Histological Observation of Some of the Endocrine Glands in the Sterile Carp-Funa Hybrid (F1), with Special Reference to the Hypophysis*

Arch. histol. jap., Vol. 42, No. 3 (1979) p. 305-318

Akira CHIBA,1 Yoshiharu HONMA,2 Sumio YOSHIE3 and Yoshio OJIMA4

Biological Laboratory1 and Department of Oral Anatomy,3 Nippon Dental University Niigata; Sado Marine Biological Station,2 Faculty of Science, Niigata University, Niigata and Department of Biology,4 Kansei Gakuin University, Nishinomiya, Japan

Received November 9, 1978

Summary. Some endocrine glands of the carp-funa hybrid were studied with a light microscope to elucidate their detailed structure and the possible causal factor of sterility in the males. Adult specimens of carp (Cyprinus carpio), gengoroh-buna (Carassius auratus cuvieri), and their hybrid (F1) were examined. The hybrid males are sterile as manifested by the failure of meiosis and seminomatous neoplasm in their testes. The hybrid females revealed well-developed ovaries, but their fertility was not tested. The hybrid hypophysis shows an intermediate condition between the parent species in the grade of ramification of the pars nervosa into the pars intermedia. Among seven types of granular cells demonstrated in the adenohypophysis, certain degenerative and anomalous changes are recognized only in the gonadotrophs of the hybrid hypophysis, especially in the female. These changes are discussed as a possible cause of sterility. A considerable amount of aldehyde fuchsin stainable neurosecretory material occurs in the cells of the nucleus preopticus and in the pars nervosa. The nucleus lateralis tuberis exhibits a histologically healthy condition.

Occasionally, the carp (Cyprinus carpio) and the funa (=crucian carp) (Carassius auratus) can mate and yield offspring under confinement. The previous studies including breeding experiment, macroscopical and microscopical anatomy, and cytological research have shown that 1) a reciprocal crossing is possible to yield similar offspring, 2) the bigeneric hybrids exhibit a condition intermediate between the parent species in morphological and cytochemical characters, 3) the hybrid males are * This paper is dedicated to the memory of the late Prof. Dr. W. BARGMANN. One of the authors, Y. HONMA,as well as his wife, Mitoko KAWAMOTO-HONMA, was intimately ac- quainted with Prof. BARGMANN through the study of piscine neuroendocrinology and also through Prof. Y. SANG and Prof. T. FUJITA. Prof. BARGMANN liked to look at and to investigate fish very much. He enjoyed visiting aquaria whenever he came to Japan. On one of such occasions, Prof. BARGMANN was deeply interested in a peculiar bony fish, the remora, and Y. HONMA, at his request, presented several sucking disks to him for morphological examination. The result was reported by Prof. BARGMANN and his associates in Z. Zellforsch. 139 (1973), while the endocrine glands of the same fish were studied and reported by Y. HONMAin Arch. histol. jap. 37 (1974).

305 306 A. CHIBA et al.: usually sterile although a small number of hybrid females are found to be fertile, and sometimes, 4) the hybrids have gonads of an intersexual character (MAKING et al., 1955; MATSUI et al., 1956; OJIMA et al., 1961; KAFUKU,1976; OJIMA, 1978). Some interesting results have been reported by OZTAN(1960, 1963), who found that the sterile hybrids of the poeciliid fish could become fertile by administration of gonadotropins and that the nucleus lateralis tuberis of the hypothalamus considered as the locus regulating the activity of the gonadotrophs was inactive in the sterile fish, judging from histophysiological criteria. However, information for endocrinological analysis on the sterility of the carp-funa hybrid is scanty, although a brief account on microscopical structure of the hybrid hypophysis was given by HONMA (1960). The present study thus intends to elucidate the detailed structure of some of the endocrine glands in the carp-funa hybrid, and especially to obtain information about the functional aspect of the hypothalamo-hypophyseal-gonadal system in this sterile fish.

