Researcher Profile and Settings

Affiliation

• Faculty of Fisheries Sciences　Marine Life Science　Aquaculture Biology

Job Title

• Associate Professor

Degree

• Studies on the steroidogenic enzyme system for ovarian estrogen biosynthesisi of Japanese eel, Anguilla japonica（Hokkaido University）

J-Global ID

• 200901088102943913

Research Interests

• oocyte maturation   sturgeon   tilapia   eel   fish reproduction   fish steroidogenesis   sex differentiation in fish   reproductive physiology in fish   Reproductive fish physiology   

Research Areas

• Life sciences / Aquaculture

Educational Organization

•  Bachelor's degree program　School of Fisheries Sciences
•  Master's degree program　Graduate School of Fisheries Sciences
•  Doctoral (PhD) degree program　Graduate School of Fisheries Sciences

Academic & Professional Experience

• 2005 - 2006 National Institutes of Natural Sciences
• 2005 - 2006 Researcher
• 2004 - 2005 科学技術振興事業団 研究員
• 2004 - 2005 Researcher
• 2001 - 2004 大学院水産科学研究科 日本学術振興会特別研究員
• 2001 - 2004 Postdoctoral Fellowships of Japan Society for the Promotion of Science
• 1997 - 2001 Center of Marine Biotechnology, University of Maryland Biotechnology Insutitute 研究員
• 1997 - 2001 Researcher

Education

•        - 1997  Hokkaido University
•        - 1997  Hokkaido University  Graduate School, Division of Fisheries
•        - 1992  Hokkaido University  School of Fisheries Sciences
•        - 1992  Hokkaido University  Faculty of Fisheries

Association Memberships

• JAPANESE SOCIETY FOR AQUACULTURE SCIENCE   JAPAN SOCIETY FOR COMPARATIVE ENDOCRINOLOGY   日本水産学会   

Research Activities

Published Papers

• Identification of maturation-inducing steroid in sturgeons via comprehensive analyses of steroids produced during oocyte maturation.

  Yuya Hasegawa, Ryohei Surugaya, Kazuki Tousaka, Shinji Adachi, Shigeho Ijiri

  The Journal of steroid biochemistry and molecular biology 238 106442 - 106442 2024/04 

  Although 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) and 17α, 20β, 21-trihydroxy-4-pregnen-3-one (20β-S) have been identified as maturation-inducing steroids (MIS) in several teleosts, to date, no MISs have been identified in sturgeons. As it remains possible that an unidentified steroid is an MIS in sturgeons, this study aimed to identify a sturgeon MIS via comprehensive analyses and maturation-inducing (MI) assay of C21 steroids. In vivo and in vitro comprehensive analyses of C21 steroids revealed that serum DHP concentrations were rapidly elevated in the oocyte maturation phase and the DHP production level was notably high among C21 steroids. MI assay indicated that the MI activity of DHP, 17α-hydroxyprogesterone (17OHP), a precursor of DHP, 17α, 20α-dihydroxy-4-pregnen-3-one (αDHP), and 20β-S was high among C21 steroids, but the MI activity of these steroids were similar. In the C21 steroids produced in ovarian follicles during oocyte maturation, 17OHP, αDHP, and unidentified compounds had a low production level, and 20β-S was suggested to be metabolized from DHP after oocyte maturation. Against this background, this study concluded that DHP is a steroid that possesses strong MI activity and is highly produced during oocyte maturation. Although this study could not identify an MIS in sturgeons by fractionation of plasma and subsequent bio assay, it was suggested that DHP is a major MIS in sturgeons.
• mRNA expression profiles of proteolytic genes during the process of ovulation in ovarian follicles of sturgeons

  Ryohei Surugaya, Yuya Hasegawa, Kazuki Tousaka, Shinji Adachi, Shigeho Ijiri

  Fisheries Science 90 (1) 41 - 51 0919-9268 2024/01 

  In sturgeon aquaculture, oocyte maturation and ovulation are induced by the administration of hormones such as luteinizing hormone–releasing hormone analog (LHRHa). However, ovulation often fails to occur, probably as a result of inappropriate hormone injection timing. It is thus necessary to understand the molecular mechanisms of sturgeon ovulation responsible for producing good-quality eggs. In this study, we investigated the proteolytic genes and their inhibitory genes in a de novo transcriptome constructed using RNA samples of ovarian follicles from Amur sturgeon (Acipenser schrenckii). We subsequently identified 14 mmp, nine adam, ten adamts, nine cathepsins, and three timp contigs by a homology search with BLASTx. We compared the messenger RNA (mRNA) levels of these genes in the ovarian follicles of anovulated and ovulated Amur sturgeons using RNA sequencing and quantitative PCR analysis. The results showed that the mRNA levels of mmp16, adam23, adamts9, and timp2 were significantly increased by LHRHa injection only in ovulated sturgeons. We also examined the expression patterns in sterlet (Acipenser ruthenus) ovarian follicles in in vitro culture. adamts9 mRNA levels were drastically upregulated by the administration of 17α-hydroxyprogesterone, suggesting that Adamts9 is a key factor in successfully inducing ovulation in sturgeons. Clarifying the functions of these four genes during ovulation will lead to improved sturgeon seedling production technologies.
• Variation in responses to photoperiods and temperatures in Japanese medaka from different latitudes.

  Ai Shinomiya, Daisuke Adachi, Tsuyoshi Shimmura, Miki Tanikawa, Naoshi Hiramatsu, Shigeho Ijiri, Kiyoshi Naruse, Mitsuru Sakaizumi, Takashi Yoshimura

  Zoological letters 9 (1) 16 - 16 2023/07/22 

  Seasonal changes are more robust and dynamic at higher latitudes than at lower latitudes, and animals sense seasonal changes in the environment and alter their physiology and behavior to better adapt to harsh winter conditions. However, the genetic basis for sensing seasonal changes, including the photoperiod and temperature, remains unclear. Medaka (Oryzias latipes species complex), widely distributed from subtropical to cool-temperate regions throughout the Japanese archipelago, provides an excellent model to tackle this subject. In this study, we examined the critical photoperiods and critical temperatures required for seasonal gonadal development in female medaka from local populations at various latitudes. Intraspecific differences in critical photoperiods and temperatures were detected, demonstrating that these differences were genetically controlled. Most medaka populations could perceive the difference between photoperiods for at least 1 h. Populations in the Northern Japanese group required 14 h of light in a 24 h photoperiod to develop their ovaries, whereas ovaries from the Southern Japanese group developed under 13 h of light. Additionally, Miyazaki and Ginoza populations from lower latitudes were able to spawn under short-day conditions of 11 and 10 h of light, respectively. Investigation of the critical temperature demonstrated that the Higashidori population, the population from the northernmost region of medaka habitats, had a critical temperature of over 18 °C, which was the highest critical temperature among the populations examined. The Miyazaki and the Ginoza populations, in contrast, were found to have critical temperatures under 14 °C. When we conducted a transplant experiment in a high-latitudinal environment using medaka populations with different seasonal responses, the population from higher latitudes, which had a longer critical photoperiod and a higher critical temperature, showed a slower reproductive onset but quickly reached a peak of ovarian size. The current findings show that low latitudinal populations are less responsive to photoperiodic and temperature changes, implying that variations in this responsiveness can alter seasonal timing of reproduction and change fitness to natural environments with varying harshnesses of seasonal changes. Local medaka populations will contribute to elucidating the genetic basis of seasonal time perception and adaptation to environmental changes.
• Morphological and molecular gonadal sex differentiation in wild Japanese eel Anguilla japonica

  Horiuchi M, Hagihara S, Kume M, Chushi D, Hasegawa Y, Itakura H, Yamashita Y, Adachi S, Ijiri S

  Cells 11 (9) e1554  2022/05 [Refereed]
  
• Regulation by Progestins, Corticosteroids, and RU486 of Transcriptional Activation of Elephant Shark and Human Progesterone Receptors: An Evolutionary Perspective.

  Xiaozhi Lin, Wataru Takagi, Susumu Hyodo, Shigeho Ijiri, Yoshinao Katsu, Michael E Baker

  ACS pharmacology & translational science 5 (2) 52 - 61 2022/02/11 

  We investigated progestin and corticosteroid activation of the progesterone receptor (PR) from elephant shark, a cartilaginous fish belonging to the oldest group of jawed vertebrates. Comparison with the human PR provides insights into the evolution of steroid activation of the human PR. At 1 nM steroid, the elephant shark PR is activated by progesterone, 17-hydroxy-progesterone, 20β-hydroxy-progesterone, 11-deoxycorticosterone (21-hydroxyprogesterone), and 11-deoxycortisol. The human PR, in comparison, is activated at 1 nM steroid, only by progesterone and 11-deoxycorticosterone, indicating increased progestin and corticosteroid specificity during the evolution of the human PR. RU486, an important clinical antagonist of the human PR, did not inhibit progesterone activation of the elephant shark PR. Cys-528 in the elephant shark PR corresponds to Gly-722 in the human PR, which is essential for RU486 inhibition of the human PR. Confirming the importance of Cys-528 in the elephant shark PR, RU486 inhibited progesterone activation of the Cys528Gly mutant PR. To investigate the physiological relevance of Gly-722 in the human PR and Cys-528 in the elephant shark PR, we studied steroid activation of the Gly722Cys human PR and Cys528Gly elephant shark PR. Compared to the wild-type human PR, there was an increase in the activation of human Gly722Cys PR by11-deoxycortisol and a decrease in activation by corticosterone, which may have been important in selection for the mutation corresponding to the human glycine-722 PR that first evolved in the platypus PR, a basal mammal.
• 17β-hydroxysteroid dehydrogenase type 12-like is associated with maturation-inducing steroid synthesis during induced oocyte maturation and ovulation in sturgeons

  Yuya Hasegawa, Shigeho Ijiri, Ryohei Surugaya, Ryuichi Sakai, Shinji Adachi

  Aquaculture 546 737238 - 737238 0044-8486 2022/01 [Refereed]
  
• Effect of diet on faecal testosterone metabolite levels in a northern fur seal (Callorhinus ursinus) expressed as ash-free dry weight.

  Mayuko Otsuki, Kaoru Kohyama, Wataru Goshima, Motoki Kobayashi, Yuya Hasegawa, Yuka Morita, Shigeho Ijiri, Yoko Mitani

  Coastal Marine Science International Coastal Research Center, Atmosphere and Ocean Research Institute, the University of Tokyo 44 (1) 1 - 6 1349-3000 2021/06 [Refereed]
  
• Gonadal expression profiles of sex-specific genes during early sexual differentiation in Japanese eel Anguilla japonica

  Hiroyuki Inaba, Seiji Hara, Moemi Horiuchi, Shigeho Ijiri, Takeshi Kitano

  FISHERIES SCIENCE 87 (2) 203 - 209 0919-9268 2021/03 [Refereed]
   

  The Japanese eel Anguilla japonica is a prominent and highly valued species in the aquaculture industry in Japan. Sex determination and sex differentiation in eels are significantly affected by environmental factors; however, the molecular mechanisms involved in sex differentiation in eels are largely unknown. In this study, to investigate the gonadal expression profiles of sex-specific genes during and after sexual differentiation in Japanese eel, we induced elvers into predominantly phenotypic males or females by rearing on a control diet or estradiol-17 beta-treated diet, respectively, during the sex differentiation period. Quantitative real-time PCR showed that forkhead box L2A (foxl2a) and cytochrome P450 aromatase (cyp19a1) were more highly expressed in ovaries than in testes, whereas the expression levels of anti-Mullerian hormone (amh) and gonadal soma-derived factor (gsdf) were significantly higher in testes than in ovaries. Furthermore, foxl2a and cyp19a1 displayed female-specific expression early in the sex differentiation process, while after slightly more growth amh and gsdf displayed male-specific expression during sex differentiation. Together, these results suggest that these genes have important roles in sexual differentiation and development in this species.
• Histological evidence of multiple spawning in wild Japanese eel Anguilla japonica.

  Akio Shimizu, Shigeho Ijiri, Hikari Izumi, Koichiro Gen, Hiroaki Kurogi, Hiroshi, Hashimoto, Hideki Tanaka, Tadao Jinbo, Hiroaki Saito, Seinen Chow

  Zoological Studies 60 (61) 2021 [Refereed][Not invited]
  
• Testosterone levels in hair of free-ranging male northern fur seals (Callorhinus ursinus) in relation to sampling month, age class and spermatogenesis

  Mayuko Otsuki, Takanori Horimoto, Motoki Kobayashi, Yuka Morita, Shigeho Ijiri, Yoko Mitani

  Conservation Physiology 9 (1) coab031  2021/01/01 [Refereed]
   

  Information about the reproductive status of free-ranging pinnipeds provides useful insight into their population dynamics, which is essential to their management and conservation. To determine the reproductive status of individual animals, blood sampling is often required despite being impractical to collect in open water. Hair as an endocrine marker has been used to less invasively assess the reproductive status of terrestrial animals. However, it is unknown whether pinniped reproductive status can be assessed from hair samples. Here, we examine testosterone levels in hair obtained from 57 male northern fur seals and used it to compare their age class and spermatogenesis during the non-breeding season off Hokkaido. We isolated testosterone from the samples using gas chromatography and measured testosterone levels using time-resolved fluoroimmunoassay. Testosterone levels in hair increased towards the breeding season. In May, testosterone levels were the highest in seals aged between 4 and 7 years, followed by those over the age of 8 years and under the age of 4 years. Spermatids, the final phase of spermatogenesis, were present in the seals sampled between April and June, even though testosterone levels were low in April. The seals with spermatids in May showed the highest testosterone levels. Our results demonstrate that seals with higher testosterone levels in May are likely to be mature males (≥4 years). Since hair can be collected using biopsy darts in the field, it will be possible to less invasively determine testosterone levels of male seals in the future.
• 17 beta-Hydroxysteroid dehydrogenase type 12a responsible for testicular 11-ketotestosterone synthesis in the Japanese eel, Anguilla japonica

  Hiroshi Suzuki, Yuichi Ozaki, Shigeho Ijiri, Koichiro Gen, Yukinori Kazeto

  JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY 198 0960-0760 2020/04 [Refereed]
   

  The production of 11-ketotestosterone (11KT), an important steroid hormone in piscine spermatogenesis, is regulated by the pituitary gonadotropins [Gths: follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh)] and it is synthesized by catalytic reactions involving several steroidogenic enzymes. Among these enzymes, the role of 17 beta-hydroxysteroid dehydrogenases (Hsd17bs) that exhibited 17-ketosteroid reducing activity (17KSR activity) responsible for 11KT synthesis is still poorly understood. In the present study, for the deeper understanding of testicular 11KT biosynthesis, we first investigated the steroidogenic pathway to produce 11KT in Japanese eel testis. In vitro incubation of the testis with androstenedione (A4) and the subsequent analysis of the metabolites by thin-layer chromatography indicated that 11KT was synthesized from A4 via 11 beta-hydroxyandrostenedione (11OHA4) and 11-ketoandrostenedione (11KA4), which indicated that the steroidogenic enzyme exhibiting the 17KSR activity responsible for converting 11KA4 to 11KT is crucial for 11KT production. Subsequently, cDNAs encoding three candidate enzymes, Hsd17b type3 (Hsd17b3), Hsd17b type12a (Hsd17b12a), and 20 beta-hydroxysteroid dehydrogenase type2 (Hsd20b2), potentially with the 17KSR activity were isolated and characterized in the Japanese eel. The isolated hsd17b3, hsd17b12a, and hsd20b2 cDNAs putatively encoded 308, 314, and 327 amino acid residues with high homology to those of other vertebrate counterparts, respectively. The Hsd17b3, Hsd17b12a, and Hsd20b2 expressed either in HEK293T or in Hepa-E1 converted 11KA4 to 11KT. Tissue-distribution analysis by quantitative real time PCR revealed that hsd17b12a and hsd20b2 mRNAs were detected in the testis, while hsd17b3 mRNA was not detectable. Furthermore, we examined the effects of Gths on the 17KSR activity and the expression of the candidate genes in the immature testis. The 17KSR activity was upregulated by administration of Gths. Furthermore, only expression of hsd17b12a among three candidates was upregulated by Gths as well as the 17KSR activity. These findings strongly suggested that Hsd17b12a is one of the enzymes with 17KSR activity responsible for 11KT synthesis in the testis of Japanese eel.
• Non-invasive monitoring of faecal testosterone metabolites expressed in unit per ash-free dry weight in a northern fur seal (Callorhinus ursinus).

  Mayuko Otsuki, Kaoru Kohyama, Wataru Goshima, Motoki Kobayashi, Yuya Hasegawa, Yuka Morita, Shigeho Ijiri, Yoko Mitani

  Japanese Journal of Zoo and Wildlife Medicine 25 (1) 29 - 34 2020 [Refereed]
  
• 17 beta-Hydroxysteroid dehydrogenase type 12 is responsible for maturation-inducing steroid synthesis during oocyte maturation in Nile tilapia

  Chak Aranyakanont, Shigeho Ijiri, Yuya Hasegawa, Shinji Adachi

  GENERAL AND COMPARATIVE ENDOCRINOLOGY 290 0016-6480 2020 [Refereed]
   

  17 alpha, 20 beta-Dihydroxy-4-pregnen-3-one (DHP) is a maturation-inducing steroid in many teleost fish. Carbonyl reductase-like 20 beta-hydroxysteroid dehydrogenase (CR/20 beta-HSD) is a candidate enzyme responsible for DHP production during oocyte maturation in various fish, including Nile tilapia. However, a novel type of 17 beta-hydroxysteroid dehydrogenase, type 12-like (17 beta-HSD12L), is responsible for DHP production during oocyte maturation in masu salmon. 17 beta-HSD12 (presumably orthologous to salmon 17 beta-HSD12L) has been detected in Nile tilapia; however, its enzymatic activity and specific ability to convert the DHP substrate 17 alpha-hydroxyprogesterone (17OHP) have not been examined. This study aimed to determine whether CR/20 beta-HSD or 17 beta-HSD12 is responsible for DHP production during oocyte maturation in the Nile tilapia. Mammalian expression vectors containing tilapia hsd17b12 or CR/20bhsd were transfected into HEK293T cells, followed by incubation with 170HP. HEK293T cells transfected with hsd17b12 exhibited a strong ability to convert exogenous 17OHP to DHP (73.8% yield). Cells transfected with CR/20bhsd or the control vector converted only 7.4% and 7.5% of 17OHP to DHP, respectively. In addition, based on LC-MS/MS analyses, 17 beta-HSD12 did not convert any substrates other than 17OHP, including DHP, adrenosterone, androstenedione, estrone, testosterone, 11-ketotestosterone, and estradiol-17 beta. CR/20 beta-HSD showed strong 17 beta-HSD oxidoreductase activity especially with adrenosterone and androstenedione. Tissue-specific hsd17b12 expression analyzed by RT-PCR showed that hsd17b12 mRNA was strongest amplification in full-grown follicles. Finally, full-grown ovarian follicles were incubated with salmon pituitary extract (SPE, 100 mu g/mL) or human chorionic gonadotropin (HCG, 100 IU/mL) to induce 2013-HSD activity in vitro, and enzyme activity was assessed by co-incubation with 100 ng/mL 17OHP for 2, 4, 8, and 16 h. Conversion of 17OHP to DHP by ovarian follicles incubated with SPE and HCG peaked at 16 h, subsequent with increased follicular hsd17b12 mRNA levels, which were significantly higher than those in control incubations. However, the levels of CR/20bhsd mRNA remained low and did not differ among time points. The present study strongly suggests that 17 beta-HSD12, and not CR/20 beta-HSD, is the 20 beta-FISD responsible for DHP production by ovarian follicles during oocyte maturation in Nile tilapia.
• Reproductive physiological characteristics of tropical Celebes eels Anguilla celebesensis in relation to downstream migration and ovarian development

  Hagihara Seishi (corresponding author), Aoyama J, Sudo R, Limbong D, Ijiri S, Adachi S, Tsukamoto K

