAE- Toyopearl column chromatography, gel filtration, hydrox- ylapatite column, and butyl-Toyopear! column. CKII was Purified with ammonium sulfate fractionation, DEAE colu- mn, gel filtration, hydroxylapatite column, and CM-Toyo- pearl chromatography. Two isoforms preparations appeared to be homogeneous on SDS-PAGE and their MW of 43K was estimated from SDS- PAGE and about 86K was estimated from gel filtration under physiological conditions, respectively. According to results of amino acid analysis, both isoforms conta- ined 380 amino acids and their enzymatic properties showed the similar results except Km values of ADP in the reverse reaction. EFFECTS OF ACETYLCHOLINE AND IONIC ENVIRONMENT ON MECHANICAL PROPERTIES OF CATCH CONNECTIVE TISSUE FROM STARFISH BODY WALL. T. Motokawa? and M. Nishimaru=. “Biol. Labee shaceuot Scie lokyo sinsit won Technol., Tokyo, “Dept. of Biol., Fac. of Sci., Univ. of Ryukyus, Nishihara. Connective tissue was isolated from the body wall of the starfish Linekia laevigata. Electron microscopical observation revealed that the connective tissue contained no muscle cells. Creep tests were performed on the isolated dermis immersed in artificial sea water (ASW). The dermis elongated with rather a constant rate of elongation. Creep viscosity was defined as "stress/(strain rate)." Acetylcholine (ACh) 10~*-10-7M caused a biphasic response: it increased the viscosity for a few minutes and then the viscosity decreased to the level lower than that before the application of ACh. The response to ACh strongly suggested that the mechanical properties of this connective tissue is controlled by nerves and thus this is catch connective tissue. Effects of ionic environment on viscosity also supported the suggestion. ASW with high-K* concentration increased the viscosity. High-Ca** ASW increased whereas low-Ca*~ ASW decreased the viscosity. The effects of ions were similar to those in sea-cucumper catch connective tissue. COMPARISON OF CALCIUM CHANNELS IN VASCULAR SMOOTH MUSCLES OF RATS AND FROGS. Y.Kobayashi, K.Shinozuka and K.Hattori. Dept. of Pharmacol., Shimane Med. Univ., Izumo. Calcium channels in membrane have an important role in intracellular signal transduction system. In the present study, calcium channels in vascular smooth muscles of rats and frogs were compared. To clarify the character of frog aorta, catecholamines contents in vasculature were measured by HPLC-ECD method. Adrenaline content was significantly higher than noradrenaline, showing apparent difference from the predominant existence of nor- adrenaline in mammalian vasculature. Adrenaline-induced contraction was slightly but significantly smaller than nor- adrenaline-induced contraction in the same concentration when the maximal contraction was taken as 100 &. Adrenaline-induced maximal contraction tended to be higher than that by noradrenaline. Calcium channel blockers, diltiazem and verapamil, induced relaxation in rat isolated thoracic aorta but not affect on adrenaline-induced contraction of frog thoracic aorta preparation. On the other hand, a calcium channel blocker peptide, omega-conotoxin relaxed frog aorta preparation, but not in rat aorta preparation. These results indicated that calcium channels in vascular smooth muscles are different between rats and frogs. EFFECTS OF COOLING ON THE HEART BEAT OF THE JAPANESE SPINY LOBSTER JN VIVO. M.Nakamura, T.Kuramoto. Shimoda Marine Res. Ctr. Tsukuba Univ., Shimoda, Shizuoka The lobster, Panulirus japonicus, had been acclimated in a perfusion chamber (25 x 40x 30ci, 20+1 °C) for month-periods. Electro- cardiogram (ECG) was taken with implanted electrodes. The Average beat rate of the heart was about 100 bpm during spring and summer or‘ about 50 bpm during winter. The effect of lowered temperature on the beat rate was studied in vivo, To cool the animal up to 15°C, cold sea water (5°C) was poured into the chamber. Speeds of cooling (0.1 -1.0°C /min) were dependent on volumes of the cold water and were measured with a digital thermometer. In spring and summer, the beat rate de- creased in proportion to decreases in tem- perature by the cooling (e.g. up to 66 bpm). However, the rate did not drop less than 60 bpm at 15 °C. In winter, the beat rate did not follow the decrease in temperature below 18°C though it did for initial few minutes. For example, the rate of 31 +5 bpm was held at 18-16°C. Correlation plots between the beat rate and the temperature obtained by 8 trials in winter showed that the beat rate decreased for 21-19 °C and increased for 19- 16°C. The negative correlation suggests that the lobster has some mechanisms which pre- vent the decrease of beat rate depending on the drop of body-temperature. Physiology 1217 EFFECTS OF COOLING ON THE HEART BEAT OF THE JAPANESE SPINY LOBSTER JN VITRO. T.Kuramoto, M.Nakamura. Shimoda Marine Res. Ctr. Tsukuba Univ., Shimoda, Shizuoka Electrical and mechanical activities of isolated hearts of Panulirus japonicus were recorded simultaneously, while warm (20+ 1 °c) or cold (8-12 °C) saline was poured into the heart via perfusion lines switched by a faucet. The effects of lowered temperature on the heart were studied in vitro. Under conditions that the initial rate of the heart beat was lower than 60 bpm, the cooling did not always reduce the rate with abolishing the second systolic contraction (SSC) while increased amplitude of electro- eardiogram (ECG). These indicated that the effect was strong on small cardiac neurons but little on large ones. Thus the high beat rate under the low temperature can be pro- duced by the large neurons, which burst at higher rates than the small neurons. Under conditions that the initial beat rate was higher than 60 bpm, the cooling re- duced both rate and amplitude of the beat (the first systolic contraction; FSC) to 50- 40 bpm and 80-60%, respectively. Whereas, ECG amplitude increased significantly with cooling. Applying octopamine (a principal pericardial hormone, 1yM) to the heart with the cold saline, the ECG amplitude further increased while FSC also was enhanced mark- edly. Since the heart tension should be feedbacked to the cardiac neurons, the en- hanced FSC appeared to prevent the decrease in beat rate by cooling. THE EFFECT OF THERMAL ACCLIMATION ON THE ELECTROCARDIOGRAM OF GOLDFISH, Carassius auratus. A.Morita and H.Tsukuda. Dept. of Biol., Fac. of Sci., Osaka City Univ. We examined the effect of thermal acclimation on the heart activity in vivo by comparison of electrocardiograms (ECG) between 10°C- and 25°C-acclimated gold- fish. Curare and atropin were used to exclude the influence of exercise and the vagal regulation, respectively. The ventricular potential difference of 25°C-acclimated fish was significantly lower than that of 10°C-acclimated fish in the control, but no difference between the two acclimation groups was found in Curare- and atropin-treated fish. The frequency of heart beat was sig- nificantly higher at lower temperatures in 10°C-acclimated fish than in 25°C- acclimated fish. This indicates tempera- ture compensation induced during thermal acclimation. The frequency was signifi- cantly accelerated by injection of atro- pin. This may be attributable to blocking the vagal regulation by action of atropin. The time for the auriculoventriclar conduction of systolic impulse decreased with temperature rising, but no Ssignifi- cant difference was found between the two acclimation groups. The auriculoventricu- lar conduction rate may be independent on acclimation temperature. EXCITATORY AND INHIBITORY PATHWAYS FROM MECHANICAL RECEPTORS TO CARDIO-REGULATORY NEURONS IN BATHYNOMUS DOEDERLEINI. K.Tanakal and K.Kuwasawa2. IDept.of Biol., Kyorin University School of Medicine, Tokyo, and 2Dept. of Biol., Tokyo Metropolitan University, Tokyo. In Bathynomus, tactile stimuli induce cardiac inhibition. This inhibition is caused by reciprocal actions between two kinds of cardio-regulatory neurons, i.e. activation of cardio-inhibitory neurons (CIs) and inhibition of the lst and 2nd cardio-acceleratory neurons (CAls and CA2s). Mechanical stimuli applied to spines of thoracic limbs and hairs on swimmerets caused EPSPs on CIs and IPSPs on CAs. We have shown that activated swimmeret movements accompanied tachycardia. In this study, we found that the activated swimmeret movements accompanied activation of CAs. Extension of the thoracic limbs and protraction of the swimmerets caused EPSPs on CAs. There are mechanoproprio- ceptors sensitive to retraction of the Swimmerets. Long-lasting positive current applied extracellularly to the receptors caused activation of CIs and inhibition of CAs, and long-lasting negative current caused activation of CAs. The results suggest that tactile receptors are involved in cardio-inhibitory circuits, and that mechanoproprioceptors are involved in both the cardio-inhibitory and -acceleratory circuits. IDENTIFICATION OF NEURONS INNERVATING THE CARDIOARTERIAL VALVE IN THE ISOPOD CRUSTACEAN BATHYNOMUS DOEDERLEINI. J.OKada and K.Kuwasawa. Dept. of Biol., Tokyo Metropolitan Univ., Tokyo. The cardioarterial valves of five pairs of lateral arteries (LA1-5) receive inhibitory innervation from lateral cardiac nerves (LCN1-5) originating from 3rd roots of the 4th thoracic to the 3rd abdominal ganglia. Activation and inhibition of LCNs induce, respectively, dilation and constriction of the valve. The former increases the haemolymph flow in the LAs, and the latter decreases that. The haemolymph distribution to LAs is centrally controlled by combinations of "open" and "close" modes of valve movements among the lateral cardioarterial valves. LCN neurons were histologically and electrophysiologically identified in the CNS. Back-filling of LCN5 with Cott and Nitt revealed candidates for somata of LCN5 neurons at the posterior half of 2nd and 3rd abdominal ganglia. Intracellular spikes in the soma and extracellular LCN5 impulses corresponded each other one to one in high Mg*t saline. Injection of Lucifer yellow into the somata showed that the neurons actually sended axons to 3rd roots of 2nd and 3rd abdominal ganglia, and that they had both ascending and descending processes in the ventral nerve cord. It is likely that the candidates for LCN5 neurons are cardioarterial valve neurons themselves. 1218 GRAVITY-RECEPTION MECHANISMS OF PARAME- CIUM Y. Mogami, R. Tatematsu & S.A. Baba Dep. of Biol., Ochanomizu Univ., Tokyo. We proposed a model of gravity reception for the negative-gravitactic behavior of Paramecium in accordance with the mechanism hypothesized for the gravity-induced changes in propulsion (Ooya et al 1992, J. exp. Biol., 163) which includes the orientation-dependent changes in membrane potential by the selective activation of mechanosensory channels differentially distributing along the antero-posterior axis of the cell. Computer simulation of the model, in combination with the fact of the potential-coupled regulation of three- dimensional ciliary propulsive thrust leading to the changes in the parameters of helical swimming trajectory, demon- strated that paramecia swim upward along a super-helical trajectory with the axis being parallel to the gravity vector. Super-helical swimming paths were re- corded from paramecia swimming under quasi-unbound conditions in a wide chamber. These results suggest that Paramecium may be capable of sensing gravity via cellular mechanoreception, and may execute gravity-sensitive migra- tion (gravitaxis) as well as gravity- sensitive regulation of propulsion (gravikinesis; Machemer et al, 1991, J. Comp. Physiol. A, 168). ELECTROPHY SIOLOGICAL COMPARISON OF THERMORECEPTOR AND MECHANORECEPTOR CURRENTS IN PARAMECIUM . T. Tominaga and Y. Naitoh, Inst. Biol. Sci., Univ. Tsukuba, Tsukuba, Ibaraki 305 A localized thermal stimulus applied to the anterior end of a voltage-clamped Paramecium produced an inward membrane current, while that applied to the posterior end produced an outward current, when its ambient temperature (Te) was 15°C. The reversal potentials for the anterior current and that for the posterior current were dependent on extracellular of Ca2+ and K+ respectively. Distribution of the thermoreceptor mechanisms in the cell and their ion dependence are identical with those of the mechanoreceptor mechanisms. Primary objective of our study is to examine whether the thermoreceptor of Paramecium share in its mechanism with the mechanoreceptor. Effect of change in external Nat, Lit, Mg?+, Mn2+ concentration on the reversal potential for the thermoreceptor current was identical with that for the mechanoreceptor current. An external application of TEA+ reduced the posterior thermoreceptor current, showing a dose-response relationship similar to that for the posterior mechanoreceptor current. On the other hand Te- dependence of the thermoreceptor current was different from that of the mechanoreceptor current. Magnitude of the receptor current evoked by simultaneous application of thermal and mechanical stimuli was an algebraic sum of magnitude of each receptor current evoked separately by each independent stimulus. Based on these results, we discussed the following possibilities in the relation ship between the thermo- and mechanoreceptor systems in Paramecium 1) The thermoreceptor channels are certainly different from the mechanoreceptor channels, though their distributions in the cell and ionic mechanisms are very similar with each other. 2) The thermoreceptor mechanism share the same ion channels with the mechanoreceptor mechanisms. Physiology THE EFFECT OF COBALT ION ON THE CONTROL MECHANISM OF CILIARY ORIENTATION IN CILLIATED SHEETS FROM PARAMECIUM. M. Noguchi and T. Shimura. Dept. of Biol., Fac. of Sci., Toyama Univ., Toyama. "Ciliary reversal" induced by Ca” is cancelled by addition of higher concentration of Co” in Triton- glycerol-extracted Paramecium. To clarify the action of Co”* on the control mechanism of cilary orientation, we examined the effect of Co”* on the ciliary response in ciliated sheets from Triton- glycerol-extracted Paramecium. Ciliary orientation reactivated in the presence of 1M Ca” ‘changed from 11 o'clock to 5 o'clock by the further addition of Co** over 2 mM (with the anterior of the cell defined as 12 o'clock). In the presence of cyclic nucleotides which also competed with the action of Ca” in the ciliary response, the competing action of Co** with Ca** became more effective. These results indicate that Co”*expels Ca** from the binding site and turns off the Ca”-dedpendent controlling mechanism of ciliary beating direction. Brief digestion by trypsin which was enough to remove the cyclic nucleotide sensitivity did not affect the competitive cancelation of "ciliary reversal" by Co”. This suggests that Ca>~- dependent regulation is essential for controlling the beating direction, and cyclic nucleotides could affect the mechanism secondarily. 22S DYNEIN OF PARAMECIUM MAY TAKE PART IN THE REGULATION OF CILIARY BEATING DIRECTION. T. Ogawa and M. Noguchi. Dept. of Biol., Fac. of Sci., Toyama Univ., Toyama. Ciliary response to either Ca” or cAMP was lost by a digestion with high concentration trypsin in the Triton-glycerol-extracted Paramecium. To determine which component of axonemal polypeptides is essential for Ca** and cAMP dependent controlling mechanism of cilia, we examined the change in SDS-PAGE pattern and ATPase activity of the axonemes during the course of thé trypsin digestion. A band of the highest molecular weight polypeptieds in SDS-PAGE pattern of axonemes disappeared by the trypsin digestion within 20 seconds. ATPase activity of ciliary axonemes also decreased and reached a minimum level (approximately 50% of activity of non-digested axonemes) within 20 seconds. Trypsin-treated axonemes were extracted with 0.6 M KCI and the extract was separated with sucrose density gradient centrifugation and fractionated. SDS-PAGE pattern of each fraction indicated that the high-molecular weight peptide was one of the heavy chain of 22S dynein. This result suggests that 22S dynein plays an important role in the regulation of ciliary beating direction. Physiology 1219 EFFECTS OF METHANOL ON THE INHIBITORY ACTION OF VANADATE AND GLYCEROL ON AXONEMAL ATPase. T. Atago and M. Noguchi. Dept. of Biol., Fac. of Sci., Toyama Univ., Toyama. Axonemal ATPase from eukaryotic flagella and cilia is inhibited by vanadate and glycerol. On the contrary, methanol induce unphysiological enhancement of the ATPase activity which accompanies inability of sliding between outer doublet microtubules. To examine the relation between the stimulating effect of methanol and the inhibitory effects of vanadate and glycerol, the ATPase activity of axonemes from Chlamydomonas flagella was measured in the presence of methanol in addition to vanadate or glycerol. Methanol which is supposed to accelerate product release from dynein:ADP-Pi activated the axonemal ATPase in some extent in spite of the presence of the inhibitors. The ratio of dynein:ADP-Pi to dynein:ADP-Vi inferred from the assumption that ATPase activity is proportional to the concentration of dynein-ADP-Pi complex increased with increasing methanol concentration. This might suggest that dynein-ADP-Vi state become unstable as well as dynein-ADP-Pi state. DISCRETE NATURE OF FLAGELLAR BENDING S. A. Baba, M Imagawa and Y. Mogami. ep. of Biol., Ochanomizu Univ., Tokyo. Present address: Dep. of Mol. Biol., Nagoya University, Nagoya Bending shapes of live and demembranated sea-urchin and starfish sperm flagella were studied at high resolution by means of digital image analysis. As reported previously (Baba, S. A., Mogami, Y. & Nonaka, K. 1990. In Biologi- cal Motion (Lecture Notes in Biomathematics 89) ed. M Alt & G Hoffmann, Springer-Verlag, Ber- lin, pp. 145-154; Baba, S. A., Hoshino, Y. & Mogami, Y. 1991. In Comparative Spermatology 20 Years After, ed. B. Baccetti, Raven Press, New York, pp. 333-336), the curvature of a flagellum tended to change abruptly from one value to another along the flagellum, whereas it remained constant within a segment of variable length. The length of the segment of this quasi-stable bend form, in which the curvature is kept con- stant transiently, varied in a beat cycle and among different flagella of the same species, but the value of that curvature itself appeared to be invariant in flagella at least within the same species. Both the maximum and the average curvature of either the principal or the re- verse bend of sperm attached to the cut edge of agar gel by the head were larger than those of sperm swimming freely, while quasi-stable bend forms appeared of common curvatures, suggesting that discrete bending described here reflects an inherent structural feature of flagella MECHANISM OF CALCIUM-INDUCED REVERSAL OF DIRECTION OF ROTATIONAL MOVEMENT IN REAC- TIVATED SEA URCHIN SPERMATOZOA. S. Ishijima and Y. Hamaguchi, Biological Laboratory, Faculty of Science, Tokyo Institute of Technology, O-okayama, Megu- ro-ku, Tokyo. Examination of demembranated sea urchin sperm has shown that Ca * in a reactiva- tion solution regulates the direction of yaw (rotation of a sperm about the axis perpendicular to the beat plane); a clock- wise (CW) yaw under the coverslip dominat- ed in high Ca *+ concentrations and an anticlockwise (ACW) yaw in low Ca con- centrations. There is a one-to-one corre- spondence of direction between yaw and roll (rotation of a sperm about its long axis); a CW yaw under the coverslip corre- sponded to a CW roll as viewed from the anterior end of sperm and an ACW yaw corresponded to a ACW roll. _Therefore, a CW roll dominates in high Ca = goncentra- tions and an ACW roll in low Ca*t concen- trations. Since the direction of roll is determined by the sense of the three- dimensional components of bending waves, sperm beat with right-handed bending waves in high Ca * concentrations and they beat with left-handed ones in low Ca¢%* concen- trations. This calcium-induced conforma-— tional change in the axoneme is perhaps caused by a reversal of the direction of propagation of the local active sliding between doublets around the axoneme. EFFECT OF ATP CONCENTRATION ON THE VELOCITY OF MICROTUBULE SLIDING IN REACTIVATED SEA URCHIN SPERM FLAGELLA UNDER IMPOSED HEAD VIBRATION. C. Shingyoji!, K. Yoshimural, D. Eshel2, I. R. Gibbons”, and K. Takahashi3. 1Zool. Inst., Fac. of Sci., Univ. of Tokyo, Tokyo, 2Pacific Biomedical Research Center, Univ. of Hawaii, U.S.A., and 3Dept. of Biology, Int. Christian Univ., Tokyo. The flagellum of a sea urchin sperm whose head is held with a suction pipette and vibrated laterally beats in synchrony with the imposed vibration. Similar synchronized stable beating was obtained in sperm demembranated and reactivated with 10 uM-4 mM ATP. We analyzed the sliding velocity of reactivated Tripneustes gratilla sperm under various vibration frequencies. At ATP concentrations lower than 100 1M, the apparent time-averaged sliding velocity of axonemal microtubules, obtained as twice the product of frequency and bend angle, decreased with the beat frequency. At ATP concentrations above 100 uM , the sliding velocity decreased with the beat frequency only below the beat frequency of the unvibrated flagella. When the beat frequency was equal to or higher than the beat frequency of the unvibrated flagella at the same ATP concentration, the sliding velocity remained unchanged. These results suggest that the sliding velocity of axonemal microtubules does not depend solely upon the local concentration of ATP, but is also controlled by an oscillatory mechanism closely associated with the mechanisms for the initiation of bending waves. 1220 Physiology FORCE-VELOCITY RELATION OF THE ATP-DEPENDENT KINESIN-MICROTUBULE SLIDING. H. Sugi!, I. Takagi!, K. Oiwa! and T. Shimizu2. 1Dept. Physiol., Sch. Med., Teikyo Univ., Tokyo and 2Biol. Eng. Dept., Res. Inst. of Polymers and Textiles, Tsukuba. When the tip of a glass microneedle, whose tip is coated with kinesin, is made in contact with a single demembranated axoneme isolated from sea- urchin sperm in the presence of ATP, the needle moves along the axoneme in one direction. If the needle is at right angles to the axoneme long axis, it moves for several microns in 10-20 s at room temperature (23-25°C) until it eventually stops moving. Judging from the elastic coefficient of the needle, the maximum force generated by kinesin- microtubule sliding is several pN. Assuming that the needle stop moving on the axoneme when the maximum "isometric" force generated by knesin-microtubule interaction balances with the force exerted by the bent needle, we tentatively obtained the force-velocity relation of the ATP-dependent kinesin-microtubule sliding by recording the needle movement with a video system. The force-velocity curve thus obtained is nearly straight and differs from that of the ATP-dependent actin-myosin sliding obtained under similar auxotonic conditions. TWO-STEP ACTIVATION OF MOUSE SPERM MOTILITY Y.Si and M. Okuno, Dept. of Biol., Coll. of Arts and Sci., Univ. of Tokyo, Tokyo. To examine the activation of mouse sperm motility, the state of flagellar axoneme in the intact sperm was further investigated using model sperm. When dry sperm from cauda epididymis was diluted into sucrose solution, the sperm showed little motility with the velocity of 13.5+3.8 ywm/sec and beat frequency of 1.5+0.4 Hz. Then, the sperm was transferred to the extraction solution for demembranation. The demembra- nated sperm was immotile in the absence of cAMP. We referred to this kind of intact sperm as "Initiated" one. On the other hand, when dry sperm was diluted into either 155 mM NaCl solution or sucrose solution containing 2 mM Ca“*, the sperm showed the beat frequency of about 9 Hz. The demembranated sperm was motile in the absence of cAMP. We referred to this kind of intact sperm as "quasi-activated" one. Alternatively, when dry sperm was diluted into the sucrose solution containing 20 mM HCO, , the sperm exhibited a vigorous motility with the velocity of 171.2+10.1 um/sec and beat frequency of 11.3+1.2 Hz. The demembranated sperm was also motile in the absence of cAMP. We referred to this kind of intact sperm motility as "Activat- ed" one. Our results indicated that the activation of mouse sperm motility took a two-step process and the conversion of quasi-activated motility to activated motility was highly dependent on the presence of bicarbonate. EFFECTS OF CALCIUM AND CYCLIC AMP ON YAWING DIRECTION OF HAMSTER SPERMATOZOA. K.Ishida’, T. Umeda and M. Okuno“~. SBept. of Urol., Teikyo Univ. Sch. Med. and*“Coll. of Arts and Sci., Univ. of Tokyo, Tokyo. Yawing direction can be easily detect- ed in hamster spermatozoa since the shape of the acrosome is asymmetry. We defined the yawing direction "right" to be the follwing hook direction of the acrosome for convenience. When the extracellular calcium concentration was below 10-4, the yawing direction of the cauda epididymal spermato- zoa of the hamster was permanentry "left" after dilution. On the contrary, the direc- tion was "right" upon dilution when the ex- Eracelluyar calcium concentration was higher than 10 *M. However, the direction gradually turned to "left" and that of every spermato- zoa became “left" finally. The direction could never be returned from "left" to "right" again by further elevation in cal- cium concentration after the spermatozoa once changed to the direction from “right” to "left". The higher the extracellular cal- cium concentration raised, the longer the direction was continued to be "right". The yawing direction was permanentry “right" when the membrane permeablecyclic AMP deri- vative, 8-bromo cyclic AMP, was present above 1mM even in calcium-free dilution medium. As well as the effect of the 8-bromo cyclic AMP, the yawing direction never changed from "right" to "left" when theophy- lline was added above 0.75mM in calcium-, and 8-bromo cyclic AMP-free dilution medium. INITIATION OF SPERM MOTILITY IN RELATION TO ACTIVITY OF MITOCHONDRIA. M. Okuno & Y. Si. Dept. Biol., Coll. Arts & Sci., Univ. Tokyo, Tokyo. Initiation of sperm motility has been considered to involve a cAMP dependent process such as phosphorylation of 15K protein in salmonid fishes. However, the role of the phosphoproteins in the axoneme was unclear. We looked at the mitochondrial activity using a fluorescent dye, DASPEI, which stains active mitochondria. Salmonid spermatozoa were immotile in the presence of KCl and appeared very low mitochondrial activity. When they were diluted in K'-free medium the mitochondria displayed strong fluorescence. Mitochondria were,also active in oyster and barnacle when spermatozoa were diluted in sea water and initiated motility. Hamster spermatozoa shows low activity in the absence of Ca’' (about 0.8 Hz in flagellar beat frequency). In this’ state, the mitochondria were relatively active. In the presence of high concentration of Ca!’ flagella were activated (about 10 Hz) and the mitochondria were also active. On the other hand, when spermatozoa were incubated with CCCP or other inhibitors of mitochondria in the presence of theophylline flagella kept intermediate motility (3 Hz) although the mitochondria were inactive. These results suggested that sperm flagellar activation involved at least two factors, one was a cAMP dependent conversion of axonemal motile system and the other the activation of mitochondria for supplement of ATP to the motile system. Physiology 1221 INITIATION OF SPERM MOTILITY IN MARINE TELEOST: ROLES OF INTRACELLULAR K* AND CA” . H. Takai and M. Morisawa Misaki Marine Biological Station, Fac. of Sci., Univ. of Tokyo. Kanagawa. : Spermatozoa of marine teleosts are immotile in the male Teproductive organ by isotonic osmolality of the seminal plasma, and that motility initiation occurs when they are spawned into hypertonic seawater (Morisawa and Suzuki 1980). Recently, it has been reported that intracellular pH(pH,) and intracellular Ca**(Ca**,) increase during the motility initiation in hypertonic solution in puffer and flounder sperm (Oda and Monsawa 1990). In addition, we found here that plasma membrane of puffer sperm was depolarized at the time of the motility initiation. From these facts, it seems possible that increases in pH, and Ca. and membrane depolarization have some roles in the initiation of sperm motility. However, it is still unclear whether pH, and membrane potential directly participate in the motility initiation process because similar membrane depolanzation and increase in pH, were observed in the immotile sperm in the presence of monensin or nigericin in isotonic NaCl solution. In addition, initiation of sperm motility did not occur when mem- brane depolarization was caused by gramicidin. Futhremore, it was found that puffer sperm which were quiescent in isotonic KCI solution, became motile by the addition of K* ionophore, nigericin. In contrast, spermatozoa were still immotile in isotonic NaCl even if nigericin was supplemented. Monensin had no effect on the sperm motility in isotonic solution. These results suggest that increase in intracellular K* is a candidate for the trigger for the initiation of sperm motility. It is possible that increase in intracellular K* and Ca” participate in the initiation of sperm motility in marine teleosts. CONTROL OF CHEMOTAXIS IN AMOEBA CELLS OF THE CELLULAR SLIME MOLD, Dictyostelium discoideum I. THE ASSAY SYSTEM T. Tani and Y. Naitoh Institute of Biological Sciences, University of Tsukuba, Tsukuba 305 Amoebae of cellular slime mold Dictyostelium have been known to exhibit positive chemotaxis to cAMP. We developed a novel assay system for quantitative examination of chemotaxis in the amoebae. The assay system consists of three thin (0.17 mm) compartments, 1) for cAMP-containing solution (18x18 mm), 2) for the reference solution (18x18 mm), and 3) for amoeba-containing solution (0.4x18 mm). These three compartments are put together placing the amoeba-containing compartment between two other compartments. Thus amoebae in the central compartment are subjected to a solution with a definite concentration gradient. Amoebae were tape-recorded and time course of change in their cell shape, moving direction, moving velocity and of their distribution in each compartment were examined on replayed images. The amoebae showed a transient stoppage of their locomotion due to retracting of their pseudopodia upon subjection to a solution with a concentration gradient of cAMP. Then they resumed their locomotion towards test solution with cAMP. Velocity of the locomotion was higher in a test solution with higher cAMP concentration by 108 M, while it became lower when cAMP concentration was as high as 10°© . Involvement of the orientation response as well as the locomotor velocity in establishment of accumulation of the amoebae are discussed. CONTROL OF ALL-OR-NOTHING TYPE CONTRACTION IN THE STALK BY CALCIUM AND RELATED SUBSTANCES IN VORTICELLA. K. Katoh and Y. Naitoh. Inst. of Biol. Sci., Univ. of Tsukuba, Ibaraki a A peritrich ciliate, Vorticella exhibits an all-or- nothing type contraction, involving coiling of its stalk and shrinkage of its cell body, in response to a stimulus. Coiling of the stalk always starts from the portion near the cell body and propagates down the stalk. Contractile element of the stalk is known to be activated by Ca?+. An injection of Ca*+ buffer-containing solution into the cell body caused coiling of the stalk to quasi- maximum extent, when [Ca*+] in the buffer was higher than 5x10-°M. Simillar injection caused no coiling when the [Ca?+] was lower than 5x10-8 M. This indicates that coiling of the stalk was induced when [Ca?+] in the cell body was over a certain threshold value (5x10-® M). 5x10-8 M Ca?* did not acivate contractile element of the stalk in triton extracted Voticella. These results suggest that Ca?* injected into the cell body induced Ca?+ release from some Ca?* storage site to evoke coiling in the stalk. An endoplasmic reticulum containing Ca?+ has been reported to be present along the contractile element in the stalk. We found that an injection of caffeine- (50 mM) and ryanodinc- (0.2-2 4M) containing solution into the cell body caused all-or-nothing type contraction, that an injection of procaine- (5 mM) and ruthenium red- (10 4M) containing solution into the cell body inhibited contraction and that an injection of IP,- (100 uM) containing solution into the cell body was not effective for evoking contraction. The "Ca?+-induced Ca?+ teleasc" might be responsible for all-or-nothing property of the stalk contraction in Vorticella. CHOLINERGIC MECHANISMS IN THE HEART OF THE CHITON (LIOLOPHURA JAPONICA). S. Matsumura and K. Kuwasawa. Dept. of Biol., Tokyo Metropolitan Univ., Tokyo. Using the chiton in the lowest molluscan taxon, we investigated effects of acetylcholine (ACh) on the heart and cholinergic blockers on the ACh effects. ACh (>107M) exerted negative chronotropic and inotropic effects on the heart. The membrane potential of the auricular myocardium during ACh-induced cardiac arrest was almost the same as the maximum diastolic hyperpolarization of a cardiac cycle. The membrane potential of ventricular muscle cells during the ACh- induced cardiac arrest was always more hyperpolarized than the maximum diastolic membrane potential of the cells. Cardio- inhibitory responses induced by stimulation of central nerve cords were blocked by methylxylocholine (MX), and blocked by tubocurarine (TC) only in the auricle. TC antagonized inhibitory ACh effects on the auricle, but not antagonized ACh effects on the ventricle. MX antagonized inhibitory ACh effects on both the auricle and ventricle. It is likely that the ACh-induced cardiac inhibition is mediated by TC-sensitive and MX-sensitive ACh receptors in the auricle, and by MX-sensitive ACh receptors in the ventricle. We may conclude that cholinergic neural inhibitory control of the heart known in other molluscs is extended to the taxon at the phylogenetic beginning of the Mollusca. 1222 Physiology EFFECTS OF BIOGENIC AMINES ON THE ADULT HEART OF THE ISOPOD CRUSTACEAN LIGIA EXOTICA H. Yamagishi and Y.Terano, Inst. of Biol. Sci., Univ. of Tsukuba, Tsukuba ee Effects of biogenic amines (serotonin, dopamine, octopamine) on the heart beat were examined in the neurogenic heart of adult Ligia. All the three amines could couse the frequency of burst discharge to increase in the cardiac ganglion and accerelate the neurogenic heart beat. The effect was less potent by dopamine than by the others. The frequency increase was concentration dependent and the threshold ranged from 10-9 (cerotonin, octopamine) to 10-8 M (dopamine). Intracellular recodings confiemed the accerelatory effects of all the amines on _ the spontaneous burst activity of the cardiac ganglion cells. Dopamine could also cause the magnitude of contraction to increase in the heart beat. With application of dopamine the heart beat first increased in frequency and then increased gradually in beat amplitude. The increase in beat amplitude lasted longer after changing perfusate to the normal saline. Intracellalar records from the heart muscle showed that the membrane depolarization underlying the potential respones’ to the ganglionic discharges gradually increased by application of dopamine. These results suggest that dopamine affects not only the cardiac ganglion but also the heart muscle EFFECTS OF CRUSTACEAN PEPTIDE HORMONES ON THE CARDIAC GANGLION AND MYOCARDIAL CELLS IN THE HEART OF HERMIT CRAB, ANICULUS ANICULUS. T.Yazawa,K.Tanaka* and K.Kuwasawa. Dept.of Biol.,Tokyo Metropolitan Univ.and *Dept.of Biol., Kyorin Univ. School of Med., Tokyo. Proctolin, crustacean cardio-active peptide, red pigment concentrating hormone, adipokinetic hormone, and FMRFamide were administered to cut-opened heart preparations. Electrical activities of small and large ganglionic neurons and myocardial cells, and contraction force of the heart were simultaneously recorded while the preparations were perfused with the peptides. Potency of proctolin was 103-104 times greater than that of other peptides. Proctolin (10-12 M - 10-& M) showed inotropic effects, but little changed heart rate, i.e. ganglionic burst rate. Intra-burst impulses of both small and large ganglionic neurons were increased. Positive tonotropic effects were elicited by proctolin at higher concentrations (>10-8 M). Membrane responses were superimposed on compound EJPs by proctolin application (10-9 M). However, current-induced electrical responses, which were blocked by either TTX or Ca?+-free salines, were not enhanced by proctolin (10-8 M). Membrane resistance of the myocardium was not changed by proctolin (10-® M). It is likely that ganglionic excitation was the most important action of proctolin in its excitatory effects on the heart. MECHANISM OF CHANGES OF ANSERINE AND CARNOSINE LEVEL AND THEIR RATIO IN RAT FAST AND SLOW TWITCH MUSCLES. M.Tanaka.Dept.of Physiol.,St.Marianna Univ.Sch.Med. ,Kawasaki. "Changes in carnosine and anserine levels and their ratio during development and the effect of endurance training on these peptides levels were studied in rat fast and slow twitch muscles. It has been reported that in the leg muscles of the chronic infected rats,the carnosine level decreases without any change in the anserine level,while tissue free histidine level increases 10-fold. The endurance training decreased both the peptides levels especially the anserine level (Carnosine; p < 0.01, Anserine; P < 0.001) ,but had no effect on the histidine nor other amino acids examined in both fast and slow twitch muscles. There are two possibilities of the decrease of both peptides levels by endurance training. One is the decrease of carnosine synthesis and the other is an increase of its degradation. REGULATION OF BUCCAL MUSCLE CONTRACTION BY A PEPTIDE-CONTAINING MOTONEURON IN THE SNAIL ACHATINA FULICA M. Yoshida and M. Kobayashi. Physiol. Lab., Fac. of Integrated Arts and Sci., Hiroshima University, Hiroshima. In the buccal ganglia of the African giant snail, we identified a pair of exci- tatory motoneurons of the radula protractor and named them B10s. Muscle fibers of the protractor responded to B10 firing with unitary excitatory junction pontentials (EJPs). Bl0-evoked EJPs and contractions of the protractor were blocked by applica- tion of a cholinergic blocker propantheline to the muscle. Contractions of the protractor induced by direct application of acetylcholine (ACh) to the muscle were also blocked by propantheline. Thus the main excitatory transmitter of B10 may be ACh. Immunohistochemical examination revealed that the substance like ACEP-1 (Achatina cardio excitatory peptide-1l), which is a bioactive peptide isolated from Achatina, was located in Bl0s. The nerve terminals in the protractor also showed ACEP-1-like immunoreactivity. Application of ACEP-1 to the protractor enhanced Bl0-evoked EJPs and contractions of the muscle, whereas ACh- induced contractions of the muscle were not affected by the peptide. These observa- tions suggest that ACEP-1 is released together with ACh from B10 in certain con- ditions and acts on prejunctional terminals of B10 to increase the release of ACh. Physiology 1223 LOCALIZATION OF NEUROPEPTIDES ISOLATED FROM A PULMONATE SNAIL, ACHATINA FULICA. M.Fujiwara-Sakata and M.Kobayashi Physiol. Lab., Fac. of Integrated Arts and Sci., Hiroshima Univ., Hiroshima. Several neuropeptides concerned with the regulation of heart beat have been isolated from the central nervous system (CNS) and the heart of Achatina fulica. They include ACEP-1(SGQSWRPQGRFa) and FMRFa. The localization of these peptides in CNS and the heart: was examined immunohistochemical- ly. In the right cerebral ganglion, more than 50 neurons about 30 1m in diameter showed ACEP-1-like immunoreactivity, and many of them also showed FMRFa-like reactivity. Axons from these cells mainly went down to the right cerebro-pleural and cerebro-pedal connectives. In the surface area of the subesophageal ganglia, a number of small neurons showed FMRFamide-like immunoreactivity. In deeper layers of the ganglia, several identifiable neurons showed FMRFamide- or ACEP-1-like reactivity. The atrium and aortic region of the ventricle had both FMRFamide- and ACEP-1-like materials. Whole mount preparation of the heart demonstrated that ACEP-1-immunoreactive nerve fibers ran to the atria abundantly, and a few small fibers entered into the ventricular wall. It seems that neuropeptides in nerve fibers in the atrium are released through small nerve fibers to the ventricle, where they exhibit their modulatory action. SEROTONERGIC PRESYNAPTIC INHIBITION IN THE GILL OF APLYSIA SPECIES. M. Kurokawa and K. KuwaSawa. Dept. of Biol., Tokyo Metropolitan Univ., Tokyo. As, in gills of Aplysia kurodai and A. juliana, we have shown that terminals of motor axons running from the abdominal ganglion to the efferent branchial vessel received presynaptic inhibition, we tried to specify the transmitter responsible for the presynaptic inhibition in this study. Fine processes immunoreactive to anti- serotonin antiserum were found to branch out from the branchial nerve and to innervate musculatures of the efferent branchial vessel. Serotonin was found to depress twitch contractions of the vessel evoked by stimulation of the branchial nerve when the branchial inner cavity was perfused with a solution containing the drug. Excitatory junctional potentials (EJPs) recorded from muscle cells of the vessel induced by motor axons arising from the abdominal ganglion were also depressed by serotonin. Methysergide antagonized the serotonergic depression of both twitch contractions and the EJPs in the vessel. The presynaptic inhibition was also antagonized by methysergide. The results may show that the presynaptic inhibition is mediated by serotonergic axons running from the abdominal ganglion. STRUCTURE AND ACTION OF A MYOMODULIN-CARP FAMILY PEPTIDE ISOLATED FROM AN ANNELID, PERINEREIS VANCAURICA. T.Takahashil, Y.Muneokal, O.Matsushima2, I. Kubota?, T.Ikeda4, H.Minakata4, K.Nomoto4, T.Nose> and W.Miki2. lrac. Integrated Arts and Sci., Hiroshima Univ., Hiroshima,“Zool. Inst., Fac.Sci., Hiroshima Univ., Higashi- Hiroshima, 3Suntory Bio-Pharma Tech Center, Gunma, 4suntory Inst. Bioorganic Res. ,Osaka and ~Marine Biotech. Inst., Shimizu. Myomodulin-=CARP family peptides are widely distributed in molluscs. In the present study, we found a member of the family in the annelid Perinerets vaneaurica The structure of the peptide is AMGMLRM-— amide. That is, the peptide has M-amide at its C-terminus, though all the molluscan peptides have L-amide. On phasic contraction of the ABRM of Mytilus by repetitive electrical stimulation,the annelid peptide, as well as the molluscan peptides, showed a potentiating effect at lower doses and an inhibitory effect at higher doses. On catch tension of the ABRM, the annelid peptide, as well as the molluscan peptides, showed a marked relaxing effect. For these effects, substitution of the C-terminal L-amide of the molluscan peptides with an M-amide seemed not to be deliterious. On the esophagus of Peritnerets,all of the family peptides showed a contractile effect. The annelid peptide was the most potent . Sub- stitution of the L-amide of the molluscan peptides with an M-amide seemed to increase the contractile potency. THREE MIP (MYTILUS INHIBITORY PEPTIDE) - RELATED PEPTIDES ISOLATED FROM THE VENTRAL NERVE CORDS OF AN ECHIUROID WARM, URECHIS UNICINCTUS. T.Ikedal, K.Nomoto!, I.Kubota2, W.Miki3, Y. Muneoka*. lsuntory Inst. Bioorganic Research, Osaka Suntory Bio-Pharma Tech Center, Gunma, auamine Biotechnology Inst. Shizuoka and 4Fac. Integrated Arts and Sci. Hiroshima Univ., Hiroshima. More than 25 species of MIPs have been isolated from the molluscs, Mytilus, Helix and Achatina. All MIPs show a potent inhibitory effect on contractions of the anterior byssus retractor muscle (ABRM) of Mytilus. From the study of structure- activity relationships of MIPs, the essen-— tial part for the activity is FVamide, and proline residue is also important. In fact, all MIPs have the —PXFV(I)amide structure in their C-terminal portions as a common sequence. Recently a MIP-related peptide, MRYFVamide, which lacks the pro- line residue, was isolated from Mytilus (Fujisawa unpublished). In the present study, we purified three peptides which are closely related to MRYFVamide from the ventral nerve cords of an echiuroid warm, Urechis unicinctus. Their structures were determined to be as follows: Ala-Arg-Tyr-—Phe-Leu-NH9 Ala-Lys-Tyr-—Phe-Leu-NH9 Ala-Lys-—Phe-Phe-Leu-NH9 These peptides show inhibitory effects on contractions of inner circular body-wall muscle of Urechis and ABRM of Mytilus. 1224 Physiology EXTENSION EVOKING DESCENDING INTER- NEURONS MODIFY THE UROPOD MOTONEURONS ACTIVITY H. Namba, T. Nagayama and M. Hisada Zool. Inst., Fac. of Sci., Hokkaido Univ., Sapporo. Uropod motor pattern of the crayfish avoidance reaction is modified during the fictive abdominal extension. We have determined the abdominal extension evoking interneurons are responsible to the modification of the uropod motor neuron activity via premotor nonspiking interneurons. Among 28 extension evoking interneurons (EEIs) so far could be picked up at the superficial lateral bundle of abdominal connective, 10 EEIs yielded enough data to characterize them according to their effects on the abdominal extension motoneurons and the uropod motoneurons. Six of them can be classified into a group of EEI with effects both on slow extensor motoneurons and uropod motoneurons. The rest (4) showed effected to the extensor motoneurons but none to the uropod motoneurons. EEI with the effects on the slow extensor motoneurons and uropod motoneurons inhibited closer and/or excited opener motoneurons. Morphologically, these EEIs projected into the dorsal region of the neuropile in the terminal abdominal ganglion. Their dendritic fields overlapped with the nonspiking interneurons but not with the sensory neurons which projected into the ventral region of the neuropile. EEIs, thus, are likely to modify the nonspiking interneurons response to the sensory stimulation of the tailfans directly but not the sensory terminals. Possibility of EEIs having also the parallel direct connection to the uropod motoneurons remains to be tested. OUTPUT EFFECTS OF ASCENDING INTERNEURONS UPON THE ABDOMINAL POSTURAL SYSTEM IN CRAYFISH H. Aonuma, T. Nagayama and M. Hisada Zool. Inst., Fac. of Sci., Hokkaido Univ., Sapporo Majority of ascending interneurons that originate from the terminal ganglion receives sensory imformations from the tail fan and they have output effects upon closer and openner motoneurons in the ganglion. About 30 ascending interneurons have so far been identified by the physiological and morphological characteristics in the terminal ganglion. These ascending interneurons project their axons to anterior ganglia, Output effects upon abdominal postural system and the structures of branches in the anterior abdominal ganglia have been studied. Intracellular recordings were made _ using microelectorodes filled with 3 % neurobiotin. Extracellular signals were simultaneously recorded using pin electrodes. They were placed on all extensor and flexor motoneurons of each abdominal ganglion and closer and openner motoneurons of the terminal ganglion. This simultaneous multirecording study revealed that majority of ascending interneurons had output effects upon extensor and flexor motoneurons. These effects are the same in all of abdominal ganglia. Neurobiotin Staining revealed that ascending axons spreaded their branches in each abdominal ganglion. These branches innervated ipsilateral neuropil of each only. These results indicated that the majority of ascending interneurons cooperatively controlled extensor and flexor motoneurons of all abdominal ganglia and that contralateral cooperation relied on _ indirect interactions in each ganglion. ACETYLCHOLINE RECEPTOR CHARACTERISTICS IN A SENSORY NONSPIKING INTERNEURON OF CRAYFISH. M. Takahata, H. Nakamura and M. Niwa. Sect. Neuro-cybem., Res. Inst. Elect. Sci., Hokkaido Univ., Sapporo. nal abdominal ganglion of the crayfish Procwnbarus clarkii Girard, was affected by perfusion of acetylcholine (ACh) agonists and antagonists using the isolated abdominal nervous system preparation. The synaptic response of LDS to electrical stimulation of the third root sensory bundle was recorded by a Lucifer-filled glass microelectrode penetrating the cell on the midline. Identification of the cell was based on its morphology revealed by Lucifer yellow injection. The synaptic delay between the sensory neurons and LDS was less than Imsec, suggesting their monosynaptic connection. The half decay time of LDS response was about 10msec at the resting potential (N=46; -70+10mV). By perfusing the preparation with Ca-free, Mg-rich (5 to 10 fold) saline, the synaptic response of LDS disappeared completely and restored later in the normal saline. The response was reduced by artificial depolarization and enhanced by hyperpolanization with the reversal potential at about -16mV. These results indicate that the LDS response is mediated chemically. Perfusion of 10mM ACh under Ca-free, Mg-rich condition caused depolarization (10- 20mV) of LDS and reduction of its synaptic response. 0.1mM nicotine showed similar effects but 1mM muscarine had no visible effect. The LDS response was blocked by 1mM D- tubocurarine and 20mM hexamethonium. However, 1mM atropine was effective as well. The results suggest that the dendritic membrane of LDS has ACh receptors which resemble the ionotropic nicotinic receptor of vertebrates but differ in atropine sensitivity. THE MECHANISM OF RHEOTAXIS IN THE WATER STRIDER, GERRIS PULUDUM. K. Taneda, M. Sakagami and T. Matsuoka. Dept. of Biol. Fac. Sci. Kochi Univ., Kochi The rheotactic behavior of the water strider, Gerris puludum was recorded on VTR. The record was played and _ projected on a _ digitizer board (Graphtec, KD3300) through a video pro- jector (Fujix, HP-40Hi). Six points of the body for analyzing the position of body center and bending angles of legs were entered using a reposed picture of the record. The digitized data were analyzed and were represented graphically. Time-changes in the bending angle of each leg and in the position of body center were measured using a blinded, antennae- or cerci- amputated specimen. Each specimen as well as untreated one showed conspicuous rheotactic behavior. There- fore, compound eyes, antennae and cerci seem to play no role in the behavior. When the specimen directed upstream, the middle legs on both sides always showed synchronized movements. While when the specimen directed downstream or at a right angle to the stream, it moved its one side of the middle leg and turned to upstream. Thus, the middle legs seem to play an important role in upstream orientation. Physiology 1225 IDENTIFICATION OF SENSORY NEURONS RESPONSIBLE FOR THANATOSIS IN THE CRICKET Gryllus bimaculatus. H. Nishino and M. Sakai, Dept. of Biol., Fac. of Sci., Okayama Univ.,Okayama. The cricket Gryllus bimaculatus shows a maintained flexion posture in response to a pressure on the prothorax (Zool. Sci. 1987, 1991). We searched the prothorax for sensory neurons receiving pressure input and triggering thanatosis. Transection of the nerve 2 (N2) emanating from the antero-ventral part of the prothoracic ganglion abolished thanatosis but that of the other nerves did not. The N2 branched off into 8 thinner bundles which innervated cuticles and apophysis on the ventral side of the neck and thorax. A total of 14 somata were found on 3 bundles near the branching point. They had short dendrites and genta projecting axons which extended to the aratnaracies suboesophageal, eeeatioracic. and metathoracic ganglia. No efferent axons were present in the N2. Mechanical stimulation of the isolated N2 including 14 neurons in question evoked both phasic and tonic spike responses. These stretch receptor-like neurons may be responsible for triggering and maintaining thanatosis. THE MUSHROOM BODY OF INSECT BRAIN PARTICI- PATES IN SPATIAL MEMORY PROCESSING. M.Migunami J.M.Weibrecht N.J.Straus- feld“. lpept. Bil@il, 5 Wee>s Sile, kanrnginiy Univ., Fukuoka and “Arizona Research Labs, Div. of Neurobiol., Univ. Arizona, Tucson. Many insects, including bees and ants, possess a well-developed spatial memory. We report that the mushroom bodies (MBs) of cockroach brain participate in the processing of spatial memory. Cockroaches were placed in an arena, the floor of which was hot (50°C) except for a small invisible cool area. The wall of the arena was decorated with visual patterns having specific geometrical relationships with respect to the hidden goal. After 3 trials, cockroaches reached the hidden goal significantly faster with such cues than in their absence. Effects of MB lesions on the ability to reach hidden goal using spatial cues were tested. Small pieces of aluminum foil were implanted into preselected brain areas. Subsequent histology determined the extent of lesions. In tests using visible goals, cockroach with bilaterally lesioned MBs reached the goal as fast as unoperated cockroaches. In a hidden goal paradigm, however, the time taken for bilaterally MB lesioned cockroaches to reach the goal was significantly longer than in unoperated cockroaches, suggesting that MBs are involved in spatial memory processing. DISTRIBUTION AND PHEROMONAL RESPONSES OF DESCENDING NEURONS IN THE BRAIN OF THE MALE SILKWORM MOTH. R.Kanzaki, A.Ikeda and T.Shibuya. Inst. of Biol. Sci. Univ. of Tsukuba, Tsukuba Male moths, Bombyx mori, display oriented zigzagging walking in response to the female's sex-attractant pheromone. Long-lasting excitation (LLE) shown by brain neurons which descend the ventral nerve cord (VNC) to the thoracic motor system (descending neurons, DNs) is one of the important signal to control the zigzagging behavior. To study the physiology and distribution of DNs in the brain of the moth, olfactory responses were recorded extracellularly from a split off section of one of the small bundles of the VNC between the brain and the thoracic ganglion. DNs contained in the bundle were stained with cobalt from the cut end of the bundle. DNs were mainly classified into 3 groups (LIL, III) according to the position of the cell body. DNs of group I and II have branches in the lateral accessory lobe (LAL) where many olfactory protocerebral neurons innervate. LLE or ‘Flip-flop (FF)' activities were typically recorded from a split off dorsal section of a small bundle of the VNC. FF activities recorded from left and right VNC had an anti- phase relationship. Our results suggest that these anti-phase FF activities to the motor center carried by group I or II DNs may be important as a zigzag-turning signal of B.mori males. THE LOCUS OF THE CIRCADIAN PACEMAKER IN LOCOMOTOR ACTIVITY OF THE HAGFISH, EPTATRETUS BURGERI. S. Ooka-Souda!, T. Kadota2, and H. Kabasawa Atomigakuen Jr. College, Tokyo, 2Dept. of Anat., Yokohama City Univ. Sch. of Med., Yokohama, 3aburatsubo Marine Park Aquarium, Miura. In a previous study, we found that a circadian pacemaker may exist in the anterior part of the hypothalmus of the hagfish. To determine the detailed location of the circadian pacemaker, we recorded the locomotor activity rhythm of hagfishes after transecting the brain with scissors and after partial destruction of brain structures with a high-frequency lesion generator (tip 0.75 mm). The cuts and the lesions were made rostral to, in the center of, and caudal to the anterior habenula after exposure of the brain surface. From histological examination of the location of the cuts and lesions, we postulated that the pacemaker might exist in the preoptic nuclei, which have retinofugal connections as shown by tracer experiments. 