MATERIALS AND METHODS

Six adult carp-funa hybrid (F1) raised in the Yamada Fish Cultural Pond attached to Hyogo Prefectural Fisheries Experimental Station were used in this study. The parent fish, six pond-cultured carp (Gyprinus carpio) obtained commercially from the suburb of Niigata City and six gengoroh-buna (Carassius auratus cuvieri) caught from the lower reaches of Shinano River in Niigata City were examined for a comparison of their endocrine structures. The size of the body, sex, and other records are sum- marized in Table 1. Immediately after decapitation, the brain with hypophysis, thyroid, interrenal gland, and gonads were removed and fixed in Bouin-Hollande-sublimate solution. Small pieces of the head kidney and gonads were immersed also in Bouin-Allen's mixture and Ciaccio's solution for detailed cytological study. These materials were dehydrated, embedded in paraffin, and cut serially 6-8μm thick in both sagittal and transverse directions. Sections were stained with the following stainings: aldehyde fuchsin (AF)-fast green-orange G, periodic acid Schiff (PAS)-fast green-orange G, azan trichrome, alcian blue (AB)-PAS-orange G, lead hematoxylin (PbH), Delafield's hematoxylin-eosin, Heidenhain's iron hematoxylin-fast green, AF-azan, AB-acid fuchsin, and PAS-PbH.

RESULTS

Ovary The three hybrid females examined have a pair of well-developed, voluminous ovaries. The gonosomatic index attaining 13.8±1.1 is comparable with that of the mature female carp (Table 1). The ovigerous folds are crowded with numerous vitello- genetic oocytes ranging from 300μm to 1mm in diameter (Fig. 1). There are im- mature yolkless oocytes, 50-180μm in diameter, and oogonial cysts intervening among the maturing oocytes. The micropyle apparatus is seldom elaborated in the Endocrine Glands of Sterile Carp-Funa Hybrid 307

Table 1. Data of the carp, gengoroh-buna, and their hybrid (F1) used in the study

maturing follicles, although no histologic anomalies are detectable in them. Fur- thermore, the collapsed follicles are occasionally found surrounded by a dense mass of small cells of stromatous origin. The epithelial lining of the ovarian cavity is constructed from ciliated tall columnar cells.

Testis Two of the three males have a pair of elongate testes with normal appearance, although an extraordinarily large, globular growth, diagnosed as seminomatous neoplasm, was found in the visceral cavity of the remaining male. No spermatogenetic

1 2

Fig. 1. A part of the ovary of a carp-funa hybrid showing maturing oocytes laden with yolk globules. Delafield's hematoxylin-eosin. ×63 Fig. 2. A part of the sterile testis of a hybrid showing pycnosis (black arrow) and abnormal meiosis (white arrow) in the spermatocytes. Heidenhain's iron hematoxylin-fast green. ×250 308 A. CHIBA et al.

cells were detected in this peculiar growth, which is exclusively replaced by large, polygonal cells having a large nucleus surrounded by rich, lightly stained cytoplasm. The histologic details of this neoplasm will be reported elsewhere. Tie testes of the former two males A are also sterile, because neither spermatozoa nor spermatids were discernible in the testicular lobules. As the result of failing meiosis, nuclear pycnosis and cell shrinkage were brought about in spermatocytes (Fig. 2). Suc- cessive absorption of the dead spermatogenetic B cells has resulted in the formation of a vacant cavity in the testicular lobules. Multiplica- tion of spermatogonia and their transformation into spermatocytes appear to be unaffect- ed, however. No appreciable changes occur in Sertoli cells, interstitial cells, and the epithelial cells lining the seminal duct in the hybrids, in comparison with those in the fer- C tile carp and gengoroh-buna.