  Journal of Fish Biology in press (3) 558 - 569 0022-1112 2020 [Refereed][Not invited]
  
• Changes in Sex Steroids and Ovarian Steroidogenic Enzyme mRNA Levels in Artificially Maturing Japanese Eel (Anguilla japonica) and Naturally Maturing New Zealand Longfin Eel (Anguilla dieffenbachii) during Vitellogenesis

  Hajime Matsubara, P. Mark Lokman, Yukinori Kazeto, Hiromi Okumura, Shigeho Ijiri, Toshiaki Hirai, Graham Young, Shinji Adachi, Kohei Yamauchi

  FISHES 4 (4) 2019/12 [Refereed][Not invited]
   

  Repeated hormone injections are routinely used to induce advanced stages of oogenesis in freshwater eels, but this approach may result in aberrant germ cell development. To investigate the underlying causes, levels of sex steroids (testosterone, T; estradiol-17fi, E2) and ovarian steroidogenic enzyme mRNAs were compared between artificially maturing Japanese eels and wild-caught, spontaneously maturing New Zealand longfin eels. The latter were employed as reference, as wild Japanese eels in advanced stages of oogenesis are near-impossible to catch. SerumT levels in artificially maturing Japanese eel changed with stage in a pattern that was comparable to that in longfin eels. Likewise, ovarian mRNA levels of most steroidogenic enzyme genes were not qualitatively dissimilar between both eel species when taking developmental stage into account. However, aromatase (cyp19a) mRNA levels, together with serum E2 levels, rapidly increased in artificially maturing Japanese eels in mid-late stages of oogenesis (gonadosomatic index, GSI = 13.8%), whereas no such increase was evident in longfin eels (GSI similar to 6.9%). In addition, sex steroid and target gene mRNA levels fluctuated drastically with each hormone injection. We contend that expression of most target genes, possibly even that of cyp19a, during induced oogenesis could be "normal", with the drastic fluctuations due most likely to hormone delivery through repeated injections. The effects of these fluctuations on gamete quality remain unknown and resolving this issue may prove fruitful in the future to further artificial propagation of anguillid eels.
• Involvement of 11-ketotestosterone in hooknose formation in male pink salmon (Oncorhynchus gorbuscha) jaws

  Hideaki Kudo, Tomoaki Kimura, Yuya Hasegawa, Takashi Abe, Masaki Ichimura, Shigeho Ijiri

  General and Comparative Endocrinology 260 41 - 50 1095-6840 2018/05/01 [Refereed][Not invited]
   

  Mature male Pacific salmon (Oncorhynchus spp.) develop a hooknose, as a secondary male sexual characteristic, during the spawning period. It is likely that androgens regulate hooknose formation. However, endocrinological and histochemical details about the relationship between androgens and hooknose formation are poorly understood. In this study, we performed assays of serum androgens, detection of androgen receptor (AR) in hooknose tissues, external morphological measurement of hooknose-related lengths, and microscopic observation of hooknose tissues of pink salmon (O. gorbuscha) at different stages of sexual maturation. Expression of the arβ gene was detected in hooknose tissues of males but not females. The elongation of these tissues was mediated directly via androgens. Serum 11-ketotestosterone (11-KT) concentrations indicated a significant positive correlation with both jaw lengths during sexual maturation of males. In the upper jaw, cartilage tissue developed during hooknose formation, and AR-immunoreactive chondrocytes were located in the rostal-vetral regions of hooknose cartilage in maturing male. The chondrocytes in maturing males before entering into rivers exhibited rich-cytoplasm with high cell activity than at other sexual development stages. On the other hand, in the lower jaw, the development of the spongiosa-like bone meshworks. AR-immunoreactivity was detected in a proportion of the osteocytes and osteoblast-like cells in the spongiosa-like bone meshworks. These results indicate that hooknose formation in pink salmon, which is associated with the buildup of a structure with sufficient strength that it can be used to attack other males on the spawning ground, is regulated by 11-KT.
• First report of the occurrence of a female Amur sturgeon Acipenser schrenckii in advanced stages of oogenesis, off the coast of Mombetsu, Hokkaido, Japan.

  Seishi Hagihara, Noriko Azuma, Kiichi Suyama, Seiji Katakura, Shigeho Ijiri, Shinji Adachi

  Coastal Marine Science 41 (1) 7 - 10 2018 [Refereed][Not invited]
  
• Expression pattern of foxl2 and dmrt1 in gonad of Amur sturgeon Acipenser schrenckii in relation to sex differentiation

  Hatsumi Okada, Seishi Hagihara, Katsumasa Yamashita, Shigeho Ijiri, Shinji Adachi

  AQUACULTURE 479 (1) 712 - 720 0044-8486 2017/10 [Refereed][Not invited]
   

  The mechanisms that govern sex differentiation in sturgeon are poorly understood. This study aimed to examine sexual dimorphic expression of foxl2 and dmrt1 in Amur sturgeon Acipenser schrenckii during molecular and morphological sex differentiation. Histologically, ovaries were first observed at 9 months after hatching (mah), reflected in an invaginated gonadal epithelium and the presence of germ cells just below the epithelium, whereas gonads with smooth epithelium and germ cells in the stroma were characterized as testis. Some fish showed undifferentiated gonads until 16 mah, but all sampled fish had morphologically differentiated sex by 19 mah. The full-length cDNAs of foxl2 and two types of dmrt1, dmrt1a and dmrt1b, of Amur sturgeon were isolated. The 2 types of dmrt1 cDNA had an identical structure from the 5'-UTR to the Y-rich domain, whereas the sequence downstream from this domain showed no homology and is considered to have resulted from alternative splicing. Foxl2 was principally expressed in the ovary and the 2 types of dmrt1 in the testis of 7-year-old Amur sturgeon, and much less so in various somatic tissues. Foxl2 expression was sexually dimorphic in morphologically differentiated gonads and was similarly dichotomous in the undifferentiated gonads of fish at 9 mah. In contrast, sexually dimorphic expression of dmrt1a and dmrt1b was not observed in gonads of the juvenile Amur sturgeon, but these genes showed testis-dominant expression in the adults. We suggest that foxl2 expression is closely related to ovarian differentiation and that two types of dmrt1 play a role in testis development and/or sperm formation rather than testicular differentiation. Additionally, we suggest that mRNA levels of foxl2 are an appropriate marker for sexing of sturgeon at an early developmental stage.
• 17 beta-HSD Type 12-Like Is Responsible for MaturationInducing Hormone Synthesis During Oocyte Maturation in Masu Salmon

  Shigeho Ijiri, Yasushi Shibata, Nonoha Takezawa, Yukinori Kazeto, Naoki Takatsuka, Erika Kato, Seishi Hagihara, Yuichi Ozaki, Shinji Adachi, Kohei Yamauchi, Yoshitaka Nagahama

  ENDOCRINOLOGY 158 (3) 627 - 639 0013-7227 2017/03 [Refereed][Not invited]
   

  The maturation-inducing hormone 17(alpha), 20(beta)-dihydroxy-4-pregnen-3-one (DHP) was first identified in the amago salmon. Although carbonyl reductase-like 20 beta-hydroxysteroid dehydrogenase (CR/20b-HSD) was reported to convert 17 alpha-hydroxyprogesterone (17OHP) to DHP in rainbow trout, we previously found that CR/20 beta-HSD messenger RNA (mRNA) was not upregulated in stimulated granulosa cells from masu salmon, which suggested that DHP is synthesized by a different enzyme. Accordingly, the current study aimed to identify the specific 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD) responsible for DHP production by granulosa cells during final oocyte maturation in masu salmon. RNA sequencing was performed on granulosa layers that were isolated from ovarian follicles at 1 month before ovulation and incubated with or without forskolin, which was used to mimic luteinizing hormone, and similar to 12 million reads were obtained, which yielded 71,062 contigs of > 100 bp. tBlastx analysis identified 1 contig (#f103496)as similar to 17 beta-hydroxysteroid dehydrogenase type 12 (hsd17 beta 12); however, because the full-length #f103496 sequence was different from hsd17 beta 12, it was termed hsd17 beta 12-like (hsd17 beta 12l). We found that mammalian cells transfected with full-length hsd171 beta 2l exhibited considerable 20b-HSD activity, as indicated by efficient conversion of exogenous 17OHP to DHP. In addition, we found that hsd17 beta 12l mRNA levels were consistently low in follicles during vitellogenic growth; however, the levels increased significantly during final oocytematuration. The levels of hsd17 beta 12lmRNA were also considerably increased in granulosa layers in which 20 beta-HSD activity was induced by salmon pituitary extract. Therefore, we suggest that hsd17 beta 12l, not CR/20 beta-HSD, is the 20 beta-HSD responsible for DHP production by granulosa cells in masu salmon during final oocytematuration.
• In vitro 11-ketotestosterone production by the ovary of the Japanese eel, Anguilla japonica.

  Yuya Hasegawa, P Mark Lokman, Shigeho Ijiri, Shinji Adachi

  Simposium Proceedings in “Fisheries Science for Future Generations” Article number 06009  2017 [Not refereed][Not invited]
  
• Detection of Japanese Eel Endothelial Cells-infecting Virus (JEECV) in Mature Japanese Eel Anguilla japonica Caught from Their Spawning Area

  Sachiko Okazaki-Terashima, Hiroaki Kurogi, Seinen Chow', Toshihiro Yamamoto, Noriaki Matsuya, Shigeho Ijiri, Noritaka Mochioka, Shinobu Tsuchiakat, Yuki Naoi, Kaori Sano, Tsutomu Omatsu, Shin-ichi Ono, Hiroshi Kuwada, Tetsuya Mizutani

  FISH PATHOLOGY 51 (2) 64 - 66 0388-788X 2016/06 [Refereed][Not invited]
   

  We examined the infection of Japanese eel endothelial cells-infecting virus (JEECV), which is the agent of viral endothelial cells necrosis (VECNE), in Japanese eels caught from the southern part of West Mariana Ridge in 2013 to know the infection status of the virus in their spawning area. JEECV was detected in the gills from a female out of five mature male and female fish by both quantitative and conventional PCRs. Additionally, the predicted polyomavirus large T like protein of the detected virus was different by one amino acid from that of the virus from farmed eels with VECNE.
• Quantitative comparisons of maternal transcripts related to cell division between good and poor quality eggs from artificially matured Japanese eel Anguilla japonica.

  Izumi H, Gen K, Horiuchi M, Matsuya N, Ijiri S, Adachi S

  Aquaculture Science 64 (1) 29 - 42 2016/03 [Refereed][Not invited]
   

  The current method of artificial egg production in Japanese eel does not always yield good quality eggs, but the molecular basis for the decrease in egg quality remains unclear. In this study, we compared the levels of 74 maternal transcripts related to cell division among good quality eggs, developing poor quality eggs (i.e., those producing a viable morula embryo without hatching), and non-developing poor quality eggs (i.e., developmentally incompetent eggs) to determine the molecular characteristics of poor quality eggs produced by artificially matured Japanese eel. Only one transcript (lsm7) had a lower level in developing poor quality than in good quality eggs, with the others being present at similar levels between the two types of egg. The levels of 11 transcripts (e.g., cdc26, psma7) were lower in non-developing poor quality than in good quality eggs. These results suggest that insufficient or reduced levels of maternal mRNA related to cell division are associated with the loss of egg quality in artificially matured Japanese eel, and that maternal characteristics other than quantity may affect the developmental competence of poor quality eggs.
• Gonadal soma-derived factor (gsdf), a TGF-beta superfamily gene, induces testis differentiation in the teleost fish Oreochromis niloticus

  Hiroyo Kaneko, Shigeho Ijiri, Tohru Kobayashi, Hikari Izumi, Yuki Kuramochi, De-Shou Wang, Shouta Mizuno, Yoshitaka Nagahama

  MOLECULAR AND CELLULAR ENDOCRINOLOGY 415 (C) 87 - 99 0303-7207 2015/11 [Refereed][Not invited]
   

  The Nile tilapia, Oreochromis niloticus, is a gonochoristic teleost fish with an XX/XY genetic system and is an excellent model for gonadal sex differentiation. In the present study, we screened novel genes that were expressed predominantly in either XY or XX undifferentiated gonads during the critical period for differentiation of gonads into ovaries or testes using microarray screening. We focused on one of the isolated 12 candidate genes, #9475, which was an ortholog of gsdf (gonadal soma-derived factor), a member of the transforming growth factor-beta superfamily. #9475/gsdf showed sexual dimorphism in expression in XY gonads before any other testis differentiation-related genes identified in this species thus far. We also overexpressed the #9475/gsdf gene in)0( tilapia, and XX tilapia bearing the #9475/gsdf gene showed normal testis development, which suggests that #9475/gsdf plays an important role in male determination and/or differentiation in tilapia. (C) 2015 Elsevier Ireland Ltd. All rights reserved.
• Characterization and expression of cDNAs encoding P450c17-II (cyp17a2) in Japanese eel during induced ovarian development

  Ting Su, Shigeho Ijiri, Hirokazu Kanbara, Seishi Hagihara, De-Shou Wang, Shinji Adachi

  GENERAL AND COMPARATIVE ENDOCRINOLOGY 221 134 - 143 0016-6480 2015/09 [Refereed][Not invited]
   

  Estradiol-17 beta (E-2) and maturation-inducing hormone (MIH) are two steroid hormones produced in the teleost ovary that are required for vitellogenic growth and final oocyte maturation and ovulation. During this transition, the main steroid hormone produced in the ovary shifts from estrogens to progestogens. In the commercially important Japanese eel (Anguilla japonica), the MIH 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (DHP) is generated from its precursor by P450c17, which has both 17 alpha-hydroxylase and C17-20 lyase activities. In order to elucidate the regulatory mechanism underlying the steroidogenic shift from E-2 to DHP and the mechanistic basis for the failure of this shift in artificially matured eels, the cDNA for cypl7a2-which encodes P450c17-II-was isolated from the ovary of wild, mature Japanese eel and characterized, and the expression patterns of cyp17a1 and cyp17a2 during induced ovarian development were investigated in cultured eel ovaries. Five cDNAs (types I-V) encoding P450c17-II were identified that had minor sequence variations. HEK293T cells transfected with all but type II P450c17-II converted exogenous progesterone to 17 alpha-hydroxyprogesterone (17 alpha-P), providing evidence for 17 alpha-hydroxylase activity; however, a failure to convert 17 alpha-P to androstenedione indicated that C17-20 lyase activity was absent. Cyp17 alpha 2 mRNA was expressed mainly in the head kidney, ovary, and testis, and quantitative PCR analysis demonstrated that expression in the ovary increased during induced vitellogenesis and oocyte maturation/ovulation. In contrast, P450c17-I showed both 17a-hydroxylase and C17-20 lyase activities, and cyp17a1 expression increased until the mid-vitellogenic stage and remained high thereafter. Considering the high level of cyp17a2 transcript in the eel ovary at the migratory nucleus stage together with our previous report demonstrating that eel ovaries have strong 17 alpha-P-to-DHP conversion activity, the failure of artificially maturing eels to produce the maturationinducing DHP may be explained by a deficiency in 17 alpha-P production due to the persistence of cyp17a1 expression after the completion of vitellogenesis. (C) 2015 Elsevier Inc. All rights reserved.
• Sexual characteristics of high-temperature sterilized male Mozambique tilapia, Oreochromis mossanbicus.

  Nakamura M, Nozu R, Ijiri S, Kobayashi T, Hirai T, Yamaguchi Y, Seale A, Lerner DT, Grau GE

  Zoological Letters 1 (21) 1 - 7 2015/06 [Refereed][Not invited]
  
• Histological characteristics of the oocyte chorion in wild post-spawning and artificially matured Japanese eels Anguilla japonica

  Hikari Izumi, Seishi Hagihara, Hiroaki Kurogi, Seinen Chow, Katsumi Tsukamoto, Hirohiko Kagawa, Hideaki Kudo, Shigeho Ijiri, Shinji Adachi

  FISHERIES SCIENCE 81 (2) 321 - 329 0919-9268 2015/03 [Refereed][Not invited]
   

  To describe the histological characteristics of the oocyte chorion in wild adult and artificially matured Japanese eels, we investigated changes in chorion thickness during artificially induced oogenesis and compared the chorion thickness and ultrastructure between wild and artificially matured eels. In artificially maturing eels, the chorion thickness and volume increased significantly with increasing follicle diameter, peaking at approximately 450 A mu m; beyond this point, the chorion thinned significantly, whereas there were no significant changes in volume. A significant positive correlation was observed between the number of salmon pituitary extract (SPE) injections and chorion thickness. In wild post-spawning adult eels, chorion thickness varied among individuals, and two had chorions that were significantly thinner than those of artificially matured eels. Ultrastructural examination revealed electron-dense layers in the chorions of wild post-spawning adult eels, as was seen in artificially matured eels. This result is inconsistent with our hypothesis that the formation of an electron-dense layer is unique to artificially maturing eels due to repeated SPE injections. These results suggest that the formation cycle of the chorion might be affected by SPE injections in artificially maturing eels, whereas that of wild eels might be synchronized with behavioral and/or environmental fluctuations that occur during the oceanic spawning migration.
• Selective depletion of rebosomal RNA from total RNA for next-generation sequencing analysis of maternal mRNA in eggs of the Japanese eel, Anguilla japonica

  Izumi H, Hagihara S, Gen K, Ijiri S, Adachi S

  Bulletin of Fisheries Sciences 北海道大学大学院水産科学研究院 65 (3) 125 - 130 1346-1842 2015 [Not refereed][Not invited]
   

  Removal of ribosomal RNAs(rRNA) from total RNA in eggs before preparation of a RNA library is required for the analysis of maternal mRNA using next-generarion sequencing ; this mRNA is essential for normal development. In mammals, the rRNA-depletion method is well-established and is already being used for the development of experiment kits. However, this method is not designed for other species, e.g., non-model organisms. In the current study, we attempted to remove rRNA from total RNA of Japanese eel Anguilla japonica, which is one of the most important aquaculture species in Japan. Removal was performed via hibridization-selection using mammalian probes from a commmercially available kit and/or an ordinally designed eel probe. Quantitative polymerase chain reaction(PCR) was performed to examine the depletion efficiency of 18S rRNA and 28S rRNA were comparative between each method, and >98% depletion was achieved. In addition, RNA-sequencing analysis using a next-generation sequencer and read-mapping analysis were carried out using the rRNA-depleted RNA, which was prepared using a mammalian probe, and mapping analysis revealed that rRNA reads were <1% of the total reads. These results suggest that an rRNA-depletion kit designed for a mammal can also be used for fish species.
• Morphological and physiological characteristics of an oceanic-migrating Japanese eel, Anguilla japonica off Fukushima, Japan.

