Department of FisheriesAssociate Professor

Last Updated :2024/07/20

■Researcher comments

List of press-related appearances


■Researcher basic information

Research Keyword

  • 生殖   性転換   水産   魚類   



  • 2022/11 - 2023/02  University of OtagoDepartment of Zoology(近畿大学 在外研究)

Educational Background

  • 2001/04 - 2004/03  北海道大学大学院  理学研究科  生物科学専攻
  • 1998/04 - 2000/03  Nagasaki University  水産学研究科  水産学専攻
  • 1994/04 - 1998/03  Nagasaki University  Faculty of Fisheries  水産学科

■Research activity information


  • Takanori Ikenaga; Tastufumi Nakamura; Tatsushi Tajiri; Minaki Tsuji; Dai-ichiro Kato; Toshinao Ineno; Yasuhisa Kobayashi; Naoaki Tsutsui; Sadao Kiyohara
    Cell and Tissue Research Springer Science and Business Media LLC 394 (3) 431 - 439 0302-766X 2023/10
  • Kobayashi, Y; Keyamura, O; Lokman, M. P; Chuda, H
    International Journal of Aquatic Biology 11 (6) 571 - 576 2023 [Refereed]
  • Akira Kanamori; Yasuhisa Kobayashi
    Frontiers in cell and developmental biology 11 1328024 - 1328024 2023 
    Mature gametes are transported externally for fertilization. In vertebrates, the gonads are located within the coelom. Consequently, each species has specific organs for export, which often vary according to sex. In most vertebrates, sperm ducts and oviducts develop from the Wolffian and Müllerian ducts, respectively. However, exceptions exist. Both sexes of cyclostomes, as well as females of basal teleosts, lack genital ducts but possess genital pores. In teleosts of both sexes, genital ducts are formed through the posterior extensions of gonads. These structures appear to be independent of both Wolffian and Müllerian ducts. Furthermore, the development of Wolffian and Müllerian ducts differs significantly among various vertebrates. Are these gamete-exporting organs homologous or not? A question extensively debated around the turn of the 20th century but now largely overlooked. Recent research has revealed the indispensable role of Wnt4a in genital duct development in both sexes of teleosts: zebrafish and medaka. wnt4a is an ortholog of mammalian Wnt4, which has functions in Müllerian duct formation. These results suggest a potential homology between the mammalian Müllerian ducts and genital ducts in teleosts. To investigate the homology of gamete-exporting organs in vertebrates, more detailed descriptions of their development across vertebrates, using modern cellular and genetic tools, are needed. Therefore, this review summarizes existing knowledge and unresolved questions on the structure and development of gamete-exporting organs in diverse vertebrate groups. This also underscores the need for comprehensive studies, particularly on cyclostomes, cartilaginous fishes, basal ray-finned fishes, and teleosts.
    NIPPON SUISAN GAKKAISHI Japanese Society of Fisheries Science 88 (6) 485 - 493 0021-5392 2022/11 [Refereed]
  • Yasuhisa Kobayashi
    Aquaculture and Fisheries Elsevier BV 7 (5) 519 - 524 2468-550X 2022/09 [Refereed]
  • Keiko Takanami; Takumi Oti; Yasuhisa Kobayashi; Koki Hasegawa; Takashi Ito; Naoaki Tsutsui; Yasumasa Ueda; Earl Carstens; Tatsuya Sakamoto; Hirotaka Sakamoto
    The Journal of comparative neurology 2022/06 
    Gastrin-releasing peptide (GRP) and its receptor (GRPR) have been identified as itch mediators in the spinal and trigeminal somatosensory systems in rodents. In primates, there are few reports of GRP/GRPR expression or function in the spinal sensory system and virtually nothing is known in the trigeminal system. The aim of the present study was to characterize GRP and GRPR in the trigeminal and spinal somatosensory system of Japanese macaque monkeys (Macaca fuscata). cDNA encoding GRP was isolated from the macaque dorsal root ganglion (DRG) and exhibited an amino acid sequence that was highly conserved among mammals and especially in primates. Immunohistochemical analysis demonstrated that GRP was expressed mainly in the small-sized trigeminal ganglion and DRG in adult macaque monkeys. Densely stained GRP-immunoreactive (ir) fibers were observed in superficial layers of the spinal trigeminal nucleus caudalis (Sp5C) and the spinal cord. In contrast, GRP-ir fibers were rarely observed in the principal sensory trigeminal nucleus and oral and interpolar divisions of the spinal trigeminal nucleus. cDNA cloning, in situ hybridization, and Western blot revealed substantial expression of GRPR mRNA and GRPR protein in the macaque spinal dorsal horn and Sp5C. Our Western ligand blot and ligand derivative stain for GRPR revealed that GRP directly bound in the macaque Sp5C and spinal dorsal horn as reported in rodents. Finally, GRP-ir fibers were also detected in the human spinal dorsal horn. The spinal and trigeminal itch neural circuits labeled with GRP and GRPR appear to function also in primates.
  • Aoshi Kobayashi; Mayuko Hamada; Masa-Aki Yoshida; Yasuhisa Kobayashi; Naoaki Tsutsui; Toshio Sekiguchi; Yuta Matsukawa; Sho Maejima; Joseph J Gingell; Shoko Sekiguchi; Ayumu Hamamoto; Debbie L Hay; John F Morris; Tatsuya Sakamoto; Hirotaka Sakamoto