Fig. 3. Diagram showing the construc- Hypophysis tion of the hypophysis in the The general structural patterns of the hypo- carp (A), gengoroh-buna (B) and their hybrid (C). The hypophysis of the carp, gengoroh-buna, and their physis of the hybrid shows an hybrid are schematically shown in Figure 3. intermediate condition between A certain close resemblance is visible among those of the parent species in them. Externally, the gland is ovoid or ellip- the grade of ramification of the tic in shape and slightly depressed. The rostral PN toward the PI. Black (PN), vertical lines (RPD), solid circles pars distalis (RPD), the smallest portion of the (PPD), and white (PI). adenohypophysis, is situated dorso-caudally to the massive proximal pars distalis (PPD), and the pars intermedia (PI) located anterior to the PPD projects antero-ventrally. From the dorso-caudal portion of the gland, the pars nervosa (PN) invades deeply into the adenohypophysis, and extensively sends its complicated ramifications into the PI. The neurohypophyseal branches in the PI are very slender and highly intricate in the carp, but they are rather thick and simple in the gengoroh-buna. The hybrid, thereby, show an intermediate condition in this context. The PN is composed of neurosecretory axons, glial elements, and capillaries. A considerable amount of AF stainable neurosecretory material is concentrated at the distal portion of the PN. Furthermore, the AF negative fibers occasionally loaded with the acidophil Herring bodies are demonstrated in the stem region of the PN. The RPD is composed of three main types of cells: two predominating types of acidophil cells, tentatively named A1 and A2, and a type of basophil cell, called B1. They are compactly arranged and intermingled with one another (Fig. 4). The A1 cell corresponding to the adrenocorticotroph is a large cell of polygonal or fusiform shape, containing a rounded nucleus with a conspicuous nucleolus. The coarsely Endocrine Glands of Sterile Carp-Funa Hybrid 309 granular cytoplasm is stained intensely with PbH and tinged bluish green by AF- fast green-orange G or reddish pink by AF-azan triple stain. This type of cell occurs solitarily or in small groups throughout the RPD, even in the neural tissue. The A2 cell, columnar or fusiform in shape, is stained yellowish with AF-fast green-orange G and deep red with AF-azan trichrome. The nucleus, usually ovoid or elliptic in shape, exhibits a wavy outline and contains a prominent nucleolus. The A1 and A2 cells tend to form a palisade layer along the adjoining neurohypophyseal branches. They are identical with prolactin cell in the staining property and the topographical distribution. The Br cell, usually fusiform or angular in shape, is small in size and number, and interspersed among the A1 and A2 cells. This cell has rather scanty cytoplasm with fine granules stained weakly with AF, AB, PAS, and aniline blue. An ovoid nucleus with a prominent nucleolus is located in the cell center. This basophil cell, regarded as the thyrotroph, occurs occasionally in the PPD. The PPD consists of two main types of cells: the acidophil cell, A3, and the basophil cell, B2 (Fig. 5). The A3 cell is arranged in groups of various sizes, intermingl- ing with masses of the B2 cell. The cell, ovoid or elliptic in shape, has a considerable amount of orangenophil cytoplasm in fine granular condition. By azan triple stain, carminophil filamentous material is occasionally detectable in the peripheral portion of the cell. The nucleus with one or two prominent nucleoli is usually ovoid in shape but sometimes exhibits a very irregular or deeply indented outline. As cell with two nuclei is often encountered, particularly in the female fish (Fig. 5). This orangenophil cell is considered as a somatotroph. The B2 cell diagnosed as a gonadotroph forms large cell groups, predominantly in the PPD. This cell, polygonal or round in

Fig. 4. A part of the RPD in a hybrid female showing three types of granular cells, PbH positive A1 cell (large, dark cell, A1), PbH negative A2 cell (predominant gray cell, A2), and B, cell with clear cytoplasm (B1). Small agranular cells (arrow) are interspersed among the granular cells. PbH. ×900 310 A. CHIBA et al.:

Fig. 5. Detail of the PPD of a hybrid female showing two types of granular cells: light A3 cells and dark B2 Cells. Note the anomalous, degenerative changes shown in the B2 cells, which are tentatively named signet ring-shaped cell (1), large colloid body containing cell (2), and shrunken cell (3). A3 cells with two nuclei are visible in this picture. PAS-PbH. ×900

shape and 10-15μm in the longer axis, is characterized by the presence of the cytoplasm containing two kinds of glycoproteinaceous granules: the cyanophil granules and the orangenophil globules ranging from l to 3μm in diameter by azan stain (Fig. 6a). The nucleus, ovoid in shape, with a prominent nucleolus is eccentrically located in the cell. Occasionally, the cytoplasm is slightly vacuolated. In addition to the typical B2 cell described above, several forms of mucoid cells considered as the same B2 cells in degenerating processes are discerned: a) a signet ring-shaped cell having extensively vacuolated cytoplasm with or without PAS positive colloid droplets and an eccentrically located flat or pycnotic nucleus (Fig. 5, 6a); b) a cell containing PAS positive colloid bodies, 5-12μm in diameter, and a densely stained flat nucleus (Fig. 5); c) a cell filled with weakly PAS positive amorphous material and an eccentrically located pycnotic nucleus (Fig. 6b, c); and d) a shrunken cell containing a pycnotic nucleus with a very irregular outline and PAS positive colloid droplets (Fig. 5, 6c). Occurrence of these degenerative forms characterizes the hybrid hypophysis; they are more numerous in the female than in the male. There is no noteworthy finding in either the cytology or histology of the hypophysis of the fish bearing a gonadal tumor (Fig. 6d). Furthermore, small chromo- phobes corresponding to so-called stellate cells and/or undifferentiated cells are occassionally encountered throughout the adenohypophysis (Fig. 4, 5). Application of PbH stain made it possible to distinguish two types of cells in the PI: a large PbH positive cell, type I1, clavate in shape and a PbH negative cell, type Endocrine Glands of Sterile Carp-Funa Hybrid 311