  Matsuya N, Hagihara S, Wada T, Ijiri S, Adachi S

  Coastal Marine Science International Coastal Research Center, Atmosphere and Ocean Research Institute, the University of Tokyo 38 (1) 8 - 11 1349-3000 2015 [Refereed][Not invited]
   

  A female silver Japanese eel Anguilla japonica (681 mm TL) was captured by bottom trawl off Fukushima on 18 October 2013. This is the first report of an oceanic-migrating Japanese eel in the western North Pacific Ocean off northern Japan. We described the status of the silvering stage, morphological characteristics, age, developmental stage of the ovaries, and the serum levels of sex steroids for the eel. The GSI of the eel was 2.33 and the mean diameter of the largest group of oocytes was 269.9 μm and these were at the early vitellogenic stage. The serum E2 level was 3.9 ng/ml and 11-KT was 17.9 ng/ml. These steroid levels were higher than the mean steroid levels of female silver eels captured in Hamana Lake, but other morphological and physiological characteristics of the eel were not much different from those of silver eels captured in river, estuary, and coastal waters.
• Identification of genes involved in gonadal sex differentiation and the dimorphic expression pattern in undifferentiated gonads of Russian sturgeon Acipenser gueldenstaedtii Brandt & Ratzeburg, 1833

  S. Hagihara, R. Yamashita, S. Yamamoto, M. Ishihara, T. Abe, S. Ijiri, S. Adachi

  JOURNAL OF APPLIED ICHTHYOLOGY 30 (6) 1557 - 1564 0175-8659 2014/12 [Refereed][Not invited]
   

  The establishment of a sexing technique at an early developmental stage is an important issue in sturgeon aquaculture, yet the mechanism of sex differentiation in sturgeons remains poorly understood. This study aimed to identify genes involved in gonadal sex differentiation and to investigate sexually dimorphic gene expression in undifferentiated gonads. RNA-seq analyses using next-generation sequencers were carried out on undifferentiated gonads of five 9-month-old juvenile Russian sturgeon Acipenser gueldenstaedtii. A total of 45686832 (8498Mb) and 79743269 (7887Mb) quality-controlled reads were obtained using Ion PGM and HiSeq 2000 sequencers, respectively, and were assembled into 338648 contigs (N50: 532 b). tBLASTx analyses identified 26 transcripts potentially involved in gonadal differentiation. Read-mapping analyses were performed to obtain relative transcript levels (RPKM values). Multivariate analyses of the RPKM values of five transcripts (gsdf, dmrt1a, dmrt1b, foxl2 and cyp19a1a) showed that the five individuals could be separated into two distinct groups. One group, comprising two individuals, had increased levels of foxl2, hsd17b1 and cyp19a1a, suggesting differentiation into females. Alternatively, levels of gsdf were higher in the other three individuals, suggesting differentiation into males. We describe the levels of 26 transcripts potentially involved in gonadal differentiation in undifferentiated sturgeon gonads and suggest that levels of gsdf, foxl2, hsd17b1 and cyp19a1a are appropriate sexing markers for sturgeon at an early developmental stage.
• Seasonal changes in oocyte maturational competence, ovulatory competence and gonadotropin sensitivity of ovarian follicles in hybrid sturgeon, bester (Huso huso x Acipenser ruthenus)

  M. Ishihara, B. Tokui, T. Abe, S. Ijiri, S. Adachi

  JOURNAL OF APPLIED ICHTHYOLOGY 30 (6) 1141 - 1148 0175-8659 2014/12 [Refereed][Not invited]
   

  Oocyte maturation and ovulation in sturgeon species are induced by injection of hormones such as the luteinizing hormone-releasing hormone (LHRH). Appropriate timing for the LHRH injection is determined by monitoring the maturational stage of the ovarian follicles. In the present study, ovarian follicles from a group of female bester sturgeon (Huso husoxAcipenser ruthenus) were sampled continuously from September to May and incubated in 100% L-15 medium (pH 8.2) containing 17-hydroxyprogesterone or salmon pituitary extract (SPE) for 48h at 15 degrees C. After incubation, ratios of oocyte maturational competence (OMC; ability of oocytes to undergo final oocyte maturation in response to maturation-inducing steroid, MIS), ovulatory competence (OC; ability of ovarian follicles to ovulate under MIS stimulation), and GTH sensitivity (ability of ovarian follicles to respond to GTH) were examined. Almost all oocytes maintained OMC throughout the sampling period. However, OC was acquired in circa December and May. The ratio of follicles that acquired ovulatory competence showed wide variation among individuals. Most females first acquired a higher degree of ovulatory competence in May, although some only did so around December. SPE-induced oocyte maturation was relatively high throughout the entire experiment, and the ovulation rate showed a tendency similar to that of the ovulatory competence. LHRH-a injections in May successfully induced ovulation in all females. Higher hatching rates resulted in females whose ovarian follicles showed an increasing trend of ovulatory competence and GTH sensitivity for ovulation in May. These results indicate that ovulatory competence and GTH sensitivity can be used as indicators to determine the timing of LHRH injections for sturgeon.
• Development and partial characterisation of an antiserum against apolipoprotein B of the short-finned eel, Anguilla australis

  Erin L. Damsteegt, Hiroko Mizuta, Yuichi Ozaki, Naoshi Hiramatsu, Takashi Todo, Akihiko Hara, Shigeho Ijiri, Shinji Adachi, P. Mark Lokman

  JOURNAL OF COMPARATIVE PHYSIOLOGY B-BIOCHEMICAL SYSTEMIC AND ENVIRONMENTAL PHYSIOLOGY 184 (5) 589 - 599 0174-1578 2014/07 [Refereed][Not invited]
   

  Despite its key role in transportation of triacylglycerides in blood, the distribution, localisation and molecular weight variants of apolipoprotein B (Apob) in teleost fish have essentially escaped study. To address this, a specific short-finned eel (Anguilla australis) Apob antiserum was produced by an immunised rabbit, purified and partially characterised. Localisation of Apob at both the mRNA (in situ hybridisation) and protein (immunohistochemistry) levels mirrored that of mammals; thus immunostaining was confined to the interstitial spaces of the liver and the vascular core of the intestinal villi. Immunostaining of proteins by Western blotting, followed by high-resolution LC-MS, indicated that peptide sequence coverage of Apob in low-density lipoproteins spanned the full-length protein. We conclude that only full-length Apob is produced by eels and that both liver and intestine are key sites for its synthesis.
• Characterization of phase-II conjugation reaction of polycyclic aromatic hydrocarbons in fish species: unique pyrene metabolism and species specificity observed in fish species.

  Yoshinori Ikenaka, Mami Oguri, Aksorn Saengtienchai, Shouta M M Nakayama, Shigeho Ijiri, Mayumi Ishizuka

  Environmental toxicology and pharmacology 36 (2) 567 - 578 1382-6689 2013/09 [Refereed][Not invited]
   

  Metabolic activity, particularly conjugation, was examined in fish by analyzing pyrene (a four-ring, polycyclic aromatic hydrocarbon) metabolites using high-performance liquid chromatography (HPLC) with fluorescence detector (FD), a mass spectrometry (MS) system, and kinetic analysis of conjugation enzymes. Fourteen fresh water fish species, including Danio rerio and Orizias latipes, were exposed to aqueous pyrene, and the resulting metabolites were collected. Identification of pyrene metabolites by HPLC/FD and ion-trap MS indicated that the major metabolites were pyrene glucuronide and pyrene sulfate in all 14 species. Differences were observed in pyrene glucuronide:pyrene sulfate ratio and in the total amount of pyrene conjugates excreted between fish species. Furthermore, a correlation was found between the amount of pyrene glucuronide present and the total amount of the pyrene metabolite eliminated. Kinetic analysis of conjugation by hepatic microsomes in vitro indicated that the differences in excreted metabolites reflected the differences in enzymatic activities.
• Geographic information system-based source estimation of copper pollution in Lake Itezhi-tezhi and metal-accumulation profiles in Oreochromis spp. from both field and laboratory studies.

  Shouta M M Nakayama, Yoshinori Ikenaka, Kaampwe Muzandu, Kennedy Choongo, John Yabe, Taro Muroya, Shigeho Ijiri, Masao Minagawa, Takashi Umemura, Mayumi Ishizuka

  Archives of environmental contamination and toxicology 64 (1) 119 - 29 0090-4341 2013/01 [Refereed][Not invited]
   

  The Copperbelt region, upstream of the Kafue River, including Lake Itezhi-tezhi (ITT), in Zambia has extensive copper (Cu) mines. In our field study, geographic information system analysis in lake sediment indicated that the northern part of the lake, i.e., the Copperbelt region, could be the source of Cu pollution. Concentrations of Cu in stomach contents between fish species were not significantly different. However, Oreochromis spp. liver showed significantly greater Cu concentrations than those in other fish species. Log liver [Cu], standard length, and nitrogen stable isotope ratio were positively correlated only in Oreochromis spp. In the laboratory study, O. niloticus and O. latipes were exposed to Cu for 4 days, and recovery phases ≤ 28 days were examined. O. niloticus showed significantly greater concentrations of Cu compared with O. latipes at all sampling points. Significantly greater concentrations of Hg in Schilbe intermedius liver than for other fish species were observed, whereas O. macrochir showed significantly greater concentrations of cadmium. In conclusion, the northern part of the lake could be the source of Cu pollution in Lake ITT. Diet may not be the reason for high Cu accumulation in Oreochromis spp. Results from both field and laboratory studies imply that Oreochromis spp. contain high concentrations of Cu under normal physiological conditions.
• Expression Patterns of Gonadotropin Hormones and Their Receptors During Early Sexual Differentiation in Nile Tilapia Oreochromis niloticus

  Hongwei Yan, Shigeho Ijiri, Quan Wu, Tohru Kobayashi, Shuang Li, Taro Nakaseko, Shinji Adachi, Yoshitaka Nagahama

  BIOLOGY OF REPRODUCTION 87 (5) 116  0006-3363 2012/11 [Refereed][Not invited]
   

  In Nile tilapia, sex-specific expression of foxl2 and cyp19a1a in XX gonads and dmrt1 in XY gonads at 5-6 days after hatching (dah) is critical for differentiation of the gonads into either ovaries or testes. The factors triggering sexually dimorphic expression of these genes are unknown, and whether the gonadotropin hormones are involved in early gonadal sex differentiation of the Nile tilapia has been unclear. In the present study, we determined the precise timing of expression of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the pituitary and that of their receptors (fshra and lhcgrbb) in the undifferentiated gonad in both XX and XY tilapia fry by quantitative RT-PCR and immunohistochemical analysis. Expression of fshb mRNA and Fsh protein in the pituitary was detected from the first sampling day (3 dah) to 25 dah in both XX and XY tilapia larvae without sexual dimorphism and increased gradually after 25 dah in the pituitary. fshra mRNA was expressed beginning 5 dah and was present at significantly higher levels in XX gonads than in the XY gonads at 6-25 dah. These results indicate that the level of Fsh protein in the pituitary was not critical for differentiation of gonads into ovaries or testes, but the expression level of its receptor, fshra, in undifferentiated gonads appeared to be involved in determining gonadal sexual differentiation. Based on these observations, it is likely that in XX gonads, up-regulation of fshra may be necessary to induce cyp19a1a expression, which stimulates estradiol-17beta (E-2) production and subsequent ovarian differentiation. On the other hand, lhb mRNA was not detected until 25 dah in the pituitaries of both sexes, and sexual dimorphism in lhcgrbb mRNA levels appeared later (10-25 dah) than that of fshra in the gonads, indicating the limited role of LH and lhcgrbb in gonadal differentiation of the Nile tilapia.
• 11-ketotestosterone Synchronously Induces Oocyte Development and Silvering-related Changes in the Japanese Eel, Anguilla japonica

  Ryusuke Sudo, Ryota Tosaka, Shigeho Ijiri, Shinji Adachi, Jun Aoyama, Katsumi Tsukamoto

  ZOOLOGICAL SCIENCE 29 (4) 254 - 259 0289-0003 2012/04 [Refereed][Not invited]
   

  To evaluate the effects of sex steroids on silvering in the Japanese eel, Anguilla japonica, the development of oocytes, eye size, digestive tract, and swim bladder were studied in relation to observations of the profiles of plasma levels of sex steroids (estradiol 17 beta, E2; testosterone, T; 11-ketotestosterone; 11-KT) during silvering for each sex and by administrating 11-KT to yellow eels. All steroids examined in the study increased in female eels after silvering had begun, whereas in males, only 11-KT increased significantly, and no statistical differences were found in plasma levels of E2 and T between eels in both developmental stages. 11-KT appeared to induce the early stage of oocyte growth, enlargement of the eyes, degeneration of the digestive tract and the development of the swim bladder. This suggested that 11-KT synchronously accelerates early development of the ovaries and the morphological changes, possibly in adaption to oceanic migration, and that 11-KT is one of the most important factors in early stages of development in the Japanese eel, as it appears to be in other anguillid eels.
• Ovarian steroidogenesis and the role of sex steroid hormones on ovarian growth and maturation of the Japanese eel

  Yukinori Kazeto, Ryota Tosaka, Hajime Matsubara, Shigeho Ijiri, Shinji Adachi

  JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY 127 (3-5) 149 - 154 0960-0760 2011/11 [Refereed][Invited]
   

  Three sex steroid hormones, estradiol-17 beta (E2), 11-ketotestosterone (11-KT), and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (DHP), are well established as primary estrogen, androgen, and progestin, respectively, in teleost fish. Japanese eel, Anguilla japonica, would be a suitable candidate to study ovarian steroid physiology of fish because the ovarian growth and steroidogenesis is dormant under laboratory condition but can be induced by administration of exogenous gonadotropic reagents. In this review, we summarized our work on the function and production of sex steroid hormones in the ovary of the Japanese eel during ovarian growth and oocyte maturation artificially induced by treatment with extract of salmon pituitary. In vitro and in vivo assays suggest that 11-KT and E2 play primary roles in previtellogenic and vitellogenic growth of oocytes, respectively, whereas DHP is essential for induction of final oocyte maturation. We also reviewed the correlation between ovarian steroidogenesis to produce these sex steroid hormones, serum titers and gene expression. (C) 2011 Elsevier Ltd. All rights reserved.
• Effect of temperature decrease on oocyte development, sex steroids, and gonadotropin beta-subunit mRNA expression levels in female Japanese eel Anguilla japonica

  Ryusuke Sudo, Ryota Tosaka, Shigeho Ijiri, Shinji Adachi, Hiroaki Suetake, Yuzuru Suzuki, Noriyuki Horie, Satoru Tanaka, Jun Aoyama, Katsumi Tsukamoto

  FISHERIES SCIENCE 77 (4) 575 - 582 0919-9268 2011/07 [Refereed][Not invited]
   

  To improve understanding of the mechanism of early ovarian development in eels, the effects of water temperature decrease on oocyte development, plasma levels of sex steroids [estradiol 17 beta (E2), testosterone (T), 11-ketotestosterone (11-KT)], and gonadotropin beta-subunit [follicle-stimulating hormone (FSH beta), luteinizing hormone (LH beta)] messenger RNA (mRNA) expression levels were investigated. A total of 27 female Japanese eels Anguilla japonica were divided into initial, control, and test (water temperature decrease) groups. Starting on 22 September 2009, eels in the test group were reared in a tank with gradual temperature decrease from 25A degrees C to 15A degrees C over 39 days, while the control group was maintained at 25A degrees C. The test group accumulated more oil droplets in their oocytes than did the other groups. Levels of sex steroids, especially 11-KT, were higher in the test group. In contrast, FSH beta and LH beta mRNA expression levels were lower in the test group. These results suggest that water temperature decrease only induced an early stage of ovarian development that was partly affected by an 11-KT increase. For further maturation, other environmental factors related to induction of gonadotropin increase appear to be needed.
• Effect of temperature decrease on oocyte development, sex steroids, and gonadotropin beta-subunit mRNA expression levels in female Japanese eel Anguilla japonica

  Ryusuke Sudo, Ryota Tosaka, Shigeho Ijiri, Shinji Adachi, Hiroaki Suetake, Yuzuru Suzuki, Noriyuki Horie, Satoru Tanaka, Jun Aoyama, Katsumi Tsukamoto

  FISHERIES SCIENCE 77 (4) 575 - 582 0919-9268 2011/07 [Refereed][Not invited]
   

  To improve understanding of the mechanism of early ovarian development in eels, the effects of water temperature decrease on oocyte development, plasma levels of sex steroids [estradiol 17 beta (E2), testosterone (T), 11-ketotestosterone (11-KT)], and gonadotropin beta-subunit [follicle-stimulating hormone (FSH beta), luteinizing hormone (LH beta)] messenger RNA (mRNA) expression levels were investigated. A total of 27 female Japanese eels Anguilla japonica were divided into initial, control, and test (water temperature decrease) groups. Starting on 22 September 2009, eels in the test group were reared in a tank with gradual temperature decrease from 25A degrees C to 15A degrees C over 39 days, while the control group was maintained at 25A degrees C. The test group accumulated more oil droplets in their oocytes than did the other groups. Levels of sex steroids, especially 11-KT, were higher in the test group. In contrast, FSH beta and LH beta mRNA expression levels were lower in the test group. These results suggest that water temperature decrease only induced an early stage of ovarian development that was partly affected by an 11-KT increase. For further maturation, other environmental factors related to induction of gonadotropin increase appear to be needed.
• Controlled reproduction in the Japanese eel (Anguilla japonica), past and present

  Ijiri Shigeho, Tsukamoto Katsumi, Chow Seinen, Kurogi Hiroaki, Adachi Shinji, Tanaka Hideki

  Aquaculture Europe the European Aquaculture Society 36 (2) 13 - 17 2011/06 [Not refereed][Invited]
   

  The Japanese eel, Anguilla japonica, is one of the mostimportant species of the aquaculture industry of EastAsia. Supply of glass eels for aquaculture is completely dependent on wild catch. However, glass-eel catches in Japan declined linearly from over 200 tons in the early 1960s to 20 tons at present. In recent years an unstable situation has occurred with glass-eel catches not meeting the demand of aquaculture farms. In order to address this rapid decline, a challenging research project for artificialproduction of glass eels was commenced in the late 1960s.Since then, through a continuing process of trial anderror, the production of second-generation larvae wasfinally achieved in 2010. Throughout the research, a new application of several substances has caused breakthroughs in artificial induction of sexual maturation and larvae rearing. This article reports on a study of controlled eel reproduction focusing onthe finding of substances that accelerated progress. Inaddition, guideline research on eel reproduction in thewild was conducted aiming at overcoming currentbottlenecks that impeded the establishment of largescale glass-eel production in captivity, by investigating maturing eels and eggs collected in their spawning area in the vicinity of the West Mariana Ridge.
• Androgens and Very Low Density Lipoprotein Are Essential for the Growth of Previtellogenic Oocytes from Japanese Eel, Anguilla japonica, In Vitro

  Taku Endo, Takashi Todo, P. Mark Lokman, Hideaki Kudo, Shigeho Ijiri, Shinji Adachi, Kohei Yamauchi

  BIOLOGY OF REPRODUCTION 84 (4) 816 - 825 0006-3363 2011/04 [Refereed][Not invited]
   

  To investigate the regulation of lipid uptake into the eel oocyte in more detail, effects of 11-ketotestosterone (11-KT) and lipid transporters (lipoproteins) were determined in vitro. Ovarian explants from previtellogenic Japanese eels (Anguilla japonica) were incubated for 28 days with 11-KT and/or with very low density lipoproteins (Vldl), low density lipoproteins (Ldl), or high density lipoproteins (Hdl) purified from eel plasma. The androgen 11-KT induced notable increases in oocyte diameter, which were accompanied by the appearance of vacuoles rather than lipid. Ldl and Hdl increased oocyte diameters, whereas Vldl did not. However, coincubation of 11-KT and Vldl, but not of Ldl or Hdl, resulted in dramatic increases in oocyte size and lipid droplet surface area. Effects of both 11-KT (oocyte size) and Vldl (lipid droplet surface area) were dose-dependent between 1 and 100 ng/ml and between 0.5 and 5 mg/ml, respectively. Interestingly, abnormal oocyte cytology under conditions of coculture with 11-KT and Vldl could essentially be prevented if Vldl concentrations were high enough (>= 5 mg Vldl/ml medium). Unlike 11-KT, estradiol-17beta had no effect on oocyte diameter or lipid droplet surface area. We conclude that Vldl is a key transporter of neutral lipids that accumulate into the eel oocyte during oogenesis and that Vldl-dependent lipid uptake is stimulated by the androgen 11-KT.
• Oceanic spawning ecology of freshwater eels in the western North Pacific

  Katsumi Tsukamoto, Seinen Chow, Tsuguo Otake, Hiroaki Kurogi, Noritaka Mochioka, Michael J. Miller, Jun Aoyama, Shingo Kimura, Shun Watanabe, Tatsuki Yoshinaga, Akira Shinoda, Mari Kuroki, Machiko Oya, Tomowo Watanabe, Kazuhiro Hata, Shigeho Ijiri, Yukinori Kazeto, Kazuharu Nomura, Hideki Tanaka

  NATURE COMMUNICATIONS 2 179  2041-1723 2011/02 [Refereed][Not invited]
   

  The natural reproductive ecology of freshwater eels remained a mystery even after some of their offshore spawning areas were discovered approximately 100 years ago. In this study, we investigate the spawning ecology of freshwater eels for the first time using collections of eggs, larvae and spawning-condition adults of two species in their shared spawning area in the Pacific. Ovaries of female Japanese eel and giant mottled eel adults were polycyclic, suggesting that freshwater eels can spawn more than once during a spawning season. The first collection of Japanese eel eggs near the West Mariana Ridge where adults and newly hatched larvae were also caught shows that spawning occurs during new moon periods throughout the spawning season. The depths where adults and newly hatched larvae were captured indicate that spawning occurs in shallower layers of 150-200 m and not at great depths. This type of spawning may reduce predation and facilitate reproductive success.
• Eel and sturgeon, unmanageable species in seeding production.