1226 Physiology GABA,-ERGIC MODULATION OF THE SPINAL LOCOMOTOR NETWORK IN THE LAMPREY. T. Matsushima’, J. Tegner’, A. El Manira?, R.H. Hill? and S. Grillner’. ' Life Sci. Inst., Sophia Univ., Tokyo. * Nobel Inst. for Neurophysiol., Karolinska _Inst., Stockholm. GABA plays an important role in the presynaptic inhibition in the spinal cord. Furthermore, GABA acts, particularly through the type B receptors, as a modulator of the somato-dendritic membrane properties of locomotor network neurons. Enhancement of endogenously released GABA by an uptake blocker (nipecotic acid) significantly reduced, in a dose- and time-dependent manner, the burst frequency of the NMDA- induced fictive locomotion in the lamprey spinal cord in vitro. The reduction was counteracted by addition of GABA,-antagonists (phaclofen or 2-(OH)-saclofen), while addition of a GABA,- antagonist (bicuculline methiodide) caused irregular activities. A GABA,-agonist (baclofen; 5-404M) alone reduced, and an antagonist alone increased, the burst frequency in a dose- dependent manner. Therefore, GABA should be released from its propriospinal sources during fictive locomotion, and continuously down-regulating the rhythmic activity through the GABA, receptors. At the cellular level, in the lateral gray matter neurons (intemeurons and motoneurons), baclofen (at the same range of concentration) suppressed voltage-dependent Ca**-currents (both HVA and LVA), and concomitantly the following Ca**-dependent K*-current. Furthermore, current-induced sub-threshold depolarization and rebound excitation were also suppressed by baclofen. On the other hand, baclofen brought about no detectable changes in the resting membrane potential, the membrane resistance, nor in the threshold level for Na*—action potentials. Voltage-dependent K*-currents (delayed rectifier and “A-current") and the inward current through the NMDA receptor/channels were not influenced. Modulatory effects of GABA could occur complementarily at both of the pre- and the postsynaptic sites, which could account for the GABA, actions in the spinal locomotor network. IONIC MECHANISMS UNDERLYING ENDOGENOUS PACEMAKER POTENTIALS OF TERMINAL NERVE- GNRH CELLS: A CURRENT CLAMP ANALYSIS. Y. Oka, Zool. Inst., Fac. of Sci., Univ. of Tokyo, Tokyo Gonadotropin-releasing hormone (GnRH)-immunoreactive terminal nerve cells have endogenous regular beating discharge mode, which may be related to their putative neuromodulator functions. Ionic mechanism underlying the pacemaker potentials was studied using intracellular current clamp recordings from whole brain in vitro preparation of a small fish brain. Addition of 1.5-3 uM TTYX to the Krebs-Ringer solution blocked spontaneous spikes, but small subthreshold membrane potential oscillations (pacemaker potentials) remained. About one third of the recorded cells showed only pacemaker potentials without spikes in normal perfusing solution. Hyperpolarizing DC current injections increased the amplitude and decreased the frequency of pacemaker potentials, and vice versa. Input resistances during both nsing and falling phases of pacemaker potentials were smaller than during the resting phase. Thus, sequential activation of inward (Na+ and/or Ca?+) and then outward (K+) current is suggested to underlie the pacemaker potentials. The pacemaker potentials were not blocked by 1mM Ni?+ or ImM amiloride (blockers of LVA-Ca?+ current) or 2mM Co?* (blocker of HVA-Ca?* current) or in Ca?+-free solution. On the contrary, they were blocked by substituting choline or TMA for Nat. They were also blocked by 5mM TEA, and the resting potential was raised to a depolarized level. The present results Suggest that the TIX-resistant persistent Na* current and the delayed rectifier K* current mainly contribute to the rising and falling phase of pacemaker potentials, respectively. THE EFFECT OF CYSTEAMINE ON THE LOCOMOTOR ACTIVITY RHYTHMS OF THE RAT C. Fukuhara’, S-I.T. Inouye~, K.Aokil. tLife eit Inst. Sophia Univ., Tokyo and Mitsubishi Kasei Inst. of “litemtescacn, Tokyo The suprachiasmatic nucleus (SCN) of the hypothalamus contains a circadian pacemaker. The dorsomedial SCN is characterized, in part, by dense somatostatin-like immunoreactivity CSS= LI), ee ES functional significance in circadian pacemaker remains unclear. A previous study revealed that 24 h SS-LI contents of the SCN in rats kept under LD and free-running conditions showed a distinct circadian rhythm. These findings suggest that SS levels in the SCN are controlled by the endogenous pacemaker. In the present study, we examined effects of Cysteamine, a SS depletor, on the locomotor activity rhythms of the rat, to explore a possible role of SS in the SCN. Cysteamine (200mg/Kg, I. PD induced permanent phase shift in subsequent free- running rhythms in rats. Administration of Cysteamine reduced SS levels in the SCN within 5 min after injection and similar reduction of SS levels was observed also 72. hrs after the injection The results of this study indicate that changes in the SS contents in the SCN induce permanent phase shifts of the locomotor activity rhythms of the rat. A NOVEL LEUCOPHORE IN THE DARKBANDED ROCKFISH, SEBASTES INERMIS is ULTRASTRUCTURE. T. Iga and A. Matsuno. Dept. of Biol., Shimane Univ., Matsue. Reflecting chromatophores in the integument of the darkbanded rockfish, Sebastes inermis, are of two distinct types, iridophores and leucophore-like chromatophores. The iridophores are smaller and non-motile, producing a metalic iridescent color. The organelles involved in the coloration of iridophores are reflecting platelets. The leucophore- like cells are larger and highly dendritic. These cells appear dull brown by transmitted light, by reflecting light they are yellowish in appearance. These cells responded to K and norepinephrine with pigment dispersion. The response was opposite to that of melanophores. The rate of the movements was markedly slow so that the full dispersion took 20-30 min. The ultrastructure of the leucophore-like cells was examined. Their Pigment granules are spherical in form, varying from 0.8-1.0 pm in diameter. They contain some fuzzy amorphous substance. Numerous intermediate filaments were observed, but microtubules were rather scarece. From these observations, these cells were recognized as a type of leucophores. Physiology 1227 CYTOSKELETON AND ITS ELEMENTS OF CULTURED IRIDOPHORES FROM FRESHWATER GOBY, ODONTOBUTIS OBSCURA H.Ishida and T.Iga. Department of Biology, Faculty of Science, Shimane University, Matsue 690. Iridophores of the freshwater tereost, Odontobutis obscura, are motile. The motility of the iridophores involves the intracellular translocation of reflecting platelets. The mechanisms of the movement of platelets remains to make clear. In order to reveal the mechanism, the cytoskeleton and its elements were examined by using the cultured iridophores. The cytoskeletons were exposed by treatment with the zwitterionic detergent CHAPS and then the iridophores were fixed with paraformaldehyde. One of these preparations was observed with scanning electron microscope. The cytoskeleton of the iridophore was composed of a filamentous system with reticular structure. The other group of preparations was treated with Rhodamin- phalloidin, anti-actin antibody, anti- tubulin antibody or antibody of the other cytoskeletal elements. Fluorescence microscopy revealed the reticular structure of actin filaments through the cytoplasm. The micro-tubular system was not observed. MOTILE MECHANISM OF THE REFLECTING PLATE- LETS WITHIN THE NEON TETRA IRIDOPHORES. H. Nagaishi and N. Oshima. Dept. of Biomolecul. Sci., Fac. of Sci., Toho Univ., Funabashi. (SS EE ESE Changes in the interference color re- flected from the motile iridophores, which are distributed in the lateral stripe skin of the neon tetra (Paracheirodon innesi), may be caused by the change in the angle of inclination of the reflecting platelets in the cells (theory of venetian blinds, Nagaishi et al., 1990). Within the irido- phores, microtubules and both thin and thick filaments are present, and these organelles are thought to be related to the movement of the platelets. To determine the organelles which par- ticipate in the platelet movements, we investigated the spatial distributions and profiles of the cytoskeletal components by the conventional electron microscopy and immunoelectron microscopy. In the ultra- thin sections of the iridophores which were cut almost horizontally, microtubules were observed nearby the edges of the platelets; they run parallel or perpendicular to the longitudinal axis of the cell. By the immunoelectron microscopy, the presence of actin in the cortical region and between the platelets was confirmed. Anti-vimentin antibodies also responded near the edges of the platelets. From these results, the role of these organelles and the mechanism which produces the motive force for the platelet movements were discussed. REFLECTION SPECTRA AND COLOR EVALUATION OF BUTTERFLY ,WINGS 1 2 M.Akimgpto , H.Namiki -Tanaka, H. Tabata , and A.Yoshida , Dept. of Biology ch. of Edu., Univ. of Waseda, Tokyo, Scientific Research Lab., Nissan Research Centex, Nissan Motor Co.,Ltd., Yokosuka, and Biohistory Research Hall, Project Office, Japan Tabacco, Inc., Tokyo. Structure and optical properties of wings of Morpho butterflies are studied, aiming at clarifying the basic characteristics of structurelly colored materials. Scanning electoron micrographs of the wing scales show that the scales have complicated lamellae structure, and light interference due to this structure is the origin of brilliant blue color of the wings. Reflection spectra of these wings are measured at various incident and viewing angles;they are characterized by (i)strong anisotropy and (ii)high excitation purity for perception of color(strong and narrow reflection band). The measurement of color is done with spectrophotometers using a specified and constant geometry. However, there are many applications where measure— ments at several or different geometries are necessary. Standard color evaluation and specification systems for ordinary color seem to be insufficient for this kind of structural color, and new systems are necessary aS a basis for developing and utilizing structurally colored materials. CHROMATOPHORE PATTERN ON INTEGUMENT OF THE JAPANESE TREEFROG, Hyla arborea japonica. Yoshihisa Kamishima Department of Biology, Faculty of Educa- tion, Okayama University, Okayama 700 Japanese treefrogs are typical arboreal frogs in Japan, and show bright green color on the dorsal skin during the active season. However, the animals kept at low temperature or under dim light show vari- ous degree of coloration from pale gray to dark brown. They also bear darker spots and bands on the anterior dorsal and femoral skin characteristic to the indi- vidual animal. Chromatophore arrangement in spot and band area is similar to other portion, but skin glands distribution in these area are dispersed. Pigment gran- ules in melanophores in the spot and band area showed slightly smaller in diameter than those in lighter area. When the excised green skin was treated with adren- ergic $-receptor agonist, isoproterenol, it took 100 to 300 min to change to gray, or to darker colors. Melanophore stimu- lating hormone caused the cells more rapid response than the neurogenic agent. All these agents, however, failed to produce the prominent dark pattern characteristic to each animal. This may suggests a pos- sibility of different responsiveness among and/or differential neuro-humoral control over the frog skin melanophores. 1228 Physiology DEMONSTRATION OF LIGHT-GUIDES IN THE EYE PHOTOPHORES OF THE JAPANESE FIREFLY SQUID. IG Gleadall!, J-M Bassot2, M Kawahara?, (e) Inamura?, Y Kito> and Y Tsukaharal. lRcars, Sendai; 2cNRS, Paris; 3toyama Med. Pharm. Univ.; Avyozu Aquarium; Sosaka Univ. This investigation is part of a wider study of the counterillumination mechanism of the firefly squid, Watasenia scintillans. _. The photophores (bioluminescent organs) in the skin and below the eyes have a distal spray of straight, membranous tubules. The function of these tubules in intact fresh eye photophores was investigated by confocal scanning laser microscopy (Bio-Rad MRC 600 system; 488 nm laser). lue fluorescence emitted from within the photophore produced an annulus of light points at the photophore surface. When the focal plane was changed. to progressivel deeper locations ('optical sectioning' the light points were no longer in focus. The diameter of the annulus remained constant but the images became progressively dimmer. This demonstrates that light is released (and stimulating light enters) only at the distal tips of the membranous tubules, which are therefore naturally occurring cite eh eee Their function in counter- illumination may be to ensure uniform emission of photophore light in a strictly defined, ventrally directed cone corresponding to that within which the squid's body would otherwise be silhouetted against downwelling sunlight. THE ROLE OF THE LATERAL EYES AND PINEAL BODY IN THE COLOUR CHANGE OF THE FRESH- WATERFISH, ZACCO TEMMINCKI. I. Takabatake, T. Saiki and T. Iga. Dept. Biol. Fac. Sci., Shimane Univ., Matsue. Physiological colour change of Zacco temmincki, in common with all other tele- osts, displays a diurnal rhythm. In this experiment, the effects of eyes and pineal elimination on the day-night colour changes OceeZee temmincki were investigated, and melanophore dispersing and aggregating activity of blood extract fractionated by HPLC were examined. In the 12L:12D experi- ments, blinding caused slight darkening response in the dorsal melanophores, as compared with intact fish, during the dark period. Melatonin contents in the blood during dark period were relatively in- hibited by blinding, while in lightness a rise of blood MSH-like substance was ob- served. Pinealectomy, in the 12L:12D ex- periment, did not affect the colour changes during the light and dark period, but in the 24DD experiment pinealectomized fish showed darkening response in contrast with normal fish. Pinealectomy caused the syn- thesis of the MCH-like substance during the dark portion of the photoperiod , while in the 24DD the synthesis of MSH-like sub- stance was activated. Pinealectomy of blind fish caused darkening response during both light and dark periods. The blood extract showed the existence of MSH-like substance. BETA-ADRENERGIC RECEPTOR SUBTYPES OF MELANOPHORES IN MARINE GOBIES. H. Katayama. Mukaishima Mar. Biol. Lab., Fac. Sci., Hiroshima Univ., Mukaishima-cho, Mitsugi-gun, Hiroshima-ken. Previously we reported that the melano- phore of an estuarine goby, Tridentiger obscurus possesses both beta-1 and beta-2 adrenergic receptors. In further confirma- tion of the previous results, I examined beta-adrenergic receptor subtypes of melan- ophores of two species of marine goby, Tridentiger trigonocephalus and Chasmich- thys dolichognathus gulosus. Denervated melanophores in split pieces of isolated caudal fins were used. The aggregation of pigment was evoked within the melanophores by verapamil or melatonin in the presence of phentolamine. Effects of several beta- agonists on the melanophores in the pres- ence and absence of beta-antagonist were photoelectrically recorded. The beta-ago- nists, adrenaline, noradrenaline, salbuta- mol and dobutamine, were all effective in evoking the dispersion of pigment within the cells. The effects of the beta-agonists were inhibited by beta-antagonists, meto- prolol, butoxamine, ICI118551 and proprano- lol. From graphical analyses of the results it was estimated that melanophores of each goby possess both beta-1l and beta-2 adren- ergic receptors, and that the ratio of beta-1l to beta-2 receptors in number is approximately 5:95 in the melanophore of T. trigonocephalus, and 30:70 in the mel- anophore of C. d. gulosus. ADP-RIBOSYLATION OF G PROTEINS BY BACTERIAL TOXINS INHIBITS THE LIGHT-INDUCED MELANO- PHORE RESPONSE OF THE XENOPUS TADPOLE. Y. Miyashita, T. Moriya, K. Asami, N. Yokg- sawa, S. Hatta, J. Arai~ and _S. Kusunoki~. Sapporo Med. Coll. 25apporo, IMEC Lab. Dai- kin Inc., Tsukuba, “LSL Co., Ltd., Tokyo Pertussis toxin, botulinum exoenzyme C3 and cholera toxin inhibited light-induced melanin aggregation in melanophores of the isolated tail fin of the Xenopus tadpole. The substrate proteins, about 26kDa, 40kDa and 42kDa, for ADP-ribosyltransferase of C3, pertussis toxin and cholera toxin, res- pectively, were present in the tail fin in- cluding photosensitive melanophores. The ADP-ribosylation of the proteins by C3 or pertussis toxin was partially inhibited by illumination, and the inhibition could be reversed by addition of guanine nucleotides In contrast, ADP-ribosylation by cholera toxin was independent of light. Furthermore, several kinds of GTP binding proteins(about 55kDa, 42kDa, 34kDa, 26kDa and 23kDa) were present in the tail fin. From the immuno- blotting for G proteins, the existence of Gs, Gi(or Gt) and Gq was confirmed. These findings suggest that the hetero- trimeric G protein close to Gi (or Gt) and the C3 substrate small molecular G protein interact with a photo-receptive substance and contribute to its signal transduction in light-induced melanin aggregation of the Xenopus melanophores. The substrate of cholera toxin might be Gs protein and the toxin appeared to induce melanin-dispersion by increasing cellular cAMP. Physiology 1229 ROLE OF NEURAL PROCESSES IN BACKGROUND ADAPTATION IN THE MEDAKA, ORYZIAS LATIPES. M. Sugimoto, N. Oshima and R. Fujii. Dept. of Biomolecul. Sci., Fac. of Sci., Toho Univ., Funabashi. In the wild type medaka, a prolonged background adaptation consists of Physiological and morphological color changes. We previously reported that an adaptation of the medaka to a black (B fish) or white background (W fish) for 10 days induced an apparent increase or decrease in the number and the size of melanophores in the scales, respectively. The melanophores in B and W fish also differed in the sensitivity to the neurotransmitter and hormone. In the present study, we have examined to what extent the nervous system affects such prolonged background adaptation, using chemically sympathectomized medaka. The fish was injected with 6- hydroxydopamine intraperitoneally in order to denervate melanophores and adapted to a black (DB fish) or a white background (DW fish) for 10 days. Melanophore responsiveness in both DB and DW fish showed the same tendency to that in B fish. Melanophores of DB fish were larger than those of DW fish, although there was no difference in the number of the cells. These results indicate that, during the background adaptation, neural processes affect the melanophore sensitivity not only to neurotransmitter but also to hormone, and have some influences on the number of melanophores, SIMULTANEOUS RECORDING OF THE CHANGES IN INTRACELLULAR LEVELS OF CA“* AND THE MOTILE RESPONSES OF MELANOPHORES IN TILAPIA. J. Toyohara and R. Fujii. Dept. of Bio- molecul. Sci., Fac. of Sci., Toho Univ., Funabashi. REALE AS US a et By using a handmade dual-wavelength BO ae fluorometer, which enabled us eae simultaneously the changes in fos and the motile response of a singe ,! ees loaded melanophore, the role re |(eny in signaling the translocation of DSpace sense within tilapia (Oreochromis niloticus) melanophores was investigated. When the aggregation of pigment was induced by sympathetic stimulation oF by alpha, agonists, an increase in [Ca“?t] always (esos vappea with the motile peers [Ca appeared highest around the mass of meee end pigment, and showed a little elevation in the periphery of the cellular projections. Mimicking norepinephrine, an ionophore, ionomycin, induced the elevation of [Ca“*], as well as the aggregation of pigment. An alpha, agonist, phenylephrine, induced pigment aggzegation, but the effect was abolished in Ca**-free saline solution, in which [Ca*t]. was normally decreased. Even in the Ca*~+t-free saline, however, Sela ie te gradually restored the level of [cay] ; to the basal level. Presumably, 2 {L@z *]j-mobjlizing mechanism, such as IP3-induced Ca liberation, is operating. renee data indicate that the in¢rease in [Ca is involved in the regulation of GHEE aggregation in melanophores. PHARMACOLOGICAL PROFILES OF THE SUBTYPES OF MUSCARINIC CHOLINOCEPTORS THAT MEDIATE PIGMENT AGGREGATION WITHIN MELANOPHORES OF THE MAILED CATFISH, CORYDORAS PALEATUS. H. Hayashi and R. Fujii. Dept. of Bio- meolecwil- Sets, PE GE Seila, Welerey Whales, Funabashi. Pigment movements in melanophores of teleost fishes are mainly under the regulation of the sympathetic nervous system. Although nerves involved in the peripheral transmission to the melanophores in several catfish species, e.g., Corydoras (Callichthyidae), Pimelodella (Pimelodidae) and Mystus (Bagridae), are normally adren- ergic, these fishes possess extra musca- rinic cholinoceptors which also mediate pigment aggregation. In this experiment, using some selective antagonists, we tried to characterized the cholinoceptor subtypes mediating aggregation of pigment in the melanophores of the mailed catfish, Cory- doras paleatus. Among some cholinoceptor antagonists, 4-DAMP, a muscarinic M selective antagonist, most effectively blocked the pigment aggregating action of acetylcholine. The results indicated that the pigment aggregation induced by the stimulation of muscarinic cholinoceptors on Corydoras melanophores is mediated by M3 subtype receptors. These observations suggest that intracellular mechanisms in the melanophores of this species are different from those in other teleosts. Namely, the inositol trisphosphate system may be involved in this process. CHANGES IN INOSITOL-PHOSPHATE LEVEL IN THE FISH MELANOPHORES INDUCED BY NOREPINEPHRINE F.Morishita, A.Shimada, M.Fujimoto and K.Yamada. Zool. Inst., Fac. Sci., Hiroshima Univ., Higashi-Hiroshima. To examine the second-messenger system mediating the pigment movement in fish melanophores, effect of norepinephrine on the inositol-phosphate level was investi- gated. For this purpose, the cultured melanophores of black-moor goldfish, Caras- sius auratus, were pre-labeled with [?H]- myo-inositol, and inositol phosphates extracted were separated by strong-anion exchange column. Norepinephrine at 100 nM transiently increased the radioactivity in inositol triphosphate and inositol tetra- phosphate fractions to 2-4 fold of control level. Alpha-antagonists, prazosin and yohimbine, reduced the NE-effect. When the cells were pretreated with 1 pg/ml pertus- sis toxin for 15 hr, the NE-induced in- crease of inositol phosphates was attenuat- ed, suggesting the involvement of GTP- binding protein in the cell response. The lipid analysis by thin-layer chromatography indicated that the increase of inositol phosphates was accompanied by decrease of the radioactivity in inositol phospho- lipids. These results suggest that stimulation of alpha-adrenoceptors increases the inositol-phosphate level in fish melano- phores. 1230 Physiology CHICKEN VISUAL PIGMENTS AND COLOR VISION T. Yoshizawa. Department of Applied Physics and Chemistry, The University of Electro- Communications, Chofu. Recently we showed that chicken iodopsin is in both principal and accessory members of double cone with or without oil droplet (green), respectively, and in a single cone with a red oil droplet, and that chicken green, blue and violet are located at single cones with yellow, clear and blue oil droplets, respectively. Since incident light to these cone pigments passes through the inner segment with or without an oil droplet, it acts like a cut-off filter. The spectrum of iodopsin was corrected with the transmittance curve of green oil droplet or the corresponding position in the principal or accessory member, respec- tively. Both the corrected spectra are so close to chicken photopic sensitivity curve that the double cone would be responsible for light intensity discrimination in daylight vision. The spectra of chicken red, green, and blue were corrected by transmittance curves of the respective oil droplets, resulting in remarkable decrease of overlaps of two spectra between chicken red and blue and between chicken green and violet. Thus almost all the wavelengths between 400 and 600 nm can be discriminated with ratios between absorptions by only two types of cone pigments. This would result in increase of the resolution of wavelength discrimination. PORPHYROPSIN AND NEW DEEP-SEA VISUAL PIGMENT WITH 4-HYDROXYRETINAL ARE FOUND IN SOME MESOPELAGIC CEPHALOPODS IN ATLANTIC. Y.Kito! K.Narita M.Seidout M. Michinomae2 iG Partridge’and Pod) a Herring? lpept. of Biol., Osaka Univ., Toyonaka,“Dept., of Biol., Konan Univ., Kobe,~Dept.of Zool., Univ. of Bristol, Bristol, ‘Inst. of Ocean. Sci., Deacon Lab., Surrey. Firefly squid, Watasenia scintillans, has three visual pigments in its retina, each pigment being segregated in different parts and based on ditferent chromophore, i.e., Al-based pigment (Amax: 484nm), A2- based pigment (Amax:500nm) and A4 pigment based on 4-hydroxyretinal (Amax:470 nm). We found that among cephalopods, collected by R/V Discovery from Atlantic, squids, Pyroteuthis, Pterigioteuthis, and octopus, Japetella had two visual pigments with Al and A4, and squid, Bathyteuthis had three pigments with Al, A2 and A4. This indicates another molecular adaptation of visual pigment to photic environment. The hypsochromic shift of absorbance spectrum is due to solvent effect in the nonpolar environment of the retinal-binding site of opsin. The Schiff base compound of 4-hydroxyretinal showed the same effect. HPLC ANALYSIS OF RETINOIDS EXTRACTED FROM THE_ PLANARIAN, Dugesia japonica. K. Azuma N.Iwasaki-, M.Azuma~“, ve Shinoz wae M. Ezaki~, S.Nakamura~ and T. Suzuki~. \oept. of Biol., Osaka Med. Coll., Takatsuki, “Dept. oe Health Sci., Osaka Kyoiku Univ. Osaka, ~Dept. of biol. and chem. Engin,, Fac. of Engin. Univ. of Gunma, Kiryu, Dept. of Biol., Fac. of pele; Univ. of Hirosaki, Hirosaki and Dept. of Pharmacol., Hyogo Coll. of Med., Nishinomiya, Retinal and retinol (including retinyl ester) were extracted from the planarian bodies and analyzed by HPLC. 11-Cis retinal was only detected in the extracts from the head piece (anterior part containing eyes). The amounts of all- trans retinal, 11l-cis retinal and all- trans retinol including the retinyl ester were O.1-1.1, 0.11-0.19, and 20-50 pmol/head, respectively. Almost all of all-trans retinol and the retinyl ester were extracted with hexane from the freeze-dried head pieces, but all-trans and 11l-cis retinals were not. These retinals were extracted from the residues after the hexane extraction by the oxime method. These results suggest that all- trans retinal is bound to an unidentified protein in the tissues and 1l-cis retinal exists as the chromophore of the visual pigment in the eye. PHOTOBLEACHING PROCESSES OF GECKO VISUAL PIGMENTS. D.Kojimal, T.Okanol, H.Imail, Y.Fukadal, Y.Shichidal, F.Crescitelli2 and T.Yoshizawa?. IDept. of Biophys., Fac. of Sci., Kyoto Univ., Kyoto, 2Dept . of Biol, Univ. of California, LA, U.S.A. and 3 Dept. of Appl. Phys. and Chem., Univ. of Electro- Communications, Chofu. The Tokay gecko (Gekko gekko), a noctur- nal lizard, has two kinds of visual pigments, P521 and P467. In spite of the pure-rod morphology of the photoreceptor cells, amino acid sequences of P521 and P467 are the most similar to the typical cone visual pigments, iodopsin and chicken green- sensitive cone visual pigment, respectively. In order to get further evidence that the gecko visual pigments are really the cone- type pigments, we investigated their photobleaching processes by means of low temperature spectrophotometry. P521 has a similar bleaching process to iodopsin. However, unlike batho- intermediate of iodopsin, that of P521 mainly converts to the next intermediate. The meta-intermediates of both of P521 and P467 are thermally less stable than those of rhodopsin. Since the less stabilities of meta-intermediates are one of the characters of cone visual pigments distinguishable from rod visual pigments, we can safely concluded that both of gecko visual pigments have functional properties of cone-type visual pigments. Physiology 1231 THE ABSOLUTE STRUCTURE OF 3-HYDROXYRETINAL USED AS THE VISUAL PIGMENT CHROMOPHORE IN INSECTA. T.Seki!', K.Isono® and M.Ito%. ‘Dept. of Health Sci., Osaka Kyoiku Univ., Osaka, ?RCAIS, Tohoku Univ., Sendai, and *Kobe Women's College of Pharmacy, Kobe. In the compound eyes of insects, two kinds of retinal congeners, retinal (ret, ) and 3-hydroxyretinal (ret,;), are used as the visual pigment chromophore. Being different from ret,, the ret, is a chiral molecule, and so the absolute structure of ret, in the eyes of fruitfly, butterfly, cicada and dragonfly was determined. Retinoids in the compound eyes of insects were extracted by the HCHO-method, and the peaks of all-trans and 11-cis ret, on HPLC were collected. The 171-cis fraction, which should have come from the visual pigment chromophore, was isomerized by exposure to white light, rechromato- graphed, and the resulting all-trans ret, was collected. The absolute structure of both the original all-trans and the photo- isomerized all-trans (originated from 11- cis) ret; was determined by HPLC using a chiral column (ChiraSpher, Merck) ‘!) . In the case of fruitfly, 11-cis ret, was exclusively (3S), but the original all- trans ret; was dominantly (3S) containing (SR) at the ratio of 10-20%. In the compound eyes of butterfly, cicada and dragonfly, by contrast, only the (3R) -all- trans and (3R)-11-cis enantiomers were detected ae 1: Ito et al, J. Nutr. Sci. Vitaminol. 38, 111-115 (1992) . OPSIN PROCESSING IN THE PROTEIN-SYNTHESIZ- ING PATHWAY OF DROSOPHILA PHOTORECEPTOR CELLS. K. Ozaki, H. Nagatani, M. Ozaki and F. Tokunaga. Dept. of Biol., Fac. of Sci., Osaka Univ., Toyonaka. The synthesis of Drosophila opsin is promoted by its chromophore, 11—cis—3—hydroxyretinal. When flies are raised on a carotenoid—free medium, no mature opsin (35 kD) is formed in their retina, but a trace amount of opsin precursor (40 kD) is accumulated. This opsin precursor possesses an oligomannose-type sugar chain, which is completely digested during the maturation after chromophore—binding. When flies are irradiated with blue light, a major part of main rhodopsin (Rh1) is converted to metarhodopsin, and the prolonged depolarizing afterpotential (PDA) is evoked in their photoreceptor cells. Under such condition, the photoreceptor cells synthesized 37 kD opsin instead of mature 35 kD opsin. The apparent molecular mass of the 37 kD opsin was reduced to 35 kD by the treatment with peptide N—glycosidase F, while it was not changed by the treatment with endoglycosidase H. This result suggests that 37 kD opsin has a sugar chain which has been partially processed in the golgi apparatus of photore— ceptor cells. The norpA mutant, which bears mutation on an eye-specific phospholipase C gene, possesses enough rhodopsin but fails to evoke receptor potential. In this mutant, mature 35 kD opsin was synthesized after blue-irradiation. Moreover, even in the wild-type flies, mature 35 kD opsin was synthesized when red or green light were given just after blue—irradiation. On the basis of the above data, we concluded that the 37 kD opsin is the metabolic intermediate sitting between 40 kD precursor of opsin and mature 35 kD opsin. In addition, it was suggested that the opsin processing could be modulated with the activation of phospholipase C, or the changes in intracellu— lar ionic condition caused by cell excitation. IN VITRO SYNTHESIS OF DROSOPHILA OPSIN. K. Katanosaka, F. Tokunaga and K. Ozaki. Dept. of Biol., Fac. of Sci., Osaka Univ., Toyonaka. Drosophila opsin (Rh1) has two potential glycosylation sites at Asn-20 and Asn-196. Although the precursor of opsin is actually glycosylated, mature opsin has no detectable oligo- saccharide chain. This fact thus suggests that the oligosaccha— ride chain is eliminated during the maturation of opsin. In order to examine the process of opsin maturation, we synthesized the opsin in a cell—free translation system using reticulocyte lysate. In the absence of pancreatic microsomal membranes, only 36k opsin was synthesized. On the contrary, three kinds of opsin with different MW (36k, 40k, 43k) in SDS-PAGE were detected when the synthesis was carried out in the presence of the microsomal membranes. Digestion of N-linked oligosaccharides with peptide N-glycosidase F (PNGase F) reduced the apparent MW of 40k and 43k opsins to 36k, indicating that both 40k and 43k opsins are glycosylat- ed, while 36k opsin is not. This behavior of 40k opsin was quite similar to that of the opsin precursor found in vivo. We next analyzed the time course of in vitro opsin synthesis. In the early stage of the reaction, 43k molecule was the domi- nant member of the glycosylated opsins, while the fraction of 40k opsin gradually increased later. In order to check the possibility that the change in MW from 43k to 40k caused by the partial trimming of the oligosaccharide chain, we blocked the initial step of the trimming with N-methyldeoxynojirimy— cin. The result that the untrimmed opsin has MW of 41k, however, suggested that the decrease in MW from 43k to 40k results from complex reactions including the usual trimming of oligosaccharide chain. Considering the extent of MW shift by the PNGase F treatment, it was also suggested that 40k opsin synthesized in vitro (and probably 40k opsin precursor in vivo, too) possesses a Slightly trimmed oligosaccharide chain. EXPRESSION OF VISUAL PIGMENTS IN MAMMALIAN CELLS. F. Tokunaga and O. Hisatomi Dept. of Biol., Fac. of Sci., Osaka Univ., Osaka. Lamprey is one of the most primitive vertebrates. The outer segment of the short photoreceptor cell of lamprey is cone type. The ceo, however, contains rhodopsin-like visual pigment (lamprey rhodopsin). We isolated a cDNA of lamprey visual pigment. The deduced amino acid sequence showed >75% identities with those of rhodopsins of higher vertebrates. The cloned cDNA was inserted into an expression vector and transfected into the mammalian cells by using calcium phosphate method. After 24-48 hr, the cells were harvested and the membrane fraction was isolated by sucrose flotation. The purified membrane fraction was mixed with 1li-cis retinal, and visual pigments were extracted with 2% CHAPS. By photo- bleaching experiments, we observed a reconstituted pigment with difference maximum at about 500 nm, which coincides with that of lamprey rhodopsin isolated from the lamprey retina. The expressed pigment, as well as lamprey rhodopsin, bleached gradually in the presence of 100 mM NH,OH in the dark. These results indicate that the cloned cDNA encodes lamprey rhodopsin, and that the expressed pigment can be used as lamprey rhodopsin. 1232 Physiology Distribution of opsin in the photoreceptors in the late pupal and newly emerged Drosophila Atsuko Matsushita, Kentaro Arikawa, and Eisuke Eguchi. Department of Biology, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236, Japan. Change of opsin distribution in the course of the development of rhabdomeric photoreceptors was examined in the late pupal and newly emerged Drosophila by electron-microscopic immunogold labeling. Numerous gold particles that represent the localization of opsin were found in the rhabdomeres of control flies (Canton-S) at 2 days before emergence, but were not found in the flies of 3 days before emergence, the time when the rhabdomeric microvilli begin to develop. Besides the microvilli, the immunoreactivity appeared in the multivesicular bodies, rough ER, and secondary lysosomes as previously reported by others. In addition, the labeling was found on some membrane-enclosed inclusion that was com- posed of electron-dense vesicles, lamellated membranes, and ribosomes. The inclusion is always found in the rER-rich cy- toplasm, where the protein synthesis takes place at a high rate. The inclusion first appears at the end of the pupal stage and disappears within 1 day after emergence: i.e. not found in the adult older than 1 day. The inclusion was not found in a mutant DPP- in which the development of the rhabdomeric microvilli stops in the late pupal stage. According to the accepted interpretation of the membrane turnover process, the inclusion described here should be cate- gorized as the secondary lysosome. However, the present re- sults indicate that the inclusion may be a structure for the tem- poral storage of over-synthesized rhodopsin, or, perhaps a structure that is involved in the synthetic pathway of the thabdomeric membrane. The antiopsin is a generous gift of Dr. T. Tanimura. Iodopsin immunoreactivity in retina and pineal organ of river lamprey, Lampetra japonica. S. Tamotsu?, T. Oishi*, K. Nakao2, Y. Fukada3, Y. Shichida3, T. Yoshizawa* and Y. Morita!. list Dept. of Physiol. Hamamatsu Univ. Sch. of Med., Hamamatsu, 2Dept. of Biology, Faculty of Science, Nara Women's Univ., Nara, 3Dept. of Biophysics, Faculty of Science, Kyoto Univ. Kyoto, *Dept. of Applied Physics and Chemistry, The Univ. of Electro- Communications, Chofu. In the present study retina and pineal organ of lamprey were investigated immunocytochemically using an antibody against the iodopsin, the chicken red- sensitive cone visual pigment. In the retina, only outer segments of the long photoreceptor cells showed lodopsin-immunoreactivities (I-IR). In the pineal organ, the I-IR photoreceptors were observed at both ventral and dorsal wall, and at the pineal stalk. Further- more, following four types of pinealocyte were identified by mean of a double immunostaining for the iodopsin and the serotonin; 1)both serotonin- and iodopsin-immunopositive, 2)serotonin- immunopositive and iodopsin-immunonega- tive, 3)reversal to the 2nd type, 4)no immunoreactivity to both antibodies. PRESUMPTIVE PHOTORECEPTOR CELLS IN THE SIPHON OF A CLAM, TAPES (AMYGDALA) PHILIPPINARUM. H. Karakisawa and K. Ohtsu. Ushimado Marine Laboratory, Okayama University, Okayama. A clam, Tapes (Amygdala) philippinarum responds to a light stimulus given locally to the siphon by retracting it and closing the shells, indicating an existence of photoreceptor cells in the siphonal region. Light- and electron-microscopical observations revealed 4 types of cells, epithelial cells, muscle cells, nerve cells and granule-rich cells (granular cells). Among them, however, no typical photo- receptor cell could be found. Instead, the epithelial cells bore striking microvilli similar to those of molluscan photoreceptor cells, suggesting that they might have a photoreceptive function and further rhodopsin might be found in the microvilli. To clarify this, an anti-squid(Toderodes pacificus) rhodopsin serum (mouse) was used in conjunction with FITC-labelled anti- mouse IgG. Specific fluorescence was detected clearly at the microvilli of the epithelial cells, and at the granular cells as well. It disappeared, however, by treating the anti-squid rhodopsin serum with Toderodes rhodopsin extracted by digitonin. It is suggested from these facts that rhodopsin exists in the microvilli of the epithelial cells as well as in the granular cells and they may function as the photoreceptor cells. ANTI-GECKO OPSIN ANTIBODY RECOGNIZING OUTER SEGMENTS OF RED-SENSITIVE CONES IN FRESHWATER TURTLE AND CHICKEN RETINAS M. Yoshida? and K. Mifune? !Inst.Biol.,Qita Univ. Oita, 2Dept. Microbiol., Med. College of Oita, Hazama, Oita. Monoclonal antibodies (mAbs) were raised to a suspension of retinal rod outer segments (ROSs) from geckos, Gekko japonicus. Hybrydomas produc- ing anti-ROS were screened with indirect immuno- fluorescence method on cryostat sections of gecko retinas. The mAbs reacted with bands cor- responding to opsins as demonstrated by SDS-PAGE of retinal homogenates solubilized with deter- gent.The mAbs labeled ROSs of all photorecep- tors, except for those of one member of type C double cells. This labelling pattern was fur- ther confirmed by using the IgG gold-silver technique at the LM level. Whole-mount prepa- rations of freshwater turtle and chicken ret- inas treated with immunohistochemical SAB reac- tion showed that the mAbs labeled OSs of cones with red oil droplet and of double cones, but not those of rods, and of green and blue cones. The raised mAbs specific for opsins of gecko rods with P521 pigment seems to recognize com- mon epitopes in the OSs of red-sensitive cones in turtle and chicken retinas. In the retinas of diurnal lizards, Jakydromus tachydromoides,both OSs of double cones and of cones with yellow oil droplet were also labeled with the mAbs. Physiology 1233 LOCALIZATION OF VISUAL PIGMENTS IN THE VERTEBRATE BRAIN. Ts Oishil, Y. Yahirol, T. Yoshikawal, H. Masuda2, Te Yoshizawa3, and M. Michinomae?. IDept. of Biology, Nara Women's Univ., Nara, 2Dept. of Cell Biol., Chest Disease Res. Inst., Kyoto Univ., Kyoto, 3Dept. of Biophysics, Kyoto Univ., Kyoto, and 4Dept. of Biology, Konan Univ., Kobe. We investigated localization of visual pigments in the extraretinal photoreceptors of frogs (Rana catesbeiana) and birds (Coturnix coturnix japonica) using immuno- cytochemistry and HPLC. The outer segments of pinealocytes in the frog pineal were well stained with anti-serum against bovine rhodopsin (Rh-As) and weakly stained with monoclonal antibodies against chicken iodopsin (Io- mAb) . There are some immunonegative cells observed. The cells in the frontal organ were immunopositive to both Rh-As and Io- mAb. Paraventricular cells in the hypo- thalamus were stained with Rh-As, but not with lIo-mAb. In the pineal of birds, pinealocytes were immunopositive to Rh-As, but the reactivity to Io-mAb was very weak Or none. And much individual variation was observed. Some cells in the hypo-thalamus were also stained. HPLC analysis revealed 1l-cis and all trans retinal in the pineal complex of the two species, and 11l-cis and all-trans dehydro-retinal were detected in the pineal of frogs. THREE-DIMENSIONAL DISTRIBUTION OF PROTEO- GLYCANS IN THE MOUSE INTERPHOTORECEPTOR MATRIX. M. Iwasaki’, A. Tawara2, M. E. Rayborn3 and J. G. HollyfieldS. 1Biol. Lab., Fac. of Sci., Fukuoka Univ., Fukuoka, 2Dept. of Ophthalmol., Fac. of Med., Kyushu Univ., Fukuoka and 3Cullen Eye Inst., Baylor Col. of Med., Houston, USA. The interphotoreceptor matrix, filling the extra- cellular compartment between the neural retina and the pigment epithelium in the vertebrate retina, has been implicated in a variety of functions, including in retinal attachment, phagocytosis, and metabolite trafficking. Proteoglycans in the mouse interphotoreceptor matrix were stained with Cupromeronic blue, and 1pm-thick sections were visualized by means of Electron Spectroscopic Imaging (ESI) technique. Analyses of stereo pairs of ES! showed extensive invagination between proteoglycan networks and apical processes of the pigment epithelium. Rinsing of the isolated Pigment epithelium with Ringer's solution did not eliminate proteoglycans near the tips of apical Processes. These results suggest that the insoluble proteoglycans play a role in retinal attachment. LIGHT-INDUCED BINDING OF PROTEINS IN THE CRAYFISH RHABDOMERIC MEMBRANES (PROCAMBARUS CLARKI/). A.Terakita*, Y.Tsukahara, T.Hariyama!, T.Seki2 and H.Tashiro, Photodynamics Res. Cent., RIKEN, 1RCAIS, Tohoku Univ., Sendai, and 2Dept. of Health Sci., Osaka Kyoiku Univ., Osaka. * Present address: Fac. of Educ., Oita Univ., Oita. Light-induced protein interaction in the process of the visual transduction in arthropods photoreceptor was investigated biochemically using crayfish retina. Irradiated and non-irradiated rhabdomeric membranes were incubated with buffer-soluble proteins extracted from the dark adapted retinas and analyzed by SDS- PAGE. A 40 kDa and a 46 kDa proteins were found to bind to the irradiated membranes but not to the non- irradiated membranes. The light-induced binding of the proteins was also observed in the intact retina. The soluble proteins of crayfish cross-reacted with blowfly rhabdomeric membranes. They were bound to the membranes with formation of metarhodopsin and dissociated from the membranes with photoregeneration of rhodopsin, showing the similar property to the 48 kDa protein in the fly retina (Bentrop & Paulsen, 1986). Immunoblot analysis was carried out by using an antiserum against the mouse arrestin (S-antigen). The antiserum cross-reacted with the 40 kDa protein but not with the 46 kDa protein. These results suggest that the binding of the 40 kDa and 46 kDa proteins to the membranes is caused by the formation of metarhodopsin, and the 40 kDa protein has similar structure to vertebrate arrestin. MUTATION THAT DISRUPTS RHABDOMERES OF RETINULAR CELLS NONSPECIFICALLY IN THE COMPOUND EYES OF DROSOPHILA K.Isono ,,T. Hariyama A K.Arikaya”, 4 -Komatsu~, D.Yamamoto’, R.Ueda*, Y.Sano”~. Res. Center for Appl. ing ormalcion Sci.,Tohoku Univ., Sendai,“Dept. of Biology, Yokohama City Univ. ,Yokohama, Dept. of Physiology, Tokyo Wemen’s College of Medicine, Tokyo and ‘“Mitsubishi- Kasei Inst.of Life Sci., Machida. A photoreceptor mutant was isolated from screening stocks obtained originally by enhancer-trap insertions. The mutation, which is tentatively named ara (all-rhabdomeres absent) eliminates photosensitivity of all three classes of the retinular cells in the compound eyes. EM study revealed that rhabdomeres are almost absent for all types of the photoreceptors. Pupal immature rhabdomere seemed to develop normally but remained small before eclosion. Within 24 hours the rhabdomeric structure undergoes degeneration. Genetic analysis showed that the gene is on the distal end of the right arm of the third chromosome where cht, another photoreceptor specific gene has been reported. Complimentation test between these mutations is in progress. 1234 Physiology PHOTORECEPTOR DEVELOPMENT IN THE PINEAL ORGAN AND THE RETINA OF THE SALAMANDER, Hynobius dunni. H. Takahama, Biological Institute, Faculty of Education, Oita University, Oita Morphological changes in the pineal and retinal photoreceptors of the salaman- der, Hynobius dunni, were examined by scanning electron microscopy after the eryofracturing and immunoreactivity of tubulin during development. At stage 39, the inner segments and budding cilia of the photoreceptor cells are visible in both organs. At stage 41, the outer seg- ments possess whorl-like membranous lamel- lae in the pineal and retinal photorecep- tors. At the hatching stage (43), the outer segments are composed of regular well-stacked membranous lamellae, and the inner segments are well-labeled for anti- tubulin and the reaction products are oriented along cell axis in the retinal photoreceptors. On the ohter hand, in the pineal photoreceptors, the outer segments possess irregular membranous lamellae and the inner segments are stained intensively but randomly for anti-tululin. These results suggest that the photoreceptors develop simultaneously in the pineal organ and retina but maturation of the pineal photoreceptors is delayed. SPECTRAL RESPONSES AND STRUCTURE OF HORIZONTAL CELLS IN THE FILEFISH RETINA K.Kawamatal, T.Osaka!, K.Ohtsuka!, H.Utiyama?, and T. Ohtsuka3 iFac. of Educa., Akita Univ., Akita, 2Dept. of Anat., Nippon Med. Sch., Tokyo, 3Natl. Inst. for Physiol. Sci., Okazaki We studied the correlation between the physiological and the morphological properties of the second order neuron in the retina of the filefish, Novodon modestus. The intracellular response of the horizontal cell (HC) to the monochromatic flashes was obtained by a glass microelectrode filled with biocytin (or Lucyfer yellow) solution, and then the same cell was injected electrophoretically with the dye. The spectral responses collected from 103 HCs were classified into three subtypes; 90 cells hyperpolarized to all the wavelengths of monochromatic stimuli, 9 cells hyperpolarized to green and blue flashes, while depolarized to red flashes, and 4 cells hyperpolarized to both blue and red flashes, while depolarized to yellow flashes. These HCs are denoted shortly by LHC, BHC, and THC, respectively. We found all LHCs inthe filefish have a maximal response at 480 nm, while it has been repoted that LHCs in the freshwater teleosts have a maximal response at around 640 nm. The intracellular dye-injection revealed functional organization of the horizontal cell layer. (1) LHC, BHC, and THC are independently coupled probably by gap junction, and each makes a functional syncytial sheet. (2) In each HC sheet, we found the somata of three subtype of HCs were arranged in regular mosaic. (3) The syncytial sheet of LHC consisting of somata and axon terminals made a distal sublayer of the INL, while the syncytial sheets of both BHC and THC, lacking axon terminals, made a proximal sublayer. POTASSIUM CHANNELS CLOSED BY LIGHT IN THE ONCHIDIUM EXTRAOCULAR PHOTORECEPTOR. T. Gotow, T. Nishi and M. Kameyama. Dept. of Physiol., Sch. of Med., Kagoshima Univ., Kagoshima. Previous studies have shown that the depolarizing receptor potential (the photo- response) of an extraocular photoreceptor, the photoresponsive neuron (A-P-]) in a mol- lusc, idium results from the light sup- pression of K currents, flowing during dark. We report here the single-channel currents suppressed by light, recorded from cell-attached patches of A-P-1. The rate of channel activity was largely unaffected by de- or hyper-polarizing the patches, in- dicating that the suppression of channel activity is caused by light itself but not by voltage changes. The single-channel conductance was estimated to be 50-100 pS, from the slope of the relation between channel current and patch polarization. The reversal potential and its shift for the single channel currents, measured from patch electrodes filled with different K solutions suggested that the light-sensi- tive channels were specifically selective for K ions. Finally, kinetics of the light-sensitive channels was investigated. These results support that the photore- sponse of the extraocular photoreceptor, A- P-1 results from the closure of specific light-sensitive K° channels. The light- sensitive channel was active in the dark- adapted A-P-1. LIGHT ADAPTATION AND CALCIUM FEEDBACK IN PRIMATE RODS. K. Nakatani’, T. Tamura? and K-.W. Yau%. ‘Inst. of Biol. Sciences, Univ. of Tsukuba, Tsukuba, 2Dept. of Ophthalmology, Kanazawa Univ. Sch. Med. Kanazawa 3%Dept. Neuroscience, Johns Hopkins Univ. Sch. Med., Baltimore, USA. It is now known in amphibians that when retinal rods are illuminated there is a net efflux of Ca?* in the rod outer segment. This Ca2* efflux leads to a decrease in free calcium, which in turn feeds back negatively on the phototransduction process to regulate the light sensitivity. Relatively little is known in mammalian rods. Using a suction pitet to record membrane current from an isolated primate rod, We examined the kinetics of the electrogenic Na*-dependent Ca?* efflux during illumination and found that the rate of this efflux, which reflects the internal free calcium concentration, declined with a time constant of about 100msec. In separate experiments including the removal of this Ca?