Fig. 6. Detail of the PPD of a hybrid female (a-c) and male (d). a. Typical B2 cell (dark cell) containg two kinds of PAS positive granules, b and c. Degenerative changes in B2 cells (dark cells). d. Light A3 cell and dark B2 cell of the PPD in the hybrid male bearing seminomatous growth. a-c: PAS-PbH, ×900; d: AB-PAS-orange G, ×1,100

I2, oval or cuboid in shape (Fig. 7). By means of azan triple stain, these cells are clearly differentiated into deep carminophil and orangenophil cells respectively. A basal prolongation of the I1 cell is in contact with neural tissue and more intensely stained with PbH than the apical swelling of the cell that contains a round nucleus. The I2 cell tends to form cell cords that border the PN. There are large acidophil colloid droplets in the PI.

Hypothalamic neurosecretory system Two neurosecretory centers of the hypothalamus, the nucleus preopticus (NP) and the nucleus lateralis tuberis (NLT), are recognized also in the hybrids. The NP is situated on either side of the third ventricle just anterior to the optic chiasma (Fig. 8), and is roughly divided into two contiguous portions: the antero-ventrally shifted pars parvocellularis (10-30μm in cell diameter) and the postero-dorsally shifted pars magnocellularis (20 to 42μm). The neurosecretory cell, polygonal or fusiform in shape, contains a round nucleus with a prominent nucleolus in the cell center. The perikaryon is stained in variable intensity by AF. Some of the cells lying close to the ependyma possess an intraventricular protrusion in contact with 312 A. CHIBA et al.:

Fig. 7. A part of the PI of a hybrid female. Two types of cells, PbH positive dark I1 cell and PbH negative light I2 cell, are distinguished in this picture. PbH. ×900 the cerebrospinal fluid (Fig. 8). It is not difficult to follow the course of the pre- optico-hypophyseal tract, for the axonal fibers are joined in a thick bundle and loaded with a considerable amount of the AF stainable material. The NLT located in the ventral region of the hypothalamus is divided into four portions: the pars lateralis, pars anterior, pars posterior, and pars inferior. The pars lateralis consists of compactly arranged large neurons (Fig. 9a). Each cell has an ovoid nucleus and rich acidophil perikaryon. Carminophil axons arising from the cells appear to be directed posteromedially. The pars anterior includes two types of cells: large (36.0μm in mean cell diameter) and small (17.3μm). They are intermingled with each other and distributed evenly in the subependymal region (Fig. 9b). Both types of cells extend their axon in a postero-lateral direction. The pars posterior is located posteriorly at the pars anterior, just ventro-lateral to the recessus lateralis of the third ventricle. The constituent cells are small, and sparsely distributed over this region. The AF stainable large granules are noticeably demonstrated in the perikaryon by a slightly foamy appearance (Fig. 9c). Small cells similar to the cells of the pars posterior scattered in the ventral portion of the recessus lateralis, are discriminated as the pars inferior. All these portions exhibit a histologically healthy condition. There are no noteworthy findings in the NLT of the hybrids in comparison with those of the parent species.

Thyroid The thyroid gland of the hybrids is seen both in the pharyngeal region and the head kidney just as those in the parent species. Each follicle of pharyngeal thyroid 20- 550μm (mean 141μm) in diameter, is surrounded by loose connective tissue and capil- Endocrine Glands of Sterile Carp-Funa Hybrid 313

laries (Fig. 10). A considerable amount of eosinophil colloid is contained in the follicular lumen. The epithelial cell, 3μm in mean height, has a flat nucleus stained deeply with hematoxylin. On the other hand, heterotopic thyroid follicles are found scattered in the lymphoid tissue of the head kidney, even in the interrenal gland area. The diameter of the follicle is comparatively small and ranged from 25- 360μm (mean 75.3μm), while the number of follicles varies from fish to fish. The lumen of each follicle is filled with colloid. These features of the gland described above may indicate a quiescent or inactive state.