  IJIRI Shigeho

  Nippon Suisan Gakkaishi 36 13 - 17 2011 [Not refereed][Not invited]
  
• Expression of androgen receptor mRNA in the ovary of Japanese eel, Anguilla japonica, during artificially induced ovarian development

  Ryota Tosaka, Takashi Todo, Yukinori Kazeto, P. Mark Lokman, Shigeho Ijiri, Shinji Adachi, Kohei Yamauchi

  GENERAL AND COMPARATIVE ENDOCRINOLOGY 168 (3) 424 - 430 0016-6480 2010/09 [Refereed][Not invited]
   

  In order to elucidate how androgens may mediate their effects on ovarian growth, we investigated the mRNA levels of two subtypes of androgen receptor (ara and arb) in the ovary of feminized Japanese eel (Anguilla japonica) during artificially induced ovarian development by quantitative real-time reverse transcriptase polymerase chain reaction and in situ hybridization. Ara mRNA levels were high from the late oil droplet stage to the late vitellogenic stage, whereas arb mRNA levels were high from the late oil droplet stage to the midvitellogenic stage. Both or mRNAs were predominantly observed in the follicle cells and the epithelial cells of the ovigerous lamellae in all stages. In the oil droplet stage, oogonia exhibited intense signals for or mRNAs. There was no obvious difference in localization pattern between ara and orb in all ovaries examined, irrespective of maturational stage. It was difficult to identify the follicle cell types that were positive for or mRNA during ovarian development. Only in post-ovulatory follicles could theca and granulosa cells be clearly identified, and ar signals were observed in both layers. The predominant localization of ar mRNA in the follicle cells suggests that androgens play important roles in oocyte growth by acting on these cells in this species. We have shown the expression profile and localization of or mRNA during ovarian development for the first time in an oviparous vertebrate. (C) 2010 Elsevier Inc. All rights reserved.
• The Relationship between the Developmental Stage of Oocytes in Various Seasons and the Quality of the Egg Obtained by Artificial Maturation in the Feminized Japanese Eel Anguilla japonica

  CHAI YU, TOSAKA RYOTA, ABE TOMOKI, SAGO KEISUKE, SAGO YOSUKE, HATANAKA ERI, IJIRI SHIGEHO, ADACHI SHINJI

  Aquaculture Science 水産増殖談話会 58 (2) 269 - 278 0371-4217 2010/06 [Refereed][Not invited]
   

  雌化ウナギ2歳魚を9月、12月または4月にサケ脳下垂体注射によって人為催熟し、得られる卵質の差を受精率、孵化率および8日後生存率で評価した。9月、12月は比較的良質な卵が得られる傾向が認められたが、4月では卵は得られるものの、その卵質は極めて悪かった。催熟前の卵母細胞を組織学的に観察すると、12月に卵黄形成開始が観察されたが、4月には退行している様子が観察された。4月に催熟を開始しても良質な卵が得られない原因として、催熟前に既に卵母細胞が退行しつつあることが考えられた。以上の結果から、雌化ウナギ2歳魚の人為催熟は9月から12月の間にかけて開始することが良質な卵を得るために適当であると結論された。
• Development of an in vitro culture system for producing eel larvae from immature ovarian follicles in Japanese eel Anguilla japonica

  Tomoki Abe, Shigeho Ijiri, Shinji Adachi, Kohei Yamauchi

  FISHERIES SCIENCE 76 (2) 257 - 265 0919-9268 2010/03 [Refereed][Not invited]
   

  To standardize conditions during the final maturation and ovulation of ovarian follicles from Japanese eel, we have developed a culture system for the production of fertilizable eggs from post-vitellogenic ovarian follicles in vitro. Post-vitellogenic ovarian follicles were incubated in culture medium supplemented with 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (DHP) with or without bovine serum albumin (BSA) to assess the effects of protein concentration. Eggs that ovulated during incubation were fertilized, and the remaining follicles were incubated in prostaglandin F(2 alpha) (PGF(2 alpha)) for a further 3 or 6 h before fertilization. Male eels were injected repeatedly with human chorionic gonadotropin. The quality of eggs obtained under the different culture conditions was evaluated after artificial fertilization in terms of hatching success. Hatching rates tended to decrease with increasing concentrations of BSA in the incubation medium in a dose-dependent manner. The addition of PGF2a drastically increased the number of eggs that ovulated, but the rate of hatching was greatly decreased compared with eggs obtained earlier by DHP incubation alone. The larvae obtained from artificially fertilized eggs produced in vitro survived for 14 days without feeding. We conclude that in vitro culture systems thus have a great potential for the acquisition of good quality eggs under tightly controlled artificial conditions, culminating in the production of eel larvae.
• Effects of reproductive stage, GH, and 11-ketotestosterone on expression of growth differentiation factor-9 in the ovary of the eel, Anguilla australis

  P. M. Lokman, Y. Kazeto, Y. Ozaki, S. Ijiri, R. Tosaka, M. Kohara, S. L. Divers, H. Matsubara, L. G. Moore, S. Adachi

  REPRODUCTION 139 (1) 71 - 83 1470-1626 2010/01 [Refereed][Not invited]
   

  In order to study the regulation of the growth differentiation factor-9 (gdf9) gene in a primitive teleost with semelparous life history, we cloned a cDNA encoding shortfinned eel Gdf9, expressed a partial peptide in Escherichia coli, and raised an antiserum to evaluate changes in Gdf9 expression during its pituitary homogenate-induced reproductive cycle. The effects of in vivo and in vitro exposure to the androgen 11-ketotestosterone (11-KT), known to affect previtellogenic (PV) oocyte growth, were also determined. Furthermore, we investigated whether Gdf9 expression was metabolically gated by treating PV fish with recombinant GH in vivo. Immunoreactive proteins of ca. 52 and 55 kDa were identified by western blot analysis. Gdf9 message and protein were most abundant in PV oocytes, and peaked slightly earlier for mRNA than for protein. Captivity resulted in reduced gdf9 mRNA levels, which were restored following pituitary homogenate treatment. As oocytes progressed through induced oogenesis, Gdf9 expression decreased. Neither 11-KT nor GH treatment affected gdf9 mRNA levels in PV fish, although GH could partially restore handling- or captivity-induced decreases in gdf9 mRNA levels. Semelparous eels thus show an expression pattern of Gdf9 during oogenesis that is similar to that seen in other vertebrates, that appears responsive to handling or captivity stress, and whose control remains to he elucidated. Reproduction (2010) 139 71-83
• Molecular Cloning, Characterization, and Evolutionary Analysis of Estrogen Receptors from Phylogenetically Ancient Fish

  Yoshinao Katsu, Satomi Kohno, Susumu Hyodo, Shigeho Ijiri, Shinji Adachi, Akihiko Hara, Louis J. Guillette, Taisen Iguchi

  ENDOCRINOLOGY 149 (12) 6300 - 6310 0013-7227 2008/12 [Refereed][Not invited]
   

  Estrogens are necessary for ovarian differentiation during a critical developmental stage in many vertebrates, and they promote the growth and differentiation of the adult female reproductive system. To understand the evolution of vertebrate estrogen receptors (ESRs) and to evaluate estrogen receptor-ligand interactions in phylogenetically ancient fish, we used PCR techniques to isolate the cDNA encoding ESRs from lungfish, sturgeon, and gar. Sequence analyses indicate that these fishes have two ESRs, ESR1 (ER alpha) and ESR2 ( ER beta),as previously reported for other vertebrate species, but a second type of ESR2 (ER beta 2) was not found as has been reported in a number of teleost fishes. Phylogenetic analysis of the ESR sequences indicated that the lungfish ESRs are classified to the tetrapod ESR group, not with the teleost fish ESRs as are the ESRs from gar and sturgeon. Using transient transfection assays of mammalian cells, ESR proteins from these three ancient fishes displayed estrogen-dependent activation of transcription from an estrogen-responsive-element containing promoter. We also examined the estrogenic potential of o,p'-dichloro-diphenyl-trichloroethane (o,p'-DDT) and p,p'-DDT as well as one of its common metabolites, p,p'-dichloro-diphenyl-ethylene (p,p'-DDE) on the ESRs from these fishes. Lungfish ESR1 was less sensitive to DDT/DDE than the ESR1 from the other two fishes. The response of lungfish ESR1 to these pesticides is similar to the pattern obtained from salamander ESR1. These data provide a basic tool allowing future studies examining the receptor-ligand interactions and endocrine-disrupting mechanisms in three species of phylogenetically ancient fish and also expands our knowledge evolution. ( Endocrinology 149: 6300-6310, 2008)
• In vitro induction of oil droplet accumulation into previtellogenic oocytes of Japanese eel, Anguilla japonica

  Taku Endo, Takashi Todo, P. Mark Lokman, Shigeho Ijiri, Shinji Adachi, Kohei Yamauchi

  CYBIUM 32 (2) 239 - 240 0399-0974 2008/07 [Refereed][Not invited]
   

  Accumulation of oil droplets in previtellogenic oocytes of Japanese eel, Anguilla japonica, could be induced in vitro. Co-treatment with very low-density lipoprotein (VLDL) and 11-ketotestosterone (11-KT) resulted in significant oil droplet accumulation. It is therefore suggested that lipids in oil droplets originate, at least in part, from VLDL, and that 11-KT plays an important role in their transfer and/or accumulation into oocytes.
• Early development of forebrain gonadotrophin-releasing hormone (GnRH) neurones and the role of GnRH as an autocrine migration factor

  E. Abraham, O. Palevitch, S. Ijiri, S. J. Du, Y. Gothilf, Y. Zohar

  JOURNAL OF NEUROENDOCRINOLOGY 20 (3) 394 - 405 0953-8194 2008/03 [Refereed][Not invited]
   

  Normal migration of the gonadotrophin-releasing hormone (GnRH) neurones during early development, from the olfactory region to the hypothalamus, is crucial for reproductive development in all vertebrates. The establishment of the GnRH system includes tangential migration of GnRH perikarya as well as extension of GnRH fibres to various areas of the central nervous system (CNS). The exact spatio-temporal nature of this process, as well as the factors governing it, are not fully understood. We studied the development of the GnRH system and the effects of GnRH knockdown using a newly developed GnRH3:EGFP transgenic zebrafish line. We found that enhanced green fluorescent protein is specifically and robustly expressed in GnRH3 neurones and fibres. GnRH3 fibres in zebrafish began to extend as early as 26 h post-fertilisation and by 4-5 days post-fertilisation had developed into an extensive network reaching the optic tract, telencephalon, hypothalamus, midbrain tegmentum and hindbrain. GnRH3 fibres also innervated the retina and projected into the trunk via the spinal cord. GnRH3 perikarya were observed migrating along their own fibres from the olfactory region to the preoptic area (POA) via the terminal nerve ganglion and the ventral telencephalon. GnRH3 cells were also observed in the trigeminal ganglion. The establishment of the GnRH3 fibre network was disrupted by morpholino-modified antisense oligonucleotides directed against GnRH3 causing abnormal fibre development and pathfinding, as well as anomalous GnRH3 perikarya localisation. These findings support the hypothesis that GnRH3 neurones migrate from the olfactory region to the POA and caudal hypothalamus. Novel data regarding the early development of the GnRH3 fibre network in the CNS and beyond are described. Moreover we show, in vivo, that GnRH3 is an important factor regulating GnRH3 fibre pathfinding and neurone localisation in an autocrine fashion.
• Sexual dimorphic expression of genes in gonads during early differentiation of a teleost fish, the Nile tilapia Oreochromis niloticus

  Shigeho Ijiri, Hiroyo Kaneko, Tohru Kobayashi, De-Shou Wang, Fumie Sakai, Bindhu Paul-Prasanth, Masaru Nakamura, Yoshitaka Nagahama

  BIOLOGY OF REPRODUCTION 78 (2) 333 - 341 0006-3363 2008/02 [Refereed][Not invited]
   

  The Nile tilapia, a gonochoristic teleost fish with an XX/XY sex-determining system, provides an excellent model for studying gonadal sex differentiation because genetic all-females and all-males are available. In this study, we used quantitative real-time RT-PCR to determine the precise timing of the gonadal expression of 17 genes thought to be associated with gonadal sex differentiation in vertebrates. Gonads were isolated from all-female and all-male tilapia before (5-15 days after hatching [dah]) and after (25-70 dab) morphological sex differentiation. The transcript of aromatase (cyp19a1a), an enzyme responsible for producing estradiol-17beta, was expressed only in XX gonads at 5 dah, with a marked elevation in expression thereafter. In contrast, mRNA expression of steroid 11beta-hydroxylase (cyp11b2), an enzyme responsible for the synthesis of 1 ketotestosterone (11-KT, a potent androgen in fish), was found in XY gonads from 35 dab only. These results, combined with the presence of transcripts for other steroidogenic enzymes and estrogen receptors in XX gonads at 5-7 dah, are consistent with our earlier suggestion that estradiol-17beta plays a critical role in ovarian differentiation in tilapia, whereas a role for 11-KT in testicular differentiation is questionable. A close relationship between the expression of foxl2, but not nr5a1 (Ad4BP/SF-1), and that of cyp19a1a in XX gonads suggests an important role for Foxl2 in the transcriptional regulation of cyp19a1a. Dmrt1 exhibited a male-specific expression in XY gonads from 6 dab onward, suggesting an important role for Dmrt1 in testicular differentiation. Sox9 and amh (anti-Mullerian hormone) showed a testis-specific expression, being evident only in the later stages of testicular differentiation. It is concluded that the sex-specific expression of fox12 and cyp19a1a in XX gonads and dmrt1 in XY gonads during early gonadal differentiation (5-6 dab) is critical for undifferentiated gonads to differentiate into either the ovary or testis in the Nile tilapia.
• The effect of female presence upon serum levels of vitellogenin in male Japanese whiting Sillago japonica

  Komei Hotta, Takayuki Watanabe, Chiho Kishida, Yukio Nakamura, Shigeho Ijiri, Shinji Adachi, Kohei Yamauchi

  NIPPON SUISAN GAKKAISHI 公益社団法人日本水産学会 74 (1) 20 - 25 0021-5392 2008/01 [Refereed][Not invited]
   

  To investigate the potential effects of female presence upon serum levels of vitellogenin (Vg) in male fish, we held male Japanese whiting Sillago japonica in the presence (Group FM) or absence (Group M) of females for three weeks. Fish were first reared for three weeks after the grouping and then sampled from each group at four-hourly intervals. Analyses were performed on serum vitellogenin levels, gonad somatic index (GSI: gonad weight/body weight x 100), and testicular histology. Serum levels of Vg in males from Group FM were higher than those of males from Group M. Moreover, GSI of Group FM males was also higher than males in Group M, although histological analysis demonstrated active spermatogenesis in the testes of males from both groups. These results suggest that the presence of females causes an increase in the serum levels of Vg and GST in male fish.
• Liver receptor homologue-1 (LRH-1) activates the promoter of brain aromatase (cyp19a2) in a Teleost fish, the Medaka, Oryzias latipes

  Yuki Ohmuro-Matsuyama, Kataaki Okubo, Masaru Matsuda, Shigeho Ijiri, Deshou Wang, Guijun Guan, Taiga Suzuki, Makoto Matsuyama, Ken-Ichiro Morohashi, Yoshitaka Nagahama

  MOLECULAR REPRODUCTION AND DEVELOPMENT 74 (9) 1065 - 1071 1040-452X 2007/09 [Refereed][Not invited]
   

  The medaka, Oryzias latipes, like other fish, have two distinct aromatase genes, the ovarian (cyp19a1) and brain (cyp19a2) forms. We previously reported that Ad4BP/SF-1, a member of the NR5A subfamily, plays an important role in the regulation of cyp19a1 expression in medaka ovarian follicles during vitellogenesis. In the present study, we investigated whether liver receptor homologue-1 (LRH-1), another NR5A subfamily member, is involved in the regulation of cyp19a2 expression in the medaka brain. In situ hybridization analysis revealed that LRH-1 was expressed in the hypothalamus, where it colocalized with aromatase (cyp19a2). We then showed by transient transfection assays that LRH-1 was able to increase expression of a cyp19a2 reporter gene in various mammalian cell lines, and that mutation of a putative LRH-1 binding site within the cyp19a2 promoter abolished this effect. Taken together, these findings suggest that LRH-1 plays a role in regulating cyp19a2 expression in the medaka brain. This is the first to demonstrate in vitro the activation of brain aromatase by LRH-1 in the vertebrate brain.
• Foxl2 up-regulates aromatase gene transcription in a female-specific manner by binding to the promoter as well as interacting with Ad4 binding protein/steroidogenic factor 1

  De-Shou Wang, Tohru Kobayashi, Lin-Yan Zhou, Bindhu Paul-Prasanth, Shigeho Ijiri, Fumie Sakai, Kataaki Okubo, Ken-ichirou Morohashi, Yoshitaka Nagahama

  MOLECULAR ENDOCRINOLOGY 21 (3) 712 - 725 0888-8809 2007/03 [Refereed][Not invited]
   

  Increasing evidence suggests the crucial role of estrogen in ovarian differentiation of nonmammalian vertebrates including fish. The present study has investigated the plausible role of FoxI2 in ovarian differentiation through transcriptional regulation of aromatase gene, using monosex fry of tilapia. FoxI2 expression is sexually dimorphic, like Cyp19a1, colocalizing with Cyp19a1 and Ad4BP/ SF-1 in the stromal cells and interstitial cells in gonads of normal XX and sex-reversed XY fish, before the occurrence of morphological sex differentiation. Under in vitro conditions, FoxI2 binds to the sequence ACAAATA in the promoter region of the Cyp19a1 gene directly through its forkhead domain and activates the transcription of Cyp19a1 with its C terminus. FoxI2 can also interact through the forkhead domain with the ligand-binding domain of Ad4BP/SF-1 to form a heterodimer and enhance the Ad4BP/SF-1 mediated Cyp19a1 transcription. Disruption of endogenous FoxI2 in XX tilapia by overexpression of its dominant negative mutant (M3) induces varying degrees of testicular development with occasional sex reversal from ovary to testis. Such fish display reduced expression of Cyp19a1 as well as a drop in the serum levels of 17 beta-estradiol and 11-ketotestosterone. Although the XY fish with wild-type tilapia FoxI2 (tFoxI2) overexpression never exhibited a complete sex reversal, there were significant structural changes, such as tissue degeneration, somatic cell proliferation, and induction of aromatase, with increased serum levels of 17 beta-estradiol and 11-ketotestosterone. Altogether, these results suggest that FoxI2 plays a decisive role in the ovarian differentiation of the Nile tilapia by regulating aromatase expression and possibly the entire steroidogenic pathway.
• Sex determination and gonadal sex differentiation in fish

  IJIRI Shigeho

  ホルモンと臨牀 54 (6) 479 - 487 0045-7167 2006/06 [Not refereed][Invited]
  
• Molecular biology of ovarian aromatase in sex reversal: Complementary DNA and 5 '-flanking region isolation and differential expression of ovarian aromatase in the Gilthead Seabream (Sparus aurata)

  TT Wong, S Ijiri, Y Zohar

  BIOLOGY OF REPRODUCTION 74 (5) 857 - 864 0006-3363 2006/05 [Refereed][Not invited]
   