* feedback, the response of a primate rod to a dim flash was found to increase by 2-3 fold, accompanied by an increase in the time-to-peak of the response. These changes can be broadly reproduced by a quantitative model of phototransduction that has the Ca2*+ feedback as one of its features. Finally, the adaptation to background light that we have observed in these cells most likely also arises from the Ca2* feedback. Physiology 1235 APPEARANCE OF IMMUNOREACTIVE PHOTORECEPTOR AND BIPOLAR CELLS IN THE NEWT RETINA DURING REGENERATION M.Niino and T.Saito. Inst. of Biol. Sci. Univ. of Tukuba, Ibaraki I It is known that the adult newts possess the ability to regenerate a functional retina following the complete removal of the original retina. The order of appear- ance of photoreceptor and bipolar cells during retinal regeneration was studied immunohistochemically. A mouse monoclonal antibody (RB-1l), specific for photorecep- tors (cone) and bipolar cell subtypes in adult newt, was generated. RB-1l immuno- reactive photoreceptors appeared in the regenerating retina before separation of two plexiform layers. The bipolar cells appeared shortly after the separation of the plexiform layer. RB-l-labelled bipolar cells were characterized by the soma lying in the middle or slightly distal level in the inner nuclear layer and the axon terminal ending at the most proximal level of the inner plexiform layer (IPL). Intracellular recording and dye injec- tion in the retinal cells showed that ON bipolar cell axons terminate in the inner half of the IPL, while OFF bipolar cells terminate either in the outer half or in the inner half. Combining intracellular staining techniques with immunohisto- chemistry suggested that RB-l-labelled bipolar cells are not ON bipolar cells, but OFF bipolar cells. GLUTAMATE RECEPTORS OF SOLITARY SPIKING CELLS IN THE NORMAL AND REGENERATING NEWT RETINA. C.Chiba, H.Sakai and T.Saito. Inst. of Biol. Sci. Univ. of Tukuba, Ibaraki. Glutamate receptors of solitary spiking cells isolated from the newt (Cynops Pyrrhogaster) retina were studied with whole-cell patch-clamp techniques. The solitary spiking cells responded to pres- sure application of L-glutamate with either depolarization (80%) or hyperpolar- ization (10%). The glutamete-induced depo- larizing response initiated spikes and hyperpolarizing response inhibited spike initiation, indicating that there are at least two types of glutamate receptors, exitatory and inhibitory, in the spiking cells. The depolarizing response was associated with a decrease in input resistance and produced by inward Currents. The hyperpolarizing response was associated with an input resistance increase and produced by outward currents. Both inward and outward currents were reversed their polarity at about O mV. The adult newts possess the ability to regenerate a functional retina following the complete removal of the original retina. Appearance of glutamate response during retinal regeneration was studied. Solitary spiking cells in the regenerating retina have shown the depolarizing response to L-glutamate before segregation into distinct synaptic layers has begun. MODULATION BY L-GLUTAMATE OF CALCIUM ACTION POTENTIAL IN DISSOCIATED HORIZONTAL CELLS FROM CATFISH RETINA. K. -I. Takahashi and D. R. Copenhagen®. “Dept. Physiol., Keio Univ. Sch. Med., Tokyo and Depts. Ophthalmol. & Physiol., UCSF Sch. Med., San Francisco, CA, U.S.A. Dissociated horizontal cells produce regenerative action potentials(APs) when depolarized by currents injected through conventional microelectrodes. This AP results from a sustained high voltage—activated(HVA) calcium current that is activated positive to -40mV. L-glutamate(100uM) reduces the amplitude and the duration of the AP. Direct measurement of an intracellular pH([pH];) of the cells with BCECF-AM reveals that L—glutamate also acidifies in a dose-dependent manner. If L-glutamate modifies the AP via [pH];, then independent modification of [pH]; should similarly affect the AP. Acidification induced by application of Na-acetate(25mM) and washout of NH,Cl(20mM) dereased the amplitude and the duration of the AP, and alkalinization induced by application of NH,Cl exerted the opposite effects, as hypothesized. These results suggested that HVA calcium channel activity was suppressed by acidification of horizontal cells induced by L—glutamate. Horizontal cells in situ have a dark resting membrane potential between —40 and -20mV, which is above the activation voltage for the HVA current. Jn situ, extinguishing a light stimulus causes a release of L— glutamate from photoreceptors and depolarizes horizontal cells from a maximum potential, which can be as high as -80mV, to the normal dark potential. No sustained APs are seen under these conditions. We hypothesize that in situ the regenerative AP is suppressed by acidification induced by L-glutamate released from photoreceptors. VISUAL RESPONSE OF NON-SPIKING GIANT INTERNEURONS IN THE BRAIN OF CRAYFISH H. FURUDATE, Y. OKADA AND T. YAMAGUCHI. Dept. of Biol., Fac. of Sci., Okayama Univ., Okayama There are five pairs of nonspiking giant inter- neurons (NGIs) in the protocerebrum of the crayfish. These NGIs have thick and long dendritic processes extending across the midline and are post synapticto the fourth order visual interneurons (SFs). Three pairs of NGIs (G1, G2, G3) running in a cluster responded with graded depolarizing and hyperpolarizing potentials to illumination on total surfaces of the ipsilateral and contralateral eyes, respectively. Although the amplitude of hyperpolarizing potentials recorded from the contralateral side of dendritic process was always larger than that recorded from the ipsilateral side, the relationship between the polarity of potentials and the recording sitesin a NGI remained to be solved. The most sensitive area to illumination occupied the posterior half of either eye. One pair (G4) of the remaining NGIs running along the cluster responded with graded depolarizing potentials to illumination on the anterior half of either eye and with graded depolarizing and hyperpolarizing potentials to illumination on the posterior half of the ipsilateral eye. These results imply that there are complex connections between the SFs conveying visual inputs from both eyes and the NGls. 1236 Physiology CELL-TO-CELL COUPLING BY GAP JUNCTIONS AMONG RETINAL AMACRINE CELLS OF A CYPRINID. S. Hidaka, M. Maehara, O. Umino, and Y. Hashimoto. Dept. of Physiol., Tokyo Wom. Med. Coll.. Shinjuku-ku, Tokyo 162 In the vertebrate retinas, responses to visual stimuli spread both vertically and laterally. Lateral spread of information is thought to be mediated by two major cell types, namely horizontal and amacrine cells. It is widely thought that all types of horizontal cells are electrically coupled in homologous sets, whereas, it is unknown whether all anatomical or physiological subtypes of amacrine cells are coupled or not. The present study was designed to answerwhat functional subtypes of amacrine cells are coupled. To address the question, we have measured tracer-coupling (Vaney, '91) among physiologically identified amacrine cells using a biotinylated compound, biocytin (Horikawa and Armstrong, '88), lower molecular weight (372Da) than the Lucifer yellow (457Da) which used previously to asses dye-coupling. Based on the responses evoked by a spot-stimuli (Chino and Hashimoto, ‘86), amacrine cells (n=90) identified in the retina of dace (Tribolodon hakonensis) were classified into seven distinct types. All types of amacrine cells showed extensive tracer-coupling to neighboring cells of the same cell morphologies by intracellular injection of biocytin. Patterns of the interconnections between coupled cells varied among the identified cell types. Localization of the innterconnected sites was strongly correlated with molphologies of the cell; soma shape, dendritic field size and stratification in the inner plexiform layer. Electron microscopic observation of the innterconnected sites revealed the presence of gap junctions. These studies demonstrate that homologous sets of amacrine cells are coupled by gap junctions and multiple clusters of such homologous sets are present on the whole retinal plane.Cell-to- cell coupling of homologous sets may occur depending on their retinal functions. Lamina-medulla projection in the compound eye in the cabbage butterfly. M. Shimohigashi and Y. Tominaga. Biol. Lab., Fac. of Sci., Fukuoka Univ., Fukuoka. The compound eye of a cabbage butterfly, Pieris rarae, has five types of spectral receptors (UV, violet, blue green and red ones). Violet, blue and green informations from each ommatidium are relayed to the lamina neurons by short visual fibers. The long visual fibers of UV and red receptors run through the lamina cartridge, terminating to the medulla. In the present study, we examined the projecting pattern of neural processes from lamina to medulla by light and electron microscopies. Axons of lamina neurons terminated at the distal level in the outer layer of the medulla. Whilst, the long visual fibers of UV receptors showed no synaptic profiles in the outer layer and had lateral branches at the middle level of the medulla. Six types of medulla neurons were found. Four of them extended their axons to the lobula complex, but the two others projected directly to the higher visual center. On the basis of the level of dendritic processes of the medulla neurons, it was supposed that spectral informations relayed by the lamina neurons are transmitted to the lobula complex via the medulla neurons, and that spectral information from UV receptor is sent directly to the posterior slope of the protocerebrum by the medulla giant neuron. POSTEMBRYONIC DEVELOPMENT OF THE OPTIC LOBE OF THE BUTTERFLY: FATE OF THE LARVAL VISUAL INTERNEURONS. T. Ichikawa. Department of Biology, Faculty of Science, Kyushu University, Fukuoka. The larval optic neuropile of the swallowtail butterfly Papilio xuthus has two distinct area, lamina and medulla, and is enclosed by two imaginal disks which are composed of neuroblasts and develop into an imaginal optic lobe dunng metamorphosis. Fate of the larval optic neuropile and larval visual interneurons have been examined by using the reduced silver impregnation method and the immunohistochemistry of y-amino butyric acid (GABA). The larval visual interneurons in the lamina could not be followed during metamorphosis, but neurons in the medulla could be followed throughout pupal development because their somata were always significantly larger than those of other neurons differentiating from the neuroblasts. The larval medulla neuropile was remained as a small frontal protrusion of the imaginal medulla. There are about 100 neurons in the larval medulla. Several neurons were immunoreactive to the GABA antibody and extended dendntic processes to the larval lamina and medulla. The medulla neurons lost those processes at the earliest stage of the pupal development and differentiated many new processes that invaded the developing imaginal medulla. The processes covered a large area of the neuropile. As the neuropiles of the optic lobe increase in volume, the dendritic processes of the GABA neurons increased in length and density. Axons of the neurons run down an optic tract between the labula and the lobula plate and extended to the lateral area of the proto- cerebrum. The results demonstrate that at least some larval visual interneurons are reorganized into imaginal optic lobe and play a new role in the visual processing of the adult butterfly. DISTRIBUTION OF GABA-LIKE IMMUNOREACTIVITY IN THE BRAIN AND OPTIC NEUROPIL OF INSECTS. X. Zhao, T. Ichikawa and Y. Toh. Dept. of Biol., Fac. of Sci., Kyushu Univ., Fukuoka. Localization of GABA-like immunoreactive neurons was examined in the brain and visual system of three species of insects: adult cockroaches (Periplaneta americana), swallowtail butterfly larvae (Papilio xuthus) and tiger beetle larvae (Cicindela chinensis). In the cockroach compound eye visual system, some medulla neurons and some fibers in the medulla neuropil were GABA- like immunoreactive. Possible centrifugal fibers originating in the medulla neuropil were’stained, but lamina neurons were not stained. In the dorsal ocellar system it was reported that possible efferent neurons might be GABAergic and their terminals contained dark vesicles together with clear vesicles. In the present study GABA-like immunoreactivity occurred in those efferent neurons. In the larval visual system of the swallowtail butterfly and the tiger beetle some medulla neurons were GABA-like immunoreactive, but no lamina neurons were immunoreactive. In both larvae possible efferent fibers were GABA-like immunoreactive. These data suggest GABA may play some _ roles in efferent control as well as in inhibitory effects of the insect visual system. Physiology DESCENDING INTERNEURONS IN THE COCKROACH PERIPLANETA AMERICANA. S.Watanabe and Y.Toh. Dept. of Biol., Fac. of Sci., Kyushu Univ., Fukuoka. Descending interneurons, which possess cell bodies in the brain, were examined in the cockroach by intracellular recording and staining. Microelectrodes were impaled at the cervical connective, and responses of interneurons to several sensory stimuli were recorded. These interneurons were classed into nine types on the basis of effective sensory stimuli. Types 1-3 neurons responded to ocellar illumination and wind to cerci and antennae. Types 1 and 2 neurons responded to illumination to ipsilateral and contralateral ocelli, respectively, whereas type 3 neurons responded to illumination to either ocellus. Types 4 and 5 neurons responded to ocellar illumination only. Type 5 neurons also responded to moving stimuli. Type 6 neurons responded to compound eye illumination and wind to cerci and antennae. Other three types responded to wind to cerci and/or antennae, but not to illumination. Of 114 neurons recorded, 108 neurons responded to wind, and 86 neurons responded to ocellar illumination. These data suggest that information about wind detected by antennae and cerci is integrated together with photic information detected by ocelli in the brain, and such integrated information may descend to the thoracic motor center to modulate locomotion pattern of the animal. BLEACHING OF THE DARK BROWN PIGMENT GRANULES IN THE BUTTERFLY EYE Y. Shimazaki, E. Eguchi, Dept. of Biol., Yokohama City Univ., Yokohama Pigment cells in the compound eye of a butterfly Papilio xuthus contain dark brown pigment granules. It is said that these pigment granules seal the light and produce light- path to retina, so that each ommatidium is optically isolated. This function of pigment granules is controlled by the pigment migration according to light and dark adaptations. Now, we present bleaching of these pigment granules themselves by light. Firstly, Both light and dark adapted eyes were fixed, dehydrated and embeded. 4 jm thick sections through a longitudinal optical axis of the ommatidia were made. The color of pigment granules in the light adapted eye was lighter than that in the dark adapted eye. The color of pigment granules in the dark adapted eye become pale within 30 min by light irradiation. Secondly, unfixed eye was frozen in liquid nitrogen, sectioned and observed with light microscope. The dark brown pigment granules become pale by light irraduation. This phenomenon was retained after eye was fixed with 2% paraformaldhyde and 2% glutaraldehyde for 3 hr. Lastly, after the removal of the retina from the compound eye, these pigment granules were isolated from the eye cup by shaking in Ringer's solution and by sucrose gradient centrifugation. These pigment granules suspended in 60% sucrose were used as sample. Spectral absorbances of the sample were examined under anaerobic condition, before light was irradiated to the sample and after light was irradiated to the sample for 30 min. Absorbance (350nm - 800 nm) of post-light irradiated sample was lower than that of pre-light irradiated sample. These results indicate a part of dark brown pigment granules themselves is bleached by light. 1237 EFFERENT CONTROL IN THE ANTERIOR LATERAL EYES OF ORB WEAVING SPIDERS S. Yamashita. Biol. Lab., Kyushu Inst. of Design, Fukuoka. The light responses of the anterior lateral(AL) eyes of the orb weaving spiders, Argiope bruennichii and A. amoena were controlled by efferent optic nerve signals transmitted from a circadian clock in the brain. At night, the amplitude of ERGs to low light intensities increased, but those to intermediate and high light intensities decreased, i.e. the slope of the intensity-response curve for the “night state" was slighter than that for the "day state". Electrical stimulation of the optic nerve produced effects on the ERGs of the AL eye similar to endogenous efferent activity. Octopamine, a putative efferent neurotransmitter, also decreased the amplitude of ERGs to intermediate and high light intensities. It was suggested that the effect of efferent signals on the green spectral mechanism was greater than that on the ultraviolet mechanism. WAVELENGTH DISCRIMINATION IN THE JUVENILE GOLDFISH. K. Ohnishi. Dept. of Physiol., Nara Medi. Univ., Kashihara Wavelength discriminations of the juvenile and the adult goldfish were measured among three spectral sensitivity maxima (450, 525 and 625 nm) of the goldfish cones using a go/no-go task. They were trained to avoid an electroshock in such a way that they move from or stay in an illuminated side ac- cording to the difference in wavelength of discriminative monochromatic lights. To prevent the fish from discriminating the lights in terms of brightness, the intensity of each monochromatic light was adjusted to produce equal stimulus ef- ficiency for discrimination on the basis of active avoidance learning rate measured at various light intensities. Juveniles and adults showed good dis- crimination ability in 525 nm/625 nm discrimination, while only juveniles showed very poor discrimination ability in 450 nm/525 nm and 450 nm/625 nm discriminations. In contrast to adults, very few juveniles trained with the lat- ter stimuli could acquire discriminative performances. These results indicate that the blue-sensitive (or blue- perceptive) mechanism of juveniles is im- mature and thus the color vision in juveniles is different from that in adults in at least visible spectra. 1238 Physiology PHOTOPERIODIC RESPONSE IN DROSOPHILA MELANOGASTER : EFFECT OF VITAMIN A-DEFICIENCY A.Okada, I.Shimizu. Center for Ecological Research, Kyoto University, Kyoto. Females of the fruit fly,D. Melanogaster ,enter an adult ovarian diapause under short photoperiods at suitable temperature(10-13°C),and sensitivity to the diapause promoting conditions is restricted to the adult stage.It was reported that carotenoid-deprivation affected the photoperiodic response in some insect species.We reared D.melanogaster (Canton-S) on a carotenoid-deficient diet and a beta-carotene-supplemented diet,and compared the diapause incidences.The result was that flies on both diets showed little differences of the diapause incidences at short photoperiod(8L) between long photoperiod(16L),but that diapause incidences of the flies on the beta-carotene-supplemented diet were higher than those of the carotenoid-deficient flies. CIRCADIAN RHYTHM IN THE RESPONSIVE- NESS OF VISUAL INTERNEURONS IN THE CRICKET OPTIC LOBE K. Tomioka, M. Ikeda and Y. Chiba. Biol. Inst., Fac. Sci., Yamaguchi Univ. , Yamaguchi The longterm record of multiple unit neural activity from the optic stalk of the cricket Gryllus bimaculatus revealed that the sensitivity of visual interneurons to 15 min light pulses given every 2 hr fluctuates in a time of day dependent manner in both in situ and in vitro preparations. This indicates that the sensitivity of visual interneurons is under the control of the circadian clock in the optic lobe. Similar circadian changes can be observed in single neuronal recordings. We have so far functionally identified five types of visual interneurons in the medulla. All of them exhibited a circadian change in their responsiveness. Two units, which did not fire spontaneously had their responsiveness to light-on increasing either nocturnally or diurnally. The other three units had a spontaneous discharge that also changed in a circadian manner. One of the three units had responsiveness increasing with the increment of the spontaneous firing frequency, while in the others the response to light pulses reversed from excitatory to inhibitory as the spontaneous activity increased. The results suggest that the circadian clock modulates not only the sensitivity of interneurons but also the visual information processing in the optic lobe. Rhythmicity of chromophore turnover of visual pigment in the Antarctic amphipod Qrchomene plebs (Crustacea; Amphipoda). T.Hariyama , A.Terakita*, and V.B.Meyer-Rochow?. IResearch Center for Applied ImEouMaL Lon Sciences, Tohoku Univ., Sendai, “Biological Inst., Facul- ty of Edu., Oita Univ., Oita, and Exp.Zool.& E.M., Univ.of the West Indies, Kingston, Jamaica 1.Retinal amounts in the compound eye of the Antarctic amphipod Orchomene plebs were assessed during conditions of continuous summer daylight every 3 hours over a period of 48 hours. The habitat of the experimental animal is the bottom of the Ross Sea (78°S; 166°E) down to depths of at least 400 m; water tempera- ture is a constant -1.8°C. A periodici- ty of twelve hours, not of twenty-four hours, was detected with relative amounts of ll-cis retinal exhibiting peaks at midday and at midnight and troughs at 7:00h and 19:00h. 2.The result that 90% of retinoid were insolu- ble in n-hexane suggests that at least 90% of the measured retinoid were at- tached to membrane-bound proteins such as opsin. 3.Selective light adaptation showed that the visual pigments were thermostable and photoregenerable. The Main absorbance peak of rhodopsin, compared with metarhodopsin, seems to be in the longer wavelengths. Detachment behavior and enhanced respiration in adult Japanese lampreys. R. Kawasaki Biomed.Technol. Niigata Univ. Niigata, 951 Japan. Combination SF attachment and detachment (initiation and elimination of sucker vacuum is necessary to lamprey feeding, nest-build- ing and mating. Anesthetized and spinalized lampreys were fixed in water at 10 C. The sucker pressure, nasal respiratory pressures and branchial EMGs were simultaneously recorded.To initi- ate detachment behavior, tactile (needles or bars) or electrical (1V,lms) stimulation was applied dorsally to the skin of the sucker funnel. The criterion for the initiation was the elimination of the pressure-negativity in the sucker cavity.The sucker was fixed by pinning it to the plate to avoid detachment, so the sucker pressure was constantly moni- tored throughout the experiment.The stimula- tion initiated enhanced respiration (syn- chronous augmentation of the EMGs and respi- ratory nasal pressure) and synchronous clos- ings (contraction of the constrictor mus- cles) of all branchial pores,and immediately followed by the decrease or elimination of sucker vacuum.The findings indicate that the enhanced respiration produces rapid movement of water from the gill pouches into the sucker cavity to eliminate sucker vacuum.The rapid detachment to escape from an unpleas- ant place may occur without involvement of annular muscles or HS. In conclusion, the functional significance of so-called "vigor- ous respiration" may not only be "cleaning or coughing" but also detachment for escape. This kind of excitatory activity of the mus- culatures which surround “branchial intes- tine" is not only in lamprey's behavior but also commonly in ‘ejection reflex' in sea squirts, salps and lancelets. Physiology 1239 THE ORIENTATION TO FOOD STIMULI BY THE TENTACLES OF THE PULMONATE SNAIL, Indoplanorbis exustus. M.Fujimoto, N.Ueda, M.Hayashi, H.Kunioka and H.Ohtani. Zool. Inst., Fac. of Sci., Hiroshima Univ., Higashi-Hiroshima. The role of tentacles on the freshwater pulmonate snail, /ndoplanorbis, for its orientation to food substances was studied. The animal was perpendicu- larly fixed in position in the experimental chamber by holding its shell on a ping-pong ball which can rotate as the animal creeps on it. This setup allows us to determine the direction of movement. Each tentacle was set in the right and left compartment of the chamber and food substance (lettuce leaves, 1 gr) was set 3 cm before the animal in either compartment. About 75 % of the intact animals showed a distinct turning response. and 85 % of them turned to the direction of the food. After amputation of either one or both tentacles, the proportion of the snails that turned in the correct direction decreased (60-80 %). The values, however, showed that they can reach food substance without the aid of tentacles. The application of lettuce extract on the tip of a tentacle caused a ipsilateral turning response. A more rapid response was also observed even from snails without tentacles when the extract was applied to the basal region of a tentacle. These results suggest that the tentacles are involved in food orientation using them for tropotaxis and klinotaxis. However, the receptor site might be located elsewhere besides the tentacles. THE ORIGIN OF AXOTOMY-INDUCED LONG-LASTING FIRING IN AN IDENTIFIED MOTONEURON OF THE CRAYFISH. A. Muramoto. Fukushima Biomed. Inst. of Environm. & Neopl. Dis., Futaba-gun. My previous experiments have demonstra— ted that transection of the abdominal nerve cord along its long-axis at any level below the 1st abdominal ganglion (Al) can always induce a prolonged excitability in an iden- tifiable motoneuron (AML) of the crayfish. The origin of this long-lasting firing (LLF) and its generation site were investigated in this study. It was found that the LLF immediately stopped by application of a co- untercurrent (bucking voltage) to the nerve cord posterior to the transection site, while a current (depolarizing pulse) could induce the LLF in the quiescent state of AML during the stimulation. Raising the stimulation strength increased the frequen- cy durino stimulation-induced LLF in AML. There was no remarkable difference between axotomy- and stimulation-induced LLFs. Depolarizing pulse to the connective above Al failed to induce a LLF in AML, but this pulse below Al always could produce such a response. It was then concluded that a prolonged firing of AML following transection must be due to axotomy of AML itself, in which its ascending axonal process runs through the nerve cord, ending to Al, and must result from a depolarization which formed in its transection site. NEUROMUSCULAR TRANSMISSION IN THE RED MUSCLE OF A PECTORAL FIN OF MUTSUGORO. T. Hidaka’, K. Kusano“, M. Tanaka“ and Y. Matsuki2. Dept. of Biol., TPac. of Gen. Edu. and 2Fac. of Sci., Kumamoto Univ., Kumamoto. Mutsugoro, Boleophthalmus pectinirost- ris, creeps around using a pectoral fin on mud flats at low tide but stays in a bur- row in the mud at high tide. A pectoral fin muscle of mutsugoro consists of the red muscle fibers. Some properties of the neuromuscular transmission were studied by the electrophysiological methods in this muscle. The single nerve stimulation elic- ited three types of junction potential, excitatory junction potential (ejp), inhi- bitory junction potential (ijp) and dipha- sic junction potential (diphasic jp). An initiation of ejp was more dominant than ijp and diphasic jp. The action potential was not initiated, even after the summa- tion of ejp was produced by the repetitive nerve stimulation. Three types of junction potential were augmented by anticholines- terase, neostigmine, and were suppressed by nicotinic ACh antagonist, d-TC. A mus- calinic ACh antagonist, atropine, was less effective than d-TC. ACh and ACh agonist, Carbachol,.also decreased all types of jun- ction potential. From the results, it was suggested that the neurotransmitter of this muscle may be ACh and the nature of the ACh receptor may be nicotinic. SPATIAL DISTRIBUTION AND ELECTROPHYSIOLOGI- CAL CHARACTERISTICS OF NEURONS ASSOCIATING WITH SPERMATOPHORE-FORMATION IN MALE CRICK- ET, TELEOGRYLLUS COMMODUS . A.Nakanishi and N.Ai. Dep.of Biol., Tokyo Gakugei Uni., Koganei, Tokyo. In adult male crickets,their cercal muscles (M1) ,muscles linked to reproductive organs (accessary glands, epididymis and ejacula- tory duct) (M2) and phallic muscles (M3) are respectively innervated by N9-1,2 and 3 branch from the 9th lateral root (N9) in the terminal abdominal ganglion (AG-V). Each branch has bilaterally about 6(N9-1) or 10 (N9-3) somata of motor-neuron in the postero- lateral region of AG-V. But in N9-2, 130 or more somata are appeared along the midline of AG-V with the bifurcating processes ex- tend symmetrically to both sides. Consider- ing from results of vital and back-fill staining,these neurons would be DUM ones for M2 muscle group. However,only 4 to 6 somata of them have been stained with their axon processes extended to only one side of the root looks like motor-neuron. By electrical activity in N9 branch, effer- ent spikes are recorded with higher freq-— uency and different amplitude in N9-2 than the others. And by electrical responses are appeared as more long lasting contraction in M3 (N9-3) than in M1 (N9-1) -. N9-2 root has in- nervation to muscle group with automatic and continuous contraction and N9-1 root with temporaly contraction for cercal muscle. 1240 Physiology RADULAR MECHANOSENSORY NEURON IN _ THE BUCCAL GANGLIA OF THE, TERRESTRIAL SLUG. .Kawahara’, M.Yano2, and H.Shimizu’. Fac. of Paamuecettleall Sei, Uniiven ot Tokyo, Tokyo, Res. Inst. of Electrical Communication, Tohoku Univ., Sendai. A radular mechanosensory neuron, RM, was identified in the terrestrial slug, Inci- laria fruhstorferi. A pair of RMs were located symmetrically on the posterior edge of the dorsal surface of the buccal ganglia. Fine neurites ramified within the buccal ganglia. Main neurites exited from buccal nerve 3s (n3) and entered the subradular epithelium. When the posterior part of radula was distorted by bending, afferent spikes of RM, which were preceded by the axonic spikes recorded at n3, were observed. his response remained after removing Ca from saline. RM was suggest-— ed to be a primary mechanoreceptor. When rhythmic buccal motor activity was evoked by food application or nerve stimulation, RM received excitatory input in the rasp phase, in which the supramedian radular tensor (smt) was active. Afferent spikes of RM, evoked by electrical stimulation to the radula, broadened synchronously with this phasic input. Spike broadening of RM resulted in an increase of the amplitude of excitatory postsynaptic potential in a motoneuron which projected to smt. These results suggested a phase-dependent senso- ry modulation in the feeding system of the slug. DISTRIBUTION OF CATECHOLAMINE-CONTAINING NEURONS IN THE CEREBRAL GANGLIA OF THE SLUG, INCILARIA FRUHSTORFERI. Y.Makino!, S.Kawaharal!, M.Yano2, H.Shimizu!. 1Fac. of Pharmaceutical Sci., Univ. of Tokyo, Tokyo, 2Res. Inst. of Electrical Communication, Tohoku Univ., Sendai. The distribution of catecholamine-containing (CA) neurons in the cerebral ganglia of the slug Incilaria fruhstorferi was studied using the formaldehyde- glutaraldehyde and glyoxylic acid histofluorescence methods. Approximately 200 CA neurons were observed in each hemiganglion. Most of CA neurons had small cell bodies (5-20 j1m in diameter), which were aggregated into five discrete clusters. Several larger CA neurons (30-50 tm in diameter) were scattered mainly on the dorsal surface. CA nerve fibers were observed in the neuropile region, cerebral commissure, and all cerebral nerve roots. In the somatic region of the procerebrum (PC), there were extrinsic CA _ fibers, which had distinctive varicosities. The extrinsic fibers entering the PC were derived from two thick bundles of CA fibers. One bundle was on the dorsal surface of the neuropile of the PC, and continued to tentacular nerve. This bundle consisted of the CA fibers from the cluster of small neurons located just around the medial edge of metacerebral giant cell and two dorsal large CA neurons. The other bundle was in the neuropile of anterio-ventral region of the cerebral ganglion. This bundle consisted of fibers from the ipsi- and contra- lateral clusters of small CA neurons located in close vicinity to the PC DISTRIBUTION OF SEROTONIN- AND FMRFAMIDE- LIKE IMMUNOREACTIVITY IN THE BRAIN OF SILK MOTH BOMBYX MORI. A.Iwamal, A.Nakagawa! and R.Kanzaki2. 1Tsukuba Research Center, SANYO Electric Co., Ltd., Tsukuba and 2Inst.of Biol.Sci., Univ. of Tsukuba, Tsukuba. Distribution of serotonin- and FMRFamide-like substances was respectively studied in the brain of silk moth Bombyx mori using immunohistochemical technique. Approximately 180 Serotonin-like immunoreactive(SLI) somata were found in the brain. Most of them were located in the optic lobe and median protocerebrum and some of them were found in the deutocerebrum and suboesophageal ganglion. FMRFamide-like immunoreactivity (FLI) was detected in more than 800 somata in the brain. These were found in the optic lobe, median protocerebrum, deutocerebrum and suboesophageal ganglion. Both SLI and FLI fibers were distributed extensively throughout the neuropil areas, especially, the optic lobes, antennal lobes, mushroom bodies, lateral accessory lobes, and central body. It was reported that the antennal lobes, mushroom bodies and lateral accessory lobes were included in the pathway of the pheromonal information processing. These results suggest that serotonin and FMRFamide might serve several functions as neurotransmitters or neuromodulators in the nervous system related to the mating behaviour. NEURONAL ARCHITECTURE OF THE MUSHROOM BODY IN THE HONEYBEE BRAIN AS REVEALED BY FMRF- AMIDE AND SEROTONIN IMMUNOHISTOCHEMISTRY. A.Iwama!, A.Nakagawa!, A.Mizukamil AND T. Shibuya2. !1Tsukuba Reseach Center, SANYO Electric Co., Ltd., Tsukuba and ?Inst. of Biol. Sci., Univ. of Tsukuba, Tsukuba. The mushroom bodies are prominent structures in the honeybee brain. To know their functional organization, we have studied the distribution of FMRFamide- and serotonin-like substances in the mushroom bodies of the honeybee brain using immuno- histochemical technique. Each mushroom body neuropil consists of two calyces connected by two stalks to two lobes. These neuropil regions contain two types of neuronal elements, the intrinsic and the extrinsic fibers. The intrinsic fibers are stratified across the stalks and lobes, based on their cell types. Serotonin-like immunoreactivitiy (SLI) was found at widespread extrinsic fibers in the lobes and stalks. While FMRFamide-like immuno-reactivitiy(FLI) was found in some layers of the lobes and stalks, equivalent to intrinsic fiber bundles. The somata and the dendrites in the calyces of intrinsic cells did not show FLI in the adult brain. However, in the pupal brain, the calyces and the somata located at the outersurface of the calycal neuropil showed FLI. The results represent the basic architecture of the mushroom bodies and suggest that FMRFamide and serotonin may serve important roles in the mushroom bodies. Physiology 1241 DEVELOPMENT OF THE CNS AND SEROTONIN- IMMUNOREACTIVE PROCESSES IN THE OPISTHOBRANCH MOLLUSC PLEUROBRANCHAEA JAPONICA. K. Ohsuga and K. Kuwasawa. Dept. of Biol., Tokyo Metropolitan Univ., Tokyo. We studied development of the CNS an distribution of serotonin-immunoreactive neurons during a period from the embryo to the young juvenile just after metamorphosis in Pleurobranchaea japonica. We observed the CNS with thin sections obtained from preparations embedded in Quetol-812 under a light optics. The basic shape in the CNS such as the cerebro- pleural, pedal and buccal ganglia and the rudiments of the rhinophore and oral veil ganglia was settled during the veliger phase. The rhinophore and oral veil ganglia were extruded from the cerebro- pleural ganglion after metamorphosis. Processes serotonin-immunoreactive to anti-serotonin antiserum were found in thick paraffin sections and whole mount preparations by means of PAP and FITC method for, respectively, paraffin sections and whole mounts. At the veliger stage serotonin-immunoreactive cells were observed in the cerebro-pleural and pedal ganglia. A cluster of immunoreactive cells was found on the larval cerebral commissure. The cluster sent processes along edges of paired velar lobes. After metamorphosis the cluster on the cerebral commissure disappeared. It is likely that the cluster was a specific neural organization for the veliger. RESPONSE OF OSCILLATORY FIELD POTENTIAL TO SOME CONDITIONED ODORS IN SLUG'S BRAIN. T.Kimura, H.Suzuki, A. Yamada, T.Sekiguchi and A.Mizukami. Tsukuba research center, SANYO ELECTRIC Co. LTD., Tsukuba. To understand the mechanisms of olfactory recognition and leaning, we analyzed odor- information flow and its expression in the slug's brain using morphological and physiological technique. An odor information caught by numerous sensory cells on tentacle tip was transferred to input mass of pro-cerebral lobe and meso-cerabral robe of brain via tentacle ganglion. The pro-cerebral lobe (PCL) was divided morphologically into three mass (cell mass, input mass and output mass). The cell mass was a cluster of cell body of intrinsic neurons, of which process elongated into output mass passing through input layer. Local field potential (LFP) was recorded from the cell mass or output mass of PCL on tentacle-brain preparation which dissected from the body. When the appetitive-conditioned odor was applied on the tentacle tip, the frequency of LFP increased during Stimulation. However, application of aversive- conditioned odor decreased the frequency. Similar results were obtained on the output-mass of PCL- tentacle preparation which was dissected from meso- cerebral lobe. These observations suggested that an olfactory stimulus sensed on the tentacle tip was transferred into PCL and recalled the memory associating with the odor in it. PROPERTIES OF DUAL EXCITATORY INNERVATION OF THE UROPOD MUSCLES IN CRAYFISH. T. Higuchi. Dept. Gen. Edu., Higashi-Nippon-Gakuen Univ. , Ishikan-Tobetsu, Hokkaido. In crayfish uropod, neuromuscular responses in muscle fibers of both tonic and phasic muscles which received dual excitatory innervation were observed in order to know the functional relationship between the two excitatory motor neurons. All of the muscle fibers investigated in such muscles exhibited no characteristic difference in their innervation pattern and in their neuromuscular responses. This suggested that the two excitatory motor neurons within the muscles equally distributed their terminals to all muscle fibers. In tonic muscle fibers, it appeared that two simultaneously observed trains of excitatory junctional potentials, which accurately reflected the activity of each excitatory motor neurons, were independent from each other, both in the absence and the presence of stimulations. The phenomena showing the direct or close interaction between the two excitatory motor neurons were not observed either centrally or peripherally except for the simple summation of the excitatory junctional potentials induced by the activity of individual excit- atory motor neurons. In phasic muscle fibers, whose excitatory motor neurons were normally silent, no peripheral interaction between the two excitatory motor neurons was also suggested by the observation of excitatory junctional potentials and active responses induced by selective electrical stimulation of individual neurons. Central interaction, however, is unknown. An interest- ing feature was the different action of the individual excitatory motor neuron which innervated the adductor exopodite. Postsynaptic events induced by one of the two excitatory motor neurons was apparently rapid fatigue, while the action of another was constant. The meaning of this difference needs to be resolved. It is likely that individual excitatory motor neurons act independently on demands of different behavioral performances. NONLINEAR ANALYSIS OF CERCAL SENSORY PATHWAY T.Shimozawa, Y.Baba and T.Shimizu. Lab. of Neuro-Cybernetics, Res. Inst. for Electronic Sci., Hokkaido Univ. Sapporo. Signal transmission pathway to giant interneuron 8-1 in the cricket cercal sensory system was clarified by using Wiener's white noise analysis. Air current stimulus was modulated with a Gaussian white noise signal of 500 Hz band width. Responses of the interneuron were recorded by intracellular electrode. The stimulus and response waveforms were stored on a digital-audio-tape recorder and collected into a workstation through GPIB interface. The linear signal transmission was estimated from the cross-correlation between stimulus and response. The 2nd order cross-correlation between stimulus white noise at two different times and response indicates the contribution of 2nd order nonlinearity to the signal transmission. Nonlinear signal transmission gave a good clue to determine the sequence of processing element in signal flow to the interneuron. Interneuron 8-1 was revealed to receive bilateral inputs. Both showed strong amplitude saturation but with different threshold. The high threshold input has 1 ms delay and was subtracted from the other at the interneuron. Subtraction of delayed signal helps to detect the rate of change of stimulus. 1242 Physiology Input pathway of cricket cercal nonspiking interneuron revealed by Wiener's white noise analysis. Y. Baba, T. Shimizu and T. Shimozawa Lab. of Neuro-Cybernetics. Res. Inst. for Electronic. Sci., Hokkaido Univ. Sapporo. Cercal sensory nonspiking interneuron, LNI-7 was studied by using Wiener's white noise analysis. Cerci were stimulated by an air current modulated with a Gaussian white noise. Band width of the white noise was 500 Hz. The first- and second- order Wiener kernels were extracted by cross-correlation between the input white noise and neural response. The extracted kernels predicted the actual neural response with mean square error of 58% in 1st order model and of 50% in 2nd order model. The first-order kernel is tri- phasic waveform with 6msec latency. The second-order kernel had elongated nine eyes which indicates a neural smoothing after a nonlinior element. At strong stimulus, small nine eyes before the elongated nine eyes appeared. When cercus contralateral to the soma was covered, the small nine eyes disappeared, whereas the first-order kernel hardly changed. The data suggest that LNI-7 has a direct, inverse high threshold small amplitude input from the contralateral cercus. CENTRAL PROJECTION OF THE COLD RECEPTOR AXON ONTO THE DEUTOCEREBRUM OF THE COCKROACH. M. Nishikawa, F. Yokohari and T. Ishibashi. Biol. Lab., Fac. of Sci., Fukuoka Univ., Fukuoka There are two types of thermoreceptive sensilla on the antenna of Periplaneta americana. One type of the sensilla contains four receptor cells: a cold, a dry ,a moist and an unknown cell. The other has a cold receptor and two or three olfactory receptors. The central projections of these receptors were examined by extracellular staining of these axons and by reconstruction of the plastic sections. One of the axons from each sensillum, which is in proximal part of the flagellum, projects into the ordinary glomerulus in the ventro-posterior and marginal region of the antennal lobe. These axons are suggested to belong to the cold receptors. The putative hygroreceptor axons project into the ventro-posterior glomeruli closely located to the cold receptor axon terminal. Two or three axons, projecting to the ventro-medial glomeruli, are suggested to belong to the olfactory receptor cells. Cold-sensitive output interneuron, which was excited by the cold air stimulation on cold receptors in the medial part of an antenna, innervated a single glomerulus in the ventro-central region of the antennal lobe. This glomerulus is different in its location from the glomerulus where the cold receptor axon terminated as mentioned above. From these observations we assumed that the cold receptor axons project into several different glomeruli corresponding to the location of the cold receptors on the antennae. EFFECTS OF NEUROTRANSMITTERS ON DUM NEU- RONS IN THE COCKROACH THORACIC GANGLIA. H. Washio. Lab. Neurophysiol. Mitsubishi Kasei Inst. Life Sci. Tokyo Effects of putative neurotransmit- ters, L-glutamic acid, gamma aminobutylic acid (GABA) and acetylcholine (ACh), on the excitability of dorsal unpaired median (DUM) neurons in the thoracic ganglia of the cockroach, Periplaneta americana, were investigated using intracellular recording technique. Bath application of these neurotransmitters modified the excitabili- ty of the DUM neurons associated with the membrane conductance change. L-glutamic acid caused a strong, but ACh had a week desensitization of the firing activity, while GABA had an inhibitory action on the excitability. No appreciable changes in Membrane potential were elicited with bath application of these neurotransmitters at the concentration of 107°M, in spite of modification of the excitability. However 10-4M L-glutamic acid caused a transient depolarization and 10-4M GABA caused a long-lasting depolarization. The results are compatible with central nervous synap- tic transmission onto thoracic DUM neurons being mediated by L-glutamic acid, GABA and ACh. DIFFERENTIATION OF DUM NEURONS ON THE LAST ABDOMINAL GANGLION IN FEMALE CRICKET, TJTELEOGRYLLUS COMMODUS. T. Ai and N.Ai. Dept.of Biol. Tokyo Gakugei Univ., Koganei, Tokyo. We have already presented that five clusters of DUM neuron in adult female cricket are oriented on the dorsal surface of the last abdominal ganglion (AG-\). The neurons of four clusters (C-1, 2,3 and 4) innervate to both the common oviduct and the lateral oviduct and the other neurons located in the fifth cluster (C-5) innervate to the tergal muscle (M-4), the intervalvular muscle (M-5,6), the targosternal muscle (M-7) and the common oviduct. In adult stage, these DUM neurons are stained by neutral red which is well known to stain monoaminergic neuron specifically. However, in the last larval inster (LLI) stage, all of five cluster on DUM neuron in AG-V) fully innervate as the same manner as that of adult, and so the DUM neuron of C-1,2 and 3 in LLI stage are stained by neutral red, but these of C-4 and C-5 are not clearly stained in some of them. Spontaneous activity of DUM neurons in LLI stage are enough recorded as same as that in adult preparation In other hand, we observed on preparat- ion in LLI stage that M7 fiber has been fully observed its striation and evoked contraction yield by electrical stimula- tion to root N7-1, but MS fiber not clearly observed its striation and evoked one also not yield Physiology 1243 MORPHOLOGY AND PHYSIOLOGICAL PROP- ERTIES OF DORSAL UNPAIRED MEDIAN (DUM) NEURONS IN THE CRICKET TERMINAL ABDOMINAL GANGLION. B. Chen and T. Yamaguchi. Dept. of Biol., Fac. of Sci., Okayama Univ., Okayama. Numerous DUM neurons are located in several clusters in the terminal abdominal ganglion (TAG) of the cricket. The morphology and physiological properties of individual DUM neurons in each cluster were studied by intracellular recording and dye injection techniques to reveal their functional roles in the sensory information processing or motor pattern generation in the TAG. So far as we have examined, various DUM neurons was divided into the type-1 and type-2 neurons. Most of the DUM neurons were the type-1 neurons, each of which bifurcated a pair of axons running through one of the nerve roots in each side and innervating the visceral muscles. This type of DUM neurons showed spontaneous impulse discharges with a high frequency, but scarcely responded to wind stimulation of the cerci and to electrical stimulation of the ventral nerve cord. The remaining neurons were the type-2 neurons, each of which bifurcated two or three pairs of axons running through two or three nerve roots in each side and innervating the skeletal muscles. These of DUM neurons responded to wind stimulation as well to electrical stimulation. Stretch Receptor Organs in the Thorax and Abdomen of Ligia exotica (Crustacea, Isopoda) Y. Takatsuki, A. Niida and T. Yamaguchi. Dept.of Biol., Fac. of Sci., Okayama Univ., Okayama Two types of stretch receptor organs(SRO) were bilaterally found through the thorax to the abdomen except for the 2nd thoracic and 6th abdominal seg- ment. Such an SRO comprised a single receptor muscle and two morphologically distinct sensory cells, each having a club-shaped dendrite or three-forked dendrites. The sensory cells with three-forked den- drites through all segments showed a slowly adapting response exclusively to stretch stimulus, while the sensory cells with a club-shaped dendrite(C-type sen- sory cell) in the abdomen responded with rapid adap- tation irrespective of degrees of stretch amplitude. Similarly the C-type sensory cells of the thorax exhibited a rapidly adapting response within the limits of physiological stimulation. However, a little larger degree than those of physiological stimulation produced grouping discharges in the C-type sensory cells throughout a maintained stretch. Concomitant impulse frequency of the grouping discharges increased with stretch amplitude. The same could be caused by injection of depolarizing current into the C- type sensory cells. These characteristics might be attributable to a transitional form between a slowly and rapidly adapting SRO in this animal. Dynamics of a Negative Feedback Loop Underlying Resistance Reflex of Leg Motor Neurones in the Locust Y. Kondoh! and P. L. Newland?, ‘Honda R&D Co. Ltd., Wako Research Center, Saitama and @Department of Zoology, Uni- versity of Cambridge, Cambridge, U.K. Imposed movements of the femoral-tibial joint of the locust metathoracic leg elicit a resistance reflex in the extensor and flexor motor neurones of the tibia. A femoral chordotonal organ (FCO) encodes movements about the femoral-tibial joint and provides feedback to the leg motor neurones that acts to main- tain a stable posture of the leg. We have analyzed the response dynamics of individual neuronal elements involved in this local reflex pathway (or feedback loop) by means of Wiener kernel methods. Intracellular recordings were made from FCO afferents and extensor and flexor motor neurones of the tibia while moving the FCO apodeme with a band-limited Gaussian white noise signal. First- and 2nd—-order kernels were computed by a cross-—correlation between the response and the white noise to define the input-output characteristics of the neurones. First- order kernels of the FCO afferents were of 6 basic types that encode position, velocity and acceleration in 2 directions. The 1st-order kernels of fast and slow extensor motor neurones were both of a low-pass type having an initial hyperpolarization followed by a small depolarization, whereas those of some slow flexors were also low-pass but had an initial depolarization. The 1st-order kernels of the fast and intermediate flexor motor neurones were differential, and initially depolarizing. Thus, the extensor and slow flexor motor neurones receive input from position sensitive FCO afferents, but with opposite directional responses, whereas the fast and intermediate flexors receive input from velocity sensitive afferents. PATTERN OF AXON GROWTH OF MECHANOSENSORY NEURONS OF MICROCHAETES ON THE DROSOPHILA NOTUM A. Usui, K.-l. Kimura. Laboratory of Biology, Hokkaido University of Education, Iwamizawa Campus, lwamizawa, Hokkaido We have followed the axon growth of microchaetes on the notum, using monoclonal antibody (Mab) 22C10 which labels neurons and accessory cells. The spatial pattern of neuronal pathways is highly reproducible (Fig.1). At 18 hr after puparium formation (APF), neurons of all microchaetes are recognized and they begin to send out axons at 20 hr-APF. At the beginning of Outgrowing, axons grow toward the particular direction. The elongating axon contacts and fasciculates with the axons of the nearest neighboring microchaetes. By 28 hr APF, all axons finish fasciculation among them, and they then follow the axons of macrochaetes and pre-existing cells, which continue to be labeled with Mab 22C10. The axon of microchaetes may easily reach distant target by following the axons of macrochaetes and pre-existing cells. 1244 Physiology MORPHOLOGY AND REGENERATION OF TENTACLE GANGLION IN SLUG, LIMAX FLAVUS. H. Suzuki, T. Kimura, A. Iwama, and A. Mizukami. Tsukuba Research Center, Sanyo Electric Co. LTD., Tsukuba. In this study we examined the morphological aspect of the regenerating posterior tentacle ganglion of the terrestrial slug, Limax flavus, to investigate the mechanism of reconstruction of neuronal networks in the ganglion. Its neural structure was studied by backfilling the tentacle nerve with hexamminecobalt chloride. The normal ganglion included 5 types of neurons. Four types of them projected axonal processes to the brain. Two types of the projected neurons shown FMRFamide immunoreactivity. The tentacle regeneration usually completed within about 1 month. That process was divided into 5 stages. At stage Ill renewal neurons elongated their neurites into the tentacle nerve. They had also FMRFamide immunoreactivity. These characteris-— tics were not observed at stage II. The present results suggest that the significant differentia- tions of the regenerated cells begin on stage Il. MORPHOLOGICAL CHANGES OF THE LATERAL LINE SYSTEM DURING T4—INDUCED METAMORPHOSIS IN THE NEOTENIC AXOLOTL, AMBYSTOMA MEXICANUM. H.-A.Takeuchi!, H.Uehara!, K.Yokota! and T.Nagai2 ‘4Dept. of Biol., Fac. of Sci., Shizuoka Univ., Shizuoka and “Dept. of PiMvsioil, 5 Weenie yoy Wai, Sela. Ox Mach s 5 Tokyo. The lateral line system functions as mechanosensor sensitive to small water displacement in many aquatic vertebrates, as well as electrosensor sensitive to weak electric field in some species. The degree of metamorphic change of the later— al line system is very different in the orders of the class amphibia: most anurans completely lose their sensory organs and nerves during metamorphosis, whereas most ulodeles show little changes. In the present study, we investigated morphologi- cal changes of the lateral line system during thyroxine(T4)-induced metamorphosis in the neotenic axolotl. T4 immersion (1.29x10-7M T4) or injec- tion (1.0nmol T4/gBW) completely metamor- phosed axolotls in 21-88 days. Their mechanosensory and electrosensory organs intensively regressed and disappeared around metamorphosis, whereas their later- al line nerves were retained. In the brainstem, transganglionically transported cobaltic lysine demonstrated the presence of the mechanosensory and electrosensory afferent fibers and the efferent neurons of the lateral line system at least 3.5 months after metamorphosis. MOTOR PATTERN GENERATION OF THE POSTERIOR CARDIAC PLATE _PYLORIC SYSTEM IN THE STOMATOGASTRIC GANGLION OF THE MANTIS SHRIMP SQUILLA ORATORIA. K. Tazaki. Biol. Lab., Nara Univ. Educ., Nara. Activity patterns of the constituent neurons of the posterior cardiac plate (pcp)-pyloric system in the stomatogastric ganglion of the mantis shrimp Squilla oratoria were studied by recording sponta- neous burst discharges intracellularly from neuron somata. These neurons were identified electrophysi- ologically, and synaptic connections among them were qualitatively analyzed. The pcp constrictor (PCP), pyloric constrictor (PY), pyloric dilator (PD), and ventricular dilator (VD) motoneurons, and the pyloric interneuron (AB) were involved in the Ppcp-pyloric system. They generated sequentially patterned outputs. Most commonly, the PCP neuron activity was followed by the activity of the PY neurons, and then by the activity of PD/AB neurons, and VD neuron. The motoneurons and interneuron were connected to each other either by electrical or by inhibitory chemical synapses, and thus constructed a characteristic neuronal circuit which was struc- turally similar to the homologous pyloric circuit of decapods. The network properties of the pcp- pyloric system in stomatopods were phylogenetically compared to those of the pyloric system in decapods. All the cell types could produce slow burst-forming potentials which led to repetitive spike discharges. Homologous cell types could be observed, but the number of neurons was variable. The main differ- ence between the two circuits was the synaptic connectivity among motoneurons: there were more electrical synapses in stomatopods, whereas more inhibitory synapses were found in decapods. AN OLFACTORY SYSTEM SPECIFIC PROTEIN IN SOCKEYE SALMON AND KOKANEE SALMON. M.Shimizul-2, H.Kudol-3, H.Uedal, A.Hara#, K.Shimazaki2 and K.Yamauchi3. 1lToya Lake Stn. for Environ. Biol., Abuta-gun, ?Res. Inst. of North Pacif. Fish., Hakodate, 3Dept. Biol., Hakodate, *Nanae Fish Cult. Exp. Stn., Kameda-gun, Fac. of Fish., Hokkaido Univ. We have begun to investigate actual functions of olfactory organs in the course of imprinting and homing behaviors in salmonid. Proteins restricted to the olfactory system (olfactory epithelium, olfactory nerve and olfactory bulb) and to the telencephalon of sockeye salmon and kokanee salmon (Oncorhynchus nerka) were compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol. Three protein bands of molecular weight 20, 24 and 40KDa were observed only in the olfactory system. A polyclonal antibody to a 24KDa olfactory protein was raised in a rabbit. The specificity of the anti- serum was examined by immunoblotting; the antiserum recognized only one 24KDa band in the olfactory system, which was absent from the telencephalon. Similar olfactory specific proteins of molecular weight 24KDa were recognized in both chum salmon (Q. kKeta) and masu salmon (Q. masou). Thus, all salmonids examined to date possess a 24KDa protein specific to the olfactory system. Physiology 1245 DIFFERENTIAL ODOR RESPONSIVENESS ON REEVE'S TURTLE ACCESSORY OLFACTORY BULB T. Hatanaka, Dept. of Biol., Fac. of Educ., Univ. of Chiba, Chiba. One hypothesis for coding of odor quality in vertebrates is that receptor neurons selectively sensitive to partic- ular odor properties are topographically segregated in the olfactory epithelium. Some evidences of topographical ordering in the olfactory bulb have been demon- strated in several species. In order to ascertain that the same olfactory discrim- inatory process was functioning in the accessory olfactory system, Reeve's turtles with developed vomeronasal system were studied. Induced wave responses. to several odorants were recorded from five points of accessory olfactory bulb (ante- rior, posterior, lateral, medial and intermediate portion of the AOB), and each response amplitude was compared. Responses at anterior portion of the AOB tended to be larger than those at other portions when iso-amyl acetate odor was applied. While, large responses were recorded at posterior portion to formic acid vapor and ammonium chloride solution. Weak responses to propionic acid odor had no tendency. So, possibility of spatial coding of odor quality on the turtle accessory olfactory system was also shown. PROPERTIES OF IP3-ACTIVATED ION CHANNEL IN FROG OLFACTORY RECEPTOR CELL MEMBRANE N. Suzuki Zool. Inst., Fac. of Sci., Hokkaido Univ. Sapporo To elucidate the role of IP3-activated ion channels in vertebrate olfactory transduction, I have studied the properties of IP3-activated ion channels in inside-out membrane patches excised from olfactory vesicle and soma membrane of frog olfactory receptor cells. In a microflow superfusion chamber, patches were perfused with low calcium intemal KCl-solution or KF-solution (Ca2+: 1x 10-8 M), in which 1 - 5.3 uM IP3 was dissolved. With Ba-external solution ( Ba2*+: 88 mM ), flickering channel openings were observed at luM IP3. The response latency varied from 24 to 1400 msec in different patches. The shortest latency, however, was comparable to that for cAMP-activated channel response to 1uM cAMP. The event histogram analysis revealed that the open time of the channels became longer in higher stimulus concentrations of IP3. The channel activity exhibited the adaptive nature and disappeared within SO - 60 sec when stimulated by 5.3 uM IP3. The unit conductance at - 85 mV(corrected for the offset potential) was about 20 pS. The analysis of success rate for recording of single channel activity in different patches from different membrane loci suggested that the channel density for IP3-activated channels was higher in olfactory vesicle membrane than in soma membrane, but was much lower than that for cAMP-activated channels. MEMBRANE CONDUCTANCES RELATED WITH DIVA- LENT CATIONS OF THE OLFACTORY RECEPTOR CILIA T. Nakamura, T. Sato and S. Miyamoto. De- partment of Applied Physics and Chemistry, The University of Electro-Communications, 1-5-1 Chofugaoka, Chofu-shi, Tokyo 182 We used the "ciliary patch" configura-— tion according to Kleen and Gesteland (1991a) to examine the total membrane con- ductance of the olfactory receptor cilium of the bull frog, and recorded the I-V curves during the application of various chemicals into the intracellular side of the cilium and integrated more than 8 curves to detect a small conductance change. First, we observed the chloride conductance gated by Canin as reported by Kleen and Gesteland (1991b), which was, however, not gated by Mg Then we used the chloride free solution to examine the blocking effect of divalent cations on the cyclic nucleotide gated channel. We found that the blocking by Ca‘t* is rather weak compared to that by Mgt. On the other hand, we could not observe the conductance change when we applied IP3 up to 50 uM into the cilium, which suggests that the electrical change induced by IP3 in the whole cell configuration reported by other group might not be due to the Ca influx Gin wey Cililsie. The overall contributions of these divalent cations to the membrane conductance in the cilia are still to be studied. CROSS-ADAPTED RESPONSES IN THE MOUSE TASTE CELL. K. TONOSAKI. Dept. of Oral Physiol., Sch. of Dentistry, Asahi Univ., Gifu. Taste stimulus adsorption is believed to occur at the taste cell microvillous membrane. Little is known about the mechanisms of taste transduction, due to the technical difficulties of inserting a glass micro electrode into the mammalian taste cell. I previously reported that the mouse taste cell response to a_ sucrose stimulus is a membrane depolarization accompanied by an increase in membrane resistance. The sucrose response increases in amplitude as the membrane is depolarized and decreases in amplitude as the membrane is hyperpolarized. The same taste cell responds to NaCl with an induced depolarization of the membrane potential accompanied by a decrease in membrane resistance. The NaCl response increases in amplitude as the membrane is hyperpolarized and decreases in amplitude as the membrane is depolarized. These results suggest that sucrose and NaCl have quite different response generation mechanisms. I now present evidence that sucrose pre- adaptation suppressed the cross-adaptation responses to NaCl. Intracellular recordings from receptor cells are appropriate to understanding cellular adaptation properties. 