Interrenal gland The interrenal gland of the hybrid is located around the wall of the postcardinal vein and its branches running through the Fig. 8. Cross section of the nucleus preopticus (pars magnocellularis) showing head kidney. The gland is composed of AF positive neurosecretory cells. two or more layers of cells arranged per- Note the protrusion of a cell in con- pendicularly to the endothelium (Fig. 11). tact with the cerebrospinal fluid of The cell, being columnar, fusiform or ovoid the third ventricle. AF. ×350 in shape, contains a considerable amount of eosinophil cytoplasm with siderophil granules. Its round nucleus has a prominent nucleolus. Chromaffin cells are easily detected as light cells interspersed among the interrenal cells, even in the prepara- tion stained with hematoxylin-eosin (Fig. 11). No essential difference was discerned in the structural design and histophysiological aspect between the male and female hybrids.

DISCUSSION

The gross morphology of the hypophysis of the carp-funa hybrid was previously described by HONMA (1960), who stated that the hypophysis of the hybrid showed a condition intermediate between those of the parent species in the invading angle and the point of the neurohypophysis to the adenohypophysis. The present study has shown the same condition in the grade of interdigitation between the PN and the PI. The peculiar sickle-shaped projection of the PI reported by HONMA(1960) was not confirmed in the present study. This discrepancy may be ascribed to indi- vidual or age differences between the specimens. The pituitary cytology of the carp, goldfish, and ironfish has been extensively documented with light and electron microscopical studies (BELL, 1938; LEVENSTEIN,1939; SCRUGGS,1951; HONMA, 1960; OLIVEREAU, 1962; KUROSUMI et al., 1963; LEATHERLAND, 1972; NAGAHAMA, 1973; KAUL and VOLLRATH,1974; CHIBA et al., 1978). With the exception of the dimorphism of 314 A. CHIBA et al.:

Fig. 9. a-c. Cross sections of the nucleus lateralis tuberis (NLT) showing its different portions. a. Pars lateralis of a female. b. Pars anterior of a male. c. Pars posterior of a male. Note the AF stainable granules in the pars posterior cells. a, b: AF-azan, ×350; c: AF-fast green-orange G, ×350 both the gonadotrophs and thyrotrophs described in the goldfish (OLIVEREAU,1962, LEATHERLAND, 1972; KAUL and VOLLRATH, 1974) and carp (IONESCU-VARO and CHRISTIAN, 1968) and follicular formation found in the RPD of the ironfish (CHIBA et al., 1978), no important differences in the glandular cells were detected among the carp, funa (including goldfish), and their hybrid. A close resemblance was recognized also in the histological design of the hypothalamic neurosecretory system (PALAY, 1960; PETER and NAGAHAMA,1976), thyroid (CHAVIN, 1956), and interrenal gland (NANDI, 1962; CHAVIN,1966) between the hybrids and parent species. There are many papers dealing with the role of the hypophysis on gonadal maturation in teleost fishes, and excellent reviews are available for reference (DODD, 1955, 1972; HOAR, 1955; PICKFORD and ATZ, 1957; DONALDSON, 1976; FONTAINE, 1976). In the hypophysectomized or chemically gonadotropin-deprived fish, spermatogonial multiplication is completely (or incompletely in some cases) blocked and transformation of spermatogonia into primary spermatocytes is also prevented. However, sub- sequent division and transformation appear to be independent of the hypophysis (BARR, 1963b; MACKAY,1973). On the other hand, the vitellogenesis is a process completely dependent on the hypophysis (BARR, 1963a; YAMAZAKI,1965; MACKAY, 1973) and the oogonial multiplication is suppressed after hypophysectomy (YAMAZAKI, 1965). Taking into consideration these facts and the present findings on the gonads, it seems that the hypophysis of the carp-funa hybrid has a potency to stimulate the gametogenesis to some extent. Then, what is the cause of sterility in the carp-funa Endocrine Glands of Sterile Carp-Funa Hybrid 315

10 11

Fig. 10. A part of the pharyngeal thyroid gland showing enlarged follicles that con- tain a large amount of dense, eosinophil colloid. Delafield's hematoxylin- eosin. ×250