  To elucidate the involvement of aromatase in sex reversal, the gilthead seabream ovarian P450 aromatase (cyp19a1a) cDNA and its 5'-flanking region were isolated and characterized. Northern blot analysis revealed that only one cyp19a1a transcript (2.0 kb) is expressed in the ovary. Four cAMP-responsive elements were identified at the 5'-flanking region of seabream cyp19a1a indicating a high potential to respond to gonadotropin signaling. Studying the seasonal profile, two expression peaks of cyp19ala transcripts in the ovarian tissues were found in July (about 15000 copies/ng total RNA) for ambisexual fish and in December (about 12 000 copies/ng total RNA) for spawning females. Starting from September, transcript levels of cyp19ala in the ovarian portions of the male-developing gonads gradually decreased. Furthermore, the ovarian portions of the female gonads expressed cyp19a1a at a significantly higher level than the ovarian portions of the male gonads after November. Taken together with levels of plasma estradiol in reversing females being significantly higher than those in developing males, the above results reinforce the importance of cyp19ala in sex reversal. In vitro exposure of ovarian fragments to gonadotropins (hCG) at 1, 10, and 100 IU/ml significantly (P < 0.05) upregulated cyp19a1a expression. Additionally, expression of cyp19a1a displayed a stronger and significant correlation with the transcript expression of ovarian Lh receptor rather than Fsh receptor during the ambisexual stage. Our results indicate that the differential expression of cyp19a1a gene is associated with sex reversal and that gonadotropin signals (particularly Lh) may serve as major players in regulating the expression of cyp19ala during the process of sex reversal.
• Cloning and characterization of a cDNA encoding cholesterol side-chain cleavage cytochrome P450 (CYP11A1): Tissue-distribution and changes in the transcript abundance in ovarian tissue of Japanese eel, Anguilla japonica, during artificially induced sexual development

  Y Kazeto, S Ijiri, S Adachi, K Yamauchi

  JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY 99 (2-3) 121 - 128 0960-0760 2006/05 [Refereed][Not invited]
   

  Cholesterol side-chain cleavage cytochrome P450 (CYP11A1:P450scc) is a crucial steroidogenic enzyme that catalyzes an initial step in the production of all classes of steroids. A cDNA encoding Japanese eel P450scc was cloned and characterized. The cDNA putatively encoded 521 amino acid residues with high homology to those of other vertebrate forms. The recombinant P450scc produced in COS-7 cells efficiently catalyzed the conversion of 25-hydroxycholesterol into pregnenolone. By northern blot, a single P450scc transcript of approximately 3.3 kb was detected in both ovary and head kidney. Transcript levels of this enzyme significantly increased throughout ovarian development artificially induced by salmon pituitary homogenate, which suggests that gonadotropic stimuli can induce ovarian expression of the P450scc gene in teleosts, as has been reported in mammals. Furthermore, RT-PCR analysis revealed that gene expression of three steroidogenic enzymes, P450scc, P450c17 and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) show distinctly different tissue-specific patterns of expression in the Japanese eel. The P450scc gene was expressed in ovary and head kidney while the sole source of the P450c17 transcript was ovary. In contrast, 3P-HSD transcript was detected in all tissues examined, brain, liver, spleen and trunk kidney, etc. These suggest that some steroidogenic enzymes are also expressed in non-endocrine tissues and could potentially regulate the local and/or circulating steroid levels in teleosts, as they do in mammals. (c) 2006 Elsevier Ltd. All rights reserved.
• Changes in localization of cytochrome P450 cholesterol side-chain cleavage (P450scc) in Japanese eel testis and ovary during gonadal development

  S Ijiri, N Takei, Y Kazeto, T Todo, S Adachi, K Yamauchi

  GENERAL AND COMPARATIVE ENDOCRINOLOGY 145 (1) 75 - 83 0016-6480 2006/01 [Refereed][Not invited]
   

  In this study, we generated and characterized a polyclonal antiserum against eel P450 cholesterol side-chain cleavage (P450scc) using a recombinant protein as the antigen. We examined the localization and abundance of P450scc by immunohistochemistry in Japanese eel testes and ovaries during artificially induced gonadal development. P450scc mRNA localization was also examined by in situ hybridization. In male eels, testicular development was induced by a single injection of human chorionic gonadotropin (HCG). In females, ovarian development was induced by weekly injections of salmon pituitary homogenate (SPH). Before HCG injection, the testis contained germ cells that were primarily type A spermatogonia. Additionally, several clusters of immunoreactive cells for P450scc were localized in the interstitial Leydig cells, but no P450scc mRNA signals were detected. This suggests that P450scc is either a relatively stable protein or it is produced by a mRNA that is present at too low a level to detect. Shortly after a single injection of HCG, expression of P450scc mRNA was stimulated and the number of immunoreactive clusters and their staining intensity were both increased. P450scc mRNA fell to an undetectable level 3 days after hormonal stimulation. Although the P450scc protein also decreased at the same time as the mRNA, it remained at a detectable level throughout this period. P450scc mRNA, but not the P450scc protein, was also detected in the spermatids and spermatozoa. The biological significance of P450scc mRNA expression at this stage is unknown. Prior to experimentation, the ovary contained oocytes that were developed to the oil-droplet stage, with several clusters of immuno reactive cells localized in the thecal layer and ovigerous lamella epithelium. Expression of P450scc, mRNA was also stimulated by SPH injections in the ovary. In contrast to the testis, P450scc mRNA was continuously detected in the thecal cell layer throughout artificially induced maturation, possibly due to a repeated stimulus by the SPH injection every week. Clusters of immunoreactive cells in the thecal cell layer increased in number as ovarian development progressed. This increase ill P450scc mRNA and protein may explain, at least in part, the increase in serum steroid hormones in female eels. The P450scc antiserum clearly immunostained interrenal steroidogenic cells in the head kidney of not only eel but also goldfish, indicating that this antibody could also be used in other teleost species. (c) 2005 Elsevier Inc. All rights reserved.
• Specific gene expression in Nile tilapia gonad during sex differentiation.

  Ijiri S, Wang D, Kaneko H, Nagahama Y

  Proceedings of the the 5th international symposium of the Asia and Oceania society for comparative endocrinology 149 - 150 2006 [Not refereed][Not invited]
  
• Changes in Serum Steroid Hormones during Ovarian Development in the Captive Common Japanese Conger Conger myriaster (Brevoort)

  CHIBA Hiroaki, IJIRI Shigeho, IWATA Munehico, NAKAMURA Masaru, ADACHI Shinji, YAMAUCHI Kohei

  Aquaculture Science 水産増殖談話会 53 (2) 189 - 198 0371-4217 2005/06 [Refereed][Not invited]
   

  マアナゴ雌の周年飼育を行い、卵巣発達過程と血中ステロイドホルモン量の変化を調べた。生殖腺体指数（GSI）は、12月から翌年6月にかけて上昇し、6月に平均4.8のピークに達した後、減少した。卵巣卵は12月から1月にかけて卵黄形成を開始し、GSIの上昇とともに卵黄形成中期まで発達したが、6月以降その大部分の個体は卵黄形成途上で退行した。脳下垂体中のアルデヒドフクシン陽性細胞数は6月まで卵黄形成の進行とともに著しく増加したが、退行卵の増加とともに減少した。エストラジオール－17βは1月の卵黄形成初期にピーク（3.53ng／ml）を迎えた後、減少し低値を維持した。テストステロンは6月の卵巣卵の退行初期に平均7.93ng／ml のピークに達した後、減少した。
• Expression of 20 beta-hydroxysteroid dehydrogenase and P450 17 alpha-hydroxylase/c17-20 lyase during hCG-induced in vitro oocyte maturation in snake head murrel Channa striatus

  G. Sreenivasulu, I. Swapna, M. K. Rasheeda, S. Ijiri, S. Adachi, K. Thangaraj, B. Senthilkumaran

  FISH PHYSIOLOGY AND BIOCHEMISTRY 31 (2-3) 227 - 230 0920-1742 2005/04 [Refereed][Not invited]
   

  Partial cDNAs encoding carbonyl reductase like 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD) and P450 17 alpha-hydroxylase/c17-20 lyase (CYP17) were isolated from the ovary of snake head murrel and they exhibited high sequence identity to the Nile tilapia and rainbow trout, respectively. A low transcript level of both 20 beta-HSD and CYP17 were detected in pre-vitellogenic follicles, while the transcript level was high in full-grown immature follicles. In hCG-induced in vitro oocyte maturation, we found a significant increase in 20 beta-HSD transcript level after 2 h. The CYP17 transcripts also showed a considerable increase following hCG-induction compared to saline-treated controls. On the other hand, Western blot analysis demonstrated no significant change in the CYP17 protein level during hCG-induced in vitro oocyte maturation. Taken together, we suggest that in addition to 20 beta-HSD, the CYP17 might have a role in the shift in steroidogenesis during meiotic maturation of snake head murrel.
• Changes in mRNA levels of ovarian steroidogenic enzymes during artificial maturation of Japanese eel Anguilla japonica

  H Matsubara, Y Kazeto, S Ijiri, T Hirai, S Adachi, K Yamauchi

  FISHERIES SCIENCE 69 (5) 979 - 988 0919-9268 2003/10 [Refereed][Not invited]
   

  Changes in the levels of serum steroids and ovarian steroidogenic enzyme mRNA were analyzed in Japanese eel Anguilla japonica induced to undergo oogenesis by chum salmon pituitary homogenate (SPH) treatment. Serum 17alpha-hydroxyprogesterone, 17alpha,20beta-dihydroxy-4-pregnen-3-one, androstenedione and estrone levels were barely or not detectable during artificial maturation. In contrast, serum estradiol-17beta (E-2) and testosterone (T) levels increased after SPH injections and peaked at the migratory nucleus (MN) stage. 3beta-Hydroxysteroid dehydrogenase mRNA levels did not change between eels in different stages of oogenesis. mRNA levels of P450 cholesterol side-chain cleavage (P450sec), P450 17alpha-hydroxylase/C17-20 lyase (P450c17), 17beta-hydroxysteroid dehydrogenase type I (17beta-HSD-I) and P450 aromatase (P450arom) were low before SPH injections. P450scc, P450c17 and 17beta-HSD-I transcript abundance increased after SPH treatment and peaked at the MN stage. P450arom mRNA levels, however, peaked at the mid-vitellogenic stage and slightly decreased towards the MN stage. We suggest that these increases in P450scc and P450c17 mRNA levels may account for high T levels at the MN stage. In turn, the high T levels may permit the production of E-2 in spite of low P450arom mRNA levels towards the end of oogenesis. Steroidogenesis in artificially maturing eels appears to proceed unlike that in other teleosts, but whether or not this is an artifact remains as yet unknown.
• Molecular characterization of the GnRH system in zebrafish (Danio rerio): cloning of chicken GnRH-II, adult brain expression patterns and pituitary content of salmon GnRH and chicken GnRH-II

  C Steven, N Lehnen, K Kight, S Ijiri, U Klenke, WA Harris, Y Zohar

  GENERAL AND COMPARATIVE ENDOCRINOLOGY 133 (1) 27 - 37 0016-6480 2003/08 [Refereed][Not invited]
   

  The zebrafish has proven to be a model system with unparalleled utility in vertebrate genetic and developmental studies. Substantially less attention has been paid to the potential role that zebrafish can play in answering important questions of vertebrate reproductive endocrinology. As an initial step towards exploiting the advantages that the zebrafish model offers, we have characterized their gonadotropin-releasing hormone (GnRH) system at the molecular level. GnRHs comprise a family of highly conserved decapeptide neurohormones widely recognized to orchestrate the hormonal control of reproduction in all vertebrates. We have isolated the gene and cDNA encoding chicken GnRH-II (cGnRH-II) from zebrafish, as well as several kilobases of upstream promoter sequence for this gene. As the gene encoding salmon GnRH (sGnRH) has been previously isolated (Torgersen et al., 2002), this is the second GnRH gene isolated from zebrafish to date. We have localized expression of these two genes in the brains of reproductively mature zebrafish using in situ hybridization. sGnRH is localized to the olfactory bulb-terminal nerve region (OB-TN), the ventral telencephalon-preoptic area (VT-POA) and, as we report here for the first time in any teleost species, the hindbrain. cGnRH-II is expressed exclusively in the midbrain, as has been found in all other jawed vertebrate species examined. Finally, the levels of both GnRH peptides in pituitaries of reproductively mature zebrafish were quantified using specific ELISAs. sGnRH pituitary peptide levels were shown to be 3- to 4-fold higher than cGnRH-II pituitary peptide. The cumulative results of these experiments allow us to conclude that zebrafish express just two forms of GnRH in a site-specific manner within the brain, and that sGnRH is the hypophysiotropic GnRH form. This work lays the foundation for further research into the control of reproduction in zebrafish, such as the functional significance of multiple GnRHs in vertebrates, and the molecular mechanisms controlling tissue-specific GnRH expression. (C) 2003 Elsevier Science (USA). All rights reserved.
• Molecular cloning and characterization of 3 beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4) isomerase cDNAs from Japanese eel ovary

  Y Kazeto, S Ijiri, H Matsubara, S Adachi, K Yamauchi

  JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY 85 (1) 49 - 56 0960-0760 2003/05 [Refereed][Not invited]
   

  3beta-Hydroxysteroid dehydrogenase/Delta(5)-Delta(4) isomerase (3beta-HSD) is a crucial steroidogenic enzyme which catalyzes an essential step in the biosynthesis of all classes of steroid hormones. Two closely related cDNAs, encoding Japanese eel ovarian types I and II 3beta-HSD, were cloned and characterized. Both cDNAs putatively encoded 375 amino acid residues sharing high sequence homology with those of rainbow trout (71%) and mammalian (approximately 45-50%) 3beta-HSD. Transient expression of types I and II 3beta-HSD in COS-7 cells revealed that both proteins possess 3beta-hydroxysteroid dehydrogenase as well as Delta(5)-Delta(4) isomerase activity for both pregnenolone and dehydroepiandrosterone, with the preference of pregnenolone over dehydroepiandrosterone as substrate, although the type I protein is more active than the type II. By northern blot analysis, a single band of the 3beta-HSD transcript of approximately 1.5 kb in length was observed in ovarian tissue and the total transcript abundance of both 3beta-HSDs remained constant throughout ovarian development artificially induced by gonadotropin-rich salmon pituitary homogenate. This lack of change in 3beta-HSD transcript abundance during ovarian development did not correlate with the fluctuation of its enzymatic activity reported previously, which may suggest that changes in 3beta-HSD activity during ovarian development may be, in part, post-transcriptionally regulated in the Japanese eel ovary. (C) 2003 Elsevier Science Ltd. All rights reserved.
• Characterization of a cDNA Encoding P-450 aromatase (CYP19) from Japanese eel ovary and its expression in ovarian follicles during induced ovarian development

  S Ijiri, Y Kazeto, PM Lokman, S Adachi, K Yamauchi

  GENERAL AND COMPARATIVE ENDOCRINOLOGY 130 (2) 193 - 203 0016-6480 2003/02 [Refereed][Not invited]
   

  A cDNA encoding P450 aromatase (CYP19) was isolated from a Japanese eel (Anguilla japonica) ovarian cDNA library. This cDNA contains a complete open reading frame encoding 511 amino acid residues. The deduced amino acid sequence is 59% and 65% identical to the catfish and rainbow trout forms, respectively, and 52-54% to mammalian and chicken forms. Non-steroidogenic COS-7 cells transfected with the eel CYP19 cDNA converted exogenous androstenedione to estrone, thus verifying its identity. Northern blot analysis indicated that there was a single 2.1 kb transcript in the ovary. A 2.1 kb transcript was also found in the brain but not in the spleen, head kidney, kidney, or liver. Throughout ovarian development induced by weekly injections of salmon pituitary homogenate (SPH, 20 mug/g body weight), the 2.1 kb transcript was barely or not detectable in the ovaries. However, signals greatly increased in intensity in oocytes in the migratory nucleus stage and then decreased slightly in the post-ovulatory ovary. These changes in transcript levels are consistent with the changes in aromatase activity of ovarian follicles, suggesting that aromatase activity in ovarian follicles is mainly regulated at the transcriptional level. In addition, fadrozole was found to significantly inhibit aromatase activity in a heterologous expression system using COS-7 cells, which indicates that fadrozole treatment could be useful to control E-2 production during artificial maturation of eels. (C) 2003 Elsevier Science (USA). All rights reserved.
• Ovarian mitochondrial cytochrome b mRNA levels increase with sexual maturity in freshwater eels (Anguilla spp.)

  PM Lokman, Y Kazeto, S Ijiri, G Young, T Miura, S Adachi, K Yamauchi

  JOURNAL OF COMPARATIVE PHYSIOLOGY B-BIOCHEMICAL SYSTEMIC AND ENVIRONMENTAL PHYSIOLOGY 173 (1) 11 - 19 0174-1578 2003/02 [Refereed][Not invited]
   

  Differential display of mRNA was used to identify an upregulated gene in ovaries of artificially maturing Japanese eels (Anguilla japonica). Accordingly, mitochondrial (mt) cytochrome b, whose transcript levels increased from early to late vitellogenesis, was isolated, cloned and sequenced. Temporal trends in artificially maturing eels were compared with those in naturally and artificially maturing New Zealand eels (longfinned eel, Anguilla dieffenbachii; shortfinned eel, Anguilla australis) by Northern blot and in situ hybridization analysis to rule out any experimental artifacts. An increase in ovarian mt cytochrome b signals was seen when comparing immature and midvitellogenic longfinned eels, but not immature and early vitellogenic shortfinned eels, from the wild. Long-term captivity yielded reduced target mRNA levels, but abundance increased after hormonal induction of vitellogenesis. These results imply that the increase in mt cytochrome b mRNA levels during artificial maturation reflects natural development, although its onset appears to be brought forward during artificial maturation in the Japanese eel. It is suggested that increased mt cytochrome b mRNA levels result from both mitochondrial replication and increased transcription, and that they reflect the build-up of machinery for enhanced ATP-synthesis at some stage of oogenesis and/or early zygote development.
• Effects of rearing water quality on expression of brain corticotropin-releasing hormone and gonadal development in goldfish (Carassius auratus)

  K Nagano, T Kawasaki, S Ijiri, T Todo, S Adachi, K Yamauchi

  FISH PHYSIOLOGY AND BIOCHEMISTRY 28 (1-4) 437 - 438 0920-1742 2003 [Refereed][Not invited]
   

  The effects of changes in water quality in a recirculating system on brain corticotropin-releasing hormone (CRH) gene expression in goldfish were examined. In the experimental group, fish were reared without water change, while in the control group, 50% of water was changed everyday. In the experimental group, total ammonia-nitrogen, nitrate and phosphate were increased over the experimental period. Brain CRH mRNA levels in the control group seemed higher than those in the experimental group, but differences were confounded by variation among individuals. There were no differences in gonadal development between both groups.
• 11-ketotestosterone potentiates estrogen-induced vitellogenin production in liver of Japanese eel (Anguilla japonica)

  H Asanuma, H Ohashi, H Matsubara, S Ijiri, T Matsubara, T Todo, S Adachi, K Yamauchi

  FISH PHYSIOLOGY AND BIOCHEMISTRY 28 (1-4) 383 - 384 0920-1742 2003 [Refereed][Not invited]
   

  Isolated hepatocytes from male Japanese eel at various stages of human chorionic gonadotropin (HCG)-induced spermatogenesis were cultured in the presence of estradiol-17beta (E2). Hepatic vitellogenin (VTG) production in response to E2 increased during testicular development. This indicates that VTG production is influenced by gonadal maturity not only in females but also in males, where serum levels of 11-ketotestosterone (11KT) increase during testicular development. Recently, it has been confirmed that significant levels of 11KT are detected in maturing female eel. Therefore, in a next experiment, effect of 11KT on E2-induced VTG production was examined in vitro. As a result, in both male and female hepatocytes, 11KT enhanced E2-induced VTG production, although 11KT alone failed to induce VTG production.
• Synthesis and possible function of 11-ketotestosterone during oogenesis in eel (Anguilla spp.)