1246 Physiology ‘YHE EFFECT OF CYCLIC NUCLEOTIDES ON HE LABELLAR SALT RECEPTOR AND IlS ADAPTATION IN THE FLESHFLY. M. Koganezawa and I. Shimada. Dept. Biol. Sci., Tohoku Univ., Sendai. The common role of cyclic nucleotides has now become noticed in the molecular mechanism of sensory transduction. Amakawa et al. (1990) found a stimulating effect of dibutyl cyclic GMP (dbcGMP) on the labellar sugar receptor of the blowfly. Why did the effect restrict to the sugar receptor? We tested the effect of several nucleo- tides on the labellar taste receptors of the fleshfly and analyzed the adaptation process. One hundred mM dbcGMP was found to be the most effective on the salt re- ceptor, while other nucleotides such as cGMP, 8bcGMP and 5'-GMP were less effective. Adaptation curve of dbcGMP was obtained by adding that of adaptive cGMP and non-adap- tive 8bcGMP. Dibutyl cGMP may stimulate the salt receptor not only by binding to a nucleotide receptor site but also by per- meating the receptor membrane. The:tendency was Similar to the result of the sugar re- ceptor by Amakawa et al., but it was on the salt receptor in our experiment. These findings may suggest a common role as a second messenger in sensory transduction inside the sugar and salt receptor cells. COMPARATIVE STUDY ON SUGAR TASTE RECEPTOR PROTEIN CANDIDATES IN_THE FLY. M. Ozakil, ~. Amakawa*, K. Ozaki! and F. Tokunaga’, ~Dep. of Bigl BacemOte seis, Osaka Univ., Osaka and“Dept. of Sci. for Human Enwironment., Fac. of Human Develop- ment and Sci., Kobe Univ., Kobe, Japan. Taste organ in the labellum of the fly is in the shape of chemosensillum, which shows less complicated structure than the taste bud in vertebrate. The chemosensil- lum includes four functionally differenti- ated taste cells. One of them, called the sugar receptor cell, was electrophysio- logically proposed to have two different types of sugar receptor site, the furanose (F site) and the pyranose sites (P site). We previously found that starch and levan selectively compete with the stimula- tive sugars for the P and the F sites, re- spectively. By using these polysaccharides as the affinity ligands, we could isolate the candidate proteins for these sites from the labellar extract on the native gel. These proteins showed the consistent sugar-binding specificities and affinities with the corresponding receptor sites, re- spectively. They were also detected in the extract of the isolated chemosensillum, which exclusively includes the sensory processes of the taste cells as cellular components. The F site candidate protein was estimated to be 27kDa and the P site candidate protein which forms a single spot on the native gel separated into the twin spots of 31 and 32kDa with SDS-PAGE. RESPONSIVENESS OF XENOPUS CHEMORECEPTORS TO BITTER SUBSTANCES. S. Yamashita. Dept. of Biol., Coll. of Lib. Arts, Kagoshima Univ., Kagoshima. Integrated gustatory responses of the glossopharyngeal (IX) nerve to various alkaloids and dipeptides were recorded in the aquatic toad, Xenopus laevis. Responses to three alkaloids except for brucine, which showed the estimated threshold at 10 M, gwere first produced approximately ax 10 M and tended to saturate at 10 M. Cross adaptation coefficients calculated from possible six pairs of four alkaloids at concentrations chosen to produce equal neural responses suggested that both receptor domains which are stimulated by strychnine-HCl and quinine-HCl are similar or mostly duplicate, being rather different from those for papaverine-HCl and brucine. When pH values of four alkaloids of a given concentration were changed by adding HCl, each response magnitude for three alkaloids except for papaverine-HCl decreased with decreasing pH, reducing to 50 % level of the maximal response magnitude at pH 3. Of fifty dipeptides used five of strong stimulatory effectiveness were Pro-Trp, Gly-Arg, Pro- Tyr, Lys-Tyr and Arg-Ile, which contained hydrophobic or basic C-terminal amino acids, Present results suggest that hydrophobicity of the stimulating substance may be responsible for an interaction between the receptor domain and the bitter stimulus. THE EMETIC RESPONSE OF TELEOSTEAN FISH. T. Naitoh and T. Kitayama. Dept. of Biol., Shimane Univ., Matsue. From the standpoint of the comparative study of emesis, we examined the vomiting ability of fish. Both Silurus asotus, which has a large stomach, and Plotosus lineatus, which lacks a stomach, vomited in response to the intraperitoneal injection of centrally acting apomorphine-HCl and copper sulfate in association with bending of the torso and pumping of the opercula. Ina subsidiary study, Odontobutis obscura also responded to apomorphine-HCl. Direct administration of copper sulfate into the stomach in S. asotus and into the upper part of the intestine in P. lineatus caused vigorous vomiting. Metoclopramide monohydrochloride effectively inhibited apomorphine-induced vomiting and vomiting by stomach stimulation with copper sulfate in S. asotus. However, the drug was not effective in inhibiting either apomorphine- induced or stomach-stimulated vomiting in P. lineatus. The effectiveness of apomorphine-HCl and the bending behavior that may raise intra- abdominal pressure suggest that fish are armed with the same mechanism of vomiting as in the higher vertebrates. The stomach is not the necessary factor to develop the emetic ability in fish. There may be a species difference in the effectiveness of antiemetics. Physiology 1247 ENCAPSULATION OF BRAINLESS PUPAE OF THE ERI-SILK WORM, SAMIA CYNTHIA RICINI S. Takahashi. Dept. of Biol., Nara Women's Univ., Nara. 60-day-old brainless pupae of S.cynthia ricini contained only about 800 hemocytes per pl. Plasmatocytes (88%) and granular cells(12%) were identified. Encapsulation in the brainless pupae were examined. When Silicon oi1(10p1) as a foreign object was injected into the hemocoel, it was encap- sulated with a thin, smooth membrane, which was presumably derived from hemolymph com- ponents. The capsule comprised an inner amorphous layer and an outer layer of flattened cells. SEM studies showed that the primary reactions were the deposition of coating substance on the foreign sur- face. During the inner layer formation, involvement of hemocytes were observed. When the foreign surface contacted with the wall (basement membrane) of organs, such as gut or fat bodies, the deposition of coating substance occurred on the surface of the organs. Sessile hemocytes were also covered with the substance. This coating layer developed to the inner layer of capsule. In developing adults, typical cellular enapsulation was detected. Thus, it was revealed that cellular responses to the foreign object were preceded by humoral responses in the brainless pupae. MODIFICATION OF GROWTH AND DIFFERENTIATION OF MOUSE BONE MARROW-DERIVED MAST CELLS CULTURED WITH GANGLIOSIDE GMs; H.Fujimaki’, N.Katayama? and K.Nohara’. ‘Environ. Health Sci. Div., Natl. Inst. for Environ. Studies, Tsukuba and *Dept. of Otorh inolaryngol. Jikei Univ. Sch. of Med., Tokyo. The characteristics of mouse bone marrow- derived mast cells (BMMC) were analogous to the mucosal type of mast cells. To examine the effects of ganglioside GMs on growth and differentiation of BMMC, mouse bone marrow cells(BMC) were cultured with various concen- trations of GMs for 3 wk in the presence of WEHI-3 conditioned medium. Although the nun- ber of total non-adherent cells was not af- fected, the number of alcian blue positive cells(mast cells) was significantly decreased at 10 and 20 wM GM3. The addition of GM; for various time intervals revealed that the decreased number of total non-adherent cells and the increase of histamine content were observed in the group added 2 weeks after initiation of culture. In the growth of BMMC induced by IL-4, a dose-related suppression was observed. However, histamine content in BMMC cultured with IL-4 showed no difference between GMs-cultured and control groups. These results indicated that the addition of GMs to the culture of BMC altered the growth and differentiation of BMMC. NEONATAL TOLERANCE IN THE DEVELOPING THYMUS: CLONAL ANERGY AND CLONAL DELETION M.Hosono, S.Ideyama and Y.Katsura. Dept.of Immunol, Chest Dis. Res. Inst, Kyoto Univ. Kyoto. VB6+ T cell receptor-bearing autoreactive T cells once-developed in the newborn thymus of Mlsa antigen-bearing mice started to de- cline at day 3 of life,and disappeared with- in the next few days.To understand cellular basis of self-tolerance, we employed the neo- natal tolerance system by injecting hemato- lymphoid cells from Mlsa mice into Mlsb new- born mice.Mechanisms of unresponsiveness of Vf6+ cells in the one-week old thymus were different among anatomical sources of the injected cells, though cells of bone marrow (BM), spleen, thymus and perioneal cavity(PC) cells induced unresponsiveness to the Mlsa deteminants. At this stage of tolerance, BM cell-induced toleance was by clonal anergy, while PCcell-induced toleance was by clonal deletion. This suggests that BM cells lack a type of cells which cause clonal deletion in the one-week-old thymus, though they con- tain a precursor of deletion-inducing cells which develope by the second month of age: Immunohistological data shows that deletion of VB6+ cells is associated with the intra- thymic presence of donor-derived B cells. ANALYSIS OF TRANSPLANTATION TOLERANCE INDUCED IN METAMORPHOSING XENOPUS TADPOLES — IV S.Tochinai, M.Ono, S.Tozaki and F.Kobari Zool. Inst., Fac. Sci., Hokkaido University, Sapporo The J-strain (JJ) clawed frog, Xenopus laevis, can easily be made tolerant against semixenogeneic (X. laevis x X. borealis; JB) adult skin grafted onto larvae before stage 57 in the presence of the thymus. Tolerance was never induced to X. borealis (BB) skin. However, when the BB skin was grafted simultaneously with JB skin, rejection of BB skin was retarded and tolerance inducibili- ty to JB skin was affected in turn. The results were corre— spondent to the size of the BB skin grafted with 2x2 mm? JB skin: when 2x4 mm? BB skin was grafted all of the BB and JB skin were rejected acutely, while no JB skin was rejected when 1x1 mm? BB skin was used (most of BB skin rejected subacutely), intermediate results were ob- tained with 2x2 mm? BB grafts. It seems that the immune reaction against BB skin perturbed tolerance induction to JB skin, indicating that the tolerance was induced in larvae after an abortive attack to B haplotype antigens. The spleen or the thymus removed from the toler— ant frog was grafted to the immunologically incompetent thymectomized frogs carrying previously grafted JB skin. Skin graft rejection initiated in the thymus-—grafted frogs, while the spleen-grafted frogs showed uninterrupted acceptance of JB skin. Although the presence of the suppressor cells in the tolerant animals is not perfectly demonstrated, it is highly probable that the suppressor activity resides in the tolerated JB skin and the spleen of tolerant animals. Attempts are being made to establish the cellular basis of the tolerant state. 1248 Physiology ROLE OF THE PROLIFERATING SPLENOCYTES IN IMMUNE RESPONSE OF NORMAL AND TOLERANT XENOPUS LAEVIS J. Sakuraoka and S. Tochinai. Zoological Institute, Faculty of Science, Hokkaido University, Sapporo We have established a simple and reliable method to quantify the proliferating cells in vivo and jin vitro by using 5'-Bromo-2'-deoxyuridine (BrdU) and anti-BrdU antibody in Xenopus laevis. When the adult or larval J- strain (JJ) frogs were grafted or injected with semixenogenic (X. laevis x X. borealis, JB) cells, splenic lymphocytes proliferated even in the frogs tolerant to JB skin. In order to examine the role of proliferating cells in these frogs, splenic lymphocytes from normal and tolerant animals stimulated with JB cells were transferred to the immunoincompetent thymectomized frogs previously grafted with JB skin. In frogs injected with splenocytes from normal frogs, the JB skin graft rejection initiated promptly and ended in about 10 days, while it took longer in the frogs injected with splenocytes depleted proliferating cells by BrdU incorporation and fluorescent light treatment, indicating that the proliferating cells were actively involved in the rejection process. On the other hand, the results were not so simple in thymectomized frogs injected with tolerant splenocytes. Interestingly, the tolerant status was broken down in many of the frogs when received splenocytes from JB cell stimulated tolerant frogs, while the tolerance was not broken down in many of the frogs injected with BrdU-light treated tolerant splenocytes. It is suggested that even in the tolerant frogs effecter cells were proliferating in response to the toleragenic JB cells along with the possibly participating suppressor cells. OBSERVATIONS OF HEMATOPOIETIC SYSTEMS IN RANA CATESBEIANA, BY SEM. N.Mimori and K.Sugiyama. Dept. of Biology, Fac. of Sci., Hirosaki Univ., Hirosaki 036 We examined the hematopoietic systems during metamorphosis of anuran amphibia Rana catesvbeiana, usng transparent and/or scanning electron microscopy. Following findings are demonstrated that ():the surface -profiles of hematopoietic cells were fundamentally same as that shown in mammalian cells. @:the phagocytosis against non-self antigen was obseved by neutrophils and by macrophages. @:characteristic inner surface features were demonstrated by the ion ecthing treatment-method of SEM. char- acteristically in neutrophils and eosino- phils. @:the mesonephros may function as a supply-source for leukocytes of larvae, however, the spleen may perhaps function as the bone marrow during the all-life of this anima) similar to marmmmalia.@i:thehypertrophy of bone marrow was clearly obseved at the middle stages of metamorphosis, and continued till the end of metamorphosis. THE LINING CELLS OF HEMOCOEL PARTICIPATE IN INTERNAL, DEFENSE OF,LAND SLUG K. Yamaguchi , E.Furuta agd A.Shimozawa Lab. of Med. Sci. and “Dept. of Anat., Dokkyo Univ. Sch. of Med., Tochigi. When microorganisms invade into the hemocoel of land slug, Incilaria fruhstorferi, the number of hemolymph cells increase in the hemolymph and then they encapsulate and phagocytose the invaders. The lining cells of the hemocoel, blood vessel and heart walls of the land slug were observed by SEM and TEM. Only the lining cells of hemocoel wall possessed numerous microvilli, high electron dense nuclei and basal lamina. These cells protruded and freed from the wall into the lumen, when non-self materials(yeasts) were inoculated into the hemocoel. They were morphologically similar to macrophages. Otherwise in tissue culture, the migrated cells from explants of hemocoel wall mainly consist of fibroblast-like cells and macrophage like cells and these cells phagocytosed yeasts. By histochemistry, the circulating hemolymph cells were strongly positive to non-specific esterase reaction, while two kinds of the migrated cells were weakly positive. These results suggest that the hemolymph cells may be derived from the hemocoel wall and the fibroblast-like cells may be able to transform to macrophage-like cells. LECTINS FROM BODY SURFACE MUCUS OF THE LAND SLU 2 1 -Furuta’ , T.Takagi” and A.Shimozawa Dept. of Anat., Dokkyo Univ. Sch. of Med. Tochigi, and Biol. Inst., Fac. of Sci., Tohoku Univ., Miyagi The characteristics of lectin activity have been shown to be exhibited by mucus substances from a variety of biological sources. In Incilaria fruhstorferi, well developed mucus glands and goblet cells are often found under or in the epidermis. The mucus agglutinated human A and B erythrocytes and the hemagglutination was specifically inhibited by low concentration of N-acethyl-galactosamine. All of this activity was completely inactivated by heating at 56°C for 30 min. By a CM-cellulose chromatography and a reverse phase HPLC, three small lectins (Y1l5a,b,c) from the mucus were purified. The molecular weight of Yl15a,b and c was 15,16 and 17kDa, respectively, by SDS-PAGE analysis. The amino acid sequence of these proteins was determined by peptide sequences analysis. Although complete sequences were not established yet, the sequences of these proteins were very similar (60-70% identical). Especially positions of 6 cysteine residues were very conservative in three proteins and these cysteines constructed three disulfide bonds in each molecule which were typical in C-type lectin domain. These results suggest that Yl5a,b and c belong to C-type lectin. Physiology 1249 SPECIES DIFFERENCES OF PHAGOCYTIC ACTIVITY IN FISH ENDOCARDIAL CELLS. H.Nakamura’ , S.Kikuchi* and A.Shimozawa. ‘Dept. of Anat., 7Dokkyo Univ. Sch. of Med., Tochigi, Kominato Lab., Fac. of Sci., Chiba Univ., Chiba. Endocardial (EC) cells of some species of fishes were reported to be phagocytic however all the reports on this phenomenon have been given by in vivo studies. We observed in vitro phagocytic activity of EC cells of several species of fishes. Hearts of fish were dessected and incubated organotypically in the tissue culture medium DM-170 containing 1% Perikan ink (carbon particles) for 2 hours at about 23°C. They were observed histologically. Animals used in this study are as follows; medaka (Oryzias latipes), guppy (Poecilia reticulatus), neon tetra (Paracheirodon innesi), lemmon tetra (Hyphessobrycon pulchripinnis), goldfish (Carassius auratus) and rose bitterling (Rhodeus ocellatus). EC cells of cyprinodontiformes (medaka and guppy) showed intense phagocytic activity. EC cells of cypriniformes (goldfish and bitterling) showed moderate and those of characiformes (neon tetra and lemmon tetra) showed weak or almost no phagocytic activity against carbon particles. Phagocytic activity of EC cells may relate to the phylogenetic status in self defense system of fish. INTERACTION BETWEEN CELL TYPES IN HEMOCYTE CLOTTING SYSTEM OF THE SPINY 'LOBSTER, PANULIRUS JAPONICUS. H. Aono. National Research Institute of Aquaculture, Mie. In order to clarify cell type specific roles in the hemocyte clotting system, hemocytes isolated from hemolymph of the spiny lobster (Panulirus japonicus) were separated into two populations, i.e., semigranular and hyaline cells (SGH eells) and granular cells by density gradient centrifugation. Clotting ability of each hemocyte population was tested in vitro. Whole hemocytes formed a cellular elot when incubated with some kinds of bacteria or lipopolysaccharides (LPS), while SGH cells did not clot nor show morphological change during incubation with LPS. Granular cells were observed to degranulate when exposed to LPS but clotting did not occur. However, medium conditioned with both granular cells and LPS strongly induced clotting of SGH cells. Lysate of granular cells also induced clotting of SGH cells. Microscopic observation showed that the lysate acts on SGH cells as a lytic factor. These results suggest that granular cells release a factor in response to LPS, and the factor induces lysis and clotting of SGH cells. THE NATURE OF MARKERS FOR RECOGNITION ON HEMOCYTES IN ALLOGENEIC PHAGOCYTOSIS OF THE ASCIDIAN, HALOCYNTHIA RORETZI S.Ohtake, T.Abe, F.Shishikura, and K.Tanaka Department of Biology, Nihon University School of Medicine, Tokyo. Small granular amebocyte (SG) phagocy- toses each other in allogeneic mixed cul- ture of the hemolymph of H. roretzi in vitro. We named this response "allogeneic phagocytosis". When washed hemocytes were cultured with glutaraldehyde-fixed hemo- cytes for 30 min, only 2-5% of SGs phago- cytosed the fixed autogeneic hemocytes (fAutoHc) but 60-80% of SGs did the fixed allogeneic hemocytes (fAlloHc). To examine the recognition mechanism, we treated the target cells, fAutoHc and fAlloHc, with B-galactosidase or trypsin before mixing with washed SGs, or added GalNAc in the mixture of SGs and targets. The treatment of fAutoHe with B-galactosidase (10-100 pg/ml) increased the number of SGs phagocy- tosing them from 4% to 48.2-68.7%. The addition of GalNAc (0.3-30 pg/ml) decreased the number of SGs phagocytosing fAlloHc from 70% to 19.6-11.2% without any sup- pression of the phagocytic ability of SGs against latex beads. The treatment of trypsin (0.01-1mg/ml) decreased the phago- cytosis against fAlloHc although it did not affect fAutoHc. These results sug- gest that SGs may see specific glycoprotein markers on the surface of hemocytes to distinguish allo-cells from auto-cells and phagocytose allo-targets. UNIQUE AGGREGATE FORMATION BY A HEMOCYTE OF TUNICATE, STYELA CLAVA. 1 and T.Sawada , S. Tomonaga®, T. Fukumoto .L. Cooper3°. ‘Dept. Anat., Sch. Med., Sch. Allied Health Sci,, Yamaguchi Univ., Ube, Yamaguchi, and 3hept. Anat. Cell Biol., Med. Sch.,Univ. California, Los Angeles (UCLA), California, USA. In tunicates, endothelial cells which seal blood vessels are absent and hemocytes may include connective tissue cells with functions other than those of vertebrate hemocytes. After culturing hemocytes of Styela clava, we found unique aggregate formation by hemocytes which consisted of a homogeneous hemocyte type (basophilic granulocytes). These granulocytes became associated and spread on matrices in oblong shape, adhering to each other and forming a flat sheets initially. These sheets became folded into a round mass which was consisted of homogeneous cell types, tightly compacted as if they were in the process of forming tissue. The nature of these granulocytes suggested that they are related to mesenchymal cells with epithelial or fibroblastic characteristics, and not those associated exclusively with immunodefence as for other hemocytes. A monoclonal antibody UB15, which reacts to a sub-population of hemocytes of Ciona savignyi and Halocynthia roretzi, exhibited a specific staining against this basophilic granulocytes. 1250 Physiology DESALINATION IN THE ESOPHAGUS OF THE SEA- WATER EEL K.Nagashima and M.Ando. Lab. of Physiol., Fac. of Integrated Arts & Sci., Hiroshima Univ., Hiroshima. The esophagus of the seawater teleost desalts the swallowed seawater. However, the mechanisms of the desalination is not clear yet. To clarify the mechanisms, net Na’, Cl. and water fluxes were measured simultaneously under various conditions. Following’osmotic gradient, water moved without accompanying significant Na’ and Cl ions; the calculated osmgtic permeability being 3-4 x 10 ml/cm¢.s.0Osm. This value is lower than that in "tight epithelia" such as frog skin and urinary bladdeg, though the tissue resistance of 70 ohm.cm“ belongs to "leaky epithelia". By altering Na* and Cl concentrations in the mucosal fluid, while the serosa was bathed with normal Ringer solution, a relationship between ion flux and its driving force was obtained. Both Na andCl fluxes were related almost linearly to their electro- chemical gradients across the esophagus, suggesting passive movements of Na and Cl However, most of these ion fluxes were coupled each other. Moreover, the couple NaCl transport was jnhibited by NaCN (10. M) and ouabain (10 * M), suggesting in- volvement of some active processes in this coupled transport. The coupled NaCl trans- port may be due_to NaCl cotransport, since bumefanide (10 ° M) and hydrochlorothiazide (10°* M) inhibited the NaCl transport. Effects of hypoxia on renal function in carp I. Kakuta and S. Murachi. Dept. of Biotechnology, Senshu Univ. of Ishinomaki, Ishinomaki. The influence of hypoxia upon renal function in fish has been studied, and investigation of blood properties and cardiac function was conducted to gain a better understanding of renal response to hypoxia in fresh water teleosts. Hypoxic conditions were induced by allowing the fish to consume available dissolved oxygen (D0) in a 10 1 experimantal tank. When the DO level fell to about 5% of oxygen saturation after several hour stress, carp had lost balance. As water flow and aeration to the tank were established, carp recovered their balance within several minutes. Glomerular filtration rate (GFR) and urine flow decreased with increased urinary concentrations of bio-components, except protein, in the course of hypoxia. Decreases in blood pH and plasma prosta- glandin Ee» ,and increases in Ht, plasma K*, Ca**, Mg**, Pi, ammonia, lactic acid and catecholamines were observed as hypoxia progressed. Decreases in heart rate (HR) and blood pressure in dorsal aorta (BPd), and increase in the duration of electrical systole in electrocardiogram were also observed. Increased GFR and urine flow, and higher values for urinary components, except protein, compared with those of the control were found in the initial post- stress stage. Increases in plasma angiotensin II, HR and BPd were also observed. They resumed to the resting levels after 12hr. IMMUNOCYTOCHEMICAL LOCALIZATION OF GLUT1, GLUT4, AND THEIR CHIMERIC GLUCOSE TRANSPORTER PROTEINS. K.TAKATA*, T.ASANO?, Y.OKA?, AND H.HIRANO?*. *Dept of Anatomy, Kyorin Univ. Sch. of Med. Mitaka,Tokyo, *3rd Dept. of Int. Med., Fac. of Med., Univ. of Tokyo, Tokyo. Facilitative glucose transporter isoforms, GLUT1 and GLUT4, have different intracellular distributions despite their very similar structure. GLUT1 is found at the plasma membrane of various tissues and cells, while GLUT4 is localized in the cytoplasmic compartments, such as Golgi apparatus in adipocytes and muscle cells. To elucidate the differential targeting mechanisms, GLUT1, GLUT4, and GLUT1/GLUT4 chimeric glucose transporters were stably expressed in Chinese hamster ovary cells. These cells were subjected to immunofluorescence and immunoelectron microscopy as well as measurement of glucose transport activity. GLUT4 was mostly localized intracellularly, especially in the trans-Golgi regions, while GLUT4 was found at the plasma membrane. These results suggest the existence within GLUT molecule of domain(s) responsible for proper intracellular targeting. The results of chimeric glucose transporters revealed that two domains of GLUT4, which are not N-terminal or C-terminal domain, determine its targeting to the intracellular compartments. IMMUNOCYTOLOGICAL DETECTION OF VACUOLAR TYPE H'-ATPASE FROM THE VANADOCYTES IN ASCIDIANS T. Uyama and H. Michibata, Mukaishima Marine Biol. Lab., Hiroshima Univ., Hiroshima. Level of vanadium in vanadocytes of Ascidia sydneiensis samea is in excess of 10 mM, which corresponds to a level more than million times higher than that in sea water. However, it is unclear that the energetistic mechanism of the accumulation of vanadium against this large gradient of concentration. Since the vacuolar contents of vana- docytes show very low pH values of approximately 2, we have examined whether vacuolar type H —ATPase exists in the vacuolar membranes, to consider a possibility of the energestistic conjugation of the ATPase with the accumulation of vanadium. As the result, immunocytological analysis revealed that the antisera specific to 72 kDa and 57 kDa subunits of vacuolar type H’-ATPase reacted with vanadocytes of A. sydneiensis samea. Immunoblotting analysis showed that the antigens recognized by the antisera were 70 kDa and 57 kDa proteins, re- spectively. RAISING OF MONOCLONAL ANTIBODY AGAINST BLOOD CELLS OF VANADIUM-RICH ASCIDIAN, ASCIDIA AHODORI J. Wuchiyama, T. Uyama and H. Michiba- ta, Mukaishima Marine Biol. Lab., Hiroshima Univ., Hiroshima. Blood cells of vanadium-rich ascid- ians can be classified into eleven types but the relationships between different types of blood cells has not been proven in spite of many cytologi- eal and morphological studies. We, therefore, intend to raise mono— clonal antibodies against several types of blood cell to follow the lineage of these cells. Among several types of blood cell in Ascidia ahodori, signet ring cells was separated using percoll density gradi- ent centrifugation and the subpopula- tion was injected intraperitoneally to balb/e mouse to immunize. The raised monoclonal antibody (AKBB3) specific to the signet ring cells of A. ahodori recognized the Signet ring cells of not only A. ahodo- ri but also A. sydneiensis samea. Molecular weight of antigen reacted with the antibody was estimated at about 77 kDa in A. sydneiensis samea and that in A. ahodori is under inves-— tigation. SPONGE SPICULES IN THE BODY OF SPONGIVOROUS SEA URCHINS R.Birenheide!, T.Motokawa! and S.Amemiya2. IBiol. lab., Tokyo Inst. of Technology, Tokyo, 2Misaki Mar. Biol. Station, Miura-shi, Kanagawa. Some sea urchin species feed on sponges but nothing is known about the possible problems caused by penetrating sponge spicules. We screened the feeding tract, coelomic organs and skeleton of the species Asthenosoma ijimai, Araeosoma owstoni, Hapalosoma gemmiferum, (Echinothu- rioida) and Diadema setosum (Diadematoida). By dissolving tissues or skeleton and subsequent filtration silicate sponge spicules were isolated from these sea urchins. Spicules of two types, i.e. raphides and styles were found. Histological sections showed the distnibution of spicules in the tissues. In the feeding tract especially pharynx and esophagus contained more raphides than styles. No specific tissue reactions on the sponge spicules were observed. In the coelom, raphides were accumulated in Stewart Organs, gills and in or near the ampulla. They were always entangled by brown bodies. Brown bodies are the main carrier of wastes in sea urchins. They leave coeloms by penetration through epithelia. The accumulation of brown bodies containing sponge spicules suggests that the spicules are too Jong to be transported through epithelia and thus are stored in coelomic cavities. From plates and spines we could isolate more styles than raphides. The spicules were often incorporated into the skeleton of the sea urchins. Physiology 1251 TWO NOVEL-FAMILY PEPTIDES ISOLATED FROM THE ABRMs OF Mytilus. Y. Fujisawal, 1. Ikedal, yY. Muneokal, o. Matsushima2 and I. Kubota3. Fac. of Integrated Arts and Sci., Hiroshima Univ., Hiroshima, 2Inst. of Biol., Fac. of Sci., Hiroshima Univ., Higashi-hiroshima and Suntory Bio-Pharma Tech Center, Gunma. Two contractile peptides were isolated from the anterior byssus retractor muscles (ABRMs) of the bivalve mollusc Mytilus edults. Their structures were determined to be as follows: Peptide 1 GPFGtTH HMI K amide (GPFG-8) Peptide 2 GPFGLWN KH G amide (GPFG-9) Both peptide 1 (octapeptide) and 2 (nonapeptide) were found to have Gly-Pro- Phe-Gly- at their N-terminal parts as a common structure, and hence they were designated GPFG-8 and GPFG-9, respectively. The peptides do not appear to be members of any other previously identified peptide family. Both the peptides evoke a weak contraction in the ABRM. However, the ABRM shows tachyphylaxis to the peptides; the second contractile response of an ABRM to the peptides is far smaller than the first response. Therefore, the peptides seem not to be physiological excitatory neuro- peptides in the muscle. They might play a role for keeping a homeostatic state of the muscle. In addition to the above peptides, three other peptides were newly isolated in this study. They were two Mytilus inhibitory peptide analogues and a related peptide. RELATIONSHIP BETWEEN ERYTHROPOIETIN AND ERYTHROID COLONY-STIMULATING ACTIVITY IN MOUSE PLASMA. $.Sakata!, Y.Enoki! and M.Ueda’.*Dept. of Physiol., Nara Med. Univ., Kashihara, Nara and *Research Inst. of Life Sci., Snow Brand Milk Products, Tochigi. A relationship between erythropoietin (EPO) and erythroid colony-stimulating activity (ECSA) in mouse plasma was exa- mined in fetal mouse liver cell (FMLC) cultures using a monoclonal antibody (MoAb) R2 raised against recombinant human EPO. Most of the ECSA in plasma from normal, anemic and hypoxic mice was neutralized by MoAb. This neutralization could be reversed by addition of excess of anemic plasma or by preincubation of MoAb with goat anti- mouse IgG antibody. Most of the plasma ECSA was bound to an immunoadsorbent column containing the immobilized MoAb, and the retained ECSA was completely neutralized by MoAb. The plasma ECSA and standard EPO showed parallel dose-response curves and additive effect on CFU-E stimulation. Based on these findings, we conclude that mouse plasma ECSA detected by the present CFU-E assay using FMLCs is mainly due to EPO. 1252 OXYGEN TRANSPORT BY HEMOGLOBIN IN DAPHNIA MAGNA. Michiyori Kobayashi!, Kenichi Ishigaki?, Michisuke Kobayashi? and Kiyohiro Imai’. 1Department of Biology, ?Information Processing Center, 3Department of Physics, Faculty of Science, Niigata University, Niigata, and 4Department of Physiology, Medical School, Osaka Univer- sity, Suita, Osaka. Using Hb from Hb-rich (Hbr) and Hb- poor (Hbp) Daphnia magna, curve-fitting Was carried out for the oxygen equi- librium curves, and Adair constants were determined. The oxygen saturation (Y) of hemo- globin Hbr and Hbp in Adair’s equation was differentiated by p to obtain the differential coefficient (Y’). The Y’ apr /Yuop ratio showed a peak (2.9) at posi- tion close to pso of Hb from Hb-rich animals. Using the monocomponent human Hbs having oxygen-affinity almost equiv- alent to those from Hb-rich and Hb-poor animals, a markedly high ratio of dif- ferential coefficient was obtained. This indicates that substitutive performance of the function of high oxygen-affinity Hb by low oxygen-affinity Hb is very dif- ficult. It is considered that for adapta- tion to a wide range of environmental oxygen condition by means of Hb, abnormal increase in Hb concentration is avoided by possession of multicomponent Hb. ELECTROPHYSIOLOGICAL STUDIES ON BINDING ACTIVITY OF AZIDE DERIVATIVE OF TETRODOTOXIN TO Na CHANNELES. Seiji UEHARA. Dept. of Biology, Saitama Med. School,Saitama. Binding activity of an azide derivative of tetrodotoxin(TTX) to Na channeles of a sciatic nerve of a bullfrog was electro- physiologically studied. The derivative was synthesized by reacting TTX and 1- fluoro-2-nitro-4-azidophenyl- g alanine, and purified with HPLC. Biological activ- ities of the derivative was 1/40 of that of TTX. 1) The nerve was incubated with the de- rivative in the dark and then photoirra- diated with a deuterium lamp. Afetr the nerve was repeatedly washed with Ringer's sol., a small decrease in the amplitude of the action potential was observed. But, photoirradiation alone also sometimes in- duced a decrease. Therefore, it could not be concluded that the small decrease was caused by the derivative. 2) Instead of photoirradiating directry the nerve, the derivative was preliminar- ily photolyzed and the nerve was incu- bated with the photolyzed derivative changing incubation time. Incubation for 60 - 90 min. induced strong inhibition of recovery of the amplitude after washing repeatedly the nerve. From these results, it can be concluded that the derivative binds covalently to Na channeles. Physiology GTP-BINDING PROTEIN IN RHABDOMERIC PHOTORECEPTORS. T.Suzuki!, K.Nagai’, K.Narita2, Y.Kito2, M.Michinomae$ and K.Yoshihara*. ‘Dept. of Pharmacol., Hyogo Coll. Med., Nishinomiya, @Dept. of Biol., Fac. of Sci., Osaka Univ., Osaka, 3Dept. of Biol., Konan Univ., Kobe and 4Suntory Inst. of Bioorg. Chem., Osaka We prepared polyclonal antibodies against polypeptides of the sequences of cholera toxin and pertussis toxin binding sites of bovine transducin and N-terminal region of Drosophila Gaq (Pab CTX, Pab PTX and Pab DGqN). Pab DGQN strongly reacted with 42kD protein localized to the head of Drosophila. We carried out immunoblot analysis to identify the G-protein of rhabdomeric photoreceptors. The Pab DGqN-positive 42kD protein was detected in rhabdomal fractions of crayfish, shrimp, octopus and squid but not in frog and bovine rod outer segments. The 42kD protein was solubilized with detergents and purified with Con A- and DEAE-columns. The purified protein was associated with 35kD protein which is positive to the antibody against GB. Pab CTX and Pab PTX did not react with the squid 42kD protein. These results indicate that major G-protein in rhabdom of photoreceptor is Gq-type. The Pab DGqN-positive 42kD protein was found also in water-soluble fraction of squid retinal homogenate. This suggests that Gaq exists in water-soluble and membrane-bound forms. PHOTOSENSITIVE STAGES IN PHOTOPERIODISM FOR WING FORM AND REPRODUCTION IN A WATER STRIDER, AQUARIUS PALUDUM. T. Harada. Dept. of Biol., Fac. of Sci., Osaka City Univ., Osaka. Long-day photoperiods induced short- winged form and reproduction, short-day caused long-winged form and diapause, and the step-wise decrease in photophase (by 15 min every 5 days) from 14.5 h to 13.5 h before the last (fifth) instar stage induced diapause in Aquarius paludum (Harada and Taneda, 1990). The step-wise decrease in the same way from 15 h to 14 h caused the long-winged form, but did not induce diapause. Insects were reared under a short-day of 9.5L-14.5D except each specific stage (12-13 days) under a long-day photoperiod of 15.5L-8.5D, i.e., during the first and second instars (group A), the third and fourth instars (group B), or the fifth instar (group C). The pro- portion of long-winged form was more than 85% in all the groups. 18.2% of the females laid eggs in group C. All the females in group A and B entered diapause. However, there was a difference in the form of the ovarioles between each of groups A, B and the group of females reared under the short-day through the nymphal stage. The sensitive stages for wing form and reproduction seem to be in the wide range of the first to fourth instar and the whole nymphal stage, respectively. Physiology CIRCADIAN OCULAR RHYTHMS IN THE JAPANESE QUAIL (COTURNIX COTURNIX JAPONICA) AND THE EFFECT OF MELATONIN INJECTION. M.Sasaki and T.Oishi Dept. of Biol., Nara Women's Univ., Nara We investigated circadian rhythms of retinal melatonin, mitotic rate in the corneal epithelium and visual pigment production in the retina, and the effects of melatonin injection on the rhythms were also observed. Retinal melatonin was high in darkness and low in light under LD cycle (LD 12:12), and the rhythm continued in continuous darkness (DD), but seems to disappear in continuous light (LL). The rhythm of mitotic rate in the corneal epithelium was very Similar to the rhythm of melatonin. The outer segments of rod cells were stained intensely by the rhodopsin anti- serum throughout 24 hours in LD cycle, LL and DD. However, some parts of inner segments (Golgi area) were stained only during light phase of LD cycle. The rhythm seems to disappear in constant conditions (high immunoreactivity throughout 24 hours in LL, and intermediate throughout 24 hours in DD). When melatonin was injected into the right eye at 8:00 for 5 days in LD 12:12, a distinct phase shift was induced in the corneal mitotic rhythm compare to the rhythm in the saline injected left eye, but the eects on the visual pigment production seem to be meagre. SPECIES SPECIFICITY OF HIBERNATION RELATED PROTEINS (HPs) IDENTIFIED IN PLASMA OF CHIPMUNKS. N.Kondo, H.Honda and J.Kondo’. Mitsubishi kasei Inst. of Life Sci., Machida, Tokyo, ‘Mitsubishi Kasei Corp. Res. Center, Yokohama. In our previous studies, novel types of protein have been identified in plasma of a mammalian hibernator, chipmunks (Tamias asiaticus). These proteins (HP-20, 25, 27 & 55) were specifically reduced or disappeared from the blood during hibernation under constant laboratory conditions. In the present study, the detection of HP-20s in the blood of various species of rodents was carried out using monoclonal antibodies (mouse) and antisera (rabbit) to HP-20s. In immunoblotting analysis, the existence of proteins reacting with the monoclonal antibodies and antisera was shown in plasma from ground squirrels (Citellus tridecemlineatus) and marmots (Marmota flaviventris), both of which are hibernating species. In plasma from other species (tree squirrels, golden hamsters, djungarian hamsters, mice and rats), these antibodies and antisera did not recognize HP-20s. From further immunoblotting studies, at least HP-20 and 25 were identified in marmots and ground squirrels. The fact that HPs were detected in two other species of hibernators indicate the specificity and physiological significance of HPs for hibernation. 1253 1254 Endocrinology ORIGIN AND MIGRATION OF LUTEINIZING HOR- MONE-RELEASING HORMONE (LHRH) NEURONS IN THE CHICK EMBRYO: A CARBOCYANINE DYE ANALYSIS. S. Murakami and Y. Arai. Dept. of Anat., Juntendo Univ. Sch. of Med., Tokyo. Recently we reported that unilateral olfactory polacodectomy resulted in the absence of LHRH neurons in the olfactory- brain axis of the operated side in the chick embryo. This suggests that LHRH neurons originate in the olfactory pla- code, after which they migrate into the brain. In the present study, to obtain a direct evidence for the migration of the LHRH neurons from the olfactory epithelium to the brain, the epithelial cells of the olfactory placode of the chick embryo (EDs 3.5-4) were labeled with Dil. The labeled embryos were incubated in ovo for 1 to 4 days. DilI-labeled cells were first de- tected in the olfactory nerve 1 day after application of Dil. Two-four days after application of Dil, labelled cells sequen- tially appeared in the rostral and medial forebrain, and in the septo-preoptic area. The distribution pattern of Dil-labeled cells closely resembled that of LHRH neurons. Double staining for LHRH and Dil showed that a part of Dil-labeled cells co-expressed LHRH immunoreactivity. These results provide evidence for the actual migration of LHRH neurons from the olfac- tory region to the septo-preoptic area. LATERAL ASYMMETRY OF LHRH CELLS IN THE BRAIN OF THE MOUSE WITH TESTICULAR FEMINIZATION MUTATION (T£m/Y) . Y. Inase, T. Machida and T. Noumura. Dept. of Regulation Biol., Fac. of Sci., Saitama Univ., Urawa, Saitama 338. The X-linked testicular feminization mutation (Tf£m/Y) in the mouse is charac- terized by insensitivity of the target cells to androgens. In the present experiment, number of immunoreactive LHRH cells was compared between the right and the left side of the brain in Tfm/Y mice. Although we have already exhibited that the number of LHRH cells are consist- ly greater in the right side of the brain than in the left side in normal male mice, no lateral asymmetry was found in the number of LHRH cells in Tf£m/Y mice. Removal of testes of both sides in Tfm/Y mice caused an increment in number of LHRH cells in the brain. In normal male mice, however, number of LHRH cells significantly decreased following bilateral orchidectomy. The results demonstrated a unique behavior of LHRH cells in the brain of Tfm/Y mice. SEX DIFFERENCE IN THE DISTRIBUTION OF LHRH NEURONS IN THE MOUSE BRAIN. W. Nishido and T. Machida Dept. of Regulation Biol., Fac. of Sci., Saitama Univ., Urawa, Saitama 338. In contrast to known sexual dimorphism present in the rat brain, no sex differ- ences have been found in volumes. of mouse hypothalamic structures. In order to clarify possible sex differences of the ‘brain in the mouse, ontogeny and distribution of immunoreactive LHRH neurons were examined in male and female mice. In male mice, LHRH neurons increased in number from 7 days of age to 30 days and thereafter decreased drastically. In adult female mice, number of LHRH neurons fluctuated during estrous cycle: largest at proestrus and smallest at estrus. Number of these neurons in adult male mice was similar to that of proestrous females. Neonatal estrogen sterilization of female mice caused as adults an increase in number of LHRH neurons in the brain. The results exhibited sexual dimorphism of the number of immunoreactive LHRH neurons in the mouse brain. GnRH IMMUNOREACTIVE SUBSTANCE IN RAT PINEAL GLAND IS A MEMBRANE ASSOCIATED PROTEIN. M.K. Park, H. Kogo, S. Kawashima, and K. Wakabayashi~. Zool. Inst., Fac. 0o SGic, Univ. of Tokyo, Tokyo 113 and Hormone Assay Center, Inst. of Endocrinol., Gunma Univ., Gunma 371, Japan Immunoreactive and bioactive GnRH in the pineal gland has been reported in bovine, rat, and ovine. A monoclonal anti- body, LRH13 is a well characterized GnRH specific monoclonal antibody (Park and Wakabayashi .1986). Using this antibody, strong immunohistochemical signal in the rat pineal gland was characterized. SDS- polyacrylamide gel electrophoresis (SDS- PAGE) and immunoblot were used to charac- terize this immunoreactive substance. Immunoblot showed that apparent molecular weight of the substance is approximately 52 K Dalton (KD). The immunochemical signal of 52 KD protein was GnRH specific because of the complete blocking with 0.5 mg of GnRH in 10 ml of LRH13 solution (1,000 times diluted). The signal was detected in the membrane fraction of the pineal homogenate, and not in the cytosolic supernatant. Treatment with NaCl (140 or 500 mM), Mg** (5 mM) or EDTA (10 mM) in 50 mM Tris-Cl, pH 7.4 could not release, but Triton X-100 (2 %) released the 52 KD protein from the membrane fraction of the pineal gland. These results suggest that the rat pineal GnRH-immunoreactive substance has a GnRH-like domain, and is a kind of membrane associated protein. Endocrinology 1255 LORDOSIS INHIBITING INFLUENCE IN THE DORSAL RAPHE NUCLEUS IN MALE RATS: EFFECTS OF NEURAL TRANSECTIONS. HORMONAL AND BEHAVIORAL STUDIES OF WINTERING FLOCK OF PINTAILS (Anas acuta) M. Wada!. A. Kishida?: R. Tanabe? and K. Fukui3. 1Dept. Gen. Educat., Tokyo Med. Dent. Univ., Ichikawa, Chiba, 2College of Agr. & Vet. Medicine., Nihon Univ., Fujisawa, Kanagawa and 3Res. Center, Wild Bird Society of Japan, M. Kakeyama and K. Yamanouchi. Dept. of Basic Human Sciences, Sch. of Human Sciences, Waseda University., Tokorozawa. To clarify lordosis inhibiting neural pathways from the dorsal raphe nucleus (DRN) in male rats, several types of cuts around DRN were performed and lordosis behavior was examined. Male rats were castrated and subjected to ventral-horizontal (VHC), dorsal- horizontal (DHC), anterior (AC) or posterior (PC) cut of DRN. Four weeks after, all animals were implanted with Silastic tubes containing estradiol, and then lordosis behavior was observed. Non- brain surgery control males showed low score of lordosis quotient (LQ). LQs in either DHC or PC males were also low, and was comparable to those in control males. In contrast, VHC males showed higher LQ value than control males. As well as VHC males, AC males displayed high LQ score. These results suggest that ventral and anterior Outputs (and/or inputs) of the dorsal raphe nucleus are involved in the lordosis inhibiting system in male rat brain. A Possible Existence of Non-Strial Pathway of Amygdala in Control of Male Rat Sexual Behavior. Y. Kondo and K. Yamanouchi. Neuroendocrinol. Lab., School of Human Sciences, Waseda University, Tokorozawa. We have reported an importance of the medial amygdala, as well as the medial preoptic area, in regulation of male rat sexual behavior. Anatomical evidence shows that the medial amygdala innervates the medial preoptic area mainly through the stria terminalis (ST). In this experiment, neural transection of the ST (STC) was made in male rats which were previously castrat- ed and received subcutaneous implantation of silastic tubes containing testosterone, and then behavioral tests were carried out. Next, the STC males received bilateral medial amygdala lesion (AMGL) followed by further behavioral tests. In the tests after STC, although most of STC males showed male sexual behavior, mount and intromission frequencies were lower than those of sham-operated males. Ejaculation was eliminated after STC. On the tests after the second brain surgery, further decrease of copulatory activity was observed in males with both STC and AMGL. The results indicate a possibility of the pathway, other than the stria terminalis, of the amygdala in neural control of copulatory behavior in the rat. Tokyo, Japan. Avian reproductive endocrinology has been mostly studied using poultry species such as chickens and turkeys or small passerine species in laboratories or in field. To understand mechanisms in avian seasonal breeding in each species, studies in different groups of birds which have different adaptational strategies are must. Fortunately we have a chance to collect blood samples from wild wintering population of pintails at Sin-hama duck-netting preserve owned by the Imperial Household. Samples were collected from male and female pintails once a month from November 1991 through January 1992 and twice in February 1992. Behavioral observation was carried out at Gyotoku Waterfowl preserve and Ueno Shinobazu Pond. Plasma concentration of luteinizing hormone (LH) in both sexes were low in November and December. Plasma concentrations of LH in males began to increase in January and became more than 1 ng/ml on February 12, but those in female were still low even on February 12. Male pintails showed courtship behavior in February but females did not respond it. HORMONAL INDUCTION OF COURTSHIP BEHAVIOR IN THE NEWT, CYNOPS PYRRHOGASTER. F.Toyoda', S.Tanaka? and S.Kikuyama? . 'Dept. of Physiol., Nara Med. Univ., Kashihara, *Inst. of Endocrinol., Gunma Unv., Maebashi, “Dept. of Biol. Sch. of Educ., Waseda Univ., Tokyo. In breeding season, the sexually matured male newt vibrates the tail in front of the female at an early stage of courtship. The vibration- behavior is elicited in sexually inert males by injection of prolactin (PRL) and/or gonadotropin (GTH) . This effect of GTH was nullified by castration. In the castrated Male receiving PRL, both testosterone propionate (TP) and dihydrotestosterone (DHT) were effective in eliciting the behavior, whereas estradiol was not effective. The male-like behavior was also observed in a considerable number of ovariectomized females with low frequencies. In these females, administration of PRL together with TP, but not with estradiol, was effective in increasing the frequency of the male-like behavior. The incidence and frequency were not affected by the difference in the sex of the partner. 1256 Endocrinology RAINBOW TROUT 3B-HYDROXYSTEROID DEHYDROGENASE /A54-ISOMERASE: cDNA CLONING AND ENZYMATIC PROPERTIES N. Sakai*, M. Takahashi, M. Tanaka and Y. Nagahama Lab. of Reprod. Biol., Natl. Inst. for Basic Biol., Okazaki 444, *Dept of Marine Biosci., Fac. of Biotech., Fukui Pref. Univ., Matsuoka 910-10 3B-Hydroxysteroid dehydrogenase/A5+-isomerase (3B- HSD) is the enzyme in the steroidogenic pathway controlling the formation of A‘-3-ketosteroids from A5-3B- hydroxysteroids. A cDNA clone encoding 3B-HSD was isolated from a cDNA library of rainbow trout (Oncorhynchus mykiss) ovarian thecal cell layers. The cDNA contained an open reading frame of 1122 nucleotides encoding a protein of 374 amino acid residues. The deduced protein has 48% homology with 3B-HSD of human and bovine, 45% homology with that of mouse and 3 B-HSD type II of rat, and 44% homology with 3B-HSD type I of rat. Expression of trout 3B-HSD cDNA in nonsteroidogenic mammalian COS-1 cells led to production of an enzyme which is capable of converting pregnenolone, 17@-hydroxypregnenolone and dehydroepiandrosterone to progesterone, 17 a-hydroxy- progesterone and androstenedione, respectively. The conversion of three A5-3B-hydroxysteroid substrates to A+-3- ketosteroids by the activity of trout 3B-HSD expressed in COS-1 cells are almost the same. The cDNA hybridized to a single species of mRNA (1.5-kb in length) in rainbow trout ovary RNA. The 1.5-kb transcript markedly increased in trout ovaries during oocyte maturation. RAINBOW TROUT CHOLESTEROL SIDE-CHAIN CLEAVAGE CYTOCHROME P450 ( P450scc ): cDNA CLONING AND mRNA EXPRESSION M. Takahashi, M.Tanaka, N.Sakai, S.Adachi and Y.Nagahama, Lab. of Reprod. Biol., Natl. Inst. for Basic Biol., Okazaki 444 P450scc catalyzes the conversion of cholesterol to pregnenolone, the initial rate-limiting reaction in the synthesis of several steroid hormones. A cDNA clone encoding P450scc was isolated from a rainbow trout ( Oncorhynchus mykiss ) ovarian thecal cell layer cDNA library. The cDNA contains an open reading frame of 1,542 nucleotides encoding 514 amino acid residues. The deduced amino acid sequence of rainbow trout P450scec shows 48% homology with that of human, and 46% homology with that of rat, bovine and pig. P450scc activity was confirmed by transfected COS-1 monkey kidney tumor cells containing P450scc cDNA and subsequent detection of conversion of 25- hydroxycholesterol to pregnenolone by radioimmunoassay. Northern blot analysis revealed a single species of mRNA approximately 1.8 kb in length. The RNA transcripts were not detected in early vitellogenic follicles but were present in postvitellogenic follicles and were abundant in postovulatory follicles. RNA EXPRESSION OF SEROIDOGENIC ENZYMES BY RAINBOW TROUT OVARIAN FOLLICLES JIN VITRO M.Tanaka, M.Takahashi, N. Sakai and Y. Nagahama. Lab. of Reprod. Biol., Natl. Inst. for Basic Biol., Okazaki 444 Postvitellogenic follicles of salmonid fishes produce predominantly 170,20B-dihydroxy-4-pregnen-3-one (17a, 20B-DP, the maturation-inducing hormone) in response to gonadotropin. Forskolin can mimic this action of gonado- tropin. Expression of mRNAs for four ovarian steroidogenic enzymes (P450scc, 3B-HSD, P450c17 and P450arom) involved in oocyte maturation was investigated in ovarian follicle layers incubated in the presence or absence of forskolin. The level of P450scc mRNA remained constant during the first 24 hr and then increased at 36 hr in the presence of forskolin, coinciding with an incease in 17a,20B-DP production. 