Fig. 11. Detail of the interrenal gland showing interrenal cells with rich, eosinophil cytoplasm and dark granules. Chromaffin cells are demonstrated as large, light cells in this picture. Delafield's hematoxylin-eosin. ×500 hybrids? Some possible answers to this question are: 1) the amount of gonadotropin released from the hypophysis may not be sufficient to complete gametogenesis, 2) the modification induced in the gonadotropin molecule by hybridization may restrict the stimulatory effect of the hormone in gametogenesis, and 3) sterility may not be affected by the hypophysis but may be caused by certain factors in the gameto- genetic cell itself, such as derangement of the meiotic mechanism, discordance of genetic and/or physiologic factors. The first speculation may well explain the condition in the hybrid females of the poeciliid fish reported by OZTAN (1960, 1963). She demonstrated that the sterile hybrid females could become fertile with gonadotropin treatment and suggested that the ablation of gonadotropic potency of the hypophysis was attributable to the decline in secretory activity of the NLT of the hypothalamus. In the case of the carp-funa hybrid, special attention should be given to the anomalous, degenerative change occurring in the putative gonadotrophs of the adenohypophysis. Is the anomalous change of the gonadotrophs responsible for the sterility of hybrid males? Two possibilities are entertained. First, if there is evidence showing that the hybrid females examined are fertile, it may be said that this anomaly does not cause the sterility of hybrid males, because the anomaly was more pronounced in the hybrid females with maturing ovary than in the hybrid males with sterile testis. Secondly, if the hybrid females studied are proved to be also sterile in spite of their developing ovaries, it may be considered that the anomaly is 316 A. CHIBA et al.: responsible for the sterility of the hybrid to some extent. After all, it is impossible to determine the causal factor of the sterility of the hybrid based only on the present results. Apparently, experimental study is neces- sary to elucidate whether or not the administration of exogenous gonadotropin can induce the sterile gonad of the hybrids to be fertile. Furthermore, electron microscopical and cytochemical studies should be carried out to understand the detailed histophysiological condition of the gonadotroph, NLT, and steroidgenic cells of the gonads, for gonadal maturation is a complicated phenomenon controled by the endocrine system. There is, as yet, no available information to confirm the second speculation. A third possible cause of sterility may be related to mechanisms of reproductive isolation. A preferable explanation from the cytogenetic point of view has been given to the problem of sterility in the carp-funa hybrid by OJIMA(1978). SONSTEGARD et al. (1976) cytologically examined the hypophysis of the carp, goldfish, and their hybrid bearing the gonadal tumor of Sertoli cell origin for understand- ing the effect of the gonadal tumor on the hypophyseal-gonadal axis. They found a remarkable hyperplasia of the gonadotrophs resulted from the presence of the tumor, in response to a lowered level of gonadal steroids. No such hyperplasia of the gonadotrophs was recognized in the present specimen bearing seminomatous neoplasm. The circulating levels of the gonadal steroids seem worthy to be examined and compared between tumorous fish and nontumorous fish.

コイとフナの雑種における内分泌腺, とくに下垂体の組織学的観察

千葉晃, 本間義治, 吉江紀夫, 小島吉雄

コイとゲンゴロウブナの属間雑種 (F1) の内分泌腺の組織構造を, 下垂体を中心に光学顕微鏡で観察し, 両親魚種との比較を試みた. 雄魚には精巣上皮腫を伴う個体もみられ, 不妊であった. 雌魚には発育の進んだ卵巣卵がみられたが, 妊性は確かめ得なった. 下垂体の形態, とくに中間葉における神経葉の分岐の程度は, 両親魚との中間の状態を示した. 腺性下垂体に検出された諸細胞のうち, 異常な退行変化が認められたのは生殖腺刺激細胞のみで, とくに雌魚で著しかったので, 雑種不妊との関連で若干の考察を試みた. 視束前核-神経性下垂体路にはアルデヒドフクシン陽性の神経分泌物が相当量検出された. また隆起部外側核には異常は認められなかった.

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本間義治 Dr. Yoshiharu HONMA 〒950-21新潟市五十嵐2ノ町 Department of Biology 新潟大学理学部 Faculty of Science, Niigata University 生物学教室 8050 Igarashi-2, Niigata 950-21 Japan