  M Matsubara, PM Lokman, A Senaha, Y Kazeto, S Ijiri, A Kambegawa, T Hirai, G Young, T Todo, S Adachi, K Yamauchi

  FISH PHYSIOLOGY AND BIOCHEMISTRY 28 (1-4) 353 - 354 0920-1742 2003 [Refereed][Not invited]
   

  The control of 11-ketotestosterone (11-KT) biosynthesis and its physiological roles were examined in female Japanese eel (Anguilla japonica) and New Zealand longfinned eel (Anguilla dieffenbachii). 11-KT was detected in serum of female eels of both species. Among various tissues from Japanese eel, the ovary had the greatest capacity to synthesize 11-KT in vitro. In addition, the oocyte diameters of eels treated with 11-KT had increased significantly. Furthermore, these oocytes were found to have an increased number of oil droplets. These findings suggest that 11-KT in female eels may be mostly of ovarian origin and that this androgen appears to play an important role in controlling pre-vitellogenic oocyte growth.
• Immunolocalization of steroidogenic enzymes (P450scc, P450c17, P450arom) in gonad of Japanese eel

  S Ijiri, N Takei, S Andachi, K Yamauchi

  FISH PHYSIOLOGY AND BIOCHEMISTRY 28 (1-4) 209 - 210 0920-1742 2003 [Refereed][Not invited]
   

  Antibodies against P450scc, P450c17 and P450arom were generated using recombinant proteins. In eel testis, P450scc and P450c17 were immunolocalized as clusters in Leydig cells. In vitellogenic eel ovary, P450scc and P450c17 immunoreactive cells were localized as clusters in the outer layer of the ovarian follicle. In contrast, P450arom seemed to be immunolocalized in the innermost follicle layer.
• The 5 '-flanking regions of CYP19A1 and CYP19A2 in zebrafish

  YK Kazeto, S Ijiri, AR Place, Y Zohar, JM Trant

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 288 (3) 503 - 508 0006-291X 2001/11 [Refereed][Not invited]
   

  This report describes the structure of the 5'-flanking regions of both the CYP19A1 and A2 genes that were isolated from the genome of the zebrafish (Danio rerio). Consensus sequences of three cAMP-responsive elements (CRE), an aryl hydrocarbon-responsive element (AhR/Arnt), a steroidogenic factor I (SF-1) site, and a TATA box were observed in the 5'-flanking region of CYP19A1 In contrast, the 5'-flanking region of CYP19A2 was located upstream of an untranslated exon and possessed consensus sequences of a single CRE, an estrogen-responsive element (ERE), a peroxisome proliferator-activated receptor alpha /retinoid X receptor alpha heterodimer-responsive element (PPAR alpha /RXR alpha), and a TATA box. Primer extension analysis revealed that the predominant transcription initiation sites for CYP19A1 and A2 transcripts were 28 and 91 bp upstream from the putative translation initiation codon, respectively. These analyses indicate that substantially different regulators, including a variety of environmental xenobiotics, control the expression the two CYP19 genes. (C) 2001 Academic Press.
• Molecular biology of the channel catfish gonadotropin receptors: 2. Complementary DNA cloning, functional expression, and seasonal gene expression of the follicle-stimulating hormone receptor

  RS Kumar, S Ijiri, JM Trant

  BIOLOGY OF REPRODUCTION 65 (3) 710 - 717 0006-3363 2001/09 [Refereed][Not invited]
   

  Molecular cloning of the channel catfish FSH receptor is reported together with temporal changes in the gene expression throughout a reproductive cycle. A cDNA encoding the receptor was isolated from the testis using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) procedures. The cDNA coded for a 662-amino acid protein that was most identical (51%-59%) to salmon gonadotropin receptor I and the FSH receptors of higher vertebrates, and less identical to LH receptors and thyrotropin receptors (45%-49% and 46%-47%, respectively). In addition, PCR analysis of the genomic DNA showed the absence of the LH receptor-specific intron. Expression of the channel catfish FSH receptor gene was highly restricted to the testis and ovary, except for a low-level expression in the spleen. Transfected COS cells expressed an active recombinant receptor as determined by the ligand-specific activation of a cAMP-responsive reporter gene (luciferase). The recombinant receptor was activated by human FSH and, to a small extent, hCG. Seasonal changes in the ovarian expression of the FSH receptor gene, examined by measuring the transcript abundance by quantitative real-time RT-PCR, showed a rise around the time of onset of ovarian recrudescence and a decrease prior to spawning. This pattern of seasonal expression of FSH receptor differs significantly from that of the LH receptor, which we reported recently. The differential expression of the two gonadotropin receptor genes, in addition to the differential secretion of the gonadotropic hormones, seem to be critical for the regulation of steroidogenesis and other gonadal physiological processes.
• Molecular biology of channel catfish gonadotropin receptors: 1. Cloning of a functional luteinizing hormone receptor and preovulatory induction of gene expression

  RS Kumar, S Ijiri, JM Trant

  BIOLOGY OF REPRODUCTION 64 (3) 1010 - 1018 0006-3363 2001/03 [Refereed][Not invited]
   

  There is little known about the molecular biology of piscine gonadotropin receptors, and information about gene expression during reproductive development is particularly lacking. We have cloned the LH receptor (LHR) in the channel catfish (cc), and examined its gene expression throughout a reproductive cycle. A cDNA encoding the receptor was isolated from the testis using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends procedures. It encoded a 696-amino acid protein that showed the greatest homology (46-50% identity) With the known LHRs and lesser similarity with FSH receptors and thyroid-stimulating hormone receptors (44-47% and 42-44% identity, respectively). In addition, two characteristics unique to the LHRs were conserved in the cloned receptor and the encoding gene: presence of an intron corresponding to intron 10 in mammals and turkey and occurrence of a double cysteine residue in the cytoplasmic tail for potential palmitoylation. The ccLHR gene was well expressed in the gonads and kidney and merely detectable in the gills, muscle, and spleen. The isolated cDNA encoded an active ccLHR protein,as the recombinant receptor expressed in COS7 cells activated a cAMP response element-driven reporter gene (luciferase) upon exposure to hCG in a dose-dependent manner. Seasonal changes in the ovarian expression of the ccLHR gene, as examined by measuring the transcript abundance by quantitative real-time RT-PCR, remained rather low during most of the reproductive cycle but was acutely induced around the time of spawning. This pattern of expression correlates well with the reported expression of its ligand (LH) in fishes and concurs with the notion that LH is a key regulator of the periovulatory maturational events.
• In vitro effects of gonadotropins on estradiol-17b production by ovarian follicles from Japanese eel.

  Seki S, Abe T, Kazeto Y, Matsubara H, Inaba K, Ijiri S, Adachi S, Yamauchi K

  Proceedings of the international symposium on advances in eel biology 222 - 224 2001 [Not refereed][Not invited]
  
• Changes in the expression of steroidogenic enzyme genes in the Japanese eel ovary during artificial maturation.

  Matsubara H, Kazeto Y, Ijiri S, Abe T, Seki S, Inaba K, Hirai T, Adachi S, Yamauchi K

  Proceedings of the international symposium on advances in eel biology 213 - 215 2001 [Not refereed][Not invited]
  
• Oogenesis in the Japanese eel.

  Adachi S, Ijiri S, Yamauchi K

  Proceedings of the international symposium on advances in eel biology 66 - 64 2001 [Not refereed][Not invited]
  
• Changes in the expression of genes encoding steroidogenic enzymes in the channel catfish (Ictalurus punctatus) ovary throughout a reproductive cycle

  RS Kumar, S Ijiri, JM Trant

  BIOLOGY OF REPRODUCTION 63 (6) 1676 - 1682 0006-3363 2000/12 [Refereed][Not invited]
   

  In vertebrates, the growth and maturation of the ovarian follicle is dependent on the appropriate dynamics of sex steroid secretion, which is dictated by gene expression of the steroidogenic enzymes. The molecular aspects of steroid regulation are poorly understood in fishes, so as a first step we determined the pattern of expression of four key steroidogenic genes throughout the ovarian cycle in an annually spawning teleost, the channel catfish (Ictalurus punctatus). The abundance of transcripts encoding 3 beta -hydroxysteroid dehydrogenase (3 beta -HSD) and cholesterol side chain cleavage (P450(scc)), 17 alpha -hydroxylase/lyase (P450(c17)), and aromatase (P450(arom)) were determined by rtqRT-PCR or ribonuclease protection assay and correlated to ovarian growth and plasma titers of estradiol (E-2) and testosterone (T) in two populations of catfish. Elevations in transcript abundance for P450(c17), P450(scc), and P450(arom) were observed at the onset of ovarian recrudescence and during early vitellogenic growth of the oocytes; however, all three decreased precipitously with the completion of vitellogenesis. Changes in the expression of these genes strongly suggest a direct correlation to E-2 and T titers. Alternatively 3 beta -HSD transcript abundance was relatively stable throughout the year. This study suggests that the genes encoding the three steroidogenic cytochrome P450s have a similar regulatory mechanism.
• Developmental changes in pituitary-thyroid axis, and formation of gonads in leptocephali and glass eels of Anguilla spp.

  Y Ozaki, H Okumura, Y Kazeto, T Ikeuchi, S Ijiri, M Nagae, S Adachi, K Yamauchi

  FISHERIES SCIENCE 66 (6) 1115 - 1122 0919-9268 2000/12 [Refereed][Not invited]
   

  Pituitary, thyroid gland and gonads in leptocephali of Japanese eel Anguilla japonica (19.8-32.6 mm in total length), A. obscura (45.0 mm), and A. bicolor pacifica (49.5 mm) and those in glass eels of the Japanese eel were histologically and immunohistochemically examined in order to observe the developmental changes of these endocrine organs in the Anguillidae. The pituitary, consisting of adenohypophysis and neurohypophysis in Japanese eel leptocephali over 22.5 mm, did not contain thyroid stimulating hormone (TSH) immunoreactive cells. Such cells were, however, detectable in the more developed pituitaries of leptocephali of A. obscura and A. bicolor pacifica and in those of glass eels. Conversely, thyroxine (T-4)-immunoreactive thyroid follicles could be detected in all specimens, both leptocephalic and glass eel. Only in glass eels, gonads were found in the body cavity, and these gonads harbored one or two primordial germ cells (PGC) per cross-section. Our results indicate that thyroid hormones (TH) production started prior to TSH production, and that TSH and TH are both secreted during the metamorphosis from leptocephalus to glass eel. Therefore, it is plausible that the TSH-TH axis is involved in the metamorphosis from leptocephalus to glass eel, but not in the early growth from preleptocephalus to leptocephalus.
• Cloning of 17 beta-hydroxysteroid dehydrogenase-1 cDNAs from Japanese eel ovary

  Y Kazeto, S Ijiri, H Matsubara, S Adachi, K Yamauchi

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 279 (2) 451 - 456 0006-291X 2000/12 [Refereed][Not invited]
   

  17 beta -hydroxysteroid dehydrogenase-I (17 beta -HSD-I) is a key steroidogenic enzyme for estradiol-17 beta (E-2) production, cDNAs encoding 17 beta -HSD-I were cloned for the first time in lower vertebrates from the ovary of a teleost, the Japanese eel. The deduced amino acid sequence from these cDNAs was approximately 50% identical to mammalian 17 beta -HSD-Is. 17 beta -HSD-I mRNA was not detected in previtellogenic ovaries by Northern blotting. However, transcript abundance increased in early vitellogenic ovaries obtained from fish artificially matured by gonadotropic treatment, but thereafter did not appear to change further. Recombinant 17 beta -HSD-I expressed in human kidney 293 cells selectively converted estrone to E-2, but androstenedione, testosterone, or E-2 were not converted to any other steroids. Although it is widely accepted that E-2 is produced from testosterone in other species of teleosts, the substrate specificity of eel 17 beta -HSD-I suggests that a steroidogenic pathway for production of E-2 from androstenedione via estrone exists in the Japanese eel ovary. (C) 2000 Academic Press.
• Cloning and functional expression of a thyrotropin receptor from the gonads of a vertebrate (bony fish): potential thyroid-independent role for thyrotropin in reproduction

  RS Kumar, S Ijiri, K Kight, P Swanson, A Dittman, D Alok, Y Zohar, JM Trant

  MOLECULAR AND CELLULAR ENDOCRINOLOGY 167 (1-2) 1 - 9 0303-7207 2000/09 [Refereed][Not invited]
   

  The thyroid stimulating hormone receptor (TSHR) mediates the pituitary control of the development, growth and function of the thyroid. The expression of the gene encoding this receptor is known only in the thyroid, lymphocytes, fibroblasts, retro-orbital tissue and fat cells. We have cloned a TSHR from the gonads of a non-mammalian vertebrate, a bony fish (striped bass, Mol one saxatilis) in the course of our search for gonadotropin receptors (follicle stimulating hormone receptor, FSHR and luteinizing hormone receptor, LHR). RT-PCR analysis demonstrated that the striped bass TSHR (stbTSHR) transcripts were abundant in both the thyroid and gonads and detectable in skeletal muscle, heart and brain tissues. The stbTSHR cDNA encoded a 779-amino acid glycoprotein hormone receptor with much higher homology (57-59%) to the mammalian TSH receptors than the mammalian LH receptors (47-49%) and FSH receptors (47%), and salmon and catfish gonadotropin receptors (42-45%). There was a TSHR-specific insertion in the extracellular domain as seen in mammalian receptors. Moreover, PCR analysis of genomic DNA indicated the absence of the LHR-specific intron in the striped bass TSHR gene. Recombinant stbTSHR expressed in COS1 cells activated reporter genes (luciferase) driven by either a cAMP response element or the c-fos promoter in response to bovine TSH, stbLH or hCG, but not human FSH. In situ hybridization studies revealed the presence of stbTSHR transcripts in the gametes but not in the follicular cells. This pattern of expression is unique and suggests a direct, albeit unknown, role for TSH in gamete physiology. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.
• Characterization of gonadal and extra-gonadal forms of the cDNA encoding the Atlantic stingray (Dasyatis sabina) cytochrome P450 aromatase (CYP19)

  S Ijiri, C Berard, JM Trant

  MOLECULAR AND CELLULAR ENDOCRINOLOGY 164 (1-2) 169 - 181 0303-7207 2000/06 [Refereed][Not invited]
   

  Cytochrome P450 aromatase (P450arom; CYP19) mediates the conversion of androgens to estrogens and its activity has been found in all vertebrates studied to date. This study describes the full-length cDNA encoding the ovarian form of P450arom and the differences in the 5'-untranslated region (5'-UTR) of the extra-gonadal P450arom transcript expressed by the Atlantic stingray (Dasyatis sabina). Elasmobranchs (cartilaginous fishes such as sharks, rays and skates) diverged from the other vertebrates more than 350 million years ago, therefore the stingray P450arom cDNA may represent an ancient form of this gene. Northern blot analysis showed that the ovarian follicle expressed transcripts of 3.1 and 1.7 kb in size which correspond to the clones isolated from a stingray ovarian follicle cDNA library. Both transcripts consisted of an identical 1.5 kb coding region and a 41 bp 5'-UTR, however the 3'-UTRs differed in the use of the most proximate and the most distal of four polyadenylation signals. COS cells transfected with the 1.7 kb cDNA had twice the aromatase activity as cells transfected with the 3.1 kb cDNA. The coding region of the cDNA predicted a 58.5 kDa protein which consisted of 511 residues. Alignment of the stingray protein indicates that the P450arom is equally identical (53-59%) to all other vertebrate forms of P450arom characterized to date, thus indicating a common ancestry. The evolutionary relationship of the stingray form of P450arom clearly predates the other forms and belongs to a unique lineage. Transcripts of P450arom were expressed in ovarian follicles (of all sizes), the testis, the pituitary, in all sections of the brain, and in the kidney. The extra-gonadal transcripts appear to encode a protein identical to the ovarian form, however, the 5'-UTR was 657 bp longer presumably due to the transcription of an untranslated 'first exon' as seen in the mammalian form of this gene. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.
• Molecular cloning and characterization of Japanese eel ovarian P450c17 (CYP17) cDNA

  Y Kazeto, S Ijiri, T Todo, S Adachi, K Yamauchi

  GENERAL AND COMPARATIVE ENDOCRINOLOGY 118 (1) 123 - 133 0016-6480 2000/04 [Refereed][Not invited]
   

  As a first step in investigating the mechanism underlying the steroidogenic shift from the production of ovarian androgens (vitellogenic stage) to that of 17 alpha-hydroxylated progestins (maturational stage) in Japanese eel during induced oogenesis, a cDNA encoding Japanese eel (Anguilla japonica) ovarian P450c17 (CYP17: steroid 17 alpha-hydroxylase/C17-20 lyase) was cloned and sequenced. This cDNA contained the complete coding region representing 510 amino acid residues, which showed high sequence homology to those of rainbow trout (74%) and mammals (45-55%). The protein encoded by this cDNA possessed high enzymatic activities of 17 alpha-hydroxylase and C17-20 lyase, thus quickly converting pregnenolone and progesterone to their respective delta(4) and delta(5) C19 products. P450c17 produced a single transcript of 2.4 kb in length, as assessed by Northern blot. Transcript levels of this enzyme significantly increased throughout artificially induced ovarian development. Considering this together with the previous data showing that C17-20 lyase activity decreased from the vitellogenic to the maturational stage, whereas 17 alpha-hydroxylase activity increased, the present data suggest that changes in C17-20 lyase activity (the production of androgens) do not depend on transcriptional changes of the P450c17gene. (C) 2000 Academic Press.
• The first report of an untranslated exon I in the aromatase gene (CYP19) of a primitive vertebrate

  Ijiri S, Trant JM

  Molecular Steroidogenesis Frontiers Science Series 29 327 - 328 1999 [Not refereed][Not invited]
  
• Molecular cloning of cDNAs encoding steroidogenic enzymes from the ovary of Japanese eel

  Kazeto Y, Ijiri S, Matsubara H, Adachi S, Yamauchi K

  Molecular Steroidogenesis Frontiers Science Series 29 323 - 326 1999 [Not refereed][Not invited]
  
• Molecular studies of steroidogenesis using primitive vertebrates (sharks and rays) as a models

  Trant JM, Ijiri S

  Molecular Steroidogenesis Frontiers Science Series 29 313 - 316 1999 [Not refereed][Not invited]
  
• Comparison of mRNA levels of ovarian steroidogenic enzymes between artificially maturing Japanese and naturally maturing New Zealand eels.