3B-HSD mRNA increased within 24 hr of incubation with forskolin, and then retumed to the initial level at 36 hr. mRNAs for P450c17 and P450arom decreased rapidly regardless of forskolin. Actin mRNA remained constant within 24 hr of incubation. Dynamic changes of mRNAs may constitute part of the mechanism underlying the increased capacity of gonadotropin- stimulated ovarian follicles to produce 17 a,20B-DP. GONADOTROPIN-INDUCED STEROID PRODUCTION IN TESTICULAR TISSUE OF THE JAPANESE EEL, ANGUILLA JAPONICA T. Kobayashi!, T. Miura!, K. Yamauchi2, N. Sakai3, M. Tanaka! and Y. Nagahama!. 'Natl. Inst. for Basic Biol., Okazaki 444, 2Hokkaido Univ. and 3Fukui Pref. Univ. In the cultivated Japanese eel, human chononic gonadotropin (HCG) induces spermatogenesis by stimulating the testicular production of 11-ketotestosterone (11-KT). The effect of HCG treatment on the pattern and yield of steroid metabolites were examined using a cell-free system. Cell-free homogenates of eel testes before and 24 hours after HCG treatment were incubated for 1 hour with 'C-labeled steroids and the metabolites were analyzed by TLC. Analysis of steroid biosynthetic pathways showed that the testicular homogenates synthesized 11-KT via the *A-pathway (pregnenolone — 17a-hydroxypregnenolone — dehydroepi- androsterone). When !4C-pregnenolone and 'C-dehydro- epiandrosterone were used as substrates, a significant increase in androstenedione production was observed in testicular homogenates obtained after treatment. In incubations with 14C-testosterone, androstenedione and 11-KT were detected as the major ; however, there was no difference in the pattern and yield of metabolites between control and HCG-treated groups. These results indicate that HCG increases 11-KT production by stimulating the activity of 3 B-hydroxysteroid dehydrogenase/AS+-isomerase, thereby accelerating the conversion of dehydroepiandrosterone into androstenedione. Endocrinology 1257 ACTIVATION OF SOMATOLACTIN CELLS IN THE PITUITARY OF THE RAINBOW TROUT BY LOW CALCIUM ENVIRONMENT. S.Kakizawa, T.Kaneko, S.Hasegawa, T.Hirano and H.Kawauchi*. Ocean Res. Inst., Univ. of Tokyo, Tokyo, *School of Fisheries Sciences, Kitasato Univ., Sanriku. Somatolactin (SL) is a putative pituitary hormone, structurally related to both prolactin and growth hormone. To clarify the possible involvement of SL in calcium regulation, we examined the effects of ambient calcium on activity of SL cells in the rainbow trout. The fish were transferred from fresh water (FW) to FW containing 10mM CaCl (Ca-FW) or to 80% sea water (SW) and from Ca-FW to FW. The pituitary was fixed 10 and 21 days after transfers. The pituitary sections were immunocyto- chemically stained with anti-chum salmon SL serum. On the adjacent section, the presence of SL mRNA was examined by in situ hybridization using a cDNA probe encoding chum salmon SL. Both cellular and nuclear cross-sectional areas were decreased when fish were transferred from FW to Ca-FW or to SW; no change was seen after transfer from FW to FW. The level of SL-mRNA decresed 10 days after transfer to Ca-FW. On the other hand, not only cellular and nuclear areas but also SL- MRNA levels increased after transfer from Ca-FW to FW. The results support a hypercalcemic role for SL. IMMUNOCYTOCHEMICAL INVESTIGATIONS OF GTH CELLS IN THE PITUITARY OF NILE TILAPIA (OREOCHROMIS NILOTICUS) DURING THE EMBRYONIC DEVELOPMENT. T.Saga !, S. Adachi 1, K. Yamauchi !, H. Kagawa 2, H.Tanaka2 and H.Takahashi!, Faculty of Fisheries, Hokkaido University. Hakodate, 2. National Research Institute of Aquaculture, Mie. In the pituitary of adult tilapia, glandular cells immunoreactive to anti-Pagrus major GTH Ip antiserum (GTH-ir cells) were localized mainly in the ventral part of proximal pars distalis (PPD) and additionally in rostral pars distalis (RPD) and in the peripheral region of pars intermedia (PI). The cells distributed in the PPD had large vacuoles in the cytoplasm, while most of the cells distributed in the peripheral PI had no vacuoles. Both small granules and large globules in the GTH-ir cells revealed immunoreactivity. During embryonic development, the pituitary first occurred at the ventral part of the third ventricle 72 hr after fertilization. In this stage, a few GTH-ir cells were detected in the centro-dorsal part of the pituitary just posterior to PRL-ir cells, but the GTH-ir cells had no vacuoles. Eighty four hr after fertilization, just before hatching, the pituitary developed further and GTH-ir cells were distributed between the PRL-ir and GH-ir cells. In this stage, the GTIlI-ir cells increased in number, but vacuoles were not observed in the cytoplasm. After hatching, the GTII-ir cells shifted their position to the ventral part of PPD. GTH-ir cells revealed a similar distribution pattern to that seen in adult pituitary 50 days after fertilization, but the cells distributed in the PPD and PI had no vacuoles. IMMUNOHISTOCHEMICAL LOCALIZATION OF MELANOCORTIN IN THE BRAIN AND HYPOPHYSIS OF THE CLOUDY DOGFISH AND CHANGE WITH ONTOGENETIC DEVELOPMENT A.Chiba and Y.Honma. “Dept. of Biol., Sch. of Dent. at Niigata, Nippon Dent. Univ., Niigata,*Sado Mar. Biol. Stat., Fac. of Sci., Niigata Univ., Niigata. Using specific antisera raised against a-melanocyte stimulating hormone(a-MSH) and adrencorticotrophic hormone (ACTH), we examined the localization of immuno- reactivity in the brain and hypophysis of the cloudy dogfish, Scyliorhinus torazame, and its change with ontogenetic development. For the purpose, 6 adults, 2 juveniles and 3 embryos were studied. In the adults, melanocortin (a@-MSH and ACTH)-like immunoreactivities were demonstrated in the brain and two adenohypophysial lobes (the pars distalis and the pars intermedia) with some topographic variations in the brain: the a-MSH- immunoreactive perikarya were found in the nucleus tuberculi posterioris, the n. sacci vasculosi, the n. lateralis tuberis, the n. medius hypothalamicus and the posterior recess organ, whereas the ACTH-immunoreactive cell bodies were localized in the n. lateralis tuberis and some parts of the posterior recess organ. Immuno- reactivity was entirely absent in the embryos (15-35mm in total length) examined. In 1- and 50-day-old juveniles, melanocortin-immunoreactive structures similar to those of adults were demonstrated, although they were less dense than in the adults. These findings suggest multiple function of melanocortin as hormone and neuro- regulator in the dogfish hypothalamo-hypophysial system INFLUENCE OF ENVIRONMENTAL SALINITY ON CHLORIDE, PROLACTIN AND GROWTH HORMONE CELLS IN OREOCHROMIS MOSSAMBICUS YOLK-SAC LARVAE F.G. Ayson, T. Kaneko, S. Hasegawa and T. Hirano, Ocean Research Institute, University of Tokyo, Nakano, Tokyo 164 Newly-hatched larvae of OF, mossambicus survive direct transfer from FW to SW (33 ppt); larvae 2 days old or older can tolerate direct transfer from FW to 25 ppt SW. Because the major adult osmoregulatory organs are still poorly developed in early larval stages, chloride cells (CC) in the yolk-sac membrane were sought, using DASPEI, a stain specific for mitochondrion-rich cells. Changes in CC size and density through complete yolk resorption were recorded following transfer of newly-hatched larvae from FEW to SW. Prolactin (PRL) and growth hormone (GH) cells in the pituitary of the larvae were also examined by immunocytochemistry. Following transfer to sw, cc size increased significantly to 2- to 4-fold over that seen in FW larvae. The mean size of PRL cells and the area of the pituitary containing PRL cells were significantly larger in FW larvae than in SW larvae. GH cell size was not affected by transfer of larvae to SW. These results suggest the possible involvement of CC in the yolk-sac membrane and of pituitary PRL cells in osmoregulation of O. mossambicus yolk-sac larvae. 1258 Endocrinology COLD ADAPTATION AND PITUITARY OF THE SAFF- RON COD, ELEGINUS GRACILIS. M. Ogawa Dept.Biol.,Saitama Univ.,Urawa,Saitama. Most of the Antarctic teleosts has been reported to have an aglomerular kidney and the antifreeze glycoprotein in their sera. Saffron cod survives the ice-laden coastal waters during the winter by possessing hi- gh concentration of antifreeze glycoprote-— in in the serum(Burchman et al.,1984). The seasonal variations of plasma osmo- lality, kidney structure and pituitary of the saffron cod were investigated. Plasma osmolality was increased during the winter than during the summer. In the winter fish, atrophy of glomerulus was ob- served. This may be related to the conser- vation of antifreeze glycoproteins for cold water. Growth hormone(GH)- and prola- ctin(PRL)-secreting cells in the pituitary were identified by immunohistochemical staining used antisera raised against the chum salmon hormones. In the summer fish, the positive immunoreactions with both GH and PRL were remarkable than those of the winter fish. Recently it is known that GH blocks the antifreeze glycoprotein mRNA transcription in the liver. However, these results may suggest the possibility of PRL clearing of antifreeze glycoprotein of the serum by increasing glomerular filtration during summer. PROLACTIN ENHANCES CELL PROLIFERATION OF EXOCRINE PANCREAS IN MICE M.Matsuda, T.Mori, M.K.Park and S.Kawashima Zool. Inst., Fac. of Sci., Univ. of Tokyo, Tokyo Prolactin (PRL) has a diverse action on various tissues in vertebrates. Recently, PRL is known to stimulate proliferation of rat pancreatic B-cells, although no report is available for PRL effects on the exocrine Pancreas. However, we have demonstrated that in mice pituitary isografting increases pancreatic weight, associated with hyperprolactinemia. The weight increase is supposed to be due to the proliferation and/or hypertrophy of pancreatic acinar cells, since B-cells constitute only a small part of the total pancreatic volume. In this study, an anterior pituitary gland was grafted under the renal capsule in 6-wk-old male SHN mice (PG mice). Pituitary grafting resulted in marked increases in the weight and DNA content of the pancreas in PG mice at 30 days compared to those in controls bearing no graft. However, DNA and protein contents per tissue weight of the pancreas were less than controls, suggesting that hypertrophy of acinar cells also contributes to the weight increase of pancreas. Rate of DNA synthesis determined by BrdU labeling was greater in both endocrine and exocrine pan- creas in PG mice than in controls. There- fore, pituitary grafting stimulates the proliferation and hypertrophy of pancreatic acinar cells as well as islet cells. PROLIFERATION OF ANTERIOR PITUITARY CELLS IN STREPTOZOTOCIN-INDUCED DIABETIC RATS. S.Takahashi. Dept.of Biol., Fac. of Sci., Okayama Univ., Okayama. Insulin is involved in the cell proliferation of fibroblasts, hepatocytes and some of cell lines. The purpose of the present study is to clarify the role of insulin in the proliferation of rat Pituitary cells. Male rats of the Wistar/Tw strain were intraperitoneally Ziven streptozotocin (STZ) at a dose of 32.5 mg/kg. STZ-treated rats showed high blood glucose levels and low serum insu- lin levels. Cell proliferation was exam- ined by the detection of bromodeoxyuri- dine (BrdU)-uptake cells. Estradiol-17B8 (504g) was given to stimulate the pro- liferation of pituitary cells. The number of BrdU-uptake cells was lower in STZ-treated rats than in controls. The responsiveness to estrogen on the cell proliferation was lower in STZ-treated rats. Insulin treatment in STZ-treated rats increased the number of BrdU-uptake cells and recovered the responsiveness to estrogen. Particularly, insulin stimulated the proliferation of prolactin cells in estrogen-treated rats. These results suggest that insulin is necessary to the proliferation of pitui- tary cells. SUPPRESSION OF THE DEVELOPMENT OF UTERINE ADENOMYOSIS BY DANAZOL TREATMENT IN MICE T. Singtripop?, T. Mori!, S. Sakamoto? S. Sassa*, M.K. Park+ and S. Kawashima?+ +Zool. Inst., Fac. Sci., Univ. Tokyo and 2Dept. Endocr., Med. Res. Inst., Tokyo Med. Den. Univ., Bunkyo-Ku, Tokyo 113 Danazol, a Synthetic gonadotropin inhibi- tor, has been used clinically in the treat— ment of uterine adenomyosis and various breast disorders. In this study, the inhi- bitory effects of danazol on the sponta- neously developed- and experimentally in- duced-adenomyosis were studied in SHN mice. Female mice were given daily injections of 0.5 ug danazol for 5 weeks during 4-9 or 9-14 weeks of age. To accelerate the gene- sis of adenomyosis, the other group of mice was ‘isografted with anterior hypophysis in- to the uterine lumen at 5 weeks of age. A half of this group was given danazol for 4-9 weeks of age. Mice receiving sesame oil only served as controls. All mice were killed at 21 weeks of age. The results showed that danazol treatment resulted in a Significantly lower incidence of Spontaneous development of adenomyosis than the control, and also inhibited the genesis of experimentally induced-adenomy- osis. Furthermore, danazol treatment caused the decrease serum levels of LH and PRL associated with hypofunction of ovaries and persistent diestrus. These results sup- port the usefulness of danazol for the cli- nical treatment of gynecological disorders except for hypofunction of ovaries. Endocrinology 1259 MORPHOLOGICAL STUDY OF MOUSE _ PITUITARY CELLS CULTURED ON COLLAGEN- OR _ POLY-L- LYSINE-COATED_ SUBSTRATA. J K.Nakatomi, S.Takahashi_ and_Y.Kobayashi, Dept.of Biol., Fac.of Sci., Okayama Univ., Okayama. In vitro stud S_necessar to stud an u ay mechanism | of Bs differentiation proliferetion of anterior pituitary cells. The purpose of this study was to clarify the effects of culture substrata_ on dissociated mouse pituitary cells. Type PeeyeS collagen-, or _ poly-l-lysine- coated PLL) glass coverslips, an non- coated glass ‘coverslips (control) were used as culture substrata. The anterior Gey cells from male ICR mice (5-s weeks of age were dissociated with 0.5% trypsin at ine for 15 min. Pituitary cells were suspended in Dulbecco modifie Eagles' medium containin 20mM HEPES supplemented with 10% feta calf serum. he dissociated cells (2.2x 10° cells/well) were seeded on ‘Gey Ea ae coat edit En Oy it : 4 ug/cm2 o pe or type collagen an O, . (0) EB £ PLL. Growth hormone {GH) ‘and prolactin (PRL) cells were immunocytochemiclly detected at 3 and 5 days of culture. We found two Eypes of GH and PRL cell, round-shaped cells and elongated cells. The relative propotions of round-shaped GH or PRL cells changed with the concentrations’ of eee ue fv collagen or PLL. The number of and PRL cells was highest in the culture condition of type I collagen-coated substrata (a ney eee This result indicats that SE I collagen is the most suitable substratum. Under this culture condition, effect of GH-releasing factor (GHRH on the mumber of GH _ cells was_ studied. PSE °M) treatment for 48hr PETE a 1.3-fold increase in the percentage of GH compared with controls. These suggest that meno Toay: of pituitary cells is affected z py culture substrata (ex. collagens Ss). cells results cultured artl ss PE EFFECT OF NEUROPEPTIDES ON CHANGES’ IN MAMMOTROPH NUMBER -FLOW CYTOMETRIC ANALYSIS- T. Shinkai and H. Ooka. Dept. of Cell Biology, Tokyo Metropol. Inst.of Gerontol. Tokyo. We developed a procedure for detection of pituitary mammotrophs with flow cytometry and analyzed the effects of neuropeptides on changes in the mammotroph number in vitro using the technique. Rat anterior pituitary cells, were cultured in 24-well plates at 8x10°*/well with 1 ml medium. After fixation of the cells with Bouin's solution, they were treated by the flow-cytometry technique. The number of mammotrophs increased in basal culture medium. The addition of GRF suppressed the increase in the mammotroph number. The addition of LHRH accelerated the increase in it. These results were similar to those of light microscopic examination. Other neuropeptides(PRL releasing peptides: PHI, substance P, angiotensin II, B-endorphin, leucine enkephalin, TRH, vasopressin, VIP, oxytocin and galanin, other peptides: bradykinin and NPY) increased in the mammotroph number up to the control level and had no effect on the changes in the mMammotroph number. These results indicate that PRL releasing factors are not always proliferation factors for the mammotroph. , PHOSPHORYLATION OF THE PIT-1 TRANSCRIPTION FACTOR IS NOT ESSENTIAL TO PROLACTIN GENE ACTIVATION IN MtT/S CELLS S. Nagata’ and Michael G. Rosenfeld2. 1Dept. Material and Biol. Sci., Fac. Sci., Japan Women’s Univ., Tokyo and Howard Hughes Med. Inst., School of Med., Univ. of California, San Diego. Growth-hormone (GH)-producing MtT/S cells switch to prolactin (PRL) production upon cultivation with estrogen and IGF-1. Western immunoblot analyses of the nuclear and cytoplasmic extracts showed no change in the Pit-1 contents in PRL gene activation. Incorporation of [85S]-amino acids into Pit-1 and nuclear translocation of the labeled Pit-1 were slightly stimulated as detected by SDS-PAGE analyses of the immunoprecipitates . After incubation of the cells for 3 hr with inorganic 32P, [32P]-Pit-1 was immunoprecipitated from the cytoplasmic extract. However, trace amount of [82P]-Pit-1 was detected in the nuclear extract. Stimulation of the cells with estrogen and IGF-1 for 20 min, 6 hr, 96 hr did not alter the amount of [82P]-Pit-1. EGF stimulated Pit-1 phosphorylation by itself and enhanced estrogen/IGF-1- induced PRL gene activation. But EGF alone could not induce PRL. Therefore, availability of phosphorylated or non-phosphorylated Pit-1 may not be essential for estrogen/IGF-1-induced PRL gene activation in MtT/S cells. Involvement of additional factors interacting Pit- 1 should be considered. INVOLVEMENT OF MAMMALIAN GROWTH HORMONE AND PROLACTIN IN INDUCTION OF VITELLOGENIN IN THE PRIMARY CULTURE OF EEL HEPATOCYTES. H.-C. Kwon, Y. Mugiya. Lab. Physiol., Fac. Fish., Hokkaido Univ., Hakodate. The role of pituitary hormones in vitellogenin (Vg) synthesis was investigated using the primary culture of immature eel hepatocytes in serum free medium. When cultured with estradiol-178 (E2, 2x10 6M) alone, little or no Vg was found in the medium throughout an experimental period of 9 days. Estradiol-178 administration to hypophy- sectomized eels also failed to induce in vivo Vg synthesis. However, cultures added E2 together with mammalian growth hormone (GH) or prolactin (PRL) induced Vg synthesis and the rate of its production gradually increased untill at least 9 days. Furthermore, even in the culture of hepatocytes obtained from immature eels primed from E2 administration, the addition of E2 alone did not show the increased synthesis of Vg, while the combination of E2 and GH or PRL stimulated Vg synthesis. Nither GH alone or PRL alone had any effect on Vg induction. These results demonstrate that the pituitary of eel may be involved in the hepatic synthesis of Vg. 1260 Endocrinology THE EFFECT OF DIFFERENT REGIONS OF BRAIN TISSUE ON CELL PROLIFERATION OF THE ADENOHYPOPHYSIAL PRIMORDIUM M. Shirai and Y. G. Watanabe Dept. of Biol., Fac. of Sci., Niigata Univ., Niigata. We previously reported that in fetal rats the developing diencephalic floor stimulated prolifera- tion of adenohypophysial primordial cells. In this study, we investigated if other parts of brain increase the rate of cell proliferation in the adenohypophysial primordium by the use of the BrDU method. For this purpose, the rostral end of the telencephalon and parts of the diencephalon ( its roof, posterior wall and chiasmal area ) were selected and their effect on the developing adeno- hypophysis was compared with the effect of the diencephalic floor. The effect of surrounding mesenchymal tissue was also studied The adenohypophysial primordia were removed from rat fetuses on day 13.5 of gestation and cultiveted with several different regions of brain. Other than the diencephalic floor facing the adenohypophysis the posterior wall of the diencephalon ( the presumptive mammillary body ) augmented the prolife- rative rate of adenohypophysial cells. Mesenchymal tissue had no effect on cell proliferation. These results suggest that the developing dien- cephalic floor specifically contains some factor(s) which is essential for the proliferation of adeno- hypophysial primordial cells. WHAT AN ANOMALY OF THE FETAL RAT TELLS ABOUT THE DEVELOPING BRAIN-ADENOHYPOPHYS!AL RALATIONSHIP Y. G. Watatabe and H. Haraguchi Dept. of Biol., Fac. of Sci., Niigata Univ., Niigata We have hitherto presented in vitro data showing that the developing diencephalic floor is involved in growth and cytogenesis of the rat adenohypophysis. In the course of a study on the adenohypophysis we have found an abnormal rat fetus in which adeno- hypophysis was out of contact with the brain. Since experiments of brain-adenohypophysis disconnection are generally difficult in mammalian fetuses, histo- logical study of such adenohypophysis is of quite interest. The results are summarized as follows: 1) The volume of this abnormal adenohypophysis was far smaller ( less than 1/10 ) than that of the normal gland. 2) When immunostained with anti- aMSH serum, the definitive pars intermedia was not ob- served. 3) A small number of immunoreactive cells were seen and after staining with antisera against ACTH, GH, TSHB and LHB. Whereas PRL cells could not be found All of these data suggest an important role of the diencephalic floor in the normal histogenesis of the adenohypophysis especially of the intermediate lobe. FINE STRUCTURAL STUDY ON THE PITULTARY—ADRENAL SYSTEM OF MICE UNDER PROLONGED EXCESS LIQUID INTAKE. S.Thara, S.Nishibayashi, S.Takeuchi, S. Takahashi and Y. Kobayashi. Dept. of Biol., Fac. of Sci., Okayama Univ. Okayama. It has been previously demonstrated that induced polydipsia(ca. 200% v/w/day) by giving a 5% glucose solution concomitant with food deprivation resulted in marked hypersecretion in cells of the mouse pituitary intermediate lobe. Despite malnutritional condition of this excess drinking animals can survive for 3 weeks or so. In the present study the effects of prolonged liquid intake on the pituitary and adreal glands were investigated. Animals were divided into 3 groups, (1) normal controls, (2) mice given a 4% powedered milk solution without food for 3 weeks and (3) mice given a. 5% glucose solution alone for 3 weeks. Their body weights increased to 115% of the initial value in group 1 but decreased to 70.9% and 62.4% in groups 2 and 3, respectively, both of which showed a marked increase in liquid consumption during the time of experiment. Morphometrical analysis of the pituitary intermediate lobe cells at the electron microscopic level indicated hypersecretion in group 3. Cell atrophy and involution of the pituitary anterior lobe were recognized. The wet weight of organs/g body weight decreased in those including the pituitary, thymus, spleen, and kidney, and no change in pancreas and testis. The adrenal glands showed weight gain/g body weight. These results indicated that prolonged liquid intake, especially in mice of 5% glucose drinking, stimulated the secretory activity of the pituitary intermediate lobe cells that due probably not to malnutrition but to polydipsia. DIFFERENTIAL DISTRIBUTION OF BIOACTIVE PEPTIDES RELATED WITH DRINKING BEHAVIOR IN THE RAT SUBFORNICAL ORGAN. = T. Yamamoto, T. Hirohama, S. Nakamura, and H. Uemura. Biol. Lab., Kanagawa Dent. Coll., Yokosuka. The distribution o loactive peptides related with drinking behavior, namely substance P (SP), Leu-enkephalin (Leu- enk), neurotensin (NT), cholecystokinin (30-39, CCK), LHRH, vasopressin (VP), and B-endorphin, was immunohistochemically studied on the rat subfornical organ (SFO), one of the drinking center in the central nervous system. Leu-enk- and SP- immunoreactive fine fibers were located on the anterodorsal half of the SFO. On the other hand, NT- and CCK-immunoreactive fibers were observed in the central portion of the SFO. Although a few thick LHRH-immunoreactive fibers were seen in the central portion of the SFO, more fibers were aligned on the ventral midline of the fornix where glial elements were condensed. A few very fine VP- immunoreactive fibers were also detected Mainly on the ependymal tissue covering ventral surface of the SFO. Endorphin- immunoreactivity was not detected in the SFO although extensive immunoreactive fibers were observed, for example, in the hypothalamus. These results suggest the differential termination of peptidergic fibers in the SFO and ecueeeenl differences within the SFO in contribution of bioactive peptides to drinking behavior. Endocrinology 1261 PRODUCTION AND CHARACTERIZATION OF SPECIFIC ANTIBODY AGAINST a-SUBUNIT OF RAT PITUITARY GLYCOPROTEIN HORMONES S. Tanaka, H. Mochida, S. Kurabuchi!, H. Hayashi, K. Wakabayashi and K. Kurosumi (Inst. of Endocrinol., Gunma Uniy., Maebashi and INihon Dental Univ., Tokyo) To obtain an antibody specific for a-subunit of rat pituitary glycoprotein hormones, we synthesized a peptide corresponding to the sequence 37-53 (Phe-Ser-Arg-Ala-Tyr- Pro-Thr-Pro-Ala-Arg-Ser-Lys-Lys-Thr-Met-Leu-Val) of the rat @-subunit. The polyclonal antibody against this peptide was generated in rabbit. This region is hydrophilic, and highly conservative among several mammalian species. This antiserum immunostained two types of cells in rat anterior pituitary: the one type contained LHB, and the other, also TSH. The anterior pituitaries of mouse, ovine, and porcine, which have identical sequence of the present peptide were immunostainable with this antiserum. However, human (45- Leu in a-subunit) and bullfrog (45-Met) were not stained with this antiserum. Similarly, using this antiserum the immunoreactive cells were found only in the anterior pituitaries of Urodele, Rhacophorus, and Hyla groups among fifteen amphibian species including Rana group examined in this study. These data suggest that this antibody is sequence specific and useful to immunological study. HEMORRHAGE AFFECTS THE ENDOTHELIN CONTENTS IN THE RAT PITUITARY. Se Nakamura!, 1, Yamamoto!, M. Naruse2, K. Naruse2, K.Aoki3, T.Hirohama', H.Uemura'. 1TBiol. Lab., Kanagawa Dent. Coll., Yoko- suka, 2Dept. of Med., Tokyo Women's Med. Coll., Tokyo, 3Div. of Biol., Natl. Inst. Radiol. Sci., Chiba. To investigate the role of endothelin (ET) in the pituitary, ET-1 and ET-3 were measured by enzyme immunoassay in rats subjected to hemorrhage via the cannula inserted into the external carotid vein. Plasma levels of arginine vasopressin (AVP) were also estimated by radioimmuno- assay. Hemorrhage (4 ml/260-275 g body weight) induced a significant elevation of plasma AVP levels. The content of ET-1 in the neural lobe [155 + 65 (n=7) vs 414 + 35 (n=9) pg/g wet weight], but not that of ET-3 [456 + 112 (n=6) vs 311 + 68 (n=5) pg/g], significantly increased immediately after hemorrhage. In the glandular lobe, hemorrhage significantly reduced both ET-1 [88.4 + 13.4 (n=8) vs 41.1 + 2.7 (n=10) pg/g, Aspin-Welch method] and ET-3 [731 + 95 (n=8) vs 361 + 66 (n=9) pg/g] levels. The present results suggest that ET-1 and ET-3 in the pituitary are involved in mediating the physiological responses to hemorrhage in the rats. APPLICATION OF A REVERSE HEMOLYTIC PLAQUE ASSAY FOR DETECTION OF HORMONE RELEASE FROM AMPHIBIAN PROLACTIN CELLS T.Kawasaki,K.Nakajima,T.Kouki and S.Kikuyama Dept. of Biol., Sch. of Educ., Waseda Univ., Tokyo. Reverse hemolytic plaque assay was in- troduced to study the response of in- dividual prolactin cells in a mixed culture of bullfrog(Rana catesbeiana) distal lobe cells to TRH(pyro Glu-His-Pro NHz2). The procedures were essentially the same as those described by Frawley et al.(1983). In the present experiment, medium 199 diluted to 75% was used for culture. Anti- prolactin antiserum used in the plaque as- say and for the immunohistochemical detec- tion of prolactin cells was produced in a rabbit immunized with highly purified bullfrog prolactin. Both rate of plaque formation and mean plaque area were in- creased according to the time of TRH(10~°M) treatment over the period of 90 min, and also in response to the concentrations of TRH(107%-107-7°M) as measured after 90 min of treatment. MOLECULAR CLONING AND NUCLEOTIDE SEQUENCE ANALYSIS OF cDNA FOR TOAD PROLACTIN N.Takahashi, K.Yamamoto, and S.Kikuyama. Dept. of Biology, Sch. of Education, Waseda Univ. The cDNA encoding C'-terminal sequence of toad (Bufo japonicus) prolactin (PRL) was specifically amplified employing polymerase chain reaction (PCR) method from cDNA encoding mRNA extracted from anterior pituitaries. Sequence analysis revealed that this clone contains 602bp nucleotides and is encoding 134 amino acids of C'-terminal region of toad PRL molecule. This region of toad PRL had Qiks, SSB, Gils, GO%w, GOB Amel Wey homologies with the corresponding region of bullfrog, salmon, sea turtle, chicken, porcine and human PRLs. Northern blot analysis carried out using this cDNA as a probe revealed that PRL mRNA consists of about 1.0 kb nucleotides. 1262 Endocrinology ELEVATION OF PROLACTIN mRNA LEVELS BY NA- TIVE AND SYNTHETIC TRHs IN THE BULLFROG DISTAL LOBE. S.Sasaki, N.Takahashi, K.Nakajima and S.Kikuyama. Dept. of Biol., Sch. of Educ., Waseda Univ., Tokyo. We have obtained a substance which has prolactin-releasing activity from the bullfrog hypothalami. This substance was identified as TRH (pyroGlu-His-ProNH2) by means of radioimmunoassay, amino acid analysis and reverse-phase high perfor- mance liquid chromatography. Northern blot analysis of prolactin mRNA in the pituitary incubated in the presence of the native or synthetic TRH(10~°M) were performed using bullfrog prolactin cDNA as a probe. AS a result, both samples similarly elevated the prolactin mRNA levels 2.5 times as high as the control value with a concomitant rise of prolac- tin levels in the medium, when measured 16h after incubation. Two forms of prolactin exist in the Xenopus distal lobe K. YAMASHITA*, K. MATSUDA*, K. YAMAMOTO>, Y. HANAOKA*, H. HAYASHI*, S. TANAKA* and T. KATO®, S. KIKUYAMA* *Dept. of Biol., Sch. of Educ., Waseda Univ., Tokyo 169-50, #Inst. of En- docrinol., Gunma Univ., Maebashi 371 and *Kirin Co., Maebashi 371. Two forms of prolactin (PRL), having antimetamorphic activity as tested with tail fragments of Xenopus larvae cultured in the presence of Ta, were isolated from the pituitary gland of Xenopus laevis by extraction with acid acetone, and high performance liquid chromatography (HPLC) on anion exchange, gel filtration and reverse-phase columns. The molecular weight of both PRLS were 23,000 and isoelectric points of xPRL I and II were als eyeuel Bio esic Sequence homology between one of the lysyl endopeptidase-digested fragments from xPRL I and that from xPRL II, which are comparable to bullfrog PRL (116-172), was 88 %. XPRL I and II showed 73 and 68 % sequences homologies with bullfrog PRL, respectively. They showed little sequence homology with GH I and II. EFFECT OF ARGININE VASOTOCIN (AVT) AND AVT-RELATED PEPTIDES ON CONTRACTION OF IN- TESTINE IN XENOPUS LAEVIS D. Kuwagaki, S. Iwamuro, S. Kikuyama Dept. of Biol., Sch. of Educt, Waseda Univ., Tokyo a We have previously found that injections of AVT to Xenopus induces defecation. This lead us to study the effect of arginine vasotocin (AVT) and AVT-related peptides such as hydrin 1 (AVT-GKR) and hydrin 2 (AVT-G) on the contraction of Xenopus in- testine in vitro. Xenopus intestine (approximately 10 mm long) was immersed in amphibian Ringers solution containing various concentrations (10°°%-10°° M) of the test substances. Changes of intestinal length were measured by using a Magnus ap- paratus. The intestine showed contraction within 10 min in response to AVT, hydrin 1 and hydrin 2, their minimum effective concentrations being 10°*, 10°” and 10°7 M respectively. The contraction induced by 10-7 M AVT, hydrin 1 and hydrin 2 was blocked by the simultaneous application of 10-@ M of [ d(CHz2)s,0-Me-Tyr*,Arg®]JAVP (V1 receptor antagonist) and [Pmp~*,0-Me- Tyr7,Arg®]AVP (V1/V2 receptor antagonist), but not of [d(CHz)s,D-Ile~,Ile*,Arg*]AVP (V2 receptor antago-nist). The results in- dicate that the effect of AVT and hydrins on the intestinal contraction is mediated through V1 type receptors. EFFECT OF NEUROHYPOPHYSEAL SUBSTANCES ON THE SECRETION OF IMMUNOREACTIVE JOINING PEPTIDE BY THE BULLFROG DISTAL LOBE S. Sugihara, S. lwamuro, and S. Kikuyama Department of Biology, School of Education, Waseda University, Shinjuku-ku, Tokyo 169-50. In amphibians, joining peptide (JP) is processed from the multiple precursor protein proopiomelanocortin (POMC) not only in the intermediate lobe but also in the anterior lobe. Secretion of other POMGC-derived peptides, such as ACTH and a-MSH, is known to be affected by neurohypophyseal substances. Development of radioimmunoassay (RIA) for JP enabled to ascertain whether the neurohypophyseal substances also influence the release of immunoreactive JP. Hemisected anterior lobes of adult bullfrog pituitaries were incubated in 67% Eagle's MEM over the period of 3, 6, 12 and 24 h. Released JP in the medium was measured by RIA. The release of immunoreactive JP increased according to the incubation period and in response to AVT, hydrin2 (AVT-Gly), MT and TRH added to the medium. In contrast, dopamine inhibited the release of immunoreactive JP into the medium. Endocrinology 1263 EFFECTS OF ARGININE VASOTOCIN AND PARA- THYROID HORMONE ON CYCLIC AMP FORMATION IN THE KIDNEY AND THE URINARY BLADDER OF THE BULLFROG. M. Uchiyama. Dept. Oral Physiol., School Dentis. Niigata, Nippon Den. Univ., Hamaura-cho, Niigata. Arginine vasotocin (AVT) and parathy- roid hormone (PTH) are major regulators of water and electrolyte excretion by the kidney and the urinary bladder. In order to elucidate the sites of actions of both hormones in these organs, the hormone— dependent cyclic AMP (cAMP) formation was studied in vitro. The basal cAMP levels are not significantly different among the segments (proximal, distal and connecting tubules and ureter) and the urinary blad- der. AVT activated cAMP production in the connecting tubules and the urinary blad- der. Marked dose-dependent activation by AVT was observed in preparations from the connecting tubules and the urinary blad- der. No activation by hPTH(1-34) was ob- served in all preparations from the neph- rons and the urinary bladder. These re- sults suggest that Vo-type receptors are present in the connecting tubules of neph- ron and the urinary bladder. The lack of hormonal activation of cAMP production ob- served in some tissues may reflect either the total absence or the presence in minor quantity of hormonal receptors associated with a cAMP-generating system in these or- gans. ISOLATION AND CHARACTERIZATION OF BULLFROG THYROTROPIN M. Sakai!, H. Hayashi, Y. Hanaoka2, S. Tanaka? and S. Kikuyama! Dept. of Biol., Sch. of Educ., Waseda Univ., Tokyo. 2inst. of Endocrinol., Gunma Univ., Maebashi Highly purified amphibian TSH was obtained for the first time from the distal lobes of Rana catesbeiana. Glycoprotein fraction was obtained from the acetone-dried pituitaries by ethanol precipitation. This was subjected to hydrophobic interaction chromatography, anion exchange chromatography, affinity chromatographies using anti- bullfrog LHB and FSHB antibodies. Glycoprotein fraction thus obtained had thyrotropic activity about 4 times as potent as bovine TSH and had no LH (germinal vesicle breakdown-inducing) bioactivity. In addition, it was confirmed that its thyrotropic activity was completely nullified by the incubation with antibody against bullfrog LHa. This fraction was subjected to hydroxyapatite column chromatography. One of the peak substances exhibited thyrotropic activity about 10 times as potent as bovine TSH. This fraction was reduced and subjected to SDS-polyacrylamide gel electrophoresis. As a result, two bands were observed at the positions of 14 and 17 kilodalton. When the reduced TSH preparation was pyridylethylated and applied to reverse phase HPLC, three main peaks were obtained. Amino acid sequence of N- terminal 13 residues of the two peaks coincided with that of a-subunit of bullfrog LH/FSH suggesting the difference in the sugar chains between the two. Amino acid sequence of N-terminal 13 residues of the remaining peak showed 46% homology with those of bovine and porcine TSHBs. Thus, it was revealed that bullfrog TSH consists of B- subunit with a considerable homology with mammalian TSHB and a- subunit presumably similar to that of bullfrog LH/FSH. ABSENCE OF METHYLATED TRH IN THE BULLFROG (RANA CATESBEIANA) BRAIN. K.Nakajima*, T.Yanagisawa*, S.Tanaka® and S.Kikuyama*. *Dept. of Biol., Sch. of Educ., Waseda Univ., Tokyo, 7Dept. of Agricultural Chem., Utsunomiya Univ., Ut- sunomiya, and *Inst. of Endocrinol., Gunma Univ., Maebashi. Recent finding that [1-Me-His*]TRH is present in the carp brain prompted us to ascertain whether the methylated TRHs exist in the frog brain. Samples were prepared from whole brain tissues of adult frogs, climax tadpoles and prometamorphic tadpoles by acid extraction. Each sample was sub- jected to reverse-phase HPLC. Standard samples of synthetic TRH, [1-Me-His~]TRH and [3-Me-His*]TRH were eluted by MeCN with concentrations of 4.5%, 4.8% and 6.9% respectively. The eluates were assayed using two antibodies against TRH, one show- ing 100% crossreactivity to both methylated TRHs and the other showing 3% and 43% erossreactivities to [1-Me-His*]TRH and [3- Me-His*]TRH. Both the distribution and amount of TRH in the eluates of each sample did not vary between the assays using the two different antibodies. In both cases a single peak with a similar immunoreactivity appeared at the position similar to that for synthetic TRH. Thus, it was concluded that the brains of adult and larval bullfrog do not contain any detectable methylated TRHs. EFFECT OF PARTIAL ABLATION OF HYPOTHALAMIC PRIMORDIUM ON THE ESTABLISHMENT OF BACKGROUND ADAPTATION. Z. Kato, K. Kawamura and S. Kikuyama, Dept. Biol., Schl. Educ., Waseda Univ., 1-6-1 Nishi-Waseda, Shinjuku-ku, Tokyo 169-50, Japan. d Our previous study indicated that in the toad, Bufo japonicus, preoptic recess organ (PRO) is the most probable candidate for the center involved in the background response. In order to confirm this, experiment was conducted using the embryos. Anterior part of the neural plate, closely situated to the anterior neural ridge, and prechiasmatic area were manually removed at the open neurula and the tail-bud stages, respectively. After metamorphosis, they were tested for the responsivenss to the background color. The animals which did not respond to the background but were permanently black, tyrosine hydroxylase (TH)-positive neurons in PRO did not develop or developed poorly, whereas in paraventricular organ and nucleus infundibularis dorsalis, TH-positive neurons developed normally. On the other hand, in those showed a normal background response, well-developed TH neurons were observed in PRO as in the sham-operated animals. These results demonstrate that dopaminergic neurons in PRO is responsible for the control of MSH secretion in the toad. 1264 Endocrinology IN VITRO RELEASE OF THYROXINE AND THYROGLOBULIN FROM TADPOLE THYROID GLAND WITH SPECIAL REFERENCE TO CYCLIC AMP RELEASE Shintaro Suzuki and Keiko Fujikura Inst.of Endocrinol., Gunma Univ., Maebashi To elucidate how cyclic AMP (cAMP) (second messenger) is related to the release of thyroid hormones and thyro- globulin (TG) in metamorphosing tadpoles we examined the release of cAMP from _ the thyroid gland. Thyroid half lobes of Rana catesbeiana tadpoles were incubated in vitro in the medium (Amphibian Ringer solution, 0.2% glucose, 0.1% BSA, 10-4M theophylline) containing bovine thyroid stimulating hormone (bTSH) or’ forskolin (adenylate cyclase activator). Thyroxine (Ts), TG and cAMP released into the incubation medium were measured by radio- immunoassay. Both bTSH and forskolin stimulated the release ol Ts, but did not stimulate the release of TG so much. A considerable amount of TG was released even from untreated thyroid gland. The amount of T.4)released into the medium increased depending on the concentration of bTSH or forskolin. The release of cAMP from the thyroid gland was also observed when bTSH or forskolin stimulated the release of T,4. These results suggest that in the release of thyroid hormones adenulate cyclase is an important trans- ducer and TG-release is regulated by another mechanism in the thyroid gland. ARTIFICIAL INDUCTION OF NEOTENY IN HYNOBIUS RETARDATUS DUNN II. FEMALE SALAMANDER CAN ALSO SEXUALLY MATURE IN THE LARVAL FORM. M. Wakahara, Zool. Inst., Fac. Scei., Hokkaido Univ., Sapporo. Hynobius retardatus Dunn, a hynobiid salamander inhabited in Hokkaido, is a unique species which had been reported to show neoteny. Unfortunately, however, the neotenic population is believed to be extinct. We are thus trying to revive the neoteny in the laboratory conditions. A large number of newly hatched laryae of Hynobius were reared in different doses of thiourea (TU). All larvae reared in TU-free and in lower concentrations of TU metamorphosed normally within 40-90 days of rearing. Several larvae in medium concentrations (0.01-0.04%) survived as long as 800 days or over in an aquatic form. Histological observations revealed a lot of bundles of morphologically mature spermatozoa in the testes of one year-old males, and full grown oocytes filled with large yolk platelets in the ovaries of two years-old females, both of which had well developed external gills and central fins. It is concluded that even female individuals become sexually mature without undergoing metamorphosis in the laboratory conditions. Therefore, it seems possible to revive the neoteny in Hynobius. CHANGES IN MONODEIODINASE ACTIVITIES DURING FLOUNDER METAMORPHOSIS E. G. de Jesus, M. Tagawa, and T. Hirano Ocean Research Institute, University of Tokyo, Nakano, Tokyo, 164 In mammals, thyroxine (Ty) is considered as prohormone that is converted to triiodothyronine (Tg) in order to exert its biological effects. Ty can also undergo monodeiodination to yield inactive reverse ‘T (rT3). During flounder metamorphosis, tissue Ty levels show a dramatic increase while T3 levels increase only slightly. However, T3 is several times more potent than T4 in promoting metamorphosis. Assays for the activity of both 5- and 5’-monodeiodinases were developed. The rates of both 5- and 5’- monodeiodination were low during premeta- morphosis, increased dramatically during prometamorphosis, remained high through metamorphic climax, and declined in post- climax and juvenile fish. , These results suggest increased turnover of both T, and T3 during metamorphosis. oat was previously reported to be absent from teleost plasma; however, not only is rT present in whole body homogenates o flounder at all stages of metamorphosis, but also changes in its concentration closely paralleled those of Ty, and T3- The importance of the monodeiodinase systems and the regulation of thyroid hormone levels during flounder metamorphosis will be discussed. EFFECTS OF THYROIDECTOMY AND REPLACEMENT THERAPY WITH THYROID HORMONE ON THE FIRST SKIN-SHEDDING IN HATCHLING SNAKES. M. Yoshihara! T. Murakami’ and C. Oguro? Dept. of Oral Physiol., The Nippon Dental Univ., Niigata, 2Toyama Univ., Toyama. It is well known that thyroid hormone stimulates molting in the lizards. On the contrary, it has been reported that thyroid hormone inhibits skin-shedding of adult snakes. In the hatchlings of the snake, Elaphe quadrivirgata, the first shedding takes place about one week after hatching. In the present study, role of the thyroid gland and thyroid hormone on the first shedding of the hatchling snakes was searched. Thyroidectomized hatchlings did not shed the skin until 15 days after hatching. In the sham-operated hatchlings, the first skin-shedding takes place nor- mally between 6 and 8 days after hatching. Thyroidectomized hatchlings injected with thyroid hormone also shed normally. On the other hand, administration of control saline in thyroidectomized hatchlings did not cause the first shedding. These results indicate that the thyroid hormone is necessary for the first skin-shedding in the hatchling snakes, contrasting to the situation in the adults. Endocrinology 1265 THE ANNUAL CHANGES IN SERUM THYROID HOR- MONE CONCENTRATION OF BOBWHITES (COLINUS VIRGINIANUS) REARED IN OUTDOOR CAGES. M.Sato H.Sakai, S.Wakabayashi, M.Shioya* and S.Sato*. Dept. of Biology, Nihon Univ. School of Dentistry, Chiyoda-ku, Tokyo 101 and *College of Agriculture and Vet. Medicine, Nihon Univ., Fujisawa 252. The relationship between the annual shift in serum thyroid hormones and the breeding and molting was examined. Ten male and female pairs of bobwhites hatched in March 1991 were housed in 10 cages under a natural daylight length and blood samples were examined, from July 1991, once every few weeks, until September 1992, together with egg laying, molting and food intake. The egg laying terminated in the middle of August 1991 and was again observed between May and August 1992. Molting of the whole body began in August 1991 and ended in December; in April, molting was partially observed and quite obvious in September 1992. No significant seasonal change in serum T4 was observed in both sexes. Serum T3, however, showed a remarkable increase in both sexes starting in August 1991 and the high values were Maintained by the middle of April 1992, lowered towards August of the same year. These results indicate that molting begins soon after the egg laying period, with which the marked increase in T3 well synchronized. This concurrence was observed earlier in males than in females. EFFECTS OF THYROIDECTOMY ON SEASONAL BREEDING OF JAPANESE MONKEYS M.Nozaki, K.Shimizu and F.Mitsunaga. Primate Research Institute, Kyoto University, Inuyama. To examine the role of thyroid hor- mones on the seasonal breeding of Japan- ese monkeys (Macaca fuscata), sexually mature females were thyroidectomized (THX; n=6) in early December, during the mid-breeding season, or they remained thyroid intact (n=4). They were housed indoors individually, and blood samples were collected three times a week to monitor gonadotropin and gonadal hormone secretions. All animals ovulated during the breeding season before operation. THX resulted in significantly earlier termination of the breeding season: Ovulatory cycles of all THX monkeys were terminated soon after THX. THX monkeys exhibited significantly high levels of plasma prolactin in comparison with those of controls. Such high levels of plasma prolactin in THX monkeys may be attributed to the increased secretion of hypothalamic TRH, which stimulates both thyrotropin and prolactin secretion. The present results suggest that thyroid hormones are involved in the regulation of the seasonal breeding of Japanese monkeys directly or indirectly by mediating the modification of prolactin secretion. ESTROGEN RECEPTOR-LIKE IMMUNOREACTIVITY WAS DETECTED TRANSIENTLY IN THE FACIAL NUCLEUS OF THE NEONATAL RAT. S.Hayashi ! and M.Yokosuka?. _—! Dept. Anat. & Embryol., Tokyo Metropolitan Inst. Neurosci., Fuchu, Tokyo and 2 Dept. Animal Reprod., Tokyo Univ. Agricul. & Technol., Fuchu, Tokyo. Estrogen receptor-like immunoreactivity (ER-LI) was detected in the brain of newborn rats by immunohistochemistry. It was detected not only in the hypothalamus, amygdala and midbrain central gray, but also in the medio-basal subnucleus of the facial nucleus. The antiserum (AS) used was raised in a rabbit in our laboratory, as the antigen a fusion protein of rat estrogen receptor (ER)-f-galactosidase construct produced in Esherichia coli cells being used. Specificity of this AS has been confirmed (Okamura et al., J.Endocr.,1992). The ER-LI in the facial nucleus was detectable only in the newborn rat, while that in the other part of the brain was detected also in the adult. Thus, the expression in this nucleus is only transient. By microinjections of a fluorescent tracer, fast blue, into the facial muscles of the newborn rat, motoneurons in the subnucleus, where the ER-LI was detected, were revealed to project their terminals to the posterior auricular muscles. However, the majority of motoneurons marked with the tracer did not contain ER-LI. No clear sex difference in the ER-LI was detected in this nucleus. Physiological roles of ER-LI in the facial nucleus of the newbom rat are presently unknown. (Supported in part by the Grants-in-Aids for Scientific Research to SH, No. 03640636, from the Ministry of Education, Science and Culture of Japan). ESTROGEN RECEPTOR (ER) EXPRESSION IN MOUSE UTERINE EPITHELIAL CELLS IN VIVO AND IN VITRO. T. Sato, T. Iguchi, *Y. Ohta and N. Takasugi. Grad. Sch. Integrated Sci., Yokohama City Univ., Yokohama; *Tottori Univ., Tottori. Nuclei of stromal cells in the mouse uterus showed a strong ER immunostaining on the day of birth (0 day of age). In contrast, ER in uterine epithelial cells appeared by day 5. In the uterine epithelial cells, ER was induced 24 h after a single injection of diethylstilbestrol (DES), 178- estradiol (Eg), testosterone or tamoxifen (Tx) given at O day. DES, Eg and Tx induced ER in uterine epithelial cells in vivo in a dose- dependent manner. A single injection of 3 ug DES significantly stimulated cell division of the epithelial cells in the uterus. In order to study the role of stromal cells in ER induction by estrogen, uteri of mice at 0 day were excised, trypsinized and separated into epithelium and stroma. The epithelial cells cultured on collagen membrane in serum-free medium in the presence or absence of Eg showed no ER immunostaining, but the stromal cells showed ER immunoreaction after 3 days in culture. When epithelial cells and stromal cells were co-cultured on either side of a collagen membrane, 15% of epithelial cells showed ER immunoreaction. These results indicate that 1) the ER induced by neonatal injections of DES is functional; 2) ER expression in the epithelial cells appears as a result of interaction with stromal cells. 1266 Endocrinology MITOTIC CHANGES IN THE UTERUS OF PREGNANT RATS DURING THE DEVELOPMENT OF THE METRIAL GLAND. , 2 2 1 Y.Ohta , T. Satoh” and T.Iguchi’. Dept. of Biol., Tottori Univ., Tottori, Dept. of Biol., Yokohama City Univ., Yokohama. gland develops in the mesometrial triangle of the uterus during the mid-pregnancy, persisting after parturition. The distri- bution of granulated metrial gland (GMG) cells and. their mitotic activities were investigated in the rat uterus during the mid-pregnancy. In the mesometrial decidua, GMG cells slightly increased in number during Days 8-12 of pregnancy and then decreased, while the cells appearing in the mesometrial triangle on Day 9 continued to increase untill Day 16, forming the metrial gland. By contrast, the mitotic change in the triangle was similar to that in the decidua. The mitotic activities increased drastically on Day 9 and then gradually decreased toward Day 16. The activities were very low within the gland. Immuno- histochemical studies revealed that proge- sterone receptor was located in decidual and muscle cells and fibroblasts, but not in GMG cells throughout the development of the gland. These results suggest that GMG cells proliferate under the control of decidual cells and/or fibroblasts in both the decidua and triangle and those pro- liferated in the decidua migrate into the triangle. DAILY CHANGE OF SEX STEROID HORMONES IN THE FECES OF HOUSE SWIFT (APUS AFINIS) H.Sakai, M.Sato, S.Wakabayashi and S.Ishii* Dept. of Biology, Nihon Univ. School of Dentistry, Chiyoda-ku, Tokyo 101 and *Dept. of Biology, School of Education, Waseda Univ., Nishi-Waseda, Tokyo 169-50. Out of the colony in Shizuoka City, 350 feces were gathered in 26 hours during the feeding period, and the contained steroid hormones were quantitatively examined. The number of feces per hour gathered at night was half that of day; the ratio of feces in which hormones were detected was 70%, amounting to three-fold of that in the day. Testosterone was detected in 42 samples, the hormone amount ranging from 7.6 to 118 pg; estradiol-17g was detected in 65 samples, ranging from 0.04 to 13 pg. Furthermore, progesterone was detected in 83 specimens, ranging from 29 to 221 pg. In 8.6% of the specimens, estradiol and progesterone were both detected. In 1.7%, testosterone and progesterone were both detected. I SHA testosterone and estradiol. The former 2 cases of hormone detection were observed both in the day and at night; the last case was observed only at night. These results indicate that the amount of hormones contained in feces do not vary by the order of hours but significantly different between day and night. INHIBITORY ACTION OF MATERNAL TESTOSTERONE ON THE REGULATION OF FETAL GROWTH K. Tsutsui Dept. of Rad. Biophys., Kobe Univ. School of Med., Kobe Fetal growth depends on maternal testosterone in the rat (Tsutsui, 1991), The present study was conducted to examine the mechanism of testosterone action and its physiological significance. Maternal plasma testosterone concentrations increased from 14 days of gestation, reaching a peak on 18 days, and decreased during 18-21 days when active fetal growth took place. Scattergrams showed a negative correlation between circulating testosterone levels of pregnant mothers on gestation day 21 and body weights of their fetuses. Artificial maternal exposure to testosterone (10 or 13 mg/one silastic plate) during 14-21 days of gestation significantly induced decreases not only in fetal weight but also in placental weight on gestation day 21. Intrauterine administration of placental lactogen to testosterone-treated pregnant rats for 7 days restrained the testosterone effect. Not only testosterone but also estradiol were effective for the decreases in fetal and placental weights under the same dose treatment, while no significant correlation existed between endogenous estradiol of normal mothers and the weight of their fetuses. Dihydrotestosterone, a nonaromatizable androgen, was inactive. These results suggest that 1) rapid fetal growth during the second half of late pregnancy is related to the decrease in circulating maternal testosterone, and 2) maternal testosterone through its conversion to estradiol decreases the placental function and consequently inhibits fetal growth. EFFECTS OF PLASMA ANDROGENS AND THEIR S- METABOLITES ON THE MOUSE SUBMANDIBULAR GLAND. K.Sawada and T.Noumura Dept. Regulat. Biol., Fac. Sci., Saitama Univ. Urawa In the mouse submandibular gland, sex difference is evident on 30 day, when the granular convoluted tubules (GCT) in the male gland rapidly grow in response to dramatically increased levels of circu- lating testosterone (T) and 50-dihydrotes- tosterone (DHT). We studied in vitro metabolism of these androgens by the gland and the effects of their metabolites on the gland. The glands of both sexes did not convert T to any metabolites, but DHT to 5a-androstane-3Q4,1768-diol (3a-diol; 8%) and SG@-androstane-3@,176-diol (38 - diol; 1%). Neonatally-castrated mice re- ceived daily treatment with these four androgens for 1-10 days from day 20. The relative occupied area (ROA) of GCT increased in both sexes by treatment with 3d-diol or OHT for 4 days, but by 38-diol or T for 10 days. The ROA gain after 10 days was in the order of DHT = 3@-diol > T > 38-diol. The mitotic activity of GCT increased in both sexes by 3Q-diol or DHT for 4 days, but in only males by 38@-diol or T for 10 days. The results suggest that these DHT metabolites have androgenic effects on the gland growth and particu- larly 3Q@-diol has a potency similar to DHT. Endocrinology 1267 ANDROGEN REGULATES 8-ACTIN mRNA EXPRESSION IN THE MOTONEURONS OF LUMBAR SPINAL CORDS IN ADULT MALE RATS. A. Matsumoto (1), Y. Arai (1), A. Urano (2) and S. Hyodo (3). (1) Dept. Anat., Juntendo Univ. Sch. Med., (2) Ocean Res. Inst., Univ. Tokyo, (3) Fac. Art & Sci., Univ. Tokyo, Tokyo. Androgen has been reported to play a key role in reorganizing the neuronal elements and synaptic connections in androgen-sensitive motoneurons in the spinal nucleus of the bulbocavernosus (SNB). As one step to clarify molecular mechanisms of androgen for neuronal plas- ticity, we examined androgenic regulation of the expression of ®-actin mRNA in the SNB motoneurons of adult male rats. Adult male rats (Wistar) were castrated and implanted with silastic tubes containing testosterone (T) or nothing. Animals were sacrificed 4 weeks later. A complementary DNA encoding chick B-actin was applied to in situ hybridization on paraffin sections of the lumbar spinal cords. Autoradio- Sraphic signals for B-actin mRNA were found to be localized on the somata and proximal dendrites of SNB neurons. The number of signals per neuron in castrates was significantly smaller than that in controls. The value in castrates given T was not significantly different from that in controls. These evidence suggests that androgen regulates expression of B-actin gene in the SNB motoneurons. SEASONAL CHANGES OF BODY WEIGHT, GONADS AND HORMONES IN DJUNGARIAN HAMSTERS KEPT OUTDOORS. A. Masuda and T. Oishi. Dep. of Biology, Fac., of Sci., Nara Women's Univ., Nara. The Djungarian hamster is an animal which shows a distinct annual cycle in some physiological parameters. In order to investigate the seasonal changes in detail under natural conditions, we kept male and female hamsters at the veranda of our university building. In most hamsters, body weight decreased in winter and started to increase in spring. Food intake per body weight was high in winter and water intake per body weight was high in summer and winter. The brown and white adipose tissues did not show clear differences among seasonal groups. Muscle weight in the winter group was less compared with that in the summer group. The weights of testes and accessory glands were small and plasma testosterone level was low in the winter group. Thus the seasonal changes of testes weight and muscle weight showed a similar tendency. The weight of heart, kidney and liver per body weight and the length of intestines were larger in the winter group than in the summer group. This is in accordance with the changes in plasma T3 level and food intake, suggesting the involvement of metabolic activity. CHANGES IN PROTEIN EXPRESSION IN PREGNANCY-DEPENDENT MAMMARY TUMORS (PDMT) DURING GROWTH AND REGRESSION IN GR/A MICE. Y. Goto, T. Iguchi, H. Nagasawa’ and N. Takasugi. Grad. Sch. Integ. Sci., Yokohama City Univ., Yokohama; *Exp. Animal Res. Lab., Meiji Univ., Kawasaki. The GR/A mouse is characterized by the development of pregnancy-dependent mammary tumors (PDMT). PDMT appear after the middle of pregnancy, reach maximal size at the end of pregnancy and regress soon after parturition. Mitotic index in the epithelial and stromal cells of PDMT decreased before parturition. Using 2- dimensional electrophoresis, protein expression in PDMT was studied during growth and regression. On Day 20 of pregnancy, 9 polypeptide spots increased in intensity, and 5 protein spots appeared newly in PDMT. Three protein spots increased in these intensity, and 2 new spots appeared at parturition. Four of these 19 proteins were not observed in normal mammary glands. The growth rate of PDMT transplanted into progesterone-treated mice was higher than in mice treated with estradiol alone or with a combination of estradiol and progesterone. The expression of specific proteins was observed in PDMT transplanted into host mice receiving estradiol or progesterone. These findings suggest that expression of specific proteins may be related to growth and regression of PDMT. EFFECT OF OVARIECTOMY ON PROTEIN EXPRESSION IN REPRODUCTIVE TRACTS OF MICE. A. Suzuki, Y. Abe, T. Iguchi, Y. Ohta* and N. Takasugi. Dept. Biol., Yokohama City Univ., Yokohama; *Dept. Biol., Fac. General. Education, Tottori Univ., Tottori. Proliferation and regression of the vagina and uterus in rodents are controlled by ovarian steroid hormones, especially estrogen. Mouse vagina and uterus regress after ovariectomy. In vagina and uterus examined 1, 2, 3, 5, 7 and 20 days after ovariectomy, their weights, epithelial thickness and the number of epithelial cell layers were significantly reduced when compared with those in intact mice at estrous stage. No significant differences in these parameters were found between estradiol-implanted ovariectomized mice and intact estrous mice. Protein expression in vagina and uterus of ovariectomized mice was examined by two-dimensional polyacrylamide gel electrophoresis in the regressing vagina and uterus. Seven and 6 proteins in vagina and uterus, respectively, showed the highest expression 2-3 days after ovariectomy. Zymography revealed that expression of metalloproteinases increased 2-3 days after ovariectomy in vagina and uterus. Matrin, one of the metalloproteinases, was secreted asa proenzyme in the uterus and as an active form in the vagina. These results suggest that metalloproteinases and some other proteins participate in regression of the vagina and uterus in mice after ovariectomy. 1268 Endocrinology TGF-8 AND INHIBIN EXPRESSION IN THE DEVELOPING RAT GONADS S.Koike! and T.Noumura. Dept. of Regul. Biol., Fac. of Sci., Saitama Univ., Urawa. ‘Pathol. & Toxicol. Res., Upjohn Pharmaceuticals Ltd., Tsukuba. To determine the expression of TGF-f and inhibin in the developing Sprague-Dawley rat gonad, gonads from the gestational day (GD) 13 through the postnatal day (PD) 21 were fixed in Metakarn solution and immunohistochemically stained with two polyclonal antibodies, one against porcine platelet TGF-f and the other against bovine follicular fluid inhibin-a subunit, respectively. TGF-f was detected in male and female germ cells throughout the perinatal period. Moderate or marked staining was observed in male germ cells from GD 16 to PD 5 and Leydig cells from GD 16 to PD 11, while in female germ cells from GD 21 to PD 11 and the interstitial cells since PD 11. In both sexes, the mesonephric tubules faintly stained from GD 13 to 18. On the other hand, inhibin-a subunit was weakly expressed in Sertoli/supporting cells on GD 14 and 15, and markedly in Leydig cells from GD 17 through 20 and granulosa cells on PD 21. These results indicate that the expression of TGF-8 and inhibin-a is stage-specific during gonadal development and that both growth factors may participate in gonadal and extragonadal differentiation. EFFECT OF NEONATAL EXPOSURE TO DIETHYLSTILBESTROL ON PELVIS AND FEMUR IN MALE AND FEMALE MICE. S. Nobata, T. Sato, T. Iguchi and N. Takasugi. Dept. Biol., Yokohama City Univ., Yokohama. Neonatal treatment of male and female mice with diethylstilbestrol (DES) causes irreversible changes in reproductive organs. Migliaccio et al. (1992) reported that total calcium in lumbar vertebrae and in femur is reduced in neonatally DES-exposed female mice. We studied changes in the pelvis and the femur of male and female mice given 5 daily injections of 3 ug DES from the day of birth. Both pelvis and femur of 12-, 15- and 18- month-old neonatally DES-treated mice were lighter than in controls. Total amount of calcium and phosphorus in both bones of neonatally DES- treated 12-, 15- and 18-month-old mice were less than in the age-matched controls. Total amount of calcium and phosphorus in pelvis and femur of 12- month-old, but not 2-month-old, female mice exposed neonatally to DES were less than in controls. These results indicate that neonatal DES exposure causes irreversible changes in bones of aged male and female mice. 3-DEHYDROECDYSONE-38-REDUCTASE IN THE LARVAL HEMOLYMPH OF THE SILKWORM, BOMBYX MORI. T. Momura and S. Sakurai, Dept. Biology, Kanazawa Univ., Kanazawa, Japan. 3-Dehydroecdysone (3DE) is the major ecdysteroid secreted from insect prothora- cic glands and exhibits no biological activity. Since 3DE is converted to ecdysone by 3f-reductase found in hemolymph and require NADPH as co-factor, the change in the enzyme activity may directly affect on the change in the concentration of ecdysteroids with biological activity, ecdysone and 20- hydroxyecdysone. Therefore, we first tried to purify the enzyme so as to understand the control mechanism of the enzyme activity. The enzyme was purified after 5 steps of purification, but the purified sample gave two bands on SDS-PAGE. Analysis with amino acid sequencer gave only one amino acid sequence, indicated that these two bands on SDS-PAGE was originated from a single protein or N- terminal of either of the bands is blocked. Enzymatic study showed that the optimum pH is 6.6, similar to the pH of larval hemolymph, and the enzyme requires NADH (Km, 5. 4yM) or NADPH (Km, 0.9yM) . PRECISE DETERMINATION OF HEMOLYMPH ECDYSTEROID TITER IN THE LAST LARVAL INSTAR OF THE SILKWORM, BOMBYX MORI S. Sakurai, M. Kaya and S. Satake, Dept. Biology, Fac. Science, Kanazawa Univ. Kanazawa, Japan. Hemolymph ecdysteroid titer was precisely determined by ecdysteroi radio- immuno assay from the onset of the fifth instar through pupation. The detectable limit of ecdysteroid concentration was approximately 50 pg/ml hemolymph. In the fifth instar, some developmental events can be observed, such as head critical period found on day 4, spinneret pigmentation on day 6 and gut purge on day 7. The ecdysteroid concentration exhibited daily change and an increase in ecdysteroid titer was observed prior to each developmental event described above. Such titer was lower than that examined so far and 10-20 ng/ml was enough to induce gut purge. Though daily release of PTTH has not been demonstrated, the present data strongly indicated that PTTH may be secreted during a specific gate of every day and such daily change in ecdysteroid titer may integulate the preparation for pupation in various tissues in larvae. Endocrinology 1269 IMMUNOBLOT ANALYSES OF THE MATERIALS IMMU- NOREACTIVE TO THE BOMBYXIN-II ANTIBODY PRESENT IN THE HEMOLYMPH OF BOMBYX MORI DURING PUPA-ADULT DEVELOPMENT H.Saegusa!, A.Mizoguchi!, A.Suzuki2, and H. Ishizaki! IDept. of Biol., School of Sci., Nagoya Univ., Nagoya; 2Dept. of Agr. Chem., Fac. of Agr., Univ. of Tokyo, Tokyo We previously obtained a monoclonal antibody against natural bombyxin-lI (bombyxin-II antibody) and examined changes in the titer of the immunoreactivity in the hemolymph during Bombyx development by a radio- immunoassay using this antibody. It was later found that the bombyxin-II antibody recognized not only bombyxin but also another 8-kD component of unknown nature. In this study, materials immunoreactive to the bombyxin-II antibody were immunoprecipitated using this antibody and immunoblotted with the same antibody. Using this system, we differentiated bombyxin and the 8-kD component in the immunoreactive material at each stage. In females, only the 8-kD component was detected throughout the pupa-adult development. In males from pupation to 1 day before eclosion, the 8-kKD component was the major component, but in newly ecdysed adults, bombyxin was predominant. Thus, bombyxin is thought to be involved in the regulation of functions specific to the male adults. The nature of the 8-kD component is being studied. NEUROSECRETORY CELLS PRODUCING SAMIA BOMBYXIN-RELATED PEPTIDES (SBRP) IMMUNOHISTOCHEMICAL LOCALIZATION AND DEVELOPMENTAL CHANGE. Y.Yagi!, A.Mizoguchi!, K.Kojima2, K.Nagata~, A.Suzuki2 and H.Ishizaki!. !Dept. of Biol., School of Sci., Nagoya Univ., Nagoya and 2Dept. of Agr.Chem., Fac. of Agr., Univ. of Tokyo, Tokyo. Bombyxin is an insulin-like peptide produced by the Bombyx brain which shows prothoracicotropic activity when administered to debrained pupae of Samia cynthia ricini. Samia genes which code for bombyxin-related peptides have been cloned and characterized. We chemically synthesized one of these peptides (SBRP-A1) based on the amino acid sequence deduced from the gene structure, and raised a mouse antiserum against the synthetic SBRP-Al. We performed an immunohistochemical study using this antiserum on Samia from the (first-instar larva to adult. Immunoreactivity was observed in the medial neurosecretory cells of brain, their axons, and axon terminals in the periphery of the corpora allata. The number of the immunoreactive cells were counted. The maximal number of the immunoreactive cells was 16 per brain hemisphere. The stainability and the average and maximal number of the immunoreactive cells did not change appreciably during the development. RELATIONSHIP BETWEEN BOMBYXIN-IMMUNOREACT- IVE PEPTIDE AND SMALL PROTHORACICOTROPIC OSIVO SOUP LE) IN MANDUCA SEXTA. A.Mizoguchi’ and L.I.Gilbert?. IDep. of Biology, Fac. of Sci., Nagoya Univ., Nagoya and 2Dep. of Biology, The Univ. of North Carolina, Chapel Hill, NC, USA. The Manduca small PTTH(Mr,7kDa) is similar in size and biologic activity to bombyxin(Mr,5kDa), a brain neurohormone of Bombyx mori which manifests PTTH activity on the prothoracic glands(PG) of Samia cynthia ricini. The presence of a bombyxin-like peptide in Manduca brain was demonstrated by immunohistochemistry and immunoblotting using a bombyxin antibody. To investigate the relationship between the small PTTH and bombyxin, a Manduca brain extract was chromatographed and the resulting fractions were examined for both PTTH activity and bombyxin immunoreac- tivity(BIR). The PTTH activity was assessed by an in vitro PG assay, and the BIR by immunoblotting. Among the fractions obtained by gel filtration with Sephadex G-50, the distributions of both activities were overlapped with slight difference. However, when the fractions containing the BIR were subjected to an anion exchange FPLC, the BIR bound to the column, while the PTTH activity did not. The BIR was eluted with an increased NaCl concen- tration. These results indicated that the Manduca neuropeptide that was recognized by the bombyxin antibody was not identical to the small PTTH. SPECIES-SPECIFICITY IN THE ACTION OF BIG AND SMALL PROTHORACICOTROPIC HORMONE OF SEVERAL SPECIES OF THE SWALLOWTAILS. I.Yokoyama and K.Endo. Environ. Biol. Lab., Biol. Inst., Fac. of Sci., Yama- guchi Univ. Yamaguchi. Lepidopteran insects have two mole- cular forms of (big and small) prothora- cicotropic hormone (PTTH) activating the prothoracic glands (PGs) to secrete ecdy- steroid(s), which is essential for growth and metamorphosis in insects. Extracts containing the big and small PTTHS were made from pupal brains of several species of papilionids, Papilio xuthus, P. machaon, P. bianor and P. helenus. The PTTHS were separated by centrifuging the extracts with a ultra- filter, UFC3LGC, passing molecules smal- ler in the sizes than M.W. 10,000. The PGs from 2-day-old 5th instar larvae of the same or other species in- sects were incubated for 2 hr in Grace's medium containing the big or small PTTH extract. The titir of ecdysteroids secreted in the incubation medium were assayed by RIA. Activity of the big and small PTTHs on the PGs were quantified by activation ratio (Ar) of 5-6 pairs. The big and small PTTHs of papilio- nids examined activated the PGs of the same and other species of 5th-instar papilionid larvae in vitro. 1270 Endocrinology ENTRAINMENT OF THE CIRCADIAN CLOCK GATING PROTHORACICOTROPIC HORMONE SECRETION IN THE ASIAN COMMA BUTTERFLY, POLYGONIA C-- AUREUM L. K.Endo, N.Kusumuto, Y.Ito and N.Matsu- shima. Biol. Inst., Fac. of Sci., Yama- guchi Univ. Yamaguchi. Prothoracicotropic hormone secretion inducing larval ecdysis from the 4th to 5th instar preceds the larval ecdysis by 32 hr at 25°C. The PTTH secretion was gated by a circadian clock in Polygonia. The acrophase-time of PTTH secretion which was obtained by subtracting 32 hr from the time of larval ecdysis came to on an almost parallel line with a line connecting the mid points of 2-hr to 20- hr light periods in 24-hr LD cycles. Groups of insects reared under 2-hr to 18-hr light photoperiodic regimens at 25°C were transferred to continuous light or to contimuous dark condition at 25°C and the acrophase-time of PTTH secretion was obtained in these groups addition to the other larval groups raised from the egg stage under resonant conditions con- sisting of an 8-hr light and an various length (2-hr to 64-hr) dark periods. The results indicated that the circa- dian clock gaing PTTH secretion may be set twice a day, at dawn and dusk. At dawn the circadian clock may be reset at CT 0:00, and again set at dusk to give the same time (CT 18:00) amid the dark period of 24-hr LD cycles. HOW DOES PARASITIC WASP INDUCE GROWTH- BLOCKING PEPTIDE IN THE PARASITIZED ARMYWORM LARVAE? Y. Hayakawa, Biochem. Lab., Inst. of Low Temp. Sci., Hokkaido Univ., Sapporo. Last instar larvae of the armyworm parasitized with the parasitoid wasps, Cotesia kariyai, do not initiate metamorphosis and, ultimately, the wasp larvae emerge from the host larvae about 10 days after parasitization. The developmental arrest can be reproduced by injection of parasitoid ovarian calyx fluid containing the symbiotic virus (polydnavirus) which is normally injected by female wasps into the host at Oviposition. A peptidergic factor, g€rowth-blocking peptide (GBP), has been purified from the larval hemolymph of the parasitized armyworm. Injection of GBP into unparasitized last instar larvae of the armyworm clearly retards larval growth and, consequently, delays the onset of pupation of the larvae. Recently, it has been demonstrated that GBP exists in plasma of the virus-injected unparasitized last instar larvae and also in plasma of the penultimate instar larvae of the armyworm. Therefore, it is reasonable to propose that infection of polydnavirus activates GBP gene expression, and the production of GBP retards a normal development of last instar armyworm larvae. ECDYSTEROIDS DURING EARLY EMBRYOGENESIS IN THE SILKWORM, BOMBYX MORI. H. Sonobe’, T. Masumoto’, Y. Mamiya’, T. Kanda’ and T. Tamura’. ‘Dept. of Biol., Konan Univ., Kobe. *"Natl. Inst. Seric. Sci., Tsukuba. Ovaries in the silkworm are the site of biosynthesis and accumulation of ecdysteroids, and these accumulated ecdysteroids are transported into eggs. First, ecdysteroid content of diapause eggs and non-diapause eggs was analyzed by both RIA and reverse phase HPLC. Nine free ecdysteroids and their conjugated forms were detected throughout early embryogenesis. In diapause eggs, most free ecdysteroids remained at original levels, but conjugated forms began to increase with the onset of the diapause. In non-diapause eggs, most free ecdysteroids, including 20-hydroxyecdysone, began to increase as embryogenesis proceeded. In contrast, conjugated forms remained at their original levels. Next, in order to examine the function of free ecdysteroids in the eggs, 20-hydroxyecdysone was injected into 20-hr prospective diapause eggs. Their developmental fate was changed from the diapause type to the non-diapause type. This fact strongly suggests that the elevation of the titer of 20-hydroxyecdysone is needed to advance embryonic development of the silkworm. PHYSIOLOGICAL SIGNIFICANCE OF 3-DEHYDROECDYSONE(3dhE) FROM CRAYFISH, Procambarus Clarkii M.Ikeda, Y.Naya. Suntory Institute for Bioorganic Research (SUNBOR), Osaka. Finding the dominant ecdysteroid 3dhE from P. clarkii, uponin vitro culture of Y-organs(1), led us to inquire what the physiological function of 3dhE is. After bilateral eyestalk ablation, the production of 3dhE in Y-organs was increased prior to ecdysone(E) and accompanied by an increase of 20-hydroxyecdys- one(20E) in hemolymph.The hormonal response of 3dhE, upon injection into P.Clarkii, was comparable to those of E and 20E. When a large amount of 3dhE* was injected into crayfish, most of the unchanged ma- terial was excreted within 1h and then its metabolite* (3-epi-E,3-epi-20E,ecdysteroids'conjugates etc.) was observed in the excretion. The major radio isotope in the body was found 3h after administration in the epidermal tissues(with carapace), and after 72h in the hepatopancreas. The epidermal isotope was found to be comprised of 3dhE,E,20E,3-epi-E,3-epi-20E,polar ecdysteroids and conjugates efc.. Ecdysteroids* were separated from the conjugates* by enzymatic digestion (Helix pomatia ). Our results concluded that 3dhE is the intimate precursor of E and results in the produc- tion of 20E in P. Clarkii. 1) H.Sonobc, M.Kamba, K.Ohta, M.Ikeda and Y.Naya, Experientia, 47, 948(1991). Endocrinology 1271 EXPRESSION PATTERN OF ECDYSTEROID RECEPTORS IN MUSCULAR SYSTEM DURING” METAMORPHOSIS OF DROSOPHILA MELANOGASTER K-I. Kimura!, D.A. Currie? and J.W. Truman 1Lab. Biol. Hokkaido University of Education, lwamizawa, 2Dept. Zoology, Univ. Washington, USA In holometabolous insects such as Drosophila melanogaster, adult musculature is rebuilt during metamorphosis. Many larval muscles die at beginning of metamorphosis. However, some larval muscles persist during metamorphosis. These persisting muscles regress once but then regrow to transform into adult ones. Also, adult specific muscles are newly formed from adult myoblasts. These processes should be regulated by ecdysteroid hormone. Ecdysteroid hormone binds to the receptor (ecdysteroid receptor; EcR) and regulates the gene expression. In Drosophila, the EcR gene encodes three EcR isofrom, EcR-A, EcR-B1 and EcR-B2. We examined the expression pattern of EcR-A and EcR- B1 in muscular system during metamorphosis, using isoform-specific antibodies. Both of EcR-A and -B1 were expressed in a Stage-specific manner during metamorphosis, respectively, that would correspond to the processes of muscle development. These results suggest that different combinations of EcR isoforms affect the developmental responce in different processes of muscle developmet. ROLE OF ECDYSTERONE IN ALTERATION OF NERVOUS SYSTEM DURING METAMORPHOSIS IN BOMBYX MORI. H. Tsujimura. Lab. Biol., Tokyo Univ. Agri. Tech., Fuch, 183 Tokyo. Hormonal requirement for the alteration of the ventral nerve cord and motor neurons dur- ing metamorphosis is examined by injecting to the pupa the ecdysterone and an imidazole compound, 1-benzy1-5-([(E)-2,6-dimethy1-1,5- heptadienylJimidazole (KK-42), which inhibits the ecdysone biosynthesis in the prothoracic gland. Many changes in the ventral nerve cord and motor neurons were inhibited by injection of KK-42 just after the pupal ecdysis, suggesting that they are controlled by a peak of ecdy- sterone titer in the pupal hemolymph. Results from several injection experiments show that the connective shortening and ganglionic fusion are stimulated by low titer of ecdysterone but inhibited by high titer of ecdysterone. On the other hand, low and high titer of ecdysterone stimulate the programmed cell death of certain larval motor neurons and the disappearance of larval dendrites and regrowth of adult ones in other motor neurons which survive and become adult neurons. These results show that alteration of ecdy- sterone titer in the pupal hemolymph controls the change of the CNS but there are 2 distinct mechanisms to respond to the hormone, which controls different part of the change. REGULATION OF DIAPAUSE HORMONE SECRETION IN THE SILKWORM, BOMBYX MORI:A TRY USING LASER MICROBEAM TREATMENT. I. Shimizu. Center for Ecological Research Kyoto University, Kyoto. The diapause hormone of the silkworm is believed to be produced in and secreted from neurosecreatory cells located in the suboesophageal ganglion(SG). However, I found, extirpataion of SG from the silkworm pupae which had been destined to produce non-diapause eggs resulted ina production of diapause eggs:the treatment caused a production of diapause in eggs which developed at the later pupal stage. On the other hand, extirpation of Brain (CC-CA)-SG complex from the non-diapause producer pupae did not produce diapause eggs as sham controls. These observation pose a question about the established thinking mentioned above about the dia-— pause hormone. Another new idea(produ- ction in SG and secreation from CC-CA) is proposed. Further a new try using laser microbeam to invesigate the hormone regu- lation mech- anism is reported pre- liminary. EXOCYTOTIC RELEASE OF NEUROPEPTIDES IN THE BRAIN-MIDGUT ENDOCRINE SYSTEM OF INSECTS Y. ENDO Dept.of Appl.Biol., Kyoto Institute of Technology, Matsugasaki, Sakyo, Kyoto In order to clarify the significance of non- synaptical release of neuropeptides, I have examined the brain-midgut endocrine system of insects, Periplaneta americana and Galleria mellonella, at the level of morphology by immunohistochemistry, scanning and transmission electron microscopy. The tissues were isolated in the saline solution and the exocytosis was stimulated by high Kt and Ca?* solution (120mM KCl, 6mM CaC], 30mM NaCl). After the fixation of tannic acid-containing glutaraldehyde and Os0x, omega-shaped figures were detected by electron microscopy. In the retrocerebral neurohemal organs (corpora crdiaca and allata), the exocytosis of neuropeptides occurred not only on the free surface of neurites, but also on the surface of neurites facing the target cells such as the glandular cells. In the muscle layer of gut, neuropeptides were also released non-synaptically. These results were comparable to the phenomenon of non-synaptical neurotransmitter release in the peripheral autonomic nerves in mammals! 2%). Some of them might be not a neurotransmission or neurosecretion in nature, but the other including neurotrophic or neuro-survival factors. 1)Y.ENDO et al. (1991) Neurosci. Lett., 126:60-62. 2)Y.ENDO (1988) Zool.Sci., 5: 965-971h 3)Y.ENDO (1988) Arch.Histol.Cytol., 51:489-494 1272 Endocrinology DISTRIBUTION OF NEUROSECRETORY CELLS IN SYNGANGLION OF THE ADULT FEMALE TICK, HAEMAPHYSALIS LONGICORNIS, WITH REFERENCE TO THE INCREASE OF STAINING INTENSITY IN INACTIVATED TICKS UNDER LOW TEMPERATURE. N. Okura, T. MOri and S. Shiraishi. Zoological Lab., Fac. Agriculture, Kyushu Univ., Fukuoka. Paraldehyde thionin (PAT) staining (a modified technique of Gomori’s paraldehyde fuchsin) revealed positive neurosecretory cell bodies (34 in number) throughout the cortex of synganglion in the adult female tick. These cell bodies were distributed in sub-groups within 11 neurosecretory centers similar to those of other ixodid ticks reported so far. By exposure to the low temperature (10°C, for i10days), active ticks were inactivated. There was a conspicuous dif- ference in staining intensity of certain cell bodies between active and inactivated ticks. Although a few cell bodies of the frontal neurosecretory center (one of the 11 centers) in active ticks were hardly stained, those in inactivated ticks had remarkably high intensity. Such high in- tensity suggests that accumulation of PAT positive neurosecretory materials within the cell bodies is caused by cessation of neurosecretory discharge. Thus, this neu- rosecretory center may play some regulato- ry roles concerning temperature dependent activity. PREMATURE EGG-RELEASING SUBSTANCE (PERS) IN ESTUARINE CRABS M. Saigusa, Coll. of Liberal Arts & Sci., Okayama Univ., Okayama The eggs of most decapod crustaceans are attached to ovigerous hairs by a funi- culus, and are ventilated by the female. When the embryonic development is complete, hatching occurs. This study focuced on a substance that remained in the medium (hatch water) where a larval release had occurred. When another ovigerous female contacted with this hatch water, she dro- pped within a few days all eggs without hatching. This egg-releasing activity clea- rly appeared associated with hatching, and the effect was eliminated by boiling and tripsin, which suggests that this substance (PERS) is protain. Gel filtration of hatch water revealed that the molecular weight of PERS is about 20,000 in Sesarma haema- tocheir, and about 29,000 in Sesarma pictum. This substance would function, by nature, as the deposition of remnants remained on the ovigerous hairs after the larval release. But the primary function must have an immediate connection with larval hatching. It might be a hatching enzyme in Crustacea, OVARIAN FACTOR(S) INDUCIBLE SECONDARY SEXUAL CHARACTERS IN ARMADILLIDIUM vulgare (ISOPOD). S. Suzuki* and K. Yamasaki+#«* *Biol. Lab. Kanagawa Pref. Col., Yokohama, **Dept. Biol. Tokyo Metropol. Univ.,Tokyo. In male A. vulgare, the androgenic gland hormone is responsible for sex differentiation, but in female there is no direct evidence of the existence of sex hormone controlling development of sexual characters. In order to elucidate the mechanism of sex differentiation in female, oostegite formation was selected as the index for the expression of secondary female character in our experiments. Young animals of both sexes (5th stage after the 4th molt) were ovariectomized or andrectomized by removing all internal reproductive organs. These gonadectomized animals attained to adult size after six months but did not form oostegites. However, they could form oostegites by the injection of the extract of vitellogenic ovaries. The ovarian extract was dialyzed against distilled water. The dialysable fraction was active for the induction of oostegite formation. The optimal amount for the activity was 1/2 ovary equivalent. These results suggest that a vitello- genic ovary contains the factors which are responsible for sex differentiation of the female A. vulgare. The isolation and characterization of the ovarian factors are now in progress. PURIFICATION AND SEQUENCE ANALYSIS OF ANDROGENIC GLAND HORMONE OF THE TERRESTRI- AL ISOPOD,._ARMADILLIDIUM VULGARE. H.Nagasawal, ¥-Hasegawa 5 K.Haino® and Y.Katakura*. locean Res. Inst., Tokyo Univ. Tokyg, 2Dept. of Biol., Keio Univ. Yokohama, “Dept. of Biol., Tokyo Metropol- itan Univ. Tokyo, “Dept. of Bioengineer- ing, Soka Univ. Tokyo. In crustaceans, male sexual characters are induced by a peptide hormone, andro- genic gland hormone (AGH), which is pro- duced by androgenic glands (AGs). AGH was first purified from whole male reproduc- tive organs of the terrestrial isopod, Armadillidium vulgare, ang some chemical properties were clarified yy Until recent- ly, however, no sequence information has been obtained. In the present study, AGH was purified again from 16,000 male reproductive or- gans, and finally about 23 pe (1.3 nmol) of AGH was obtained. Because the N-termi- nus of AGH was blocked, it was digested separately with lysyl endopeptidase, chymotrypsin and endoproteinase Glu-C. The resulting peptide fragments were se- quenced. Though the complete amino acid sequence of AGH has not yet been obtained by aligning the sequences, the following partial sequences were identified; MTTQMG- KLR, LKVVGQDSNEIHFRVK, KSYSERVGVPVASLRFLF- DGRRINDEETPK and EMENDDVIEVYQEQTG. 1) Y.Hasegawa et al., Gen. comp. Endocri- nol, Gi, LOL L987). Endocrinology 1273 CALC1 TON IN- IMMUNOREACTIVE SUBSTANCE PRESENT IN THE CELLS OF MIDGUT CAECUM OF AMERICAN CRAYF1!SH Y. Nosé, N. Suzuki and Y. Sasayama Dept. Gre Iul@uhs 5 Fac. Ge SOAs » Toyama Umiv., Toyma 930, Japan Calcitonin induces a hypocalcemia in mammals. Recently, we found calcitonin- immunoreactive cells in the midgut caecum in some crustaceans. In the present study, the positive substance contained in those cells of the American crayfish (Procambarus clarki) was studied. At first, the midgut caeca were homogenized with 0.1 M acetic acid. The homogenate was separated centrifugally to two fractions (molecular weights (MW): 3,000-10, 000 and 10, 000-30, 000). These fractions were subjected to SDS-PAGE, to estimate the MW of the substance. For this purpose, immunoblotting method using anti-salmon calcitonin antiserum was applied. As a result, two positive bands were found around 4,000 MW and 17,000 MW. In the next step, fractions containing these extracts were injected into the pericardial cavity of the crayfish. Several hours after, Ca, Mg, Na, K, Pi and glucose level of the hemolymph were examined. There was no Significant difference in those minerals between the injected group and the control. On the other hand, glucose level in the group which was administered with the fraction containing larger extract in the MW size was Significantly lower than the control. These results seem to be interesting considering the physiological role of calcitonin-immunoreactive substance in the crayfish. EFFECTS OF ADMINISTRATION OF GOLDFISH CALCITONIN ON THE SERUM CA CONCENTRATIONS IN THE GOLDFISH K. Ukawa, N. Suzuki and Y Sasayama Dept. Gur IesL@l sy, Fac. Ge SOAs 5 Toyama Univ., Toyama 930, Japan It has been well known that calcito- mins of the eel and salmon show hypocal- cemic activity when administered to mam- mals. Nevertheless, roles of calcitonins in those fishes have not been clear. We developed a new calcitonin from the gold- fish. In the present study, using goldfish ealcitonin, effects of administration on the serum Ca levels in the goldfish were Studied (Experiment I). One nmol of the goldfish calcitonin was administered per 100 g body weight. Furthermore, we raised anti-goldfish calcitonin polyclonal anti- serum in guinea pigs. This antiserum was administered to the goldfish to know whe- ther serum Ca level is affected or not (Experiment II). The antiserum was diluted to 100 times, and 10 ul of which was ad- ministered per 100 g body weight. In those two experiments, both calcitonin and its antiserum were administered through a can- nula into the arterial bulb. Blood was taken through the cannula periodically Giulio tiger Gh loses} ayel Irons, i, untel sese(o)m ohio chial vessels during 1 hr in Expt. II. As a result, in both experiments, no differ- ence in the serum Ca level was recognized between the experimental groups and the eontrols. These results suggest that some conditions may be necessary for calcitonin to express its hypocalcemic effect, and that calcitonin level in the blood may be so low as being negligible in the goldfish SEXUAL DIMORPHISM AND STRAIN DIFFERENCE IN MOUSE ANOCOCCYGEUS MUSCLE. Y. Fukazawa, A. Suzuki, T. Iguchi, N. Takasugi and H. A. Bern*. Dept. Biol., Yokohama City Univ., Yokohama; *Dept. Integrative Biol. and Cancer Res. Lab., Univ. California, Berkeley, CA, U.S.A. The anococcygeus muscle (AcM) is a paired, thin sheet of smooth muscle inserting on the rectum, having a tendinous origin largely on sacral vertebrae. The cross-sectional area of AcM in the juxtarectal region in 90-day-old male mice was significantly larger than that of females in 3 strains: BALB/cCrgl, ICR/Jcl and C57BL/Tw. The AcM area in female mice showed a strain difference: BALB/c > ICR>C57BL. The AcM area in 60-day- old ICR male mice castrated at 30 days of age was significantly smaller than in intact males, and that in ovariectomized females was significantly larger than in intact females. Implantation of a testosterone or estradiol pellet (12 mg) on the day of operation inhibited the change in AcM area. ICR female mice exposed neonatally to diethylstilbestrol had significantly larger AcM than controls. Expression of androgen receptor (AR) and estrogen receptor (ER) in AcM of 15- to 120-day-old mice was studied immunohistochemically in ICR and C57BL mice. The AcM in both strains showed positive AR and ER immunostaining at 15 days. In ICR mice, both AR and ER in AcM disappeared by 90 days. AR and ER (male) and ER (female) were present in the AcM of C57BL mice at 90 days. The results suggest that androgen and estrogen play an important role in sexual dimorphism of the mouse AcM. DISTRIBUTION OF EEL ATRIAL AND VENTRICULAR NATRIURETIC PEPTIDE IN EEL HEART: ANALYSIS BY IMMUNOCYTOCHEMISTRY USING MOLECULAR- SPECIFIC ANTISERUM. Y. Takeil, N. Nemoto and T. Kameya3 locean Res. Inst., Univ. Tokyo, Tokyo and 2Electronmicrosc. Lab. and 3Dep. Pathol., Kitasato Univ. Sch. Med., Sagamihara. The radioimmunoassay (RIA) specific to eel atrial or ventricular natriuretic peptide (ANP or VNP) measured ANP almost exclusively in eel atria, but VNP was measured comparably both in atria and ventricles. Immunohistochemistry using the specific antiserum localized ANP only in eel atria and VNP in both atria and ventricles. However, the atrial staining was much greater than that expected from the concentration measured by the RIA. This indicates some crossreaction of VNP antiserum to ANP despite no crossreaction in the RIA. The pretreatment of VNP antiserum with 10-8M eel ANP eliminated the crossreaction without affecting crossreaction to VNP. The immunogold electronmicroscopy using the pretreated antiserum revealed that atrial granules contained both ANP and VNP, whereas ventricular granules contained only VNP. The double-antibody method using gold particles with different sizes showed that ANP and VNP co-localized in the same atrial granules. 1274 Endocrinology UROTENSIN-LIKE IMMUNOREACTIVITY IN THE NERVOUS SYSTEM OF LOWER VERTEBRATES AND INVERTEBRATES §.0ka', A.Chiba’ and Y.Honma?. 'Dept.of Biol. ,Nippon Dent.Univ., Niigata, *Sado Mar.Biol.Stat.,Fac.of Sci.Niigata Univ. ,Niigata. Using specific antisera raised against urotensins(U) I and II, immunohistochemical examination was carried out on the nervous system of some lower vertebrates and invertebrates from the phylogenetic point of view in the caudal neurosecretory system. Both UI- and Ul]-immunoreactivities were demonstrated in the brains of two amphibians (Rana catesbeiana and Hynobius sadoensis) and two primitive bony fishes (Lepidosiren paradoxa and Polypterus senegalus), but only Ul-immunoreactivity was found in the brain of hagfish (Paramyxin atami). In the Polypterus spinal cord, UJI-immunoreactivities occurred in the terminal part, whereas UJ-immunoreactivities were widely distributed in the greater part of cord excepting for terminal region. No immunoreactivities were seen in the spinal cord of Hinobius and Paramyxine, but Ul-immunoreactivities were detected in the nerve cord of amphioxus (Branchiostoma floridae) and in the cerebral ganglia of the crab (Gaetice depressus) the squid (Todarodes pacificus) and the sea hare (Aplysia kurodai). These results suggest a long history of the caudal neurosecretory peptides, especially UI or its related molecules, in association with the evolution of the central nervous system. CO-LOCALIZATION OF LH& AND PROTEOGLYCAN-— LIKE IMMUNOREACTIVITY IN THE PITUITARY OF MALE AND FEMALE RATS. N.Hama , A.Sato“, M.Nishizyka’, T.Hari- gaya, N.Ooshima“, and Y.Arai Sch. Agr., Meiji Univ., Kawasaki, “Dept. Biomol. Sci., Fac. Sci., Toho Univ., Narashino, Dept. Anat., Juntendo Univ. Sch. Med., Tokyo. The anterior pituitary of adult female rats contained cells that were immunocyto- chemically stained with an monoclonal antibody raised against proteoglycans (PGs) in the brain of rats. Since ultrastructure of PG-like immunoreactive (irPG) cells resembled that of gonadotropin (GTH) cells, co-localization of leteinizing hormone (LHB) and irPG was studied. All of the irPG cells were LHB-immunoreactive in adult female rats, whereas some were immunonega-— tive to the anti-PG. These results showed that irPG cells were a population of GTH cells. During the estrous cycle, frequency of co-localization, namely, irPG-LHB cells per LH®B cells, varied, being greater at proestrus. As for adult male rats, irPG was not seen in the anterior pituitary. By contrast, in castrated male rats that were treated with estradiol-178 for 2 weeks, irPG cells were numerous. Most of these cells were LHB-positive, some being LHUB- immunonegative. These finings indicate that estrogen increased the PG-like immu- noreactivity in the anterior pituitary. The irPG-substance in the pituitary may be involved in the reproduction. DISTRIBUTION OF PROTEOGLYCAN-LIKE IMMUNORE- ACTIVITY IN THE ANTERIOR PITUITARY OF MALE AND FEMALE RATS. A.Satol, N-Hama“, M.Nishizuka, N.Ooshimal, T.Harigaya“~, and Y.Arai~. ~Dept. Biomol. Cie.) hac. (Ser... Lonos Unwin REPRE OLD | Sch. Agr., Meiji Univ., Kawasaki, & ~Dept. Anat., Juntendo Univ. Sch. Med., Tokyo. Distribution of the immunoreactivity specific to an anti-proteoglycan (PG) monoclonal antibody, raised against rat brain PGs, in the anterior pituitary of rats was studied. In adult female rats, PG-like immunoreactivity (irPG) was seen in the cytoplasm of glandular cells. By contrast, irPG were not seen in adult male rats. Similarly, irPG was seen in the female on postnatal day 41, but not in the male. However, irPG were seen in both male and female rats on postnatal day 5 and 15. These results indicate that appearance of the irPG in the anterior pituitary was sexually different. However, existence of the irPG was inherently programmed in both the male and female rats. The appearance seemed suppressed in adult male rats. Castration for 2 weeks and treatment with testosterone following to castration decreased the irPG in females. By con- trast, castrated male rats that were treat- ed with estradiol-178 for 2 weeks had numerous and intensely stained irPG-cells. These findings indicate that the appearance of the irPG depends on estrogen. rt re- mains unsolved whether androgen suppressed the appearance the irPG. Cell Biology and Morphology 1275 INTERSPECIFIC TRANSPLANTATION OF GERMINAL MICRONUCLEUS IN PARAMECIUM K. Oka and K. Mikami. Res. Inst. for Sci. Educ., Miyagi Univ. of Educ., Sendai. Ciliates have two types of nucleus, the somatic macronucleus (mac) and the germinal micronucleus (mic). To investigate a relationship between species belonging to a genus Paramecium, the mics were transplanted into cells of other species. In the present work, amicronucleate cells of P. caudatum were transplanted with the mic of P. trichium. The transplanted mic was retained in the cytoplasm for a while. In the cytoplasm, some of the mics apparent- ly divided. However, the mic division did not couple with cytokinesis so that the re- nucleated clone contained amicronucleates, uni-micronucleates and two or more micro- nucleated cells. Whereas the shape of the mic of P. trichium is spherical before transplantation, the transplanted mic some- times looked abnormal; spindle or rod shape. Amicronucleate cells grow very slowly after mic removal. These amicronucleate cells recover its fission rate after reimplantation of P. caudatum mic. However, they did not recover the rate after im- plantation of P. trichium mic. The result shows that the mic of P. trichium did not perform its function in the cytoplasm of P. caudatum. When ordinary micronucleate cells of P. caudatum was transplanted with the mic of P. trichium, the behavior of the implanted mic was almost the same as that mentioned above. MICRONUCLEAR DIVISIONS AFTER MACRONUCLEAR REMOVAL DURING CONJUGATION OF PARAMECIUM. K. Mikami. Res. Inst. for Sci. Educ., Miyagi University of Education, Sendai. Each cell of Paramecium caudatum has a germinal micronucleus (mic) and a somatic macronucleus (mac). When the macs were removed from both cells of a conjugating pair before metaphase of of the lst meiotic division, the mics did not divide. When the Macs were removed at the metaphase or the anaphase, however, the mic repeated division so that it produced more number of nuclei than usual 4. In an extreme case, a mic divided 4 times and produced 16 nuclei. The result shows that the mic is able to repeat division in the absence of the mac. To know whether the mac is necessary for mic division during the postzygotic process, the mac was removed at the stage of syn- karyon or two postzygotic nuclei. Then, mic (synkaryon) divided twice or more times. In an extreme case, 32 nuclei were produced. These results show the mic is able to divide in the absence of the mac after meta-— phase of the 1st meiotic division and lead to the following hypotheses: the informa- tinal factors required for mic division have been prepared prior to the mac removal, or the factors are prepared by the mic itself. When cells were treated with Actinomycin D (100ug/m1) at the metaphase or postzygotic stage, mic division was inhibited in most of the cells. Although the results seem to support rather the later hypothesis, so far, we cannot exclude the former hypothesis. BEHAVIOR OF MICRONUCLEI TRANSPLANTED BETWEEN THE TWO DIFFERENT EUPLOTES DURING CONJUGATION. K.Sato. Dept. of Biol., Naruto Univ. of Educ., Naruto. A study in which micronuclear transplantation was performed reciprocally between the two different species of Euplotes (E. octocarinatus, E. patella) has been reported (Proceedings of the 62 Annual Meeting of the Zoological Society of Japan, 1991). In the stock derived from the operated E. octocarinatus cells into which E. patella micronuclei were transplanted (called Tmp(K18)-0(1-17)9), alive progenies from this 60 homotypic conjugants was not obtained, but spherical macronuclear anlagen developed. Also ina stock of the opposite transplantation combination, called Tmo(1-14)-p(K18)12, 56 exconjugants were isolated, but they died out. Though meiosis, eight nuclei stage after meiosis and small swelling macronuclear anlagen were observed, extra nuclei were also observed in the exconjugants. From results, the transplanted micronuclei derived from the different species can undergo meiosis and other nuclear divisions in the cells of the different species, although macronuclear anlagen could not develop. If the informations for meiosis and the other nuclear behavior are produced by macronuclei, the informations seem to resemble closely between the two Euplotes. ELECTRON MICROSCOPY ON MEIOSIS OF MICRO- NUCLEUS IN Tetrahymena. Y. Suganuma. Biol. Lab., Narasahojogakuin College, Nara. Mitosis of the micronucleus in Tetrahymena thermophila is a "closed acentric" type in which no centriole exists and the nuclear membrane progresses without breakdown. In first, second and third prezygotic divisions, chromatin loosen into thin threads and loosely congregate to form chromosomes with low density of electrons. Cap-shaped kinetochores can be found in the nucleus during its division, which consist of a core with high electron density (external diameter, 210 nm; thickness, 20 nm) and a halo of low electron density around the core (thickness, 20 nm). The chromosomes are attached to the inside of the core. Microtubules (KM) are attached to the halo, which show polar alignment. The other side of the KM ends at the polar region of the nucleus. As the KM becomes shortened, chromosomes separate and shift in both polar directions. During anaphase of the third prezygotic division, a part of the elongated nucleus nearest to the cell-to-cell junction is positioned parallel to the junction area. This part of the nucleus remains within the junction area even after the division and becomes a migratory pronucleus. On the other hand, the other part of the nucleus which has elongated towards the macronucleus approaches the joint region after the division, becoming a stationary pronucleus. Chromatins within both the pronuclei are thick and short with high electron density and are scattered within the nuclei. However, when a part of the migratory nucleus migrates into the partner cell, the chromatins immediately loosen and become thinner. After migration, the chromatin becomes a loose configuration like chromosomes. 1276 Cell Biology and Morphology ULTRASTRUCTURAL COMPARISON OF PARORAL REGION OF AMICRONUCLEATE AND MICRONUCLEATE CELLS DURING CONJUGATION IN PARAMECIUM TETRAURELTA. Y. Yashima. Dept. of Biol., Sch. of Lib. Arts and Sci., Iwate Med. Univ., Morioka. When the meiosis was completed during conjugation in Paramecium tetraurelia, 8 meiotic products were observed and only one of them located in the paroral region survived. In order to understand this mechanism, the morphology of the paroral region and that surrounding the nuclei of the conjugants which were present between the stage of nuclear moving into the paroral region and nuclear exchange were examined by electron microscopy. A meshwork structure composed of microtubules was present in the paroral region of the conjugant that consisted of 3 types of combinations with amicronucleate cells (A cell) and micronucleate cells (M cell) in the stage prior to the nuclear migration into the partner cell. Moreover, microtubules were present around the micronucleus located outside the paroral region in the telophase of the secondary meitotic nuclear division. At the stage of micronuclear exchange, various kinds of vesicles in size and shape, i.e.,rough endoplasmic reticulum, discoidal vesicles, golgi apparatus, coated vesicles were present directly behind the migratory nucleus in the paroral region of both the A cells and the M Cells. These results suggest that the formation of the meshwork structure may be controlled by the macronucleus,and the nuclear migration into the paroral region may be controlled by microtubules. DISTRIBUTION AND PROLIFERATION OF CORTICAL UNITS IN THE CILIATE PARAMECIUM TRICHIUM. M. Okubo, T. Kosaka and T. Takahashi. HOO IL ANS Bo 5 CK Cat Seals, bhiicoglniyma Univ., Higashi-Hiroshima. The cell surface of Paramecium is cov- ered with many cortical units longitudi- nally arranged in kineties. In these units main components are cilium(c), basal body(bb), kinetodesmal fiber(kd) and lattice. Each unit of the morphostatic cell may contain one or two bb. It, howev- er, has not been cleared yet whether all of them are ciliated or not. The purpose of this work is to examine the detail morphology, cytogeometrical distribution and proliferation processes of the units by silver impregnation technique and by scanning electron microscopy. According to the number of contained bb and c, the cortical units could be classified the following three types which distributed in the specifically localized field of the cell; 2bb-2c unit in the ventral left anterior field, 2bb-1c unit in the field nearby posterior pole of the cell, and 1bb-1c unit in the rest field. Moreover, the mixed field of 2bb-1lc and ibb-1ec units was also observed in the region near the border of their fields. These cortical units proliferated in all over the cell surface during cell division except for the certain parts of the 2bb-2c unit and 1bb-1lc unit fields. The present work indicated that there were four modes in the proliferation processes of the units. ORAL ASSEMBLY IN THE .CILIATE GLAUCOMA SCINTILLANS. M.Suhamal and K.Matsumoto2. 1Fac. of Liberal Arts, Shikokugakuin Univ., Zentsuji and 2Zool. Inst., Fac. of Sci., Hiroshima Univ., Higashihiroshima. We have investigated the assembly and alignment of basal bodies during develop- ment of oral apparatus(OA) in the right- (RH) and left-handed(LH) cells of Glaucoma scintillans by SEM observation. Observa- tion was done mainly on isolated OAs and peeled pellicles with anlage of OA. At the lst step of fromation of membranelles basal bodies arranged in a row at the left edge of rudimentary field were commonly seen in RH cells, and then each basal body became pair, adding a basal body to its left side (cell's). The 3rd-7th rows of basal bodies were orderly added to the left side. The basal body displacement occurred at the anterior ends of 3 membranelles(Ml, M2, M3) during oral development in RH cells; at the anterior tip of M2, 6 basal bodies moved toward M3 and made a peculiar sculptured appearance, while the sculptures of Ml and M3 were not intricate in appearance. The sculpture of M2 in many LH cells was found at the posterior end, indicating 180 rota- tion of M2. Cilia grew from the anterior end of eacn membranelles in RH and LH cells regardless of the direction of alignment of basal bodies. The bundles of membranellar basal microtubules connecting between Ml, M2 and M3 were found at the late period of oral development. REGENERATION OF LONGITUDINAL UNEQUALLY SECTIONED FRAGMENTS IN THE STICHOTRICH CILIATE ONYCHODROMUS QUADORICORNUTUS. T. Sato, T. Kosaka and T. Takahashi Zool.inst., Fac. of Sci., Hinroshimasunive. Higashi-Hiroshima. For interpretation of pattern formation in ciliate, Frankel(1989) proposed an application of the cylindrical coordinate model(CCM) which has been developed from polar coordinate model for patterning. According to this model, it is considered that if one-half or more of the cell cir- cumference is removed, the fragment might undergo mirror-image duplication of the surface structures in the regeneration. The problem, however, as to whether the presumption is correct has been remained to examine. We, therefore, examined the morphogenetic fate of longitudinal un- equally sectioned fragments (right:left = 2:1) of the large stichotrich edaiace Onychodromus (max. ca. 600um in length). About 70% of right large fragments and about 15% of left small ones regenerated to normal cells within 2 days after the operation. All of the other fragments did not regenerate and died; that is, mirror- image duplication were not observed in the present operations. These findings indi- cated that it may difficult to application of the CCM hypothesis to the pattern formation in the regeneration of the longitudinal unequally sectioned fragments of this ciliate. Cell Biology and Morphology 1277 IMMUNOLOGICAL EXAMINATION OF THE CYST WALL SPECIFIC PEPTIDES IN A STICHOTRICH CILIATE, HISTRICULUS CAVICOLA. M. Himura and T. Matsusaka. Dept. of Biol. Sci., Fac. of Sci., Kumamoto Univ., Kumamoto. A rabbit antiserum raised against isolated cyst wall of a ciliate, Histriculus cavicola specifically decorates, immuno- electron microscopically, the 3 layered cyst wall, ecto- meso- and endocyst, and also autophagic vacuoles. Several peptide bands was recognized by the antiserum on the Western blot of the cyst wall. Especially, 4 of these peptide bands, 180, 54, 48, and 37 kD, showed intense reaction to the antiserum. As the 180 kD peptide has been known to locate in the endocyst, antibodies against the other 3 peptides were affinity purified from the immuno-blots to determine the localization of these 3 peptides within the cyst. Affinity purified antibodies against 54 kD peptide cross-reacted with the 48 and the 37 kD peptides and decorated the ecto- and the mesocyst. The same results were obtained from the antibodies purified from 48 and 37 kD peptides. To determine possible involvement of specific sugar residues or polysaccharides in the cross-reaction, Western blot filters were exposed to 4 lectins, Con A, WGA, PNA, and DBA and were also examined by PAS-test. Only 54 kD peptide was recognized by DBA, indicating the involvemen of N-acetyl galactosamine in the peptide. The other 2 peptides, however, were neither recognized by the lectins tested nor reacted to PAS-test. These results suggest that sugar residues are not involved in the cross reaction. The results of partial proteolysis of the 3 peptides using protease V8 indicated that given 2 peptides out of the 3 contained a few common peptide fragments but that no common fragment among 3 peptides were present. Some of these fragments were recognized by the antiserum. These results may indicate that the 3 peptides may contain common epitopes, and that the common epitopes may be peptides. ANALYSIS OF SPECIFIC ANTIGENS IN ACAN- THAMOEBA BY MONOCLONAL ANTIBODIES. Y. Hirukawa!, H.Ohba?., J. Chiba? & T. Tsuruhara!. 