  Matsubara H, Kazeto Y, Lokman P. M, Okumura H, Ijiri S, Young G, Adachi S, Yamauchi K

  Proceedings, the 6th International Symposium on Reproductive Physiology of Fish 181 - 181 1999 [Not refereed][Not invited]
  
• Pretreatment reproductive stage and oocyte development induced by salmon pituitary homogenate in the Japanese eel Anguilla japonica

  S Ijiri, T Kayaba, N Takeda, H Tachiki, S Adachi, K Yamauchi

  FISHERIES SCIENCE 64 (4) 531 - 537 0919-9268 1998/08 [Refereed][Not invited]
   

  Induction of the ovarian development was investigated in three groups of eels with respect to the importance of the pretreatment reproductive stage. Initially, the developmental stages of oocytes varied widely among these groups; silver eels had the most advanced oocytes which were in the early stage of vitellogenic growth, whereas oocytes from feminized eels were in the oil droplet or early vitellogenic stage and those from cultivated eels were in the oil droplet stage. Eels received weekly intramuscular injections of salmon pituitary homogenate (SPH) at 20 mu g per g body weight (low-dose treatment). The ovarian follicles of silver eels developed the fastest and reached the final maturational stage after, on average, 10 weeks of treatment. The oocytes of feminized eels and cultivated eels developed more slowly, requiring approximately 17 and 19 weeks of treatment, respectively. All silver eels were induced to the final maturational phase (100%), but lower ratios were seen for feminized and cultivated eels (64 and 29%, respectively). Feminized eels were also subjected to a high-dose treatment of SPH (20 mu g per g body weight). High-dose treated feminized eels were induced to the final maturational phase in less time (11 weeks) and with greater efficiency (71%) than low dose-treated feminized eels. It was shown that eels whose follicle diameter was over about 180 mu m could be stimulated to develop through the final maturational phase. This result indicates that the majority of feminized eels may be expected to reach the final maturational phase after SPH treatment. However, a 100% rate to reach the final maturational phase could not be attained, possibly due to immunoreaction to SPH, the stress of injections, or other yet unidentified factors. We conclude that silver eels are the best candidates for artificial maturation. Even though feminized eels were not the most responsive group, their abundance makes them an important resource. Moreover, we recommend that eels should be induced to the final maturational phase during a relatively short period of SPH treatment (about two to three months) for artificial maturation to be successful.
• Ovarian development and serum steroid hormone profiles in hatchery-bred female catfish Clarias macrocephalus (Gunther) during an annual reproductive cycle

  JD Tan-Fermin, S Ijiri, H Ueda, S Adachi, K Yamauchi

  FISHERIES SCIENCE 63 (6) 867 - 872 0919-9268 1997/12 [Refereed][Not invited]
   

  Ovarian development e.g. gonadosomatic index, oocyte diameter, fecundity, histology, and related steroid hormones e.g. testosterone (T), estradiol-17 beta (E-2), 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (DHP), were examined in captive female catfish Clarias macrocephalus during an annual cycle to establish the optimum season for its artificial propagation. Results showed that captive C. macrocephalus had a group-synchronous pattern of ovarian development, as indicated by the presence of oocytes at all stages of development throughout the annual cycle. Mean gonadosomatic index (GSI; 11-13%), oocyte diameter (1.54-1.56 mm), fecundity (80-110 eggs/g body weight), and serum T levels (36-37 ng/ml) were lowest in January-April, suggesting that it is not the optimum season to induce C. macrocephalus to spawn during these months. Serum E-2 levels were lowest in January (7 ng/ml), and highest in December (20 ng/ml). Serum DHP levels were below detectable limits (< 0.02 ng/ml) throughout the year, supporting the observation that final maturation and ovulation do not occur in this species under captive conditions. Changes in various reproductive parameters and steroid hormone levels indicate that January-March, April-June, July-September and October-December correspond to the refractory, preparatory, spawning and post-spawning periods, respectively, of the annual cycle. The results of the present investigation can be used as a guide for the controlled breeding and commercial aquaculture of C. macrocephalus in the Philippines.
• Eel ovarian 20 beta-hydroxysteroid dehydrogenase: molecular cloning and gene expression during artificial maturation of the Japanese eel, Anguilla japonica

  PM Lokman, Y Kazeto, S Ijiri, S Adachi, K Yamauchi

  ADVANCES IN COMPARATIVE ENDOCRINOLOGY, TOMES 1 AND 2 1517 - 1521 1997 [Not refereed][Not invited]
   

  The production of 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (DHP), considered to be the maturation-inducing steroid in the Japanese eel, requires involvement of the enzyme 20 beta-hydroxysteroid dehydrogenase (20 beta-HSD). Furthermore, 20 beta-HSD activity is conferred by the enzyme carbonyl reductase (CR), which has been cloned in several mammalian vertebrates. We investigated the molecular cloning of cel ovarian CR-like 20 beta-HSD and analysed its expression by Northern hybridisation in artificially maturing females to clarify the previously demonstrated failure to produce DHP in vivo in spite of high substrate-supported DHP production in vitro.
• Molecular cloning of the cDNA encoding P450c17 from the Japanese eel ovary and development changes in its expression

  Y Kazeto, S Ijiri, S Adachi, K Yamauchi

  ADVANCES IN COMPARATIVE ENDOCRINOLOGY, TOMES 1 AND 2 581 - 583 1997 [Not refereed][Not invited]
   

  A cDNA encoding P450 17 alpha-hydroxylase/C17-20 lyase (P450c17) was isolated from Japanese eel (Anguilla japonica) ovary. The deduced amino acid residues from the cDNA showed high homology to that of rainbow trout Northern blot analysis revealed that the mRNA of P450c17 had a single transcript which was 2.4kb in length and increased during ovarian development. This result coincides with the ability of ovarian follicles to synthesize 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (DHP) from exogenous steroid substrates.
• Changes in serum steroid hormones and steroidogenic ability of ovarian follicles during artificial maturation of cultivated Japanese eel, Anguilla japonica

  S Ijiri, Y Kazeto, N Takeda, H Chiba, S Adachi, K Yamauchi

  AQUACULTURE 135 (1-3) 3 - 16 0044-8486 1995/10 [Refereed][Not invited]
   

  Serum levels of oestradiol-17 beta (E(2)), testosterone (T) and 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (17 alpha,20 beta-DHP) were assessed by radioimmunoassay during induction of sexual maturation of female Japanese eel (Anguilla japonica) by chum salmon pituitary homogenate (SPH) treatment. SPH stimulated vitellogenic growth in about 50% of the eels. Serum E(2) levels in eels which responded to SPH remained low for the first 10 weeks after the start of SPH injections, and tended to increase after 12 weeks, Levels increased further after completion of vitellogenesis. Serum levels of T increased gradually as SPH treatment progressed, and remained higher than E(2) levels during the first 12 weeks. A further increase in T levels occurred after completion of vitellogenesis. 17 alpha,20 beta-DHP was not detected in serum during the experimental period. In vitro production of E(2), T and 17 alpha,20 beta-DHP by ovarian follicles in response to forskolin (an activator of adenylate cyclase), and changes in the ability of follicles to convert exogenous pregnenolone of 17 alpha-hydroxyprogesterone (17 alpha-OHP) to E(2), T and 17 alpha,20 beta-DHP, and exogenous T to E(2), were examined using 18 h incubations. Forskolin had no effect on the production of any measured steroid by follicles at any stage of development. Production of E(2), T and 17 alpha,20 beta-DHP in the absence of exogenous substrates was low. The ability of follicles to produce E(2) from pregnenolone, 17 alpha-OHP or T was low during vitellogenesis, followed by an increase during the migratory nucleus stage. The ability of follicles to produce T from pregnenolone and 17 alpha-OHP gradually increased during vitellogenesis, followed by a further increase or a slight decrease during the migratory nucleus stage. The conversion of pregnenolone or 17 alpha-OHP to 17 alpha,20 beta-DHP increased during vitellogenesis, followed by either a further increase or a decrease during the migratory nucleus stage. A good correlation existed between serum levels of E(2) and aromatase activity (the ability of follicles to convert T to E(2)). E(2) production by follicles was shown to depend largely on aromatase activity, while T production by follicles appeared to be depend largely on SPH-induced pregnenolone production. Increased aromatase activity at the migratory nucleus stage may inhibit 17 alpha,20 beta-DHP production and spontaneous final oocyte maturation and ovulation.
• CHANGE OF LIPOVITELLIN DURING IN-VITRO OOCYTE MATURATION IN JAPANESE FLOUNDER PARALICHTHYS-OLIVACEUS

  T MATSUBARA, S ADACHI, S IJIRI, K YAMAUCHI

  FISHERIES SCIENCE 61 (3) 478 - 481 0919-9268 1995/06 [Refereed][Not invited]
   

  The change in molecular weight in the native form of lipovitellin during in vitro oocyte maturation was examined by gel chromatography in Japanese flounder, Paralichthys olivaceus, a marine teleost which lays pelagic eggs. The major protein peak at 400 kDa in the gel chromatogram of post-vitellogenic oocyte homogenate disappeared, and a new 140 kDa peak was observed in ovulated eggs. Both 400 kDa and 140 kDa yolk proteins were immuno-reactable to the specific antiserum against female specific-serum protein, suggesting that the 400 kDa yolk protein was lipovitellin of this species and the 140 kDa one was a proteolytic derivative from the 400 kDa lipovitellin. The time course analysis of the proteolysis of the lipovitellin during in vitro maturation demonstrated that the lipovitellin of 400 kDa was cleaved into 140 kDa during the later half of oocyte maturation. In combination with the result of SDS-PAGE analysis, this seems to be caused by the proteolytic cut-off of the 108 kDa peptide which constructs 400 kDa lipovitellin.
• DIURNAL PERIODICITY IN TESTICULAR ACTIVITY IN THE JAPANESE FLOUNDER PARALICHTHYS-OLIVACEUS

  M MATSUYAMA, M YONEDA, H TAKEUCHI, H KAWAGA, M KASHIWAGI, K TABATA, Y NAGAHAMA, S IJIRI, S ADACHI, K YAMAUCHI

  FISHERIES SCIENCE 61 (1) 17 - 23 0919-9268 1995/02 [Refereed][Not invited]
   

  Testicular activity, as represented by the amount of each type of testicular germ cell in the seminal lobules, and the serum levels of three major male teleost steroids-testosterone (T), 11-ketotestosterone (11-KT), and 17alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-DP)-were investigated during the daily spawning cycle of the male Japanese flounder Paralichthys olivaceus. B-type spermatogonia and spermatocytes increased in number from the lowest level at 22:00 (186 cells/lobule), and peaked at 10:00 (292 cells/lobule). The number of spermatozoa increased from 14:00 (370 cells/lobule) and peaked at 22:00 (782 cells/lobule). In contrast, spermatid numbers decreased from 14:00 (579 cells/lobule) and showed the lowest level at 22:00 (348 cells/lobule). These results indicate that spermatogenesis (spermatogonial proliferation, the formation of spermatocytes and spermatids) occurs between 22:00 and 10:00, and spermiation (release of spermatozoa into the lobular lumen from the cysts) occurs between 14:00 and 22:00. Thus, spermatogenesis and spermiation occur on a daily basis in the male Japanese flounder. Serum T and 11-KT levels showed no significant fluctuation throughout the day. Serum 17alpha,20beta-DP was maintained at a low or undetectable level throughout the experimental period, but showed a surge (110 pg/ml) at 22:00. This short-lived 17alpha,20beta-DP surge corresponded to the peak of spermiation. This study demonstrates, therefore, the existence of a diurnal periodicity in (1) spermatogenesis and spermiation, and (2) 17alpha,20beta-DP production.

Books etc

• 魚類の性決定・性分化・性転換 : これまでとこれから = Sex determination, sex differentiation and sex change in fishes-past, present and future

  菊池, 潔, 井尻, 成保, 北野, 健, 日本水産学会 (Joint editor第6章 ティラピア・チョウザメの性分化)
  恒星社厚生閣 2021/03 (ISBN: 9784769916635) xiv, 242p
• ウナギの科学

  塚本, 勝巳 (Contributor)
  朝倉書店 2019/06 (ISBN: 9784254485028) x, 226p, 図版 [8] p
• 魚類学の百科事典

  井尻 成保 (Contributor生殖腺の性決定と性分化)
  丸善 2018/10 (ISBN: 9784621303177)
• 海をまるごとサイエンス

  井尻 成保 (Contributor第4章殖えない魚を殖やしたい：難種苗生産魚種への挑戦)
  2018/08 (ISBN: 9784303800017) 127 42-54
• 魚類学

  井尻 成保 (Contributor第14章生殖)
  恒星社厚生閣 2017/09 388 155-178
• Eel Biology

  Adachi S, Ijiri S, Kazeto Y, Yamauchi K (ContributorOogenesis in the Japanese eel.)
  Springer-Verlag 2003 (ISBN: 4431004580) 497 301-318

Conference Activities & Talks

• ティラピア、チョウザメ、ウナギの性分化  [Not invited]

  井尻 成保

  平成30年度日本水産学会秋期大会、シンポジウム「魚類の性決定・性分化・性転換 –これまでとこれから–」  2018/09
• Molecular mechanism of maturation-inducing steroid synthesis during oocyte maturation  [Not invited]

  IJIRI Shigeho

  International Fisheries Symposium  2016/11
• Molecular mechanism of maturation-inducing steroid synthesis during oocyte maturation in Japanese eel  [Invited]

  IJIRI Shigeho

  Towards reproduction of eel in captivity to support sustainable aquaculture  2016/10
• Molecular mechanism of the maturation-inducing hormone production in cultured ovaries of the Japanese eel, Anguilla japonica  [Invited]

  IJIRI Shigeho

  International Symposium on Fish Endocrinology  2016/06
• 水産重要魚種、ウナギとチョウザメの増養殖研究におけるNGSの利用  [Invited]

  井尻 成保

  NGS現場の会 第四回研究会  2015/07
• ウナギ増養殖研究の最前線  [Invited]

  井尻 成保

  平成27年度資源・素材学会北海道支部春季講演会  2015/06
• Molecular mechanism of maturation-inducing hormone production during final oocyte maturation in the japanese eel.  [Invited]

  Ijiri, S, Su, T, Kato, R, Kazeto, Y. Kagawa, H, Adachi, S

  10th International Symposium on Reproductive Physiology of Fish.  2014/05
• Dimorphic expression pattern of sex differentiation-related genes in morphologically undifferentiated gonads of Amur sturgeon and Russian sturgeon  [Invited]

  Ijiri S, Hagihara Seishi, Yamashita R, Suzuki H, Adachi S

  Diversification in Inland Finfish Aquaculture II  2013/09
• Identification of a novel type of 20beta-hydroxysteroid dehydrogenase responsible for maturation-inducing hormone production during oocyte maturation in masu salmon  [Not invited]

  Shigeho Ijiri, Yasushi Shibata, Nonoha Takezawa, Yukinori Kazeto, Naoki Takatsuka, Erika Kato, Yuichi Ozaki, Shinji Adachi, Kohei Yamauchi, Yoshitaka Nagahama

  17th International Congress of Comparative Endocrinology  2013/07
• ニホンウナギ増殖研究の過去と現在  [Invited]

  井尻 成保

  日本機械学会北海道支部総会付帯特別講演会  2012/03

MISC

• アムールチョウザメ17β-水酸基脱水素酵素タイプ12-likeの酵素活性および卵成熟期における発現動態

  長谷川祐也, 井尻成保, 駿河谷諒平, 酒井隆一, 足立伸次  日本水産学会大会講演要旨集(CD-ROM)  2021-  2021
• ニホンウナギ生殖腺における新規性分化関連遺伝子の探索

  堀内萌未, 稲葉駿, 萩原聖士, 板倉光, 山下洋, 久米学, 塚本勝巳, 井尻成保, 足立伸次  日本水産学会大会講演要旨集(CD-ROM)  2021-  2021
• 天然ニホンウナギの生殖腺の性分化 口頭

  堀内 萌未, 中司 大智, 長谷川 祐也, 萩原 聖士, 板倉 光, 山下 洋, 久米 学, 寺島 佑樹, 塚本 勝巳, 井尻 成保, 足立 伸次  東アジア鰻学会第2回研究発表会  2019/03/31  [Not refereed][Not invited]
  
• 北海道沿岸における2015年のチョウザメ科魚類の異常漁獲および生殖腺の特徴

  萩原聖士, 須山喜市, 東典子, 堤尚信, 西尾朋高, 市村政樹, 三坂尚行, 鈴木渉太, 宮城大助, 古市明文, 市川卓, 松原創, 川崎琢真, 宗原弘幸, 高橋英祐, 山羽悦郎, 井尻成保, 足立伸次  日本水産学会大会講演要旨集  2016-  16  2016/03/26  [Not refereed][Not invited]
  
• 北海道産チョウザメ類保護と養殖産業化のための種苗生産体制整備

  足立伸次, 井尻成保, 萩原聖士, 三坂尚行, 古市明文, 市村政樹  ノーステック財団 研究開発助成事業2016 成果報告書  27  -29  2016  [Not refereed][Not invited]
  
• Transcriptional profiling of olfactory imprinting in the olfactory center of Pacific salmon

  MinowaY, Hagihara S, Abe T, Ijiri S, Adachi S, Kudo H  Chemical Senses  41-  (9)  e145  2016  [Not refereed][Not invited]
  
• Gonadal soma-derived factorはティラピアの精巣分化を誘導する

  井尻成保, 金子裕代, 泉ひかり, 倉持有希, 水野翔太, 足立伸次, 長濱嘉孝  日本水産学会大会講演要旨集  2015-  2015
• Acipenser medirostrisとされている北日本に来遊するチョウザメ属魚類はA.mikadoiである

  萩原聖士, 東典子, 井尻成保, 足立伸次  日本水産学会大会講演要旨集  2014-  100  2014/03/27  [Not refereed][Not invited]
  
• サクラマスの卵成熟誘起ステロイド産生を担う20βHSD遺伝子の同定

  竹澤野葉, 井尻成保, 柴田安司, 風藤行紀, 鈴木博史, 足立伸次, 山内皓平, 長濱嘉孝  日本水産学会大会講演要旨集  2013-  2013
• ナイルティラピア遺伝的雄仔魚の雌化誘導に伴う性分化関連遺伝子群の発現変動

  李爽, 井尻成保, ヤン ホンウェイ, 西澤朋実, 足立伸次, 長濱嘉孝  日本水産学会大会講演要旨集  2013-  2013
• 自然エネルギーを利用した北方型循環養殖システムの開発

  安部智貴, 加藤良平, 金子拓未, 安井肇, 井尻成保, 足立伸次  日本水産学会大会講演要旨集  2012-  118  2012/03/26  [Not refereed][Not invited]
  
• GSDF(gonadal soma‐derived growth factor)はメダカ遺伝的雌の雄化を引き起こす

  木寺智則, 内川拓, 白石絵吏, 松田勝, 井尻成保, 北野健  日本水産学会大会講演要旨集  2012-  34  2012/03/26  [Not refereed][Not invited]
  
• 自然エネルギーを利用した循環養殖システムの開発

  加藤良平, 安部智貴, 金子拓未, 木村暢夫, 藤森康澄, 安井肇, 井尻成保, 足立伸次  日本水産学会北海道支部大会講演要旨集  2011-  58  2011  [Not refereed][Not invited]
  
• Effects of reproductive stage, GH, and 11-ketotestosterone on expression of growth differentiation factor-9 in the ovary of the eel, Anguilla australis

  P. M. Lokman, Y. Kazeto, Y. Ozaki, S. Ijiri, R. Tosaka, M. Kohara, S. L. Divers, H. Matsubara, L. G. Moore, S. Adachi  REPRODUCTION  139-  (1)  71  -83  2010/01  [Not refereed][Not invited]
   

  In order to study the regulation of the growth differentiation factor-9 (gdf9) gene in a primitive teleost with semelparous life history, we cloned a cDNA encoding shortfinned eel Gdf9, expressed a partial peptide in Escherichia coli, and raised an antiserum to evaluate changes in Gdf9 expression during its pituitary homogenate-induced reproductive cycle. The effects of in vivo and in vitro exposure to the androgen 11-ketotestosterone (11-KT), known to affect previtellogenic (PV) oocyte growth, were also determined. Furthermore, we investigated whether Gdf9 expression was metabolically gated by treating PV fish with recombinant GH in vivo. Immunoreactive proteins of ca. 52 and 55 kDa were identified by western blot analysis. Gdf9 message and protein were most abundant in PV oocytes, and peaked slightly earlier for mRNA than for protein. Captivity resulted in reduced gdf9 mRNA levels, which were restored following pituitary homogenate treatment. As oocytes progressed through induced oogenesis, Gdf9 expression decreased. Neither 11-KT nor GH treatment affected gdf9 mRNA levels in PV fish, although GH could partially restore handling- or captivity-induced decreases in gdf9 mRNA levels. Semelparous eels thus show an expression pattern of Gdf9 during oogenesis that is similar to that seen in other vertebrates, that appears responsive to handling or captivity stress, and whose control remains to he elucidated. Reproduction (2010) 139 71-83
• 紅藻(ツルシラモ)を用いた魚類循環飼育水の水質浄化の検討

  増田淳史, 山本大輔, 山羽悦郎, 井尻成保, 安井肇, 足立伸次  水産増殖  57-  (4)  661  2009/12/20  [Not refereed][Not invited]
  
• Eel and sturgeon, unmanageable species in seeding production

  Shigehiro Ijiri  NIPPON SUISAN GAKKAISHI  75-  (5)  866  -867  2009/09  [Not refereed][Invited]
  
• In vitro induction of oil droplet accumulation into previtellogenic oocytes of Japanese eel, Anguilla japonica

  Taku Endo, Takashi Todo, P. Mark Lokman, Shigeho Ijiri, Shinji Adachi, Kohei Yamauchi  CYBIUM  32-  (2)  239  -240  2008/07  [Not refereed][Not invited]
   

  Accumulation of oil droplets in previtellogenic oocytes of Japanese eel, Anguilla japonica, could be induced in vitro. Co-treatment with very low-density lipoprotein (VLDL) and 11-ketotestosterone (11-KT) resulted in significant oil droplet accumulation. It is therefore suggested that lipids in oil droplets originate, at least in part, from VLDL, and that 11-KT plays an important role in their transfer and/or accumulation into oocytes.
• Growth and differentiation factor-9 in the ovary of the eel (Anguilla SPP.)-stage dependence and hormonal control of expression-

  Lokman P M, Kazeto Y, Ijiri S, Tosaka R, Kohara M, Divers S L, Matsubara H, Adachi S  6 th International Symposium on Fish endocrinology  2008/06  [Not refereed][Not invited]
   

  ウナギ卵巣に発現するGDF-9遺伝子を単離・発現変化を調べた。（Lokman P M, Kazeto Y, Ijiri S, Tosaka R, Kohara M, Divers S L, Matsubara H, Adachi S.）ポスター発表
• Immunohistochemical characterization of gonadal soma-derived growth factor (GSDF) in male undifferentiated gonad of the Nile tilapia.