'Dept.Biol., fokyo Gakugei Univ.,Koganei and 2Dept. Biol. Sci. & Tech., Sci. Univ. of Tokyo, Noda. We have developed hybridoma cell lines which secrete murine monoclonal antibodies to the cysts and trophozoites of Acanthamoeba polyphaga (ATCC 30871, Page 3a). The cross-reactivity tests of the monoclonal antibodies were performed by using other Acanthamoeba strains (A culbertsoni, A. astro- nyxis & A.castellanii) in enzyme-linked immuno- sorbent assay and immunoblotting experiments. These analyses revealed that the monoclonal anti- bodies recognized primarily the polypeptides of an comparative low molecular weight of homologous amoeba. And we also observed the binding mono- clonal antibodies to the cyst and trophozoites of the same strain by an indirect immunoperoxidase technique for light microscopy. Further biochemical and morphological works are in progress to study the characteristics especially in cell differentiation. PHYSIOLOGICAL AND MORPHOLOGICAL CHARACTERIZATIONS OF ACANTHAMOEBA HATCHETTI-ISOLATED FROM SOIL SAMPLES. K. Watanabe!, H. Horikami*, K. Ishii? and T. Tsuruhara1!. Dept. Biol., Tokyo Gakugei Univ., Koganei and 2Lab. Biol., Hosei Univ., Tokyo. Acanthamoeba species are a small free-living amoebae and are found very widely in nature and 18 different established species are now described, but Acanthamoeba hatchetti is known only from brackish-water sediments in USA. We have isolated two strains of Acanthamoeba hatchetti from soil samples taken in Tokyo and Naha, and have successfully established pure culture lines starting from single cysts. These strains are able to grow and encyst on agar plates containing artificial seawater (salinity: 30 %o). Their cysts are small and refractile with thick ectocyst and have the three- or four- endocyst arms (mean cyst diameter:11.44.m) which gives a triangular shape. We have now investigated their patterns of isoenzymes to compare with other strains. DISINFECTION AGAINST ACANTHAMOEBA’S CYST FROM HU- MAN KERATITIS. H. Horikami!, K. Ishii!, H. Yamaura?, and Y. Ishibashi?. 1 Lab. of Biol., Hosei Univ., Tokyo, 2 Dep. of Parasitol., Tokyo Women’s Med. Coll., To- kyo, 3 Dep. of Ophthalmol., Tsukuba Univ., Tsukuba-shi. The cysticidal properties of various test solutions were examined against 2 strains of Acanthamoeba isolated from keratitis. The cysts(2-3 weeks old, 10° cells) of A. polyphaga, strain Fuks and A. castellanii, strain Toks were soaked overnight into 1.5 ml of test soln in plastic microtube at room temper- ature, and then washed 4 times by centrifugation (600Xg/3 min) with amoeba saline. The sediment was inoculated on non-nutrient agar plate smeared with 5 % yeast extract and 10 % glucose and incubated at 30°C for 10 days. The plates were examined daily for amoebic growth. 1) 0.2% Osvan soln(DAIGO), 30 % ethanol, 1 % commercial sodium hy- pochlorite soln "HAITER”(KAO CORP.), 0.1 % sodium hypochlorite soln “Antiformin”"(WAKO CHEMICALS), 1 N NaOH soln, 0.1 % thymol soln and 1 % picric acid soln inhibited perfectly the excystment, especially 4 % HAITER and 10 % Antiformin induced cytolysis, but the double cyst walls were maintained. On the other hand, 50 % glycerin soln, 1 N HCI soln, 1 M soln of LiCl , NiCl, BaCl5, ZnCl,, and MgCl, were not effective. 2) In spite of treatment of cysts with 0.1 % soln of HAITER or 0.05 % soln of Antiformin was not able to inhibit the excystment, addition of 10 % of glucose, sucrose or mannnitol into the test soln inhibited perfectly the excyst- ment This phenomenon could not induced with yeast extract or amino acid. The cysticidal action of chlorine on Acanthamoeba was reported by Jonck- heere and Voorde(1976). In comparison with their chlorine content, our re- Sults show that the content was at least 100 times lower with the addition of a sugar, and this new disinfectant might be more safety. 1278 Cell Biology and Morphology FOOD VACUOLE FORMATION IN PARAMECIUM FED WITH STREPTOBACILLUS. S.Mishima. Biol.Lab., Coll.Gen.Educ., Ibaraki Univ., Mito. Previously we reported that the food vacuoles produced by Paramecium fed with streptobacillus(SB) were not spherical but they had a strange shape such as a string of cells or a bundle packing the strings. Further it was found that Paramecium pro- duced spherical food vacuoles including SB when mono- or oligo-saccharide was added to the culture. In the present study we examined the shape of food vacuoles when added with a mixture of SB and polystyrene latex particles. Formation of abnormal vacuoles(10-15%) such as apparently spher- ical ones with long-ranged cells sticking out of their surface or vacuoles peaked at both longitudinal ends like an American football were observed. The SB prepara- tion contained long strings of more than 6 cells at the proportion of 10%. Since the diameter of an ordinary food vacuole corresponds to the length of a string 4 cells, it is supposed that formation of these abnormal vacuoles might occur when a vacuole incorporates longer SB than the diameter of a vacuole, whereas Paramecium given saccharide can produce a spherical vacuole by incorporating them curved along its half circumference even in the case of long SB. EFFECTS OF HEAT-SHOCK ON REGENERATION IN THE CILIATE PSEUDOUROSTYLA LEVIS: SDS-PAGE ANALYSIS OF THE PROTEINS IN THE REGENERATING AMICRO- AND MICRONUCLEAR FRAGMENTS T. Takahashi. Zool. Inst., Fac. of Sci., Hiroshima Univ., Higashi-Hiroshima. For analysis of the somatic functions of micronuclei in the multinuclear ciliate Pseudo- urostyla, effects of heat-shock on regeneration of amicro- (ami) and micronuclear (ml) fragments were examined. At 2h after the cell division many Gl cells were transected into two frag- ments at posterior to the mouth, and then they were maintained at 23 C. At 1.5h after the operation these fragments were treated at 38.5 C for 20min. Subsequently they were fixed for silver impregnation with protargol technique, or were transferred to sample buffers for SDS-PAGE at constant intervals of lh. About 300 frag- ments per lane were needed to prepare for the SDS-PAGE samples. Regeneration of non-heat- shocked ami and ml fragments completed about 5- 6h after the operation. In contrast, completion of regeneration delayed for about 3-4h In heat- shocked both ami and mi fragments. SDS-PAGE indicated that each of lanes contained more than 50 bands, but any specific proteins for the mouth part formation could not be determined Moreover, about 8KD protein was always observed as major band In mi fragments, but faintly In ami fragments. The problem as to whether this difference {is related to the somatic functions of micronucle! remains to be analyzed in the future. RESTORATION OF THE REDUCED FISSION RATE OF THE JUMYO MUTANT OF PARAME- CIUM TETRAURELIA. Y. Tokusumi!, H. Fujisawa!, Y. Takagi! and K. Kaji2 'Dept. of Biol., Nara Women's Univ., Nara, 2Tokyo Metropol. Inst. Gerontol., Tokyo. The jumyo mutant of Paramecium tetraurelia has been used for the assay of mitogenic substances by the restoration of its reduced fission rate in daily reisola- tion culture. We have shown that 2% or more fetal bovine serum (FBS) was mitogenic for the jumyo mu- tant, as well as the cell-free conditioned medium of a variety of Paramecium cultures. We here tested for the effect of mammalian growth factors (TGFa, EGF, PDGF, IGF, b-FGF) and the cell-free conditioned medium of Tetrahymena. TGFa and EGF were some- what effective at very high concentrations (50 ~ 300 ng/ml), although no growth factors were effective at 20 ng/ml. The cell-free fluid of T. pyriformis and T. thermophila concentrated 100-fold by ultrafiltration was also effective. When wild type cells were used, their nutritionally reduced fission rates were restored by a concentrate of culture medium whether or not it included cells of Paramecium or Tetrahymena, but not restored by FBS. There is yet much to do before we conclude decisively, because the fission rate of the jJumyo mutant is such unstable as to become suddenly high without any mitogens. FUNCTION OF TETRAHYMENA EXTRACT FOR INDUCTION OF MATURATION OF XENOPUS OOCYTES E.Ogawa!, M.Fujishima!, M.Yamashita?, Y.Nagahama? and N.sagata?. 1Biol. Inst., Fac. of Sci., Yamaguchi Univ., Yamaguchi, 2Lab. of Reprod. Biol., Natl. Inst. for Basic Biol., Okazaki, and 3Div. of Mol. Genet., Inst. of Life Sci., Kurume Univ., Kurume. Reinitiation of meiosis (maturation) of Xenopus oocyte can be induced if Tetrahymena extract is injected into them. The activity of this meiosis- reinitiation-inducing factor (MRIF) differed from M-phase-promoting factor (MPF), because action for induction of germinal vesicle breakdown (GVBD) of MRIF needed the recipient oocyte protein synthesis. However, MPF-activity appeared in the MRIF-injected oocytes. Immunoblots using antibodies for Mos protein and PSTAIR sequence of cdc2 showed that MRIF induced appearance of Mos and dephosphorylation of cdec2 in the oocytes in the presence of the oocyte protein synthesis. This result suggests that primary function of MRIF in the oocyte is induction of Mos protein, and then the Mos induces dephosphorylation of cde2 to induce active-MPF and GVBD. Cell Biology and Morphology 1279 INBREEDING OF THE CILIATE PARAMECIUM BURSARIA. S. Matsuda, T. Kosaka and T. Takahashi. Zool. Inst., Fac. of Sci., Hiroshima Univ., Higashi-Hiroshima. Two breeding systems, inbreeding and outbreeding, are known in ciliates. This study was made to examine how these breed- ing systems function as a sexual reproduc- tive strategy in a natural population of ciliates. Using Paramecium bursaria syngen 1, which is considered a typical outbreeding species, the effect of in- breeding (sib-cross) on the viability of exconjugants was studied. Four Fl clones belonging to different mating types (I, II, III and IV) were used. They originat- ed from a cross between parental stocks OK-312(1) and OK-223(IV). When any two complementary mating type II, III or IV were mixed together, the viability of the exconjugant cells (F2) was over 90%. However, when the mating type I clone was used in crosses with any of the other mating types, the viability of exconjugant cells was under 75%. The cause of the low viability remains unclear. The viability of outcrossing (conjugation between two stocks originated from different places) was generally high. Thus, the results show that with occasional exception in- breeding as well as outbreeding could work to maintain viable populations of P. bursaria in nature. A BIOLOGICAL CLOCK SYSTEM OF MATING TYPE REVERSALS RHYTHM IN PARAMECIUM MULTIMICRONUCLEATUM, SYNGEN 2. S. Kamiya! and I. Miwa’. ‘Dept. of Biology, Fac. of Sci., Ibaraki Univ. *Biol. Lab., Coll. Gen. Educ., Ibaraki Univ. , Mito. Cells of P mulomicronuclentum change the mating type two times in a day, they express the mating type III for a part of a day and the complementary mating type IV for the remainder of the day. Cells of a stock used in this experiment changed the mating type from III to IV about 11 hours after the onset of the dark period when they were put in the various light and dark cycles. The length of the period of mating type IV depended on the dark period. Therefore their timekeeping system is supposed to be a clock of sandglass type, which begins to time with the signal at onset of darkness. But the mating type reversals rhythm was continued under the both conditions of constant light and darkness. Thus, an endogenous circadian oscillator also controls the rhythm of mating type reversals in P multimicronucleatum. In addition, according to a result of an experiment of low temperature treatment, it became clear that they needed a certain reaction during the light period in order to change the mating type from III to IV. Ina light pulse experiments, the timing of mating type transition was delayed when they were exposed to a light pulse at the first half of the dark period, but it was not delayed when they were exposed to a light pulse at the latter half of the dark period. Now we are continuing to investigate the effect of many drugs of known specificity on mating type reversals of P multimicronucleatum. EARLY MATURE MUTANT ON THE DEVELOPMENTAL CLOCK IN PARAMECIUM BURSARIA I. Miwa!, S. Takaya? and Y. Tomioka®. Biol. Lab., !Coll. Gen. Educ., *Fac. of Sci., 9Fac. of Edu., Ibaraki Univ., Mito An exconjugant clone of Paramecium has a well-defined life cycle, consisting of the period of immaturity, maturity and senility. During the immaturity period, Paramecium is unable to mate even under appropriate conditions. The duration of the immaturity period can be measured by the developmental clock which depends on the number of fissions after conjugation. The exconjugant clones of P bursaria show adolescence more 30 fissions after conjugation during in the transition from immaturity to maturity, in which they express the one of two genes determined mating type. We isolated recently a early mature clone after treated with 2 pg/ml nitrosoguanidine (MNNG) for 5 hours. This clone exhibited mating reactivity 15-20 fissions after conjugation, and genetic control of the early mature character was shown by Mendelian segregation. Homozygote of the early mature gene exhibited mating ability 8-12 fissions after conjugation. Though the length of adolescence in wild type stocks was 12- 14 fissions, that of the early mature mutant was shortened was 2-4 fissions. P. bursaria exhibits circadian rhythm of mating reactivity. Then we investigated the correlation of both clocks of development and circadian. The stocks shown short period ciradian rhythm of photoaccumulation became to mature earlier than the stocks shown long period of circadian rhythm. But this early mature mutant showed almost the same circadian period as the wild type stocks shown short period. COLD RESISTANCY OF SOME CNR MUTANTS IN PARAMECIUM CAUDATUM. K.Hiwatashi. Dept. of Biotech., Senshu Univ. of Ishinomaki, Ishinomaki, Miyagi. We have reported (Hiwatashi & Skoblo, 1991) a CNR mutant discovered in a natural stock of Paramecium caudatum collected in Russia. Since CNR mutants are defective in a vital function of paramecium cells, the discovery of natural stock containing a CNR gene suggests that the CNR gene may in some respect give a selective advantage for the stocks containing it. Resistancy to low temperature was tested for the Russian CNR stock, since the stock was collected in a cold district. Five differ- ent CNR mutants, cnrA, cnrB, cnrD, cnrD and the Russian CNR were cultured in daily isolation lines at three different temper- ratures, 5°, 10° and 25°C. At the temper- ratures of 10° and 25°C, no remarkable difference was seen among stocks of wild type, CNRs and their Fl heterozygotes, but at 5°C, stocks of cnrC€ and the Russian CNR showed a significant cold resistancy com- pared with other CNR stocks. However, since a few wild-type stocks and the stock of cnrC homozygote expressing wild type also showed the cold resistancy, the cold resistancy of the above resistant CNR stocks seems not to be controlled directly by the CNR genes involved. 1280 Cell Biology and Morphology PRODUCTION OF MONOCLONAL ANTIBODIES SPECIFIC FOR HOLOSPORA OBTUSA OF PARAMECIUM CAUDATUM. H.Dohra and M.Fujishima. Biological Institute, Fuculty of Science, Yamaguchi University, Yamaguchi. The gram negative bacterium H.obtusa is a macronuclear specific symbiont of the ciliate P.caudatum. This bacterium changes its morphology in good accordance with the growth of the host cells; reproductive short form and infectious long form. When the host cells starve in the stationary phase of the growth, the reproductive short forms cease binary fissions, elongate themselves and differentiate the infectious long forms. To detect bacterial stage-specific substances, in the present study, we intended to get monoclonal antibodies(mAbs) specific for the infectious long forms. We isolated infectious long forms and their whole cells, sonicated brei and purified proteins from 2D-SDS- and native- PAGE gels of the infectious long forms were injected into mice as the antigens. As a result, seven mAbs including infectious form specific ones were obtained. We show intra-cellular localities and molecular weights of the antigens by indirect immunofluorescences and immunoblots. DIFFERENTIATION FROM REPRODUCTIVE FORM TO INFECTIOUS FORM OF ENDOSYMBITOTIC BACTERIUM HOLOSPORA OF PARAMECIUM IS INDUCED BY INHIBITION OF THE HOST PROTEIN SYNTHESIS. M. Fujishima and N. Hirakawa. Biol. Inst., Fac. of Sci., Yamaguchi Univ., Yamaguchi. Reproductive short form (1.5 ~m in length) of an endonuclear symbiont Holospora obtusa of Paramecium caudatum grows by binary fissions in the host macronucleus when the host grows. When the host starves, however, the bacterium also ceases the fission and differentiates into the infectious long form (13 ~im in length). Short forms-bearing paramecia in the log phase of growth were suspended in different mediums at 25°C: (1) culture medium, (2) phosphate-buffered saline and (3) culture medium-containing 1 ug/ml emetine, and then their bacterial morphology was observed. In (1), no bacterial elongation was induced. In (2) and (3), the elongation began at 48 h. In (3), the elongation was enhanced more than that in (2), notwithstanding that the hosts were not starved. These results suggest that induction of the differentiation of the infectious form by the host starvation may be due to the deterioration of the host protein synthesis. REESTABLISHMENT OF SYMBIOTIC ASSOCIATION BETWEEN ALGAE-FREE PARAMECIUM BURUSARTA AND SYMBIOTIC ALGAE FROM GREEN PARAMECIA. K. Kimura, T. Kosaka, and T. Takahashi Zool. Inst., Fac. of Zool., Hiroshima Univ., Higashi-Hiroshima. The incubation of algae-free(AF) Parame- cium bursaria with symbiotic algae(SA) from green paramecia results in the rees- tablishment of symbiotic association of them. The mechanism of this event, howev- er, is hardly clear. Recently, we found that SA remarkably changed their morpho- logical feature with the growth of host paramecia. In this work, therefore, examined the relationship between the reestablishment of symbiotic association and the growth phase of host paramecia. When the log and stationary phase AF paramecia were incubated with SA from log growing green paramecia, more than 95% and about 60% of AF paramecia were reinfected with the symbiotic algae. On one hand, when the SA from stationary phase green paramecia were mixed with log and station- ary phase AF paramecia, successful infec- tion rate was about 10% and 20%, respec- tively. It has been also found in this work that the SA can be cultured with CA medium. When the cultured SA were incu- bated with log and stationary AF parame- cia, rates of reestablishment of symbiotic association were about 90% and 50%. These findings suggest that the reinfection ability of symbiotic algae may change with the growth phase of the host. SYMBIOTIC CELLULOSE DIGESTION SYSTEM IN THE LOWER TERMITE: ROLE OF BACTERIA. I. Yamaoka and R. Murakami. Biol. Inst., Fac. of Sci., Yamaguchi Univ., Yamaguchi. It has been clear that the cellulose digestive system in the lower termite consists of the complicated relation between host and the symbionts. Especial- ly bacteria play an important role. One of them bacteria make the anaerobic condition of the hindgut lumen for intes- tinal protozoa and the other supply the metabolites to the symbionts. In here it suggests that one of the metabolites of the intestinal protozoa H gasses are reduced into CH gasses by the methanoge- nic bacteria in the hindgut. Their bacte- ria adhere to the surface of the hindgut epithelium and live in some species of the intestinal flagellates. They are examined by the fluorescence microscope and the electron microscope. Cell Biology and Morphology 1281 THE ALIMENTAL CANAL IN THE HIGHER TERMITES: ULTRASTRUCTURE OF THE MIDGUT AND THE MIXED SEGMENT. G. Tokuda, R. Murakami and I. Yamaoka. Biol. MInst., Fac. of Sci., Yamaguchi Univ., Yamaguchi. The alimental canal of the higher termite ( Nastitermes takasagoensis and Odontotermes forsanus ) were examined by the electron microscope. The midgut epithelia in both species were similar in the fine structure and consisted of columnar cells and regenerative cells formed a mass. In the columner cells the nucleus situated at the cell apex (a side of the lumen ) and invaginations of the cell membrane observed at the cell base ( a side of basal lamina ). Large and with the well developed crista mitochondria were observed into the cell invaginations. Large phagosome was observed in the cytoplasm of each cell. The mixed segment observed in the N. takasagoensis was not observed in the O. formosanus. Another difference of the fine structure of both species was also clear. SYMBIOSIS BETWEEN AN EOLID NUDIBRANCH CUTHONA SP. AND A HYDROZOA AGLAOPHENIA CUPRESSINA LAMOUROUX T. Yamasu, Dept. of Biology, Div. of Gen. Educ. Univ. of the Ryukyus, Nishihara, Okinawa. Living specimens of an eolid nudibranch, Cuthona sp. were collected on the colonies of a thecate and zooxanthellated Hydrozoa, Aglaophenia cupressina Lamouroux found abundant all the year round on patch reefs at Cape Zanpa in Okinawa Island. The colonies of the hydroid collected were cut into small size and reared in containers (30 cm in diameter and 12 cm in depth, together with 2 litter of sea water) kept aerated under fluorescent lump illumination (1500 lux) in the laboratory for about 4 days before they were given as food. The nudibranch fed on polyps and hydrocauli of the hydroid. Zooxanthellae ingested were stored within the liver cells in cerata when the animals were kept fed continuously. Animals fed on food showed dark brownish green color in the cerata. When starved for 2 days, however, the cerata turned to orange indicating digestion of the zooxanthellae took place. Light and electron microscopies exmined with starved animals revealed that small number of the zooxanthellae were remained still in the liver cells. Adult animal spawned an egg mass every day for more than 1 month after copulation. The number of eggs in a mass were over 1200 in fully matured adult. Hatched veligers provided with well developed digestive and nervous systems including paired eyes and few radular teeth. They clung to food hydroid soon after release. After casting off the velar cillia and larval shell, they metamorphosed to a vermi-form juvenile in the next day. A pair of cerata developed at first. Sexual maturity was attained for about 1 month after the hatching. Unlike the case of symbiosis in nudibranch, Pteraeolidea lanthina (Kempf,1984) which fed on another zooxanthellated hydroid, Myrionema cuppressina, this species seems to show a somewhat temporary association with symbionts ingested. AN ACOEL FLAT WORM SPECIES RELATED CLOSELY TO THE SPECIES CONVOLUTRILOBA RETROGEMMA HENDELBERG AND SSON OCCURS IN OKINAWA ISLAND,RYUKYU ARCHIPELAGO. K. Ishikawa! and T. Yamasu? !Coll. of Sci.and Div. of Gen. Educ., Univ. of Ryukyus, Nishihara,Okinawa. We collected two acoel species very similar to the species Convolutriloba retrogemma Henderberg and Akesson (1988) at two habitats, Cape Zanpa in Okinawa Island and Sesoko Island in 1991 and 1992. Both species were found in low tidal zone where colonies of a Hydrozoa Myrionema amboinense Pictet grew. Onc of them, the larger one, is much more similar to C. retrogemma than the smaller one, having not only two transparent spots at eye field and a straight nozzle of seminal bursa but also coinciding well in size and external morphology. A tremendous number of adult worms of this species were also found in an outdoor aquarium in Sesoko Marine Science Center of University of the Ryukyus in Sesoko Island. However, no worms have released any progeny upto now. The smaller species, ranging from 0.62 to 2.43 mm in length, on the other hand, released so many progenies from the ventral caudal part that total number of progenies released by an active individual amounted more than 14 during two week rearing in the laboratory, at an average rate of 0.8 / day / individual(14 worms were used). Neither transparent spot at eye field nor sexual organ provided in C. retrogemma and the larger one, have been found in all specimens collected (270 in total number) in both habitats so far as examined. Both species have similar uni-cellular green symbionts with 4 flagella (Prasinophyceae). Motile phase of symbionts separated from the smaller species seems to be rather shorter in comparison with those in the larger one or other acoel species which have also similar flagellated green symbionts. Whether the larger species might release its progeny or not should be watched carefully in further observation. DEVELOPMENT OF THE VERMIFORM EMBRYO OF DICYEMIDS (MESOZOA) . H. Furuya, K. Tsuneki and Y. Koshida Dept. of Biol., Coll. of Gen. Educ., Osaka Univ., Toyonaka. The development of vermiform embryos within the axial cell of Dicyema acuticephalum was studied microscopically on the fixed and stained specimens. An agamate devides equally and its daughter cells remain in contact with each other. One of them divides again equally to produce the prospective axial cell. This undergoes extremely unequal divisions at least three times, and smaller cells degenerate and disappear. From the five cell stage onward, the division pattern becomes bilateral type. At the final stage of embryogenesis, the prospective axial cell devides equally to produce two daughter cells, one of which is in- corporated in the axial cell to form the agamate. The fully formed embryo is composed of one axial cell and 18 peri- pheral cells. In addition, preliminary survey was carried out to determine the survival time of nematogens and infusoriform larvae of D. japonicum in vitro in the host octopus urine and also in seawater at 14 C. The nematogens have been alive for ten days in the urine, but they died whithin five days in seawater. The infusoriforms became immobile only within two days in both the urine and seawater. 1282 Cell Biology and Morphology ULTRASTRUCTURE OF THE MARGINAL ADHESIVE ORGANS OF THE FRESHWATER PLANARIANS. S. Ishii. Division of Cell Science, Research Laboratories, Fukushima Medical College, Fukushima. A comparative study of the marginal ad- hesive organs of freshwater planarians (Turbellaria, Tricladida) was carried out using 5 species collected in the suburbs of Fukushima City (Dugesia japonica, Pha- gocata vivida, Ph. kawakatsui, Polycelis auriculata and Bdellocephala brunnea). All the organs examined were of duo-gland type similar to those reported in marine forms (Tyler,1976), consisting of long necks of viscid and releasing glands whick perforate through the cytoplasm of special modified epidermal cells, anchor cells. The secretion granules of the vis- cid glands were large ovoid dense granu- les showing multifarious ultrastructural features of possibly species-specific varieties. On the contrary, the secretion granules of the releasing glands were small and less specific in features.The anchor cells were of insunk type and pro- vided with many surface microvilli with specially developed fibrilar cores which continued to the cell webs. The adhesive organ of Bdellocephala brunnea had specially developed areas of adhesion, but ultrastructurally they were basically not different from those of the other species. THE BOTRYOIDAL TISSUE IN LEECHES, HIRUDO MEDICINALIS UNDER 6°C H.Inamura Dept. of Biol.,Tokyo Med. Coll., Tokyo The structure and function of botryoidal tissue were studied using electron microsc- opy at temperatures under 6°C,instead of the previously reported 25°C.Hungry leeches were fed blood from rabbits once in 2 mont- hs.After 5 day,the leeches had consumed four to five times their own weight in blood with a weight loss of 15 percent,as compared to a 35 percent weight loss for leeches at 25°C.Blood in the crop showed fresh vermilion colors and erythrocyte,in contrast to blackish colors at 25°C. In botryoidal cells at 25°C,rER was report- ed to clearly develop around nuclei,and in the apical and lateral cytoplasm. There was lots of microvilli in the lumenal surface of cells.Cells consisted of three granule types ;Ll-granules were oil-drople- ts,L2-g. were ACPase activity,and L3-g. were like a lysosome.Under 6°C,rER :did not develop.In the lumenal cytoplasm there were lots of vesicles with low density,and microvilli decreased.The apical cell membr- ane was indented like a tubule with high density,and was a similar shape at 6°C and at 25°C.A high numbers of L2 and L3 granules were observed,but Ll-g. were not observed. After 1 to 4 weeks,cells showed a similar shape. Therefore we can conclude that metabolism in botryoidal cells is affected by tempera- ture. THE DEVELOPMENT AND THE REGENERATION OF THE GENITAL ORGANS IN THE LAND PLANARIAN, BIPALIUM PENNSYLVANICUM. Y.Shirasawa and N.Makino. Dept.of Biol. Tokyo Med. Coll., Tokyo. The development of the genital organs, ovary, testis and copulatory apparatus of the larva and the regeneration of these organs in the cut pieces of the matured individual have been examined, morphologi- cally and histologically, in the land pla- nNarian, B.pennsylvanicum which reproducts only sexually. In the three-weeks larva, we observed several masses which are com- posed of basophilic cells in the parenchy- ma between the ventral nerve cord and the intestine. The anterior small masses make a pair, the middle larger several ones are close to the intestine, and the posterior is the largest, in which the disintegra- tion of the intestinal wall is most re- markable, and the stainability of the basophilic cells is the strongest. In the next stage, the anterior masses differen- tiate into ovaries, the middle into testes and the posterior into copulatory appara- tus. In the regenerating cut pieces,which regenerate well without reference of body levels, the head pieces regenerate testes and the anlage of the copulatory apparatus in eight weeks. The pre- and the post pha- ryngeal pieces regenerate ovaries simi- larly. A half of the tail pieces regenera- te heads and pharynges but not the genital organs. THE ENDOCRINE CONTROL SYSTEM OF THE FUNCTIONAL HERMAPHRODITISM OF THE SLUG (LIMAX MARGINATUS). N. Seo and N. Makino, Dept. of Biol., Tokyo Med. Coll., Tokyo Until now we have obtained the follow- ing on the endocrine control of sexual growth due to neurosecretory cells(NCs) in the cerebral ganglion(CG) and the suboeso- phageal ganglion(SG) of Limax marginatus. The CG and SG at the mature stage were effective in development of male and fema- le phases of ovotestes(OT). Female germ cells (GCs) defferentiated under an unhor- monal condition. Aldehyde fuchsin positive NCs were classified Type] — X according to their size and distribution. TypeVl NCs appeared during the oviposition stage. Others appeared in a less mature stage and increased in number, the number reached a Maximum in the mature stage. Endocrine effects on the OT were class- ified, the provocation of differentiation of male GCs,the stimulation of progressive spermatogenesis, the provocation of ovipo- sition and the acceleration of vitello- genesis. It can be said that NCS produce two stimulating hormones of OT, one being the male differentiation hormone(MDH), the another being the oviposition hormone(OH). The functional hermaphroditism is retained due to autodifferentiation of female GCs and differentiation of male GCs by MDH, and by, parallel development of male and female GCs by increase in MDH and the hormone of stimulative vitellogenesis. Cell Biology and Morphology 1283 STRUCTURE OF THE OVARY AND OOGENESIS IN SCHIZOMUS SAWADAI (ARACHNIDA; SCHIZOMIDA) K.Miyazaki!, R.Ueshima2 and T.Makioka@. Dept. of Biol., Keio Univ., Yokohama, and 2Inst. of Biol. Sci., Univ. of Tsukuba, Ibaraki. In most orders of the Arachnida, struc- ture of the ovaries and the modes of oogen- esis share some unique features (Makioka, 1988). In the Schizomida, however, few studies have been done on these subjects. In the present study, we report structure of the female reproductive system and oogenesis in a Japanese schizomid, Schizo- mus Sawadai. The adult female reproductive system con- sists of a single sac-like ovary, paired lateral oviducts, a common oviduct and a genital pore, all of which are localized in the abdomen. No oogonia are found in ovaries of all the examined adult speci- mens. Very young oocytes are embedded within the dorsal ovarian epithelium, but the larger ones protrude outward from the lateral and ventral ovarian epithelium into the hemocoel, connected with the ovary by cellular stalks. A real connection between the ovarian lumen and the genital pore through the oviducts indicates that mature oocytes on the stalks should be ovulated into the ovarian lumen and sent toward the genital pore. Structure of the female reproductive system and mode of the oogene- sis in S. sawadai are basically identical with those in many other arachnids, espe- Cially those in orders of the Pedipalpi. COMMON FEATURES IN OVARIAN STRUCTURE OF SOME JAPANESE PENICILLATE DIPLOPODS. K. Yahata and T. Makioka. Inst. of Biol. Sci., Univ. of Tsukuba, Tsukuba. In a European penicillate diplopod, dagurus, Kubrakiewicz (1989) described the following structural features of the adult ovary as the characteristics peculiar to the penicillates; existence of a distinct germarium including oogonia and absence of cellular connections between the Ovarian epithelium and the oocytes growing in the ovarian lumen. In adult ovaries of some Japanese penicillates, we found not only the germaria, but also the cellular connections between the ovarian epithelium and the oocytes. Eudigraphis nigricans, E. takakuwai, E. kinutensis, BE. sp., PB. shinoharai, and PB. sp. showed mostly similar structural features on adult ovaries despite some minor differences possibly due to their sizes. A germarium composed of oogonia, very young oocytes and interstitial cells was usually located in the centre of the ventral ovarian epithelium. Paired germ areas consisting of young oocytes and young somatic cells were serially arranged in the ventral ovarian epithelium, from each of which areas several elongated somatic cells rose to be connected with some larger oocytes floating in the ovarian lumen. These connecting structures would be common not only among penicillates, but also to other diplopods. POSTEMBRYONIC DEVELOPMENT OF THE OVARY IN A NOTOSTRACAN, TRIOPS LONGICAUDATUS (BRANCHIOPODA, CRUSTACEA). H. Ando and T, Makioka. Inst. of Biol. Sci., Univ. of Tsukuba, Tsukuba. Two remarkably different types of ovaries have been known in the branchiopod crustaceans: one type seen in the Notostraca and Conchostraca and the other in the Anostraca and Cladocera. The ovary in the former type branches into many ovarioles, on the tips of which the germaria including oogonia are located. An egg-follicle with a growing oocyte and nurse cells is protruded from each germarium to the hemocoel. On the other hand, the ovary of the latter type does not branch. The germarium is localized at a particular part of the ovary, and the oocytes grow in the ovarian lumen. We studied postembryonic development of the ovary in anotostracan, Triops longicaudatus, to know how the ovary of the former type, unique among the Crustacea, is formed and to find origins of some differences between the types. By the 4th instar, a pair of ovaries appeared as young germ cell-masses on both sides of the gut. At about the 11th instar, each ovary became tubular, having in the ovarian epithelium many germ cell- masses or germaria with the egg-follicles. This ovary already showed the outward migration of the growing egg- follicles as one of the particularities of the former type. Formation of the ovarioles began later at about the 15th instar, and spawning eggs at about the 18th. STRUCTURE OF THE FEMALE REPRODUCTIVE SYSTEM IN ARGULUS JAPONICUS (CRUSTACEA: BRANCHIURA) . K.Ikuta and T.Makioka. Inst. of Biol. Sci., Univ. of Tsukuba, Tsukuba. The adult female Argulus japonicus has a single sac-like ovary lying above the alimentary canal in the thorax. A long germ zone or germarium including many oogonia runs in the dorsal ovarian epithelium along the median line. A pair of wide and flat-— tened oviducts start from the antero-dorsal end of the ovary and run along both sides of the ovary. Either of oviducts is con- nected with a genital pore at the end of the last thoracic segment, and the other ends blindly. Growing oocytes are found, not in the ovarian lumen, but on the outer surface of the repeatedly folded ovarian epithelium, protruded into a narrow hemo- coelic space surrounded by the oviducts and the alimentary canal. These oocytes are tightly covered with the extended basement membrane of the ovarian epithelium. Neither follicle cells nor nurse cells are found around the oocytes. Mature eggs are ovu- lated into the ovarian lumen, transported into and stored in the oviduct connected with the genital pore. A pair of sperma- thecae are located in the abdomen, opened just outside the genital pore. Some basic features on structure of the ovary and oogenesis in A. 3 j seem similar to those in many chelicerates and pentastomids rather than those in many other crustaceans. 1284 Cell Biology and Morphology STUDIES ON THE ENDOPARASITES OF ANURA 5.0N THE NEMATODA;RHABDIAS NIPPONICA IN LUNG Of RANA Y.Sasaki and N.Makino. Dept. of Biology.,Tokyo Med. Coll., Tokyo. The nematoda, Rhabdias nipponica is known to show the hetelogony.This worm of parasitic stage are fe- nale. Eggs are deposited by the female,hatch out, and the hatched larvae develop and grow up to free living males and females.We tried to culture of this hatched larvae. Under the environment of water as a basis, larvae were in high spirit for two or three days but after five or ten days,their half died. Only a few larvae lived fourteen days. Many larvae didn’t grow and their body length was about 500un. Sometimes big larvae were observed, their body length was about 700 um and they were two times as body width as many larvae. It seems that big larvae grow. And then hatched larvae were cultured under various following solution ; frog’s ringer, Earl’s balanced salt solution and medium 199 . FBS was added 5% or 10% to culture media to achieve a final concentra- tion. And antibiotics were that a mixture of peni- cillin and streptomycin was added to culture media to achieve a final concentration of 100 or 50 units penicillin and 0.1 or 0.05 mg streptomycin per ml medium. In the each culture media,survival of larvae were the same number of days with water as a basis. It is interesting that a few adult worms were sur- vived about one month in some culture media. But eggs of this females showed a poor rate of hatching. CYTOCHEMISTRY AND ULTRASTRUCTURE OF "MYCE- TOMES" OF THRIPS (INSECTA, THYSANOPTERA } Tsutsumi, M. Matsuzaki~ and K. Haga Inst.of Biol.Sci.Univ.Tsukuba, Tsukuba. Lab.of Biol.,Fac.of Educ. ,Univ.Fukushima, Fukushima. Idolothripine thrips, Bactrothrips brevitubus possesses the structures named mycetomes" in the oocyte or egg, which have been accepted as the intracellular symbiont from the morphological appearance. It was, however, revealed through our study that the "mycetome" of thrips is different from the structures identified as intra- cellular symbiont in the other insects in some features, such as stainability to basophilic dyes [hematoxylin, Schiff's reagent (Feulgen's reaction) etc.]. Here, we examine the "mycetome" in detail cyto- chemically and ultrastructurally, aiming at the characterization of it. From the present study, the features of "mycetome" are summed up as follows: 1) The "mycetome" is composed of numerous small granules filled with electron- dense material and larger ones with myelin figures inside. 2) The "mycetome" shows positive stain- ability to the dyes of which specific uptake into lysosome is demonstrated (neutral red, acridine orange etc.). 3) High activity of acid phosphatase is detected in the "mycetome" (metal precipitation or azo coupling method). These results may suggest that the thysanopteran "mycetome" should be not symbiont but an aggregation of lysosomes. CHARACTERISTICS OF THE PHOTORECEPTIVE CELL CANDIDATE IN THE BUTTERFLY GENITALIA Y. Miyako, K. Arikawa, and E. Eguchi. Dept of Biol, Yokohama City Univ, Yokohama Two pairs of extraocular photoreceptive sites (Pls and P2s) were identified in the butterfly genitalia. Previously we reported that a distinguishable ovoid structure (ca.30x40pm) containing a cell body of a sensory neuron (photoreceptor candidate;PRC) exists in both sexes' Pls of Papilio xuthus?. At the distal Part of the sensory neuron(PRC) there are several distal processes(DP) and a plenty of tubular membranes (TM) protrude from the tip of the DPs. They form an entangled thread ball-like structure. In the present study to examine the effect of light to the ultrastructure of PRC, the genitalia of the intact female Papilio xutus were illuminated by white light of ca.20000lux for 4 hours, then Pl was examined by electron microscopy. As a result in the distal part of the PRC tightly packed TM disappeared and a number of vesicles and membranous fragments became abundant in several specimens. In addition the nerve which innervates to Pl of each sex, mN6-2 for male and £N6-3 for female was identified. While recording the photoresponse from the nerve of mN6-2 or from £N6-3 with isolated Pls, nervous tissue in Pis were Surgically isolated from the cuticular surface and exess tissues were removed. Resulting piece of tissue that still respond to the light flash with the sustained train of spikes was fixed, embedded in Epon and examined by electron microscopy. As a result the preparation contained the PRC. The results suggest that the PRC is the genital photoreceptor. 1.Arikawa et al. Nature, 288,700-702, 1980 2.Miyako et al. Zool mag, 7,1028, 1990 MORPHOLOGICAL CHANGES AND MITOTIC ACTIVITY OF DUODENAL MUCOSA IN FASTED XENOPUS K.Fujikura, T. Sakai, and Z. Itoh. Inst. of Endocrinol. Gunma Univ., Maebashi. The epithelial cells of the intestine continuously proliferate, but they are constantly maintained in its size. The mechanism involved here is not well known, but stimuli brought by food are considered to be one of the important factors. In the present study, we used Xenopus as an experimental animal because it remains healthy without taking food for long period. We examined morphological changes and the mitotic activity in the duodenal epithelial cells of Xenopus after they were fasted for 14, 28, 42, 77, 109 and 151 days, and observed the effect of re-feeding. It was found that the mean height of villi in the duodenum measured by means of a microscopic image analyzer gradually decreased with time. On the 28th day of fasting the mean height had significantly decreased and was only 1/2 of the control by day 151. But the mean number of villi in the circumference of the duodenum did not change at any days of fasting. On the other hand, the mean frequency of mitosis detected by immunohistochemical staining with bromo deoxyuridine labeling rapidly decreased after fasting for 14 days, and remained at 20-30% of the control all experiment days thereafter. However, re-feeding caused a rapid increase in the frequency of mitosis, which reached 2.5 times after 5 days of re-feeding. These findings suggest that the growth of epithelial cells and frequency of mitosis of the duodenal mucosa are significantly affected by food intake. Cell Biology and Morphology 1285 HISTOCHEMICAL STUDY ON THE DERMAL CHROMATO- PHORE UNIT FORMATION IN THE FROG, HYLA ARBOREA. 2 1 M.Yasutomi and Y.Yokota . Biol. Lab., Aichi Med. College, Aichi and “Biol. Lab., Aichi Pref. Univ., Nagoya. In the frogs of the genus Hyla, the der- mal chromatophore unit (DCU) was formed during metamorphosis and the animals have ability for rapid color change (physio- logical color change). When metamorphosis began, the fibroblasts, which were seen under the basal lamina (BL) in the tadpole, invaded into BL and open space (stratum spongiosum;SS) was formed. Chromatophores such as melanophore, xanthophore and irido- phore, were also present under BL in the tadpole and migrated through BL at metamor- phic stage and reached to SS. Then, DCU was formed. The sections of the back skins were incubated with peroxidase-labelled lectins (ConA, WGA, RCA and PNA). RCA and PNA bindings were observed in SS. Immuno- electron microscopic study using anti- laminin IgG showed the existance of laminin in BL. The direction of the neu- ral crest cells migration was determined by galactose bearing proteoglycans and laminin (Milos et al.,1986; Bronner-Fraser, 1987; Martin-Green & Erickson,1987). These facts suggest that the migration of the chromatophores and the DCU formation involve the interactions between chromato- phore surface components and galactose bearing proteoglycans and laminin. THE EFFECTS OF TPA AND CHOLERA TOXIN ON FROG MELANOPHORE PROLIFERATION IN CULTURE. S. Takeuchi, H. Suzuki, M. Yabuuchi and Y. Kobayashi. Dept. of Biol., Fac. of Sci., Okayama Univ., Okayama. The present study was undertaken to compare and correlate the effects of 12-O-tetradecanoyl- phorbol acetate (TPA) and cholera toxin(CT) on frog me lanophore proliferation. Dermal melanophores were isolated from the dermis of a tree frog (Hyla arborea) and cultured in diluted Leibovitz-L15 medium supplemented with 20% FBS and 16nM TPA and/or 2nM CT. TPA and CT concomitantly induced the cell proliferation at a maXimal rate and the cells finally piled up as previously shown. TPA alone in the absence of CT did not result in optimal proliferation and the contact inhibition was observed. These results suggest that mitogenic effect of TPA on frog melanophore was greatly enhanced by the addition of CT. Under our experimental conditions, proliferating cells shared a common morphological features which included dispersion of melanosomes in the cell. This response resembled that induced by CT and hence appears to be cAMP mediated, however, CT did not induce melanophore proliferation. Further analysis are required to determine the exact role of protein kinase C and protein kinase A in regulating proliferation of frog melanophores. RETINOIC ACID DIRECTLY AFFECTS PROLIFERA- TION AND DIFFERENTIATION OF FETAL RAT FORE- STOMACH EPITHELIAL CELLS IN PRIMARY CULTURE H. Fukamachi. Zool. Inst., Fac. of Sci., Univ. of Tokyo, Tokyo. We have previously reported that fetal rat forestomach epithelial cells prolifera- te in primary culture. We have also shown that retinoic acid (RA) inhibits forestom- ach epithelial keratinization in organ culture. Here we examined whether RA directly affects forestomach epithelial cells in primary culture. Forestomach epithelial cells were cultured as previously reported. MTT assay showed that their proliferation was suppressed when more than 2 mcg/ml of RA were added to the culture medium. Thus, the effect of RA on the epithelial differ- entiation was examined by adding RA on day 4 in culture when the cells have prolifer- ated and formed cell sheets. In control cultures, the cells exhibited keratiniza-— tion on days 6 to 7. Their keratinization was suppressed when 20 mcg/ml of RA were added to the culture medium composed of F12 and growth factors, but such effect was not observed when MDCK152 was used instead of Fl2. EXpression of AE3 cytokeratin antigen was also suppressed in RA-added cultures. We thus conclude that RA directly affects both proliferation and differentiation of fetal rat forestomach epithelial cells, but that many other factors are involved in the control of the epithelial keratinization. PRIMARY CELL CULTURE OF APHID DERIVED CELLS H. Harada and H. Ishikawa. Zool. Inst., Fac. Sci., Univ. of Tokyo, Tokyo. Aphids, as pests of various crops and the most important group of plant virus vectors, have attracted investigators’ interest for a long time. In spite of every effort made to establish a cell line of aphid, there is no succesful report, yet. Aphids have bacterial endosymbionts which are harbored by bacteriocytes in the fat body. Endosymbionts are indispensable to normal growth of aphids, and do not have free living phase any longer. In an attempt to study this host- endosymbiont interaction in the aphid symbiotic system, we tried to establish the primary cell culture system of this insect. We were successful in establishing a culture system free from gut microbes, which enabled us to culture the aphid cell and its endosymbiont without antibiotics. Among seven insect media tested, the TC-100 medium exhibited the best survival of aphid cells. Five percent FBS included in the TC-100 insect medium activated the protein synthesis of aphid cells, and promoted cell attachment. On the contrary, the isolated endosymbionts in the medium with FBS were broken down after aggregated to each other within several days. 1286 Cell Biology and Morphology CELL CULTURE OF KERATINOCYTES FROM CHICK EMBRYONIC TARSOMETATARSAL SKIN AND THE DECREASE OF pH IN THE CONDITIONED MEDIUM. K. Mikami-Takei, H. Endo and A. Obinata. Dept. Physiol. Chen., Fac. Pharmaceu. Sci., Teikyo Univ., Kanagawa. It was impossible in MEM+FCS medium to culture chick embryonic keratinocytes but in BGJb medium, which has been used in organ culture of chick embryonic skin with supplementary chicken serum for several weeks though they keratinized autonomously and their rates of increase were rela- tively low. When cultured keratinocytes were mixed with skin fibroblasts, they keratinized more quickly but with fibro- blasts that ceased proliferation by hard X-ray-irradiation or by treatment with mitomycin C, keratinization was reduced. These data suggest that the chick kerati- nocytes culture requires a milieu of which the cells come from and fibroblasts sup- port keratinocytes culture effectively. It is also observed that marked pH de- crease in the conditioned media of kerati- nocytes with fibroblasts. Any increase of organic acid was not detected by HPLC analysis. Amino acid analysis revealed the decrease of glutamine in the condi- tioned media. The reason why mix culture decreased pH of media is unknown, but this Phenomenon implies some interaction be- tween keratinocytes and fibroblasts. FCS INDUCED CELL DEATH IN VITRO Takeshi Kurita and Hideo Namiki. Dept. of Biol., Sch. of Educ., Waseda Univ., Tokyo. Serum is commonly added to defined basal media as a source of nutrients and macro- moleculars essential for cell growth. Fetal calf serum (FCS) has been believed as the best one for cultures and used most commonly. It is well known that FCS often displays growth inhibition and cytotoxic-effects. The mechanism of the FCS cytotoxity is however not clear and not investigated well. We found high content of FCS in the basal medium induced cell death in several types of cultured cells. In order to estimate’ the morecular-weight of the toxic factor(s), FCS was separated into two fractions by ultrafiltration (YM2 M.W.1,000cut, Amicon). The macromolecular fraction was supplemented inorganic salts and nutritents and its osmotic-pressure was adjusted with MEM. Thus reconstituted medium (UF-FCS) didn’t induce cell death. On the other hands, high consentration of the FCS filtrate («1,000) induced cell-death. These data suggest FCS containes low-molecular-weight(<1,000) factor(s) which causes cell growth inhibition and cell death. As far as we tested, all type of cells in culture necrosed in FCS and well grew in UF-FCS. DISASSEMBLY OF F-ACTIN FILAMENTS IN HUMAN ENDOTHELIAL CELLS ON TYPE V COLLAGEN K. Yamamoto+, M. Yamamoto+-~, and Ths Noumura~ +Dept. Cell Biol., Tokyo Metropol. Inst. Gerontol., Tokyo, “Dept. Regul. Biol., Fac. Sci., Saitama Univ., Urawa. Human endothelial cells cultured on type V collagen attached temporarily to the substrate and formed F-actin filaments. However, the cells failed to proliferate and gradually detached from the substrate. After 24 h, the cells on type V collagen lacked discernible F- actin filaments and exhibited dots in small aggregates of F-actin. In addition, the cells expressed little or no proteins as focal adhesions, including vinculin and f, integrin. In contrast, the cells on fibronectin and type I collagen developed complete F-actin filaments, exhibited sufficient vinculin and B integrin, and grew logarithmically from 4 days. On the other hand, human smooth muscle cells formed complete F-actin filaments, revealed typical focal adhesions, and started to grow rapidly after 24 on type V collagen as well as on fibronectin and type I collagen. Thus, the disassembly of F-actin filaments in focal adhesions was observed as a specific phenomenon in human endothelial cells cultured on type V collagen and may result in the detachment of the cells from type V collagen. AUTOCRINE REGULATION OF THE MIGRATION OF HUMAN FETAL SKIN FIBROBLASTS. H. Kondo and Y. Yonezawa, Dept. of Exp. Biol., Tokyo Metropol. Inst. of Gerontol. Tokyo. It was recently reporte that growth factors in addition to extracellular matrix (ECM) were extremely important to cell migration. We found out using Stenn's method, which was devised as a model sys- tem of wound healing, that adult-donor skin fibroblasts migrated more slowly in serum-depleted medium than in serum- supplemented medium. On the other hand, human fetal skin fibroblasts migrated in serum-free medium as well as in serum- supplemented medium. In other words, human fetal skin fibroblasts migrate autonomous- ly.’To determine what factors regulate the migration of human fetal skin fibroblasts, we investigated whether monensin, an in- hibitor of ECM secretion and suramin, a competitor of growth factors at the receptor level suppressed fibroblast migration. The following results were obtained. Monensin inhibited the migration of human fetal skin fibroblasts. And, addition of type I collagen abolished the inhibition by monensin. Suramin also suppressed fibroblast migration. This inhibitory effect was partially recovered by addition of PDGF or bFGF. These results indicated that the migration of human fetal skin fibroblasts was regulated by growth factors and ECM which were supplied by an autocrine manner. Cell Biology and Morphology 1287 EFFECTS OF SIMIAN VIRUS 40 LARGE T- ANTIGEN GENE ON CELL IMMORTALIZATION. N. Yanai, R. Okuyama, and M. Obinata. Dep. of Cell Biol., Res. Inst. for Tuberculosis and Cancer, Tohoku Univ., Sendai. We established several cell lines from transgenic mice harboring tempera- ture-sensitive Simian Virus 40 large T- antigen gene. Those cell lines exhibit- ed continuous proliferation under the permissive temperature with their dif- ferentiation phenotypes. A liver cell line (TLR2) had potencies to produce albumin and was induced P450IAl1 by 3- methylcholanthrene. A tubule cell line (TKC2) were induced intracellular cAMP by arginine vasopressin. At the nonper- missive temperature, the degradation of T-antigen was observed, and cells could not proliferate and successively dead of apoptosis. According to previous works revealed that cell immortalization is caused by mutations of recessive immor- talizing genes. Presented result may lead a hypothesis that SV40 T antigen cause not only cell proliferation but also blocking of cell death, then pre- vent a senescent stage before an immor- talization. SUPPRESSION OF UV-INDUCED CELL TRANSFORMA- TION BY A CONTACT-INHIBITORY FACTOR IN MOUSE C3H10T1/2 CELLS. T.Yamaguchi?., and K.Nakasone THace Gent Educ. and *Fac. Sci., Ehime University., Matsuyama. (“Present address: Japan Inter- national Cooperation Agency, Tokyo) 2x , A membrane fraction of C3H10T1/2 (cl.8) cells prepared by homogenization and successive centrifugation was effective to inhibit the proliferation of a sparsely seeded growing culture of the same cell line which was sensitive to the contact inhibition of growth. The cells in conflu- ency were irradiated with UV, plated sparsely and treated with the membrane fraction during the transformational damage-fixing period (1 week) after the irradiation. The culture was continued with a weekly medium renewal for 6-8 weeks and transformed foci were detected. The transformation frequency was expressed as the ratio of the transformed foci to the number of surviving colonies using a formula of Poisson distribution. The transformation frequency was sig- nificantly decreased in a dose-dependent Manner by the membrane fraction added, suggesting the suppression of carcinogene- sis by the inhibition of cell prolifera- tion during the damage-fixing period after irradiation: A similar condition might be assumed to exist within the body where a damaged cell was surrounded by other cells of the same tissue. IN VITRO MODEL FOR THE PERIPHERAL AUTONOMIC NERVOUS SYSTEM : DIFFERENTIATION OF NEURITES T.MARUYAMA and Y.ENDO Dept.of App].Biol., Kyoto Institute of Technology, Matsugasaki, Sakyo, Kyoto In order to construct a model in vitro for the peripheral autonomic nervous system, we have tried to co-culture a pheochromocytoma cell line (PC-12) as a Neuronal element, and a smooth muscle cell line (SM-3) and insulinoma cel] line (In-R1-G9) as target cells.* PC-12 cells elongated several thin processes in this system, which appeared to be somewhat different in shape each other. It suggested the possibility of differentiation of them into axons and/or dendrites. An immunocytochemical approach using monoclonal antibodies agaist microtubule-assocated proteins (MAP1,2) showed that some neurites have no immunoreactivity of MAP2, whereas most of them have MAP1-immunoreaction. Occurrence of axon-like neurites without. MAP2 immunoreactivity was seemed to be related with their contact or adhesion to the other cells. Alternatively, length of neurites was also seemed to be related with the differentiation of neurites. Cells were also cultured by embedding in the typel collagen gel, just like in vivo conditions. Although the collagen gel caused a conspicuous change of cell shape, we could not find the difference of MAP distribution. xy. ENDO, T. MARUYAMA, Y.SASAKI (1991) Biomed Res 12:211-214 DEVELOPMENT OF NERVOUS SYSTEM IN THE TELEOST FISH, ORYZIAS LATIPES WITH SPECIAL REFERENCE TO RADIATION EFFECTS. Y. Hyodo-Taguchi and Y.Ishikawa. Div. of Biol. Natl. Inst. of Radiol. Sci., Chiba. The process of normal development of nervous system in the brain region of the medaka, Oryzias latipes and its radiation effects were preliminary studied. Embryos and fry of inbred strain HO4C and albino medaka were used. To examine development of nervous system, we made whole-mount specimens and the nerve fivers were stained immunohistochemically by using antineurofilament protein(NFP) antibodies. Divisions of the brain into the fore-, mid-, and hindbrain became well-defined at St.22. Mauthner cells(MC), nucleus of fasciculus longitudinalis medialis (FLM) and FLM were identified by NFP staining at St.24. At the same stage, 4-5 pairs of the neurons of reticular formation and reticulospinal nerve fibers (RF) were observed in the hindbrain region. Until hatching stage, all cranial nerves were formed. If doses of 2-6 Gy of radiation (X-rays, heavy ion and Y-rays) were given to embryos in early developmental stages, disorder of bilaterally symmetrial structures of nervous system was often occurred in the embryos with morphological anomaly. 1288 Cell Biology and Morphology RADIOBIOLOGY OF THE GUPPY, LEBISTES RETICULARIS (PETERS), THE RADIATION DEATH INDUCED BY WHOLE-BODY IRRADIATIONS WITH X-RYAS AND CARBON ION PARTICLES. H.Ohara, Y.Kohda, T.Kanai, Dept. Gen. Ed., Okayama Univ., Okayama, and Div. Accel. Res., Natl.Inst.Radiol.Sci., Chiba. The young guppies, being aged about 2-3 months after birth, were received whole body irradiations with graded dosis of 200 kVp of X-rays and 135 MeV/n of carbon ion beams produced by the RRC at RIKEN(Inst. Chem. Phys.). The fishes were observed for the post-irradiation occurrence of radiation death over 6 months of period. The possible reasons of radiation death were also explored by histological examinations in various tissues and organs. The main results were as the followings. More than 95 % of fishes died after irradiation with 15 Gy of heavy ions, while more than 60 % of fishes were survived by 25 Gy of X-rays. The most sensitive tissues/organs were identified as hematopoetic kidney tissues and those of epithelials in skin, gill, intestine, and oral cavity. Testis, liver, and glomerulus in kidney were also identified as being sensitive. Remarkable loss of blood cells was found in the head kidney , but loss of villi was not so distinct even with higher dosis of heavy ion irradiation. The damage of epithelial tissues in skin and oral parts was much more severe than those of intestine. Accordingly, some fishes died apparently from the starvation of foods due to the damage of oral cavity epithelials. This can be identified as "oral death" , which has already defined as the most important reason of radiation death in mammals to be induced with almost same level of radiation dosis. RBE for cabons was evaluated as some of 2.0 -2.2 in occurrence of radiation death at 20 C. IMP-DISTRIBUTION ON PLASMA MEMBRANE OF FISH EGGS BEFORE AND AFTER CORTICAL ALVEOLUS BREAKDOWN T. Ohta! and E. Kinoshita?, Dept. of Life Sci., Aichi Univ. of Educ., Kariya, *Fac. of Integr. Arts and Sci., Hiroshima Univ., Hiroshima. Cortical alveolus breakdown (CABD) of the rose bitterling (Rhodeus oce/latus ocellatus) eggs occurred in fresh water and continued for about 20 min. Insemination in fresh water induced polyspermic fertilization in all dechorionated and non-activated eggs. At 15, 30 and 60 min after activation treatment (AT, immersing into fresh water). dechorionated eggs were inseminated. The results showed that polyspermic fertilization rates decrease with the time after AT. Membranes of CA were added to egg plasma membrane (EPM) with CABD. We focussed on the change in IMP-distribution on the plasma membrane to clarify for the decrease in the fertilization rates. In non-activated eggs, few IMPs were distributed on PF surface of EPM and many IMPs existed on the PF surface of CA. The diameter of IMPs was about 13 nm. In eggs 15 min after AT, EPMs were dotted with aggregates of slight increased IMPs. In the eggs 30 and 60 min after AT, IMP-distribution on EPM was similar to that of non-activated eggs, in spite of addition of CA IMPs. No changes in IMP-distribution which seem to result in decreased fertilization rates were observed. CTENII FORMATION IN REGENERATING CTENOID SCALES IN THE JAPANESE FLOUNDER, PARALICHTHYS OLIVACEUS. Ss. Kikuchi? ,H. Nakamura* and A. Shimozawa~ {Kominato Lab.,Fac. of Sci., Chiba Univ., Chiba, #Dept. of Anat., Dokkyo Univ. Sehr of Med., Tochigi. Squamation process of regenerating ctenoid scales of the Japanese flounder Paralichthys olivaceus were studied histologically with special reference to ctenii formation. Scales were plucked off from ocular sides of fish and after several intervals, regenerating scales were observed by light microscopy (LM) and scanning electron microscopy (SEM). For SEM, aldehyde-fixed specimens were immersed in a solution of diluted sodium hypochlorite to remove soft tissues which covered ossified scales. On 5th day of regeneration, minute calcium deposition was stained with alizalin red. By SEM, 2 to 6 circuli and small ctenii were already found on the edge of the scale plates. Subsequent ctenii formation was observed between pre-formed ctenii and this formation patern “might make cone-shaped ctenii of ctenoid scales. SYNAPTOLOGY OF SPINAL MOTONEURONS IN BONY FISH : COMPARISON BETWEEN LARGE AND SMALL NEURONS. A. Matsumura. Dept. of Biol., Natl. Def. Med. Coll., Tokorozawa. The author previously presented the ultrastructure of large motoneurons dis- tributed from the lateral side of the central canal to the anterior horn of the spinal cord in goldfish, Carassius auratus. In the present study, the neurons distrib- uted in the same area and the cross- sectional area of the cell body up to 440 pm? were determined as small motoneurons, and the fine structure waS examined by comparing it to large motoneurons using a transmission electron microscope. In the densities of the cell organelles, the S/F ratio ( large cells, 1.49; small cells, We 3) was not largely distinguished between the neurons, but the covering ratio of the cell body by terminal boutons was 46.1% in the large neurons and 19.8% in the small ones and the ratio of the small neuron was less than half of the large ones. Even considering the size of the cell body, there were large quanti- tative differences in the total synaptic inputs between the large and small neurons. This study was co-worked, with Dr. K. Saito. Cell Biology and Morphology 1289 A HISTOLOGICAL STUDY ON THE NERVE INNERVA - TION IN THE ORAL CAVITY OF CLEMMYS JAPONI - CA. H. Ishihara. Biolog. Lab., Fac. of Sci & Engineer., Aoyama {rakuin Univ., Tokyo. The morphology of nerve innervation and nerve endings in the oral cavity of Clemm- YS japonica was investigated according to the silver impregnation method devised by the present author. Oral cavity walls were constituted of tunica muscularis, tela submucosa and epithelium. It was found that the nerve plexus in oral cavity consisted of both autonomic and central nerve fibers showing wavy course. Emerged from fundus plexus, the nerve bundles were observed to enter tunica muscularis and to form plexus myentricus. The nerve bundles originated from plexus myentricus, after repeated ramifications and anastomosis came to form fine net-work, and then constitute a fine terminal reticulum were seen. Further in this net-work, many ganglion cells were detected here and there. Cap- sulated Slomerular corpuscles were found to occur in the smoothe muscle layer. In the tela .submucosa, the nerve fibers branched from the secondary net-work, further again branched out the fibers which terminated in this layer were detected. Fine neurofi- brils running freely in all directions were very often found. In addition, fine nerve fibers were observed to enter the epithe- lium and to end there. A COMPARATIVE STUDY ON THE MITRAL AND GRANULE CELLS OF THE OLFACTORY BULB. N. Iwahori, Dept. of Anat., Fac. of Med., Nagasaki Univ., Nagasaki. The intrinsic organization of the olfactory bulb (OB) was studied in the mouse and arctic lamprey with special reference to the morphology of the mitral (MC) and granule cells (GC). The OB in the mouse showed clear laminar organization. The mouse MC had primary and secondary dendrites: the former traveled superficially to be distributed in the olfactory glomeruli, while the latter extended in an arched manner forming the external plexiform layer. The processes of the GC traveled superficially to terminate mainly in the external plexiform layer. The lamination of the OB in the arctic lamprey was much more obscure than that of the mouse OB, and the external plexiform layer could not be identified. The MC of the arctic lamprey had several primary dendrites but secondary dendrites were not observed. The GC in the arctic lamprey had several long superficial processes which terminated in the olfactory glomeruli forming tufted endings. Thus, the morphology of the MC is closely related to the laminar patterns of the OB, especially to the external plexiform layer, and also to the terminal areas of the GC processes. REARING ENVIRONMENT EFFECTS ON SYNAPSES IN VOMERONASAL SYSTEM M. Ichikawa: Dept. of Anatomy and Embryology, Tokyo Metropolitan Inst. for Neuroscience, Tokyo The effects of differential rearing on synaptic morphology were examined in the granule cell layer of accessory olfactory bulb (AOB) of adult rats. Forty-day old male rats were housed in one of three ways: individually (isolated condition-IC); with 4 males per cage (unisexual condition-UC); or with 2 males and 2 females per cage (social condition-SC). After 2 months, the animals were prepared for electron microscopy. Two type of synapses were Classified; (1) perforated (P) synapses, which are characterized by discontinuities in the postsynaptic thickening, and (2) nonperforated (NP) synapses. The length of synaptic contact zone (SCZ), and area (A) and length of perimeter (LP) of presynaptic and postsynaptic terminals were measured in each synapse. In the P synapses, the length of SCZ were significantly greater in the UC and the SC than in the IC. In the presynaptic terminals, the A was greater in the UC and the SC than in the IC, and the LP was longer in the UC than in the IC while, in the postsynaptic terminals, there was no difference in the A and the LP among three groups. In the NP synapses, there was no statistically significant difference in both the A and the LP of presynaptic and postsynaptic terminals among three conditioned groups while the length of SCZ was larger in the SC than in the IC. These results demonstrated that the exposure to different rearing conditions, in which the pheromonal environment can be substantially different, can induce striking morphological changes in the presynaptic terminal and the SCZ of P synapses in the AOB of adult rats. DIFFERENT EFFECTS OF ESTROGEN ON THE INTRACELLULAR LAMININ IN THE ANTERIOR PITUITARY OF FEMALE AND MALE RATS T. Kikuta and H. Namiki Dept. of Biol., Sch. of Educ., Waseda Univ., Tokyo Through immunocytochemistry we (1988) have demonstrated the quantitative sexual difference of laminin positive LH cells in the rat anterior pituitary at puberty. In the case of the female, the number of laminin positive cells decreased at the age of 40 days, nevertheless, that of male rats increased at the same age. And we (1991) reported the instant decrease of laminin positive cells of prepubertal female (non-OVX) rats provided estrogen. In the present study, we examined changes in number of the positive cells of 30 and 60 day-old male and 25 day- old ovariectomized female rats given steroid hormones (30 days male: estradiol-17f or testosterone; 60 days male and 25days female: estradiol-17$). As for 30 days male, laminin positive cells increased 1 to 5 day after either the treatment of estradiol or testosterone, and arrived at maximal level at day 5, as well as intact 60 days, and then slightly decreased at day 7. But for 60 days male, estradiol did not seem to have any effects. In the ovariectomized female, behavior of the positive cells is similar to the non-OVX, although, the decrease slightly delayed. We have thus concluded that the sexual difference of laminin positive cells, that is, decrease in number of positive cells in female rats and increase in male rats, are due to the difference of the pituitary responsiveness to sex steroids between male and female. 1290 Cell Biology and Morphology G-S CONNECTION (INTIMATE APPOSITION OF THE GLOMUS AND SMOOTH MUSCLE CELLS) IN THE AMPHIBIAN CAROTID LABYRINTH T. Kusakabe. Dept. of Anatomy, Yokohama City Univ. School of Med., Yokohama Intimate apposition of the glomus and smooth muscle cells (g-S connection) was found in almost all glomus cells of the carotid labyrinths in juvenile ullfrogs, Rana catesbeiana, 1 month after metamor- phosis. There were three types of g-s con- nection: between 1) the tips of many slender processes (0.1-0.2um in width) without dense-cored vesicles of glomus cells and smooth muscle cells, 2) between relatively thick processes (1.0-1.5um in width) of glomus cells with dense-cored vesicles and some cell organelles and smooth muscle cells, and 3) between some tongue-like projections of smooth muscle cells and the flat surface of the glomus cell. In some cases, a single glomus cell formed g-s connections with two or three smooth muscle cells. Exocytosis was often observed at the g-s connection. Enclosed by a supporting cell, afferent and efferent synapses with typical membrane thickenings were found on the glomus cells with g-s connections. Reciprocal synapses were also observed. On the basis of these findings, the second and third types of g-s connection are presumed to participate in the vascular regulation of the carotid labyrinth. ASSESSMENT OF THE BILATERAL SYMMETRY OF THE MYELINATED NERVE FIBER NUMBERS IN THE MOUSE RAMUS STAPEDIUS, R.JUGULOHYOIDEUS AND R.STYLOHYOIDEUS. M.Aikawa, E.Furuta and A.Shimozawa Dept. of Anat., Dokkyo Univ. Sch. of Med., Tochigi. Myelinated nerve fiber count was performed on the mouse r.stapedius (RSd), r.jugulohyoideus (RJh) and r.stylohyoideus (RSh) of n.facialis from both sides of the individual to examine bilateral symmetry. The mean fiber number in the right RSd was 75.0+16.9, and in the left RSd it was 77.1 +21.9 (mean+s.d., n=8). The mean fiber number in the right RJh was 14.0+2.6, and in the left RJh it was 13.4+2.4 (n=12). The mean fiber number in the right RSh was 14.9+2.9, and in the left RSh it was 17.0 +3.4 (n=9). The differences in the fiber number between the right and left nerves in the same individual were 9.9+4.2 (range 6-18), 1.9+1.4 (range 0-4), and 4.1+3.1 (range 0-10), respectively, for the RSd, RJh and RSh. The intra-animal relation between the fiber numbers from the right and left nerves was close in the RSd and RJh, but was not close in the RSh. The correlation coefficient was 0.88 for the RSd and 0.58 for the RdJh. These data show that the myelinated nerve fiber numbers in the right and left of the same individual are approximately equal in the RSd and RJh, but are unequal in the RSh. A DISTRIBUTION AND LOCALIZATION OF SEROTONIN IMMUNOREACTIVE CELLS IN THE TRACHEAL EPITHELIUM OF HYNOBIUS TOKYOENSIS. Y. Kikuchi. T.Gomi, A.Kimura and K. Kishi. Dept. of Anat., Sch. of Med., Toho Univ. Tokyo. The trachea of Hynobius tokyoensis were studied by the immunohistochemical method to determine the occurrence, localization and distribution of serotonin. Serotonin immunoreactive cells (SIC) in the trachea were only solitary cells; they were columnar, fusiform and cuboidal in shape between and/or under the ciliated cells, and they were identified in all spheres of the trachea. Iinyea cranial area developed sphincter, a large number of SIC were seen. The number of SIC in this area was about 5 per 100x100um?. But their numbers decreased in the caudal area of the trachea gradually, and non- cilliated cells between capillaries, such as the respiratory portion in the lung, were seen in the dorsal part of the trachea, SIC were not identified, and these structures were located near the lung. SIC corresponded to neuroendocrin cells of the mammalian lung, they may be paraneuron cells or APUD-type cells. These cells, may be related to regulation of hypoxia or other chemical and physical stimulation. The sphincter-developed area near the air entrance seemed to be related to these regulatory systems. THREE DIMENSIONAL POSITION OF THE EPITHELIAL CELLS OF THE QUAIL LUNG A.Kimura?, D.Adriaensen?, T.Gomi+, J-P. Timmermans?, M.H.A.De Groodt-Lasseel?, D.W.Scheuermann2, Y.Kikuchi*? and K.Kishi? 1Dept. of Anat. | Sch. soxeemed: Toho Univ., Tokyo; 2Lab. of celle Bion and Histol., Univ. Centre of Antwerp, Antwerp, Belgium Birds’ respiratory systems manage breathing with an air sac; This differs from the respiratory systems of mammals, reptiles, and amphibians. A computerized three-dimensional image processor and a resin casting method were used to analyzed the quail's airway system. An analysis of the three-dimensional position of the bronchi system was attempted. Location of the epithelial cells of the lung were further examined. The divergence of the secondary bronchi was chiefly exhibited on the dorsal side by the resin casting specimen. Moreover, a lot of anastomosis was exhibited in the secondary and third bronchi. By light and electron microscope, two types alveolar epithelial cells were found: Type II alveolar epithelial cell groups, which included OLBs, existed near the airway; moreover, Type I alveolar epithelial cells, which covered capillaries, were located in the respiratory area. This was able to be confirmed visibly through a three- dimensional reconstructing imaging system. Cell Biology and Morphology SURFACTANT PRODUCING CELLS IN BRONCHIOLES AND TERMINAL BRONCHI OF THE RAT LUNG. T.Gomi?, D.W.Scheuermann?, D.Adriaensen2, J-P.Timmermans2, M.H.A.De Groodt-Lasseel2, A.Kimura!, Y.Kikuchi?, K.Kishi?. 1Dept. of Anat. Sch. of Med. Toho Univ., HokyoreesLab. of cell Biol. and Histol. , Univ. Centre of Antwerp, Antwerp (Belgium) It is well known that the type II alveolar pneumocyte is the main secretor of alveolar surfactant. However, the site of origin of the bronchiolar lining layer is largely uncertain. Using a fixative containing glutaraldehyde, paraformaldehyde and tannic acid, an ultrastructural study of all the cell types present in bronchioles and terminal bronchi of the adult rat lung was performed. In the investigated area, the non-ciliated cells (Clara cells) are dome-shaped and contain large amounts of smooth endoplasmic reticulum and mitochondria. The Clara cells also harbour a variable amount of osmiophilic lamellated bodies (OLBs). The fine-structure of these OLBs appears compatible with bimolecular leaflets of lipoproteins. The OLBs are often aggregated into large complexes and do not seem to be membrane-bound. At the luminal surface of Clara cells, images of OLBs suggesting secretion are obvious. It can, therefore, be postulated that the Clara cell plays a role in the synthesis and secretion of bronchiolar surfactant... Finally, it is noteworthy that a small amount of inclusion bodies, comparable to those found in Clara cells, was also observed in ciliated cells and in the rarely found goblet ceils of rat lung bronchioles. 1291 1292 Behavior Biology and Ecology ECOLOGICAL STUDY OF REPRODUCTION OF Oulastrea cispata IN OKINAWA. Y. Nakano! and K. Yamazato2 1Sesoko Marine Science Center, Univ. of the Ryukyus, Okinawa, and 2Dept. of Biology, Col. of Sci., Univ. of the Ryukyus, Okinawa. Oulastrea crispata, which is distinguished by a black skeleton, lives patchily in depressions in shallow reef flats or in turbid, muddy bays where few hermatypic corals occur. No gonads were observed in polyps before July, 1992, using a microscope, but gonads with mature eggs and sperm were observed from July onwards. Each polyp was hermaphrodite, with ovarial and testicular regions in all twelve mesenterial filaments. Eggs within the gonads were of different sizes. Mature white eggs without zooxanthellae were about 130 pm in diameter. Eggs were spawned from midnight to dawn, sank to the bottom, and changed into planulae without zooxanthellae before noon. The spawning season was longer than in other faviid corals. O. crispata continued to spawn eggs every few days starting in July regardless of lunar phase. Another type of planula was observed that was the same size or smaller than mature eggs of Oulastrea. A few planulae of this type, with zooxanthellae and nematocysts, were released during the rest periods between spawnings. These planulae may stem from mesenterial filaments and represent a form of asexual reproduction. GROWTH RELATED MIGRATION OF THE INTERTIDAL SNAIL, MONODONTA LABIO CONFUSA, WITH A CONSIDERATION TO A RELATION TO THE FOOD AMOUNT A. lijima. Toho Univ., Chiba. In the intertidal rocky shore at Kominato, Pacific coast of Boso Peninsula structure of the shore shows mozaic pattern which consits of stable rock surfaces and pachy cobble fields. Species composition of the benthic organisms between two bottom structures is quite defference. Molluscan grazers are very abundant in both bottom stuructures, but most species prefer either bottom structure except the trochid snail Monodonta labio confusa which very numerous in the middle inter-tidal zone not only of the stable rock surface but also of the cobble field. It was observed by recapture of marked snails that juveniles of the snail inhabited in the cobble fields and migrated to the stable rocks after it reached about 5mm in shell hight. The analysis of chlorophyll amount of epilithic microalgae showed the amount tended to be higher on the stable and macroalgae-free rock surfaces. The food amount may be an important factor for the distribution of the Monodonta snail. AGGREGATION FOR SPAWNING IN THE BREEDING SEASON OF THE SEA STAR, ASTERINA MINOR. H. Tominagat and M. Komatsu2. ‘Kanazu High Sch., Fukui and ?Dept. of Biol., Fac. of Sci., Toyama Univ., Toyama. ——— It is known that in the laboratory con- dition the hermaphroditic sea star, Asterina minor aggregates for spawning (Komatsu et al., 1979). However, no de- tailed observation has been made for the spawning behavior in the natural habitat. In the present study,field observation was carried out using a 1 m2 guadrant at Echizen Coast in Fukui Pref., During the breeding season, aggregate behavior, density and biomass were studied. Asa result, it was revealed that (1) this sea star bred between late May and early June, (2) the size of maturity was more than 100 mg wet weight and 4 mm major_radius. Furthermore, density (11.4/m“), biomass (13114 mg) and the ratio of overlap (14.9 %) are large in the breeding season than in the non-breeding season. Judging from these results,it was sug- gested that mature, large sea stars aggre- gated just before the spawning, overlapped each other for about 2 weeks, and then dis- persed quickly after spawning. Self-fer- tilization was confirmed by an isolated experiment. This breeding behavior may give a high rate of cross-fertilization for a high degree of efficiency in breeding. ORIENTATION AND POSITIONAL ARRANGEMENT OF ZOOIDS IN FOUR DIFFERENT ASCIDIAN COLONIES. Y. Taneda and T. Watanabe. Dep. of Biol., Fac. of Educat., Yokohama Natl. Univ. ,Yoko- hama. From the structural organization, ascid- ian colonies are divided into four differ- ent types. Polycitor proiiferus forms a colony without both common vascular system and common cloaca. Aplidium yamazii posse- sses common cloaca, while does not common vascular system. In eee ee 5 ae 315 Harmonie Mamie Sis occ cane bo aioe wdoeek 275 Hasegawa, Yoshihisa ................... 375, 765 1310 Hashimoto, Ryuhei .......................5. 223 asumin Masatomernene ee ereee eee eee CREE rer 1093 Hayashiiensichi@nssa- pane ete eeere at 199 ISENO, WETS 5c ccoscoccncsncec0c0ocsteK 193 Hedricks Jerny e358. 2a a eee 995 Hihara,, Fuywore.cecaare nce eee eas 397 lSHieIO), WEVA coccogsvodcanodscn0000 633, 1061 Mirata: Junk@n. feccedclehorcee ceric ee eeeece 207 Prose JE WICH. nce cies crn ee oe 405 Bushida.VOmico: j22c5255-ecke eee 1087 Horita: PAKUSMI 2.5 .eccuus oieric eee 413 Horiuchi Shintt- 22-7265 eee eee 211 Muang ZN sos ch vena d che nee oe eae 113 ERUYS: JROMY. osseaicted nocjeomaennaeeeeer 859, 875 Ely odo; SUSUMU js)... 52 o ese eee eer 157 I Ichikawa, Tomoyuki ........................ 305 ligo, Masayuki «acc sates. scenes 1047 ino, WenwhikoO. . ssc ec eee 119, 337 Tkdta; Takaaki rias.okcccnccuatiacnne ty soap aee 947 Ikeda, AinOsht js ssectscn oan seen ie ane 397 Inoue, Chiemit..«.5..c..0¢00.5.0 ee eee 757 Till; WaSWO | cs... eee eee 649 U Wemuraisearuko! ocecs cose ocmbitieiseeet 619 Wenos Satoshi s ees enc ontseeoee tenet cose 125 WerumovsMichacl)Vig ct aeetmet cited: 17 Wkeshima;)Attsumil ......1...).-4s5e eee 733 Wrano pAKihISal yo. csc04 000 oc see eee 157 Vv Van Minnen, Jim. «.-......<23...0..\. sae eee 533 W Wakabayashi, Katsumi ................. 175, 791 Wassersug, Richard J. ...................... 713 Watanabe: Hiroshi ........+.s2seee eee 405 Watanabe, Kazuo .............00.00 «pee 749 Watanabe, Mamoru ....................---s- 133 Watanabes Yuichi'\G: <5.>.¢-2 eee eee 625 Weissman, Irving, L. ....... 52 .eeeeeeee eee 989 Williams; Glynne|.-....-->- 224 eeee eee 315 Y Yada, Takashi: 23). ...0..0....0..000e eee 143 Yamagami, Kenjiro ...............-....-..-- 891 Yamaguchi, Kazuhito ....................... 113 Yamaguchi, Tsuneo ..........: sees eee 53 Yamamoto, Masamichi ..................... 211 Yamanaka, Hidetoshi ....................-. 1009 Yamanouchi, Korehito ....................-- 223 Yamashita, Shigeki .........-..-94eee eee 887 Yano, Jumji 0.060. oe os eon ee 89 Yasumasu, Iku ....5...........- eee 329 Yasuyama, Kouji..............- eee eee 53 Yazji, Mansour E: L. ....9.ida:4seee Pee 1037 Yoshida, Masako ..........0..50. etna 211 Yoshizawa, Hideki .........:.....0¢9seeeeeeee 785 Yur Hon-Tseny osc... 0s 00s 659 Z Zairin, Muhammad Jr. ...................6- 607 Zelezna, Blanka ............«:s;egseees seen 925 Zerani, Massimo . ....0s,....02.05 5.0 090 639 Zhang, Hongwei .................. 113, 551, 665 1313 AUTHOR INDEX Abe, Hiroshi 1149 Abe, Shin-Ichi 1198 Abe, Takeyuki 1138 PND CAYAUIGHI © ook ds cinev nen ect te Sl MRL Adachi, Shinji Adachi, Takehiro Adriaensen, Dirk Agata, Kiyokazu Ai, Naohiro Ai, Takuya Aikawa, Masuo Akasaka, Koji Akazome, Yasuhisa Akimoto, Makio Akiyama, Kazuyo Akiyama, Tadashi Akiyama-Nambu, Fumiko Amakawa, Taisaku Amako, Daisuke Amanai, Kazuhito Amano, Minoru Amemiya, Shonan Amikura, Reiko Ando, Hironori Ando, Hitoshi Ando, Masaaki Aoki, Kazuko Aoki, Kiyoshi Aono, Hideaki Aonuma, Hitoshi Arai, Junichirou Arai, Kiyoshi Amal AO nc.c 4 mmnyrans ninvean seas 8 Arai, Tomoko Arai, Yasumasa Araki, Isato Ariizumi, Takashi Arikawa, Kentaro.............. Arita, Tomikazu Ariyoshi, Nobuyuki Asada, Nobuhiko Asami, Kouichi 1189 1200 1160, 1251 (Abstracts) ANSATION METS AKIN 15) sara oat -aeoraichdearon HSL 1156 INSVNO 5 UO NUON) soancscssneneannoeanacde 1250 RISO AS a OME ZOE ES orercvst ss arncecrayse crsiayakenvishorevonsmottte 1154 , 1199 Asashima, Makoto .............. 1155 1156, 1199 , 1249 Ashida, Kazunori .......................05. 1192 iWon AshidaMasaakils. 5.40.5 ..0505.deeiadeeed: 1134 2S ee Atacos;moshihirojnasscse eee eee 1219 IeAh -Atysoni FelixiGinis 2c ccis sce aescere cache eee 1257 meo “Azuma, IKatsut sss ssc acces sateen ee 1230 ASG) “Azuma; Masami . 22.1... astdastebeba asks 1230 , 1242 1242 1290 : G4 BabaeShojiWAgig.c ssa. scehonce cen tee 1218, 1219 (45; BalbalMadashigiwiee isco beso c cvine eB aeteee des 1197 leon “BawatlakakOs 7Fiscss edison vce no Se ee 1208 1138 Baba, Yoshichika .............. 1181, 1241, 1242 1295)" Bannoreloshikazu.4.-5..-254 05a 1136 4 OR Bassotewien=Mianereeeeeee eee eee 1228 WAG — iwerm, ISlOwweMGl AN, sesceecenacascnoucseccaean 1273 See Besshommomokoi-ns-c seen een eee 1155 , 1141 Bireivheide, Ruediger...................... 1251 W203; ~ Breyaule cer h cc octets s apocn Ae ANE 1134 , 1299 1188 1200 g 1283 Cervello, Milchiorre ....................... 1178 1250. “Chen SBin. & Reis cao. cc Ae ose 1243 1261 ChibaWAkirawk coccwwd oe ceecwaedetee 1257, 1274 12265 1 Ghibay Chikafumiles eee eee eee eee ene eee 1235 1249) * (Chibahomeeiek. cb occucaice os tteeee 1171, 1277 1224 Chiba, Kazuyoshi .......................... 1197 1228 Chiba, Yoshihiko .................... 1295, 1238 OT SS Chinospyukihinopeecereee reer eee ceeEe cee: 1160 pilel4G, ~—"- @hinzeis Nasu), -eAssaacseeet hs. sabe ee 1152 Id Coopers hs porcccccan tices ATBES REY 1249 mlan4 CopenhagensDavidiRe aia. --n- ce eee - 1235 1192 Crescitelli, Frederick ....................... 1230 1iSS (Currie MDouglasvAc .. cei es. coc I. EE 1271 , 1284 1301 1199 D 1133 Dan-Shokawa, Marina ..................... 1300 22S Daintdes Masashiieemeeeeeaeen eee dee 1293 1314 De Groodt-Lasseel, Marie H. A...... 1290, 1163, 1174, Deguchi, Ryusaku de Jesus, Evelyn Grace T................... Diaz, Maria Rosario M. .................... Dohra. Hideo! 2.3 .5)6ent elec’ a eee Ebina, Yukiko Ebitani, Noriaki EB ouchinbisukemeereeeeee eee nee Eguchi, Goro Eguchi, Sachikol ...22: 2 ess.c.0 use ae ee El Manira, Abdeljabbar .................... Emori, Yasufumi Endo, Hiroyoshi Endo, Katsuhiko ..................... Endo, Makoto Endo, Yasuhisa Enoki, Yasunori Eshel Dania) cvccc eke: cheer eee Ezaki, Masanobu Fujit; RYOZ00 2204.2 acee nck eee eee JOH UIA, INCU coscoccccvssoavc0an00¢ Fujimaki, Hidekazu .....................05. Fujimoto, Hirokazu ...................0005- Fujimoto, Hirotaka FujimotosKengoee-eee eee ee eer Fujimoto, Masaaki ................... Fujino, Hideki Fujisawa, Chiemi Fujisawa, Hiromi Fujisawa, Hirosuke Fujisawa, Toshitaka ....................0005 Fujisawa, Yuko Fujisawa-Sehara, Atsuko ..................- Fujishima, Masahiro Fujita, Keisuke Puyita; YOSHIAKI. 5 aciencnocn Mee aes adele Fujiwara, Akiko Fujiwara, Keigi Fujiwara, Shigeki Fujiwara, Yoshihiro .................. Fujiyama, Shizuo Fukada, Toshiyuki 1229, 1184, 1140, 1195, 1173, 1291 1207 1264 1155 1280 1286 1147 1251 1219 1229 1247 1133 1239 1187 1135 1164 1150 1196 1141 1302 Fukada, Yoshitaka ............. 1182, 1230, 1232 Fukamachi, Hiroshi ........................ 1285 Fukatsu, Takema’ «0.6 .c0dcs.¢0. ee 1211 Fukazawa, YUGO) «..020.5-45ace6 soe eee 1273 Fukuda, Kiyok0) 7 52..4.242 9-2 eee 1300 FukudaVut...0. 20.0040 60656 ee eee 1202 Fukuhara! Chiaki .........-...0+-- ee eeeeeee 1226 Fukui sAkimasa: ..............+0..¢s2 eee 1155, 1156 FukuisKazuji. 20... ..000 6250. See 1255 Fukumitsu, Takashi ........................ 1133 Fukumoto, Tetsuo ......................... 1249 Fukuta, Keiko) i: .....0..00. 00. --e eee 1297 Fukuzawa, Toshihiko ...................... 1207 Furudate, Hiroyuki ........................ 1235 Furukawa, Kentichi’ .......... 0-2 eee 1148 Furukohri, Takahiro ......................- 1301 Furusawa, Mitsuru .......................-. 1187 EurutaEmiko) ).5;.....+.-50: see 1248, 1290 Furuya, Hidetaka ..........-.:eeeeen eee 1281 Furuya, Sigehisa ..................... 1165, 1166 G Gibbons, Ian Ri 24... 600..00 eee 1219 Gilbert, Mawrence I. 3.....-.5.. cease 1269 Gleadalli Tan'G: >... ...023.. 3200.06 ee 1228 Gomi! Toshiakiv, 53.4... 2.0 1290 Goto, Mutsuo 2 .......000000. oe ee 1303 Goto, Taichiro) «.sc.0.5000.0 2+ «sn eee 1294 Goto, Yoshihide: ......005...0..:0.5 eee 1267 Gotow, Tsukasa) ....<..s:000 20000 ee 1234 Grillner; Stem)... oc. 30.00. 208 oo ee 1226 Grygier, Mark Ji. ......00...00000 0000 eee 1305 H Haga, KaZu0 onc cc eecuinje cee oe eee 1284 Haga, Nobuyuki. .....:..0.0.. 00+ see 1212 Hagiwara, Yasuko. 2... 600.0 scene ee eee 1200 Haino-Fukushima, Kazu ............. 1172, 1274 Hama, Natsuko) 2..5.)(50. .s\.0<0.00 0 eaten 1274 Hamaguchi, Miyako S. ............... 1177, 1178 Hamaguchi, Satoshi ......................5. 1196 Hamaguchi, Yukihisa .......... 1177, 1178, 1181 1219 Hanaoka, Yoichi ............... 1139, 1262, 1263 Flara AKIDIKO) \...c.cciine cise. eee 1170, 1244 Fara , ITO aI. jee s0ye.0,00ciessieiww shee ee 1294 FATA MENT Wen wink snes ene sean Bekele 1195 Fara. Masayuki ..:....0scc-eeeeees ee eueeee 1138 Marada\, Hosamil :.<..2..:..6ie.. Sa 1285 lama aU SSClt ion el siete snvec asc ics ome ete oe 1146 laradatMetsuO: «oc. caccswsce ands eeetucke 1252 Haraguchi, Hisashi ......................... 1260 Iancayay Voshio® .....¢022)26 1.2 eh tas. daee 1274 Harigaya, Wakako ...................0.008- 1138 Hariyama, Takahiko ................. 1233, 1238 Haruguchi, Yoshiko ......................5. 1165 laruimibeatSuO) . 22.22. .sccascescen ne? 1204, 1205 Iasegawas Kazu. ..ccevec ec eeeeece ee es 1195 Hasegawa, Sanae ................... eee eee 1257 Hasegawa, Yuriko ................... 1135, 1272 Hasegawa, Zutaro .....................008. 1155 Hashimoto, Koichiro .....................5. 1172 Hashimoto, Naohiro ....................... 1169 lashimotos YOKO :<..issc0< codec. sees ce lade 1236 latanakaewlsune@) s..0...s50+s+o seen 1245 Hatsumi, Machiko ......................... 1188 Rlattamshinicht. 2c. .sccccccccccds sss: Maes 1228 RMATCONIPINCISUKE? ocgc5 ccc oo hae cote cosa ee 1216 Hayakawa, Tsuyoshi ....................... 1175 Hayakawa, YOICHI. ....:0..s0s.00s. 5. sites. 1270 Hayakawa, Yoshinori ...................... 1294 Hayashi, Hiroaki......... 1139, 1261, 1262, 1263 Hayashi, Hiroshi .......................005. 1165 Ifayashindinoshi) 22. 4.6.262-c.2-22s sees ees e- 1229 Hayashi, Kensuke .....................-00- 1200 Hayashi; Mikal io. .20se%0ct ere JES 1239 Flayashi,;Shinjil ©... .sceeeeees cect ees wate ee es 1265 Hayashi, Susumu ..................2-e-0 ees 1294 emine Peter dn te). c2c2ccorn-kee teense ote 1230 irdakay Soh (Aes oh eek ssn dc AGRE oR 1236 irdaka OOD: s.5 sa0 088s has cee PRRs 1239 CAA VONKIW ES a2 s22500c0c07< cde RGR 1202 SUCH EIdEO . ascecaceres vere nc Seale . 1147 Higuchi, Takashiro .......................05- 1241 |ShiqoriNe), Iti $0) oooadeneoauevseopodobogceauE 1200 Elikosakas Akira! 2h) Qocccc cc ciose ccc se das ess 1193 PLTVRUISSE IME Ms aed otis von ee ee 1226 HimmuanasMasaon ss. s2.c5 0c. . soho tenn scene 1277 Hino, Akiva)... 22 hence cee cacw nee llay! 1181, 1303 TINO GAY OMe nadine. d ode c vig Maas oe 1147 Hirabayashi, Tamio ............ 1141, 1160, 1168 1169, 1173 lebimil, IENGMERS, Gosoooscdnodouspanooneeseoe 1181 irai® Moshiaka) s-secccec sek p cole cede fhe 1162 Hirakawa, Noriko ....................0 eee ee 1280 Hiramotoy WUKIO Geek eos ancsesces Se ha 1177 inanoW Hiroshi. ss 3-42.20. ..ceeien eee 1143 Ikegami, Susumu .................... 1167, 1179 ikenishityWohijiteeese ee eee eee eee 1173, 1187 Ikezaway Hiromi 425-2 ee ee eee eee 1304 Ikwita, Kyosuke® ..c0% coe nassau Seen eee 1283 ImatukuyMichioy--- ese e eee 1305 ameArSeaNyiel, IMUINO) apecccaccaaaccgca000ncnocoac 1219 Imai. FinOOmiciees doc ute canon oA One 1230 Imai: KiyohitOs.j.cced.essnaseceste ee eeeee 1252 maby as KWaZu@) m.ccacicenis nc ceneee ee 1145, 1206 Inagaki, Masaki ................ 1143, 1145, 1146 Inamori, Megumi ....................+---5- 1300 Imamura, Hiroko ........................00- 1282 Inamuras©OSamlieeeee eee eee eee eee 1228 Inase SWOSMINON. ae. a5 ctisoe oo no RRO 1254 Inohayar Neils fete een eee 1157 Inokuchi, Tomofumi ....................... 1139 Wao, SRYGENKO) 55 000c0nscagns0n¢onacnn 1173, 1174 INOUC>, YASUO! 2665000 se ete eee 1173, 1174 Inouye Shinitidy .3.2 50.02. sheer ee 1226 TS@ka A CHINOW Rye a.c, os ts ssecnsicnee SOREN 1203 Ishibashi; Nakaakiy).... 0+ +.c0- eben 1242 Ishibashi, Yasuhisa......................05- 1277 TshidanAidekiies «..c:0:0¢-s:csevorw oi re 1227 Ishiday Katsumi) .eeeeee eee 1154 Tuchi; Ichiro, 8 ....50...420...ese eee 1157, 1171 Iuchi, Yoshihito ...................... 1163, 1164 Iwahori, Nobuharu .......................-- 1289 Iwai; Yukiko:.2 .. e.0 s0c.++.0 + oe ee 1171 Iwama, Akifumi ..................... 1240, 1244 Iwamatsu, Takashi ....... 1160, 1170, 1173, 1209 Iwamuro, Shawichi......................-.- 1262 Iwao; Megumi... 2.2.62. 2.0.+ oe eee 1156 Iwao; Yasuhiro ........... 4700 eeee 1161, 1206 Iwasaki, Masayuki ..................-...-5- 1233 Iwasaki, Naohiko ...........:..+sce seen 1230 Iwase, Tomoko. ........ 6.0.0.0. 000 dee 1159 Iwashita;, Shintaro).....0..... se sseee eee 1169 Izumi, Susumu.............++++++0. eee 1141 J Ji Zai-Stwsiwee occ sncrcanaasis eee 1198 Jikumaru, Shouta ...................0000eee 1206 JinguhyipYouichiss..... 2.55... 0-5 1150 Jongwatiwes, Somchai ...................5 1211 Juchault, Pienre! .... o..00... 05. Ge ee 1135 K Kabasawa, Hiroshi .................000.00s 1225 Ieado tas MEtSUO) .iojectcce science ta ol 1225 Reapawval IUTOMIKO oi... ee ee ote 1257 Kageyama, Tetsuo ...................e eee 1175 Gain aAZUMIKO) oe os cect ote weve dee de ae 1278 Reap imassITOKO: 0.60. jee ee hee ovate 1162 Kakeyama, Masaki......................... 1255 IRENE NIG AS) 00) eS Oe 1257 Rev PAU eS is corre eee 1250 Kamata, Yasuyuki ............. 1165, 1166, 1167 CAMS WARN ORWMA «pce ese ace HA Rae AE 1273 Kameyama, Masaki ..................-.---- 1234 Kamidochi, Mika .....................0005. 1215 Kamimura, Ryuichi ........................ 1210 Kamimura, Shinji ..................ee eee eee 1144 Kamishima, Yoshihisa ..................... 1227 AMM aA SHINO Na sisi ca ee eee See cl 1279 iene. (Qe) <0) tag aoe eee Banh 1190 Ream atSUakans .... oe: se wad ce ee ol IER 1288 Kanbarasirojil sco... de taen ddl, eeu. 1211 Kanda, Masahiko .....................00005 1160 Kean al Miyuki. 2. cece cae eee ADA MEMABles 1166 KReamd a mbOshiOne:. ..0cccee een wn otidhicles «Mae 1270 Ream KOM MASA OY ce ecie a cieressnscvoirrrcseue fein ees Shae 1176 Kaneko, Nobuaki ........................0. 1206 Kaneko, Tomoko .................... 1208, 1214 Kaneko SOV Opii isch... cie cece ea ee cee bale wd 1257 IAMS KOMIK ONG ccicjeeicice ae ereve es ehhleldelele ott 1182 Kang, Wonkyung ....................00005- 1141 Kani-e, Toshihiro .......................00. 1160 RAMON VCASWIATKO! 5d. oye ccis iors oie ole bleclatetolnn «Sle Bie 1202 Kanzaki, Ryohei ..................... 1225, 1240 Kanzawa, Nobuyuki ....................... 1147 Karakisawa, Hideyuki ..................... 1232 Reary aR MISK OY Sei e ciesee ele en Mali Maile 5 wattle 1137 Katagiri, Chiaki ...................... 1180, 1199 IKgrizweatal, (Clive) Gagoseedsso0000G0050405000c 1142 Katakura, Yasutoshi ................. 1135, 1272 Katanosaka, Kimiaki....................... 1231 Katayama, Heizaburo ...................... 1228 Katayama, Noboru ......................05. 1247 Katayama, Tomoe ....................-005: 1302 Kato MKOehin sensei. sno bie EL 1179, 1181 Keaton MakashiQ' 252 0.0cccccicsles oe BORER 1267 ATOR KY OPPS eet osie neresiesaae ae daa Gch 1263 Kat OMIA SKE give recs. roreibne dhe teehee 1172 Kat OW MIKA OMG aso: esis ereceie ordiaie w leas wo ohd Boek 1221 IKETION, IMBVHIKO) -5555050000000000000005 1212, 1151 KatOMsSEtSWKO GS icc: chscivsnciecespuocso ey S Ole dee 1135 Keats usPYOSNIMN AO sits. acs: ccenovencsoccd hap eh SANS 1162 Keats um ViOichi sei ecccces ncameataeeoden 1134 Katsura, Yoshimoto .....................00. 1247 Katsuyamial, YOuI miner aces. acsscdeeriered- 1195 AWA SOKO NSS S oars. ela iiaceneics TR a Ae 1208 KWawalharas vA Kia). soca erses caddis dete Melde 1203 Kawahara, Masahiko ...................... 1228 Kawahara, Shigenori ....................... 1240 Keawiai Miia ISee ooo salocmats ona one do 1299 Kawakatsu, Masaharu ..................... 1298 Kawamata, Kunihiko ...................... 1234 Kawamoto, Manabu ....................... 1166 Kawamura, Kazuo ................... 1195, 1196 Kawamura, Ken-ya ............-.......0-5- 1181 Kawamura, Kosuke ........................ 1263 Kawamura, Yuuki ................... 1146, 1147 Kawasaki, Ryoji .............0.: eee eee eees 1238 IRGWWERAl GL, Were ooccnccncncnnaceoonsucsee 1261 Kawasaki, Yukishige ....................... 1184 Kawase, Eihachiro ......................--. 1172 Kawashima, Selichiro ................ 1254, 1258 Kawashita, Hiroto ......................... 1143 Kawauchi, Hiroshi ......................... 1257 IXBNEL, IMERETS Ch cis Gee aeee Geers oxtbise Bet olor 1268 KeinosHiroomiliaseew... . os. 00 ee A 1136 Kettoku™ Masakomeee =] eee eee Eee eneee 1166 Kikuchi, Shin-ichi .................... 1249, 1288 Kikuchi, Yasuhiro ................... 1290, 1291 Kolkutasoshitenigeeeeeeeehceene eee eeeeee 1289 Kikuyama, Munehiro ...................... 1177 Kikuyama, Sakae .............. 1139, 1255, 1261 1262, 1263 Kimura, Akihiko ..................... 1290, 1291 Kimura, Kazuyoshi ........................ 1211 Kimura, Ken-ichi .............. 1189, 1234, 1271 KoimuraZKOUKL siicccos cee cine ke ee oe 1280 Kimura, Masashi ........................... 1212 Kimura, Sumiko ..................... 1146, 1147 Kamunal Pakeyit ce ec ncrnaceome eee oe. 1293 Kimura, Tetsuya ..................... 1241, 1244 KinohMHinoakat so. ode scen sles 1205 KimoshitawEijit ccc ce aceace secs occa te alseh 1288 Konoshitatikeiyya.. .as.4.-.5.-42 eee 1156, 1159 Kinoshita, Masato ......................000- 1209 Kinoshita, Tsutomu .................. 1156, 1159 Kirinoki, Masashi .......................... 1168 KaiShie KiyOshie. i cesieasces temeees ace 1290, 1291 1318 KishidasAkihiroiees ee eeeeee eee eee eee 1255 Kishimoto, Koichi ......................2.. 1161 Kishimoto, Takeo .............. 1142, 1161, 1176 Kishimoto, Yasuo ...................00000e 1183 KoitaryNobuyukiteneeesere nee eee eee eee 1179 Kitajima Ken 2c cnc cmcnnc ones ceeeneee 1173, 1174 Kitamunatsumieceeeeeeee eee eee 1189 Kitayama, Tetsuya .......................5. 1246 ISO, ON eo eee enh anadoobodode ar 1298 ICOM GU esoa ee cack nvan eee ee 1228, 1230, 1252 Kiyomoto, Masato ................... 1158, 1159 KobanmhumikoOpere sss eee eee eee ete eee 1247 Kobayakawa, Yoshitaka ................... 1190 Kobayashi, Hiroaki ........................ 1182 INobayashis) Kant=-----eee ae eee eee 1203 Kobayashi, Ken-ichiro ..................... 1139 Kobayashi, Makoto .................. 1222, 1223 Kobayashi, Michisuke ...................... 1252 Kobayashi, Michiyori ...................... 1252 Kobayashi, Minoru ........................ 1152 Kobayashi, Satoru ................-...-.-5- 1188 Kobayashi, Seitaro ................... 1185, 1186 Kobayashi, Takaaki .................. 1137, 1145 Kobayashi, Takanori ................. 1300, 1303 Kobayashi, Takuro .....................0-0- 1197 Kobayashi, Tohru .....................0000- 1256 Kobayashi, Wataru ....................-00- 1181 Kobayashi, Yasuo.............. 1259, 1260, 1285 IX@ RWI, YOUU 54550009000 s900ac0c0n0g0000 1216 KodamayRyujlj eee eeeeeeeeee ee ener eee 1189 Koganezawa, Masayuki .................... 1246 Koo, SHirOshil.).. ...nacnenin eens Coos 1254 Kohama, Kazuhiro ......................--. 1147 Kohda, Yasutoshi ....................0..05- 1288 Kohno, Hiroyuki ......................0 eee 1138 KOtke Satoshi. iis wei wowona wae Mee 1268 Iowan, SOS WO s55009000s0950000000005000- 1134 Koizumi OSamulyee sete eee 1190 IXOpunne. IDEN 3555500000000 90000050000006 1230 IX@WRTEL, IMMNENS! G55005500200000G04Gu050000e 1269 Kojima, Manabu K......................065 1181 Kokubu, Tomokuni ........................ 1143 KeOmatsu VARIA 5.5.65 oscr cause nce wie arbiters 1233 Komatsu, Miéko ..................... 1292, 1299 Komatsu, Satoshi ....................00eeee 1143 Kominami, Tetsuya .....................00. 1178 Kondo TOshih Se sececmie-e oeeeeeete aaee 1286 Keon OM IUIN oasis vis dro.sisie Veewreciowae an Mes 1253 Kondo, Noriaki. ............5... 0222 eee 1253 Kondo, Yasuhiko: ..............-- ..eeeeeeeee 1255 Kondoh, Yasuhiro .......-.... 2p eeeeeeeEe 1243 Konishi; Kooichi:......... 012) 2252 eee 1305 Kosaka Toshikazu ............. 1276, 1279, 1280 Koshidas, Yutaka .........--..500eeeee 1281, 1182 Kotani, Susumu). .....4.5)..... eee 1150 Kouki, Tom: sic 52.00.02 000000 00 e 1261 Kouyama, Hiroshi ......................... 1138 Koyama} Eiki Wis:40c......:.5 eee 1201 Koyama, Hiromichi ........................ 1191 Kubo, Toshiyuki. ..........:.-:+e Reese 1210 Kubota; Hiroshi ............)..sseeeeeeeeae 1175 Kubota; Ichiro. <.. <2 ...¢5....0. eee 1223, 1251 Kubota; Shin ..........-.0.... «eee 1190 Kubota, Yasuo .................. S¢:esepee ee 1208 Kubota, Yukahiko .....-.-- sense 1208 Kudo;, Hideaki... =. ...........0..:00 1008 e ee eee 1244 Kudo, Shigeharu ........................... 1170 Kumakura, Masahiko ...................... 1203 Kunioka; Hiroshi’ .............- eee 1239 Kuno; Shinnichi .........5..-s. sees 1168 Kurabuchi, Shingo ................... 1203, 1261 Kuraishi, Ritsu .........:...0..% cee 1158 Kuramoto, Taketeru ................. 1216, 1217 Kurasawa, Yasuhiro .....................-. 1151 Kurita, Takeshi, .......0%.00.0.9t2. see 1286 Kuroda, Hideyo ..................... 1175, 1206 Kuroda, Masaaki .................... 1149, 1150 Kuroiwa, Atsushi .................... 1200, 1201 Kurokawa, Makoto ........................ 1223 Kurokura, Satoshi ............. seen 1210 Kurosawa, Yoshikazu ....................-- 1213 Kurosumi, Kazumasa ...................... 1261 Kusakabe, Takehiro ................. 1140, 1193 Kusakabe; Tatsumi ...:......0. scenes 1290 Kusano, Kazuhiko .............05en 1239 Kusano, Toshihisa ......................00. 1211 Kusada, Hidenori .......................... 1206 Kusumoto, Naoko ................-.20e005- 1270 Kusunoki, Sinichirou ....................... 1228 Kuwagaki, Demmi .......................4. 1262 Kuwahara, Tsutomu ....................055 1185 Kuwasawa, Kiyoaki ............ 1217, 1221, 1222 1223, 1241 Kwon, Hyuk-Chu .......5.....s0. note 1259 KwanyO-Yuw ec ccan cesses yctee ce 1152 Kyozuka Kel-ichiro ..................0 eee ee 1174 L WECM ONG=SU yoo. ce eee ese e eee eenmeuebens 1157 M IMabUGMIISSE1: bs. s1e5c6 cc lesa eee 1145, 1207 Machida, Kazuhiko ........................ 1137 Ifa Ini aya 0) oicscccssacscasensuorovevstererave. spol diate ove 1254 Maehara, Michiyo ....................0000- 1236 Maekawa, Kotano: .....0..6ccewe cd ee bee sce 1198 Makabe, Kazuhiro W. ......... 1140, 1192, 1193 IIT ANKIKO) ess svisenscoreniareens nt Sede ct 1190 Makino, Naoya ..............-. 1282, 1284, 1196 Makino; OsShimanl ....00..2000.00s000. onsen 1240 Makioka, TOSHIKL 0.05: cece ee ble pelele ore 1283 Mamiya, Yoshitaka ..................0.005. 1270 IMfamabe BAC aS. byercie:.-sverecoreirere Sila ewe aay lee 1147 Mandelkow, Eckhard ...................... 1144 Marikawa, Yusuke .....................0005 1192 NMilatiny Gilli yee os. ececeysyeatersrersre ADEE AS 1135 Maruo, Fumiaki ..................... 1171, 1182 Mar O Ka MINN ON Ais. cseseccsrece scare esas clvioare ales oxsbe 1159 Maruyama KCI 2 )s6.c.)--eicresee-coe Hedbek s weellal 1138 Maruyama, Koscak ............ 1146, 1147, 1148 Maruyama, Tatsuya ........................ 1287 MiasudarvAtsUkOs cs.) csochearccne Shee. Manas 1267 IMe@RUGE), Jeli Satoshi joc as «0 soeloupeererr as» cess Tamate, Hidetoshi B. Tamotsu, Satoshi Tamura, Koji Mamuras SachiKOy. cae oss 5-1.5 sehen senk «cots Tamura, Toshihiro Tamura, Toshiki Tanabe, Kazuyuki Tanabe, Ryota Tanaka, Akira Tanaka, Haruo Tanaka, Hideaki Tanaka, Hideki Tanaka, Kosuke Manaka wKUMIOe:. cs sce. 4 eee whanakaw Masaaki. cc: seeps ope: Tanaka, Midori Tanaka, Minoru Tanaka, Mitsuo WWanakarRy Ol ns caccs.: dadedaaad este Tanaka, Shigeyasu Tanaka, Shingo Manakatl, Shojity. 206. cee se soos cates Tanaka, Takashi Tanaka, Tetsuya Mian dai Ofleee Peis coec eis a EEE cee Taneda, Yasuho Tani, Tomomi Tanigawa, Hisashi Manimura,Teiichi ...........idacdadadd.-assee MashirowhideOrenw eee eee eee eee Tatematsu, Ritsuko Tatunami, Shinobu ......................... MawarawAkihikoin gg. .006.50 teeta oda Tazaki, Kenro Tazawa, Eigoro Tegner, Jesper Terakita, Akihisa Teramura, Wataru Terano, Yasuhiro Terasaki, Asako Teshirogi, Wataru .................... Timmermans, Jean-Pierre ............ Titani, Koiti Tochinai, Shin 1217, 1138, 1139, 1255, 1262, 1184, 1290, 1222 1249 1239 1222 1133 1192 1261 1263 1227 1226 1148 1185 1291 1135 1326 Toh YoOshihitoOpest ee eee eee eee 1236, 1237 Tokudas'Gakw o.ncs2c20 eee ee 1281 Tokumoto, Toshinobu ..................... 1162 Tokunaga, Fumio .............. 1182, 1231, 1246 Tokusumi, Yumiko ........................ 1278 Tominaga, Hideyuki ....................... 1292 Tominaga, Takashi......................... 1218 Tominaga, Yoshiya ...................-.-.. 1236 WOMINO -SHITOY hotest ee 1141 Momiokas Kenyie---- eee eee 1238, 1295 Tomioka YuKie> «..:..6c..cle ee 1279 Tomitas Hideo... eee 1209, 1214 Tomonaga, Susumu ........................ 1249 Tonomura, Yasuko .....................0- 1211 Monosakia Neichit-.- eee eee eee 1245 Moratanis| Satoshi \s..cce eects eee oe 1176 Toriyama, Masaru ..................-2-00-+ 1176 Toriyama, Mayumi ....................-.--- 1141 Tosujis Hiroakiey.aceeee. eee ee 1145 TOIT, SWOT 5 5000000000d00c0000G00000C 1146 Toyoda, Fumiyo ................--.--0--+-s 1255 Toyohara, Haruhiko ....................-.. 1209 Toyohara, Jun isc cecrcen 1229 HoyoizumibRy uel eee eee eee eee eee 1184 Moyoshimayp Oko) a. ee eee eee 1143 Tozakil Shizuka ic neem ee 1247 Truman, Jamesews s.005..62ne7.c eeeeoeee 1271 ‘suchiya,. Takeshiltyycicctirn rare eee eee 1302 Msuchiyas TEiZO Wee cesses ene ee 1215 Psujie Lakashive..pr.cesnn ent eeee aoe 1184 Tsujimura, Hidenobu ...................... 1271 Tsukahara; JUuNZo..4:ennncc eee ee 1159 Tsukahara, Masatoshi ....................-- 1151 Tsukahara, Yasuo .................... 1228, 1233 Msukuda) Aaroko ...cccccic ee ee 1217 TSW) I, NREVAUIWKO) coococccoccnbd0soo0000000 1281 Tsuruhara, Takashi ........................ 1277 Tsuruta, Tatsuhiko ......................... 1167 MSUSUE="NIOCOO «hora. arerensts ls toterarasoromele eo 1135 Tsutsui, Kazuyoshi .................0ee ee eee 1266 aisutsumisladaakil cnn eee 1284 TUTON NAVIET Phe Siete eennce win eee ree 1303 U Wehiday Katsuhisa s...cehiee exter se eens 1303 Uchiyama, Hideho ................... 1155, 1156 Uchiyama, Hiroyuki ....................... 1206 Uchiyama, Hiroyuki ....................... 1234 Uchiyama, Minoru .......................-. 1263 WedarBrnA Gen. weariness ee 1176 Weda Hiroshi... 285)... ee 1170, 1244 Weda, Masatsugu <5... eee 1251 Veda, Ry ..102.6:00c einen nee eee 1233 Weda; Shui )..:..:0.:.0:..:..0. eee 1239 Wehara, Hiroko 2.00.00. \o0nn neh eee 1244 Ueharas Seiji: 22. wen). ene ee 1252 Ueki, Tatsuya 0:00.) 1193 Wemura; Haruko .........7.05 eee 1260, 1261 Weno, Naoto: oilec icc ee 1156 Weshimas Rel. eee 1283, 1300, 1303 Ukawa, Ken-ichi «...... ...:.-..:.:...0%1 eee 1273 Umebachi, Yoshishige ..................... 1134 Umeda; Takashi: «............. eee 1220 Umino, Osamu -..........0./..:.0 0 eee 1236 Urano; Akihisal ..c:.c:.:.:000:..0 ee 1267 Wrase, KOKO ~. ....:01s ser se 1194 Wsami, Mihoko .....\. <0. «eee 1138 Ushiyama, Akira ...................0.00000- 1197 Usui; AKIKO 2h. cicccnrscron ote eae 1243 Usui, Kazuya.) ooo. ect cintee ee 1189 Usui, Noriko d..0.:e.:e.n%00 ee 1180 UWto, Noribiko «.-.-.0.:0.0.% 000k ee ee 1176 Utsugi, Kazuo 2... cops 1297 Wyama, Taro ..s:0.:.).1.)-n ences eee 1250, 1251 W Wada, Hiroshi ................. 1301, 1302, 1303 Wada, Masaru® . ..:0 coco nrete Oe 1255 Wada Shigeo’... chee eee 1144 Wakabayashi, Katsumi ......... 1183, 1254, 1261 Wakabayashi, Shuichi ................ 1265, 1266 Wakahama, Ken-Ichi ...................... 1188 Wakahara, Masami .................. 1154, 1264 Wakamatsu, Yuko ....05...5<. dene 1209 Washio, Hiroshi. .....:........0. ose 1242 Washitani-Nemoto, Setsuko ................ 1177 Watabe, Shoji ....ccc.s omen eee 1138 Watanabe, Akihiko! .......:...%ees eens 1201 Watanabe), Kazuo «..)..:...05 otaveentnoenenene 1200 Watanabe; Ken’ 2.0... 0.0005... e eee 1277 Watanabe; Kenji ............... Se eaeweee 1178 Watanabe, Kenji <......... 00.5 seen 1186 Watanabe, Satoshi ..................0.0000s 1148 Watanabe, Shinichi .....................0.5 1237 Watanabe, Tomohiro Widtanabe. YOKO) sccccscecu00t cons vecieehes Watanabe, Yoshio Watanabe. YUICHI! 2... .0.00ceese sees cece ces Wataya, Yusuke Weibrecht, Josette M. Wuchiyama, Junko......................05. Yabuuchi, Masafumi ....................... WiaSieMYOSHIMASA) ..... 260 5eee seine as cena Yago, Nagasumi Yahata, Kensuke Yamaai, Tomoichiro Yamada, Akira Yamada, Atsushi Yamada, Kazumi iat aAC AMOI ee cin vais oie cuneieninvacinve ec ee aces Yamada, Osamu Yamagami, Kenjiro Yamagishi, Hiroshi Yamaguchi, Kelichiro ...................... Nama cuchibakeO syne acese. sees cee: Yamaguchi, Tsuneo .................. Yamahama, Yumi Yamakawa, Yoshio Yamamoto, Daisuke Yamamoto, Hiroaki........................ Yamamoto, Kazutoshi Yamamoto, Kiyotaka Yamamoto, Mari Yamamoto, Masamichi Yamamoto, Takashi Yamamoto, Toshiharu Yamamoto, Yoshimi ....................... Yamanouchi, Korehito ..................... Yamaoka, Ikuo Yamasaki, Kenji Yamashiki, Naoko Yamashiro, Hideyuki Yamashita, Kaoru Yamashita, Masakane.......... Yamashita, Satoru Yamashita, Shigeki Yamasu, Kyo Yamasu, Terufumi Yamauchi, Kohei 1260 1202 1229 1215 1222 1287 1176 1164 1139 1255 1246 1237 1281 1327 1244, 1256, 1257 Miamaura, ELISASII sac -sys)suesssecsrevoe SN oe 1277 Namazakis Hinoko)aveeeeeneeeeeeae sneer 1134 Meamazakin Wen as cticechee ates 1143 Yamazaki, Tadashi......................04. 1196 Yamazato, Kiyoshi ......................... 1292 Yanagimachi, Ryuzo ......................- 1180 Yanagisawa, Tadashi ....................... 1263 anal NO DUA Mercier rece eet te ee 1287 RYAN OMINeM=1Clih ee kas socusntesiens RAT hee 1141 VWAO, MAGI copacosoncooogsonnneunsoaes 1240 BVA OR SYA OM rience icrsvessroxcistseceoare od PRE ct 1169 BY¥ashimmas PY O1 Chit ees, icesicusrevss vorccecrson eee 1276 RYAN se VAUIT es eersjctcy (elec natininlee mente altos 1233 Nasu cietsukoy tere acsier ear acne 1194 DASUCIRS Ada Ota ce aecisae acl asec eter. 1194 Yasumasu, Ikuo ......... 1137, 1143, 1165, 1166 1167, 1168 Wasumasus shigeki senses ase eer 1157 VASUO, IaNOKOSM 5occuccco0ocsc00s00c0undoe 1193 Yasutomi, Masumi ................... 1188, 1285 Yau KGNPoWiat on ach cecsamesincenee seen: see 1234 Yazaki, Kazumori.............. 1143, 1145, 1146 Wazawar SMM eee ei oc ciccgncyewme vale ee 1139, 1170 Wazawab Mohnu® ccs...