  KURAMOCHI Yuki, IJIRI Shigeho, KANEKO Hiroyo, KANEKO Hiroyo, KOBAYASHI Tohru, NAGAHAMA Yoshitaka, NAGAHAMA Yoshitaka, ADACHI Shinji  Program & Abstracts. 5th World Fisheries Congress, 2008  2008
• ニホンウナギのアロマターゼおよび3β-水酸基脱水素酵素の生殖腺における発現解析

  風藤行紀, 小原真由子, 武井則雄, 井尻成保, 足立伸次, 山内晧平  日本水産学会大会講演要旨集  2008-  2008
• 性分化期のティラピア生殖腺における性分化関連遺伝子の発現変化

  井尻成保, 金子裕代, 王徳寿, 足立伸次, 長濱嘉孝, 長濱嘉孝  日本水産学会大会講演要旨集  2007-  2007
• ニホンウナギの卵質評価マーカータンパクの検索

  佐合慶祐, 佐合洋祐, 小林亨, 長濱嘉孝, 小林弘子, 井尻成保, 足立伸次, 山内晧平  日本水産学会大会講演要旨集  2007-  2007
• GENE EXPRESSION PATTERN IN TILAPIA GONADS DURING SEX DIFFERENTIATION

  IJIRI Shigeho, KANEKO Hiroyo, WANG De-shou, ADACHI Shinji, NAGAHAMA Yoshitaka  Proceedings of the Japan Society for Comparative Endocrinology  (21)  47  -47  2006/11  [Not refereed][Not invited]
  
• Gene expression profile in Nile tilapia gonad during sex differentiation

  S Ijiri, D Wang, Y Nagahama  JOURNAL OF EXPERIMENTAL ZOOLOGY PART A-COMPARATIVE EXPERIMENTAL BIOLOGY  305A-  (2)  135  -135  2006/02  [Not refereed][Not invited]
  
• ティラピア性分化期の生殖腺における性分化関連遺伝子の発現変化

  井尻成保, 金子裕代, 王徳寿, 足立伸次, 長濱嘉孝  日本比較内分泌学会大会及びシンポジウムプログラム・講演要旨  31st-  2006
• GENE EXPRESSION PATTERN IN TILAPIA GONADS DURING SEX DIFFERENTIATION

  IJIRI SHIGEHO, KANEKO HIROYO, WANG DE-SHOU, ADACHI SHINJI, NAGAHAMA YOSHITAKA  Proc Jpn Soc Comp Endocrinol  (21)  47  2006  [Not refereed][Not invited]
  
• 性分化期のティラピア生殖腺における発現遺伝子

  井尻成保, 王徳寿, 松田勝, 長浜嘉孝  日本水産学会大会講演要旨集  2005-  48  2005/04/01  [Not refereed][Not invited]
  
• Induction of oogenesis by injections of salmon pituitary homogenates in hypophysectomized immature Japanese eel

  Matsubara H, Senaha A, Takei N, Shin D H, Ijiri S, Todo T, Adachi S, Yamauchi K  Zoological Science  1603  -1603  2003/12  [Not refereed][Not invited]
   

  脳下垂体を摘除したニホンウナギ雌に人為催熟を施したところ卵成長が進行した。（Matsubara H, Senaha A, Takei N, Shin D H, Ijiri S, Todo T, Adachi S, Yamauchi K.）Zoological Science 2003; 20: p1603.ポスター発表
• 魚類の卵形成におけるアンドロゲンの役割

  瀬名波絢子, 松原創, 井尻成保, 足立伸次, 山内晧平  道立水産孵化場と北大水産学部の研究交流会 魚の研究 to be continued  2003/02  [Not refereed][Not invited]
   

  アンドロゲンがニホンウナギおよびベステルの卵形成に及ぼす影響を調べた。（瀬名波絢子、松原創、井尻成保、足立伸次、山内晧平.）ポスター発表
• EFFECTS OF CORTISOL ADMINISTRATION ON SEX DETERMINATION, BODY COLOR AND GROWTH IN FISH(Endocrinology,Abstracts of papers presented at the 74^ Annual Meeting of the Zoological Society of Japan) :

  Takahashi Akari, Takahashi Takayuki, Ijiri Shigeho, Adachi Shinji, Yamauchi Kohei  Zoological science  20-  (12)  1603  -1603  2003
• CHANGES IN IMMUNOLOCALIZATION OF STEROIDOGENIC ENZYMES (P450SCC, P450C17, P450AROM) IN GONAD OF THE JAPANESE EEL(Endocrinology,Abstracts of papers presented at the 74^ Annual Meeting of the Zoological Society of Japan) :

  Takei Norio, Ijiri Shigeho, Adachi Shinji, Yamauchi Kohei  Zoological science  20-  (12)  1603  -1603  2003
• Synthesis and possible function of 11-ketotestosterone during oogenesis in eel (Anguilla spp.) (vol 28, pg 353, 2003)

  H Matsubara, PM Lokman, A Senaha, Y Kazeto, S Ijiri, A Kambegawa, T Hirai, G Young, T Todo, S Adachi, K Yamauchi  FISH PHYSIOLOGY AND BIOCHEMISTRY  29-  (3)  253  -253  2003  [Not refereed][Not invited]
  
• Oogenesis in the Japanese eel, Anguilla japonica

  S Adachi, S Ijiri, Y Kazeto, K Yamauchi  EEL BIOLOGY  301  -317  2003  [Not refereed][Invited]
  
• The GnRH system in zebrafish (Danio rerio): Developing a model system for functional studies of GnRH multiplicity.

  KE Kight, Steven, SR, S Ijiri, D Alok, U Klenke, Y Zohar  BIOLOGY OF REPRODUCTION  66-  299  -300  2002  [Not refereed][Not invited]
  
• Changes in steroidogenic enzyme mRNA levels in ovarian follicles of artificially maturing Japanese eel

  Matsubara H, Kazeto Y, Ijiri S, Abe T, Inaba K, Seki S, Hirai T, Adachi S, Yamauchi K  The 25th Symposium on the Japan Society for Comparative Endocrinology  2000/12  [Not refereed][Not invited]
   

  人為催熟に伴うニホンウナギ卵濾胞における5種のステロイド合成酵素遺伝子量を比較した。(Matsubara H, Kazeto Y, Ijiri S, Abe T, Inaba K, Seki S, Hirai T, Adachi S, Yamauchi K) The 25th Symposium on the Japan Society for Comparative Endocrinology. 2000 ; 15 : p10.ポスター発表
• 人為催熟および天然ウナギ卵巣のステロイド合成酵素発現量の比較

  松原創, 風藤行紀, Lokman P M, 奥村浩美, 井尻成保, 足立伸次, 山内晧平  魚の研究 to be continued  1998/11  [Not refereed][Not invited]
   

  前卵黄形成期、卵黄形成初期および中期における人為催熟ニホンウナギと天然ニュージーランドロングフィンウナギ卵巣の３種のステロイド合成酵素遺伝子量を比較した。（松原創、風藤行紀、 Lokman P M、奥村浩美、井尻成保、足立伸次、山内晧平）ポスター発表
• MOLECULAR CLONING OF OVARIAN 17β-HYDROXYSTEROID DEHYDROGENASE AND CHANGES IN ITS EXPRESSION DURING SEXUAL MATURATION IN THE JAPANESE EEL

  MATSUBARA Hajime, KAZETO Yukinori, IJIRI Shigeho, ADACHI Shinji, YAMAUCHI Kohei  Proceedings of the Japan Society for Comparative Endocrinology  13-  56  -56  1998/07  [Not refereed][Not invited]
  
• Isolation and characterization of cytochrome P450 aromatase (CYP19) from a cartilaginous fish, the Atlantic stingray

  S Ijiri, JM Trant  FASEB JOURNAL  12-  (8)  A1426  -A1426  1998/04  [Not refereed][Not invited]
  
• MOLECULAR CLONING OF THE cDNA ENCODING P450 17α-HYDROXYLASE/C_<17-20>LYSE (P450c17) FROM THE JAPANESE EEL OVARY AND DEVELOPMENTAL CHANGES IN ITS EXPRESSION IN THE OVARY

  KAZETO Y., IJIRI S., ADCHI S., YAMAUCHI K.  Proceedings of the Japan Society for Comparative Endocrinology  12-  44  -44  1997/07
• GONADOTROPIN INDUCES AN INCREASE IN 17α,20β-DP RECEPTOR CONCENTRATION AND THE DEVELOPMENT OF OOCYTE MATURATIONAL COMPETENCE IN A TELEOST, THE HIRAME, Paralichthys olivaceus.(Developmental Biology)Proceedings of the Sixty-Third Annual Meeting of the Zoologiacal Socistry of Japan :

  Yoshikuni M., Nagahama Y., Adachi S., Ijiri S., Yamauchi K.  Zoological science  9-  (6)  1162  -1162  1992

Research Grants & Projects

• Exploration of the follicle-stimulating hormone-releasing hormone in the hypothalamus

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2023/04 -2026/03 

  Author : 神田 真司, 大久保 範聡, 井尻 成保
• Studies on regulatory mechanisms underlying steroidogenic enzymes expression that governs sex differentiation, oocyte growth and maturation in teleost

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2022/04 -2026/03 

  Author : 井尻 成保, 木村 敦
• Molecular studies on sex differentiation, oocyte maturation, ovulation, and egg quality loss in sturgeons

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2021/04 -2025/03 

  Author : 足立 伸次, 井尻 成保

   

  本研究では、チョウザメの性分化、卵成熟、排卵および卵質悪化の分子機構解明を推進し、各ステージに特徴的な分子マーカーを利用して、早期性判別、安定的良質卵生産技術および全雌生産技術を確立することを目的としており、本年度は下記の3項目について、分子機構解析と関連技術開発を行なった。 １）性分化：最近、コチョウザメにおいて雌特異的な増幅を示すプライマー（AllWSex2）が見出された。本年度は、アムール、ダウリア（カルーガ）、ミカドチョウザメおよび各種雑種において、本プライマーを用いた遺伝的性判別が可能であることを検証できた。さらに、チョウザメ類における濾胞刺激ホルモン（Fsh）と性分化との関係を明らかにするために、組換えFshを作製し、レポーターアッセイにより生物活性を有することを確認した。 ２）卵成熟および排卵：チョウザメの場合、生体外培養で卵成熟能および排卵能を確認した後、生殖腺刺激ホルモン放出ホルモン（GnRH）注射することで卵を得ているが、タイミングが不適切な場合は良質卵が得られない。これまで、プロスタグランジン合成関連遺伝子発現を指標とした採卵適期推定法を検討してきたが、適当な指標はみつからなかった。そこで、GnRH注射の直前に採取したアムールチョウザメ卵濾胞サンプルを用いて、RNA-seq解析を行なったところ、いくつかの候補遺伝子を見出した。これら遺伝子のうち、Wntシグナルに関与する2個の遺伝子の発現動態を調べたところ、排卵個体ではGnRH注射直前に発現ピークが存在することが判明した。 ３）卵質悪化：これまで、ニホンウナギにおいて、母性mRNA量および局在の差異から、孵化までの胚発生過程で異常が生じる不良卵の分子生物学的特徴の一部を明らかにしている。本年度は、様々な卵質のアムールチョウザメ卵を得ることができた。
• 条鰭類を網羅する卵成熟誘起ホルモン産生分子機構の解明

  日本学術振興会：科学研究費補助金 基盤研究（B)

  Date (from‐to) : 2018/04 -2022/03 

  Author : 井尻成保
• ウナギ雌化と食味に優れた大型雌ウナギの生産技術の確立

  農研機構：イノベーション創出強化研究推進事業

  Date (from‐to) : 2018/04 -2021/03 

  Author : 稲葉博之
• チョウザメの性分化および卵成熟／排卵の分子機構に関する研究

  日本学術振興会：科学研究費補助金 基盤研究（B)

  Date (from‐to) : 2017/04 -2021/03 

  Author : 足立伸次
• 環境依存的性決定の分子機構とその普遍性の解析

  日本学術振興会：科学研究費補助金 基盤研究（B)

  Date (from‐to) : 2016/04 -2019/03 

  Author : 北野健
• バキュロウイルス遺伝子導入系の魚類生殖生理研究における利用技術開発

  日本学術振興会：科学研究費補助金 挑戦的萌芽研究

  Date (from‐to) : 2016/04 -2019/03 

  Author : 井尻成保
• Generation of transgenic Medaka producing a transporter targeting to fish oocytes

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2015/04 -2018/03 

  Author : HIRAMATSU Naoshi, IJIRI Shigeho, KAWAKAMI Koichi

   

  The aim of this study is to obtain the first technical basis for the development of a bio-reactor system through the production of model transporters in transgenic fish, with the final goal of which constructs a system transporting various effective materials across generations (i.e., from mother to egg or larva) in fish. Various expression constructs were generated by combining transporters, fluorescent proteins, a linker, trans-genic vector systems. Trans-genic medaka expressing fluorescent model transporters, as well as a model transporter with a linker, were produced as the second (F1) and the third (F2, the fluorescent transporter alone) trans-genic generations. This study provided fundamental knowledge for the next examination toward a massive bio-production of the transporters.
• ティラピア生殖腺の性分化開始機構の解明とその応用

  日本学術振興会：科学研究費補助金 基盤研究（B)

  Date (from‐to) : 2014/04 -2018/03 

  Author : 井尻成保
• Sea urchin has steroid hormone metabolism ?

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2013/04 -2016/03 

  Author : Ura Kazuhiro, TAKAGI Yasuaki, IJIRI Shigeho

   

  It is still unclear that the endocrine system such as synthesize and/or embolism of steroid hormones during the period of gonadal growth in sea urchin. This study aimed to identify genes involved in gonadal growth and to investigate gene expression in the period of gonadal growth in sea urchin. RNA-seq analysis using next-generation sequencer was carried out on growing gonads of the Northern sea urchin, Mesocentrotus nudus. BLASTn analysis identified 22 transcripts potentially involved endocrine factors such as nuclear receptor (NR) and 21 transcripts of P450 in the growing gonad. However, the homologous genes as ER, AR, P450scc, P450c17, P450arom were not observed in sea urchin. In these analysis, it is suggested that sea urchin has no sex steroid hormones as same as vertebrate. These results suggested that sea urchin synthesized steroid hormones as same as other insects.
• チョウザメの生殖統御技術開発のための性分化、卵成長および卵成熟の分子機構解析

  日本学術振興会：科学研究費補助金 基盤研究（A）

  Date (from‐to) : 2012/04 -2016/03 

  Author : 足立伸次
• 魚類最終成熟誘起ステロイド産生メカニズムの定説を覆す

  日本学術振興会：科学研究費補助金 挑戦的萌芽研究

  Date (from‐to) : 2012/04 -2014/03 

  Author : 足立伸次
• 超雄・超雌作製と早期性判別を実現するための性連鎖DNA配列段階的同定法の確立

  日本学術振興会：科学研究費補助金 挑戦的萌芽研究

  Date (from‐to) : 2011/04 -2014/03 

  Author : 井尻成保
• 最新の生理生態情報に基づくウナギ大量種苗生産技術の実現

  日本学術振興会：科学研究費補助金 基盤研究（S）

  Date (from‐to) : 2008 -2014 

  Author : 塚本勝巳
• 魚類養殖及び放流事業の性統御を可能にする遺伝的雌雄判別技術の開発

  科学術振興機構：地域イノベーション創出総合支援事業「シーズ発掘試験」

  Date (from‐to) : 2009/04 -2010/03 

  Author : 井尻成保
• Mechanism of previtellogenic oocyte growth in fish

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2007 -2009 

  Author : ADACHI Shinji, IJIRI Shigeho, TODO Takashi

   

  Estrogen treatment may promote oocyte growth during ovarian differentiation in eel and sturgeon. Androgen treatment accelerates previtellogenic oocyte growth in eel. Somatic growth may promote oocyte growth between estrogen and androgen-dependent phase in fish.
• ティラピアの生殖腺の性分化に関わる遺伝子の特定と機能解析

  日本学術振興会：科学研究費補助金 若手研究B

  Date (from‐to) : 2006/04 -2008/03 

  Author : 井尻成保
• 催熟ウナギ雌の脳下垂体の成熟への関与に関する内分泌学的研究

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2001 -2003 

  Author : 井尻 成保

   

  昨年度までの脳下垂体除去雌ウナギの催熟実験によって、通常のウナギ人為催熟には、ウナギ自身の脳下垂体は必ずしも必要ではないことがあきらかとなった。本年度は、主要なステロイド合成酵素の抗体を作製し、タンパクレベルでの発現制御の解明、および未だ単離されていないステロイド合成酵素の単離を目指した。1)20β-HSD遺伝子の単離:他魚種で20β-HSDとして単離されているカルボニル還元化酵素はウナギでは20β-HSD活性を持たなかったことから他のタイプが存在すると仮定して、機能発現クローニングを試みたが20β-HSD活性を持つ遺伝子を単離するには至らなかった。2)P450c11の免疫組織化学的局在:ウナギP450c11組替タンパクに対する特異抗体を作製し、ウナギ組織を免疫染色した結果、頭腎のステロイド合成細胞および精巣のライディッヒ細胞が良好に染色されたが、卵巣に陽性細胞は観察されなかった。この時期卵巣のP450c11 RNAの発現は高い為、矛盾する結果となった。3)P450scc、P450c17、P450c11のmRNAの局在:卵巣ではscc、c17ともにmRNAはタンパクと同時期、同部位に発現していた。精巣では催熟開始直後に発現が高まるも、以降ほとんど発現は認められず、scc、c17ともタンパクが一貫して発現している様子とは異なった。精巣のc11に関しては催熟開始9日目にmRNA、タンパクともにほぼ同時期に発現していた。以上、これまでにニホンウナギのFSH、サケFSH、P450scc、P450c17、P450arom、P450c11の特異抗体を作製し、免疫組織化学的観察を行った。これまでの結果、ウナギ雌の人為催熟にはウナギ自身の脳下垂体は必ずしも必要でなく、つまり様々なステロイド合成酵素は注射されるサケ下垂体によって発現が促進されている者と考えられた。そのステロイド合成酵素の発現制御も各々異なるとが示唆された。

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