    Science advances 8 (9) eabk0331  2022/03 
    Vasopressin/oxytocin (VP/OT)-related peptides are essential for mammalian antidiuresis, sociosexual behavior, and reproduction. However, the evolutionary origin of this peptide system is still uncertain. Here, we identify orthologous genes to those for VP/OT in Platyhelminthes, intertidal planarians that have a simple bilaterian body structure but lack a coelom and body-fluid circulatory system. We report a comprehensive characterization of the neuropeptide derived from this VP/OT-type gene, identifying its functional receptor, and name it the "platytocin" system. Our experiments with these euryhaline planarians, living where environmental salinities fluctuate due to evaporation and rainfall, suggest that platytocin functions as an "antidiuretic hormone" and also organizes diverse actions including reproduction and chemosensory-associated behavior. We propose that bilaterians acquired physiological adaptations to amphibious lives by such regulation of the body fluids. This neuropeptide-secreting system clearly became indispensable for life even without the development of a vascular circulatory system or relevant synapses.
  • Masaru Nakamura; Ryo Nozu; Shigeo Nakamura; Mikihiko Higa; Ramji K Bhandari; Yasuhisa Kobayashi; Ryo Horiguchi; Toru Komatsu; Yutaka Kojima; Ryosuke Murata; Kiyoshi Soyano; Satoshi Ogawa; Toshiaki Hirai; Hajime Matsubara; Toshinobu Tokumoto; Tohru Kobayashi; Hirohiko Kagawa; Shinji Adachi; Kohei Yamauchi; Yoshitaka Nagahama
    Galaxea, Journal of Coral Reef Studies Japanese Coral Reef Society 24 (1) 5 - 17 1883-0838 2022/01
  • Effect of synthetic androgen treatment on the gonad of juvenile longtooth grouper (Epinephelus bruneus)
    Yasuhisa Kobayasahi; Takamasa Morishita; Hisashi Chuda
    International Aquatic Research 13 163 - 171 2021/08 [Refereed]
  • Asuka Hirooka; Mayuko Hamada; Daiki Fujiyama; Keiko Takanami; Yasuhisa Kobayashi; Takumi Oti; Yukitoshi Katayama; Tatsuya Sakamoto; Hirotaka Sakamoto
    Scientific reports 11 (1) 13315 - 13315 2021/06 
    Bombesin is a putative antibacterial peptide isolated from the skin of the frog, Bombina bombina. Two related (bombesin-like) peptides, gastrin-releasing peptide (GRP) and neuromedin B (NMB) have been found in mammals. The history of GRP/bombesin discovery has caused little attention to be paid to the evolutionary relationship of GRP/bombesin and their receptors in vertebrates. We have classified the peptides and their receptors from the phylogenetic viewpoint using a newly established genetic database and bioinformatics. Here we show, by using a clawed frog (Xenopus tropicalis), that GRP is not a mammalian counterpart of bombesin and also that, whereas the GRP system is widely conserved among vertebrates, the NMB/bombesin system has diversified in certain lineages, in particular in frog species. To understand the derivation of GRP system in the ancestor of mammals, we have focused on the GRP system in Xenopus. Gene expression analyses combined with immunohistochemistry and Western blotting experiments demonstrated that GRP peptides and their receptors are distributed in the brain and stomach of Xenopus. We conclude that GRP peptides and their receptors have evolved from ancestral (GRP-like peptide) homologues to play multiple roles in both the gut and the brain as one of the 'gut-brain peptide' systems.
  • Ryosuke Murata; Ryo Nozu; Yuji Mushirobira; Takafumi Amagai; Jun Fushimi; Yasuhisa Kobayashi; Kiyoshi Soyano; Yoshitaka Nagahama; Masaru Nakamura
    Scientific reports 11 (1) 11117 - 11117 2021/05 
    Vertebrates usually exhibit gonochorism, whereby their sex is fixed throughout their lifetime. However, approximately 500 species (~ 2%) of extant teleost fishes change sex during their lifetime. Although phylogenetic and evolutionary ecological studies have recently revealed that the extant sequential hermaphroditism in teleost fish is derived from gonochorism, the evolution of this transsexual ability remains unclear. We revealed in a previous study that the tunica of the ovaries of several protogynous hermaphrodite groupers contain functional androgen-producing cells, which were previously unknown structures in the ovaries of gonochoristic fishes. Additionally, we demonstrated that these androgen-producing cells play critical roles in initiating female-to-male sex change in several grouper species. In the present study, we widened the investigation to include 7 genera and 18 species of groupers and revealed that representatives from most major clades of extant groupers commonly contain these androgen-producing cells, termed testicular-inducing steroidogenic (TIS) cells. Our findings suggest that groupers acquired TIS cells in the tunica of the gonads for successful sex change during their evolution. Thus, TIS cells trigger the evolution of sex change in groupers.
  • 瀬戸内海東部におけるアイゴの年齢・成長と産卵.
    山本昌幸; 棚田教生; 元谷 剛; 小林靖尚; 片山知史
    水産海洋研究 84 (3) 178 - 186 2020/09 [Refereed]
  • Naoaki Tsutsui; Yasuhisa Kobayashi; Kouichi Izumikawa; Tatsuya Sakamoto
    Crustacean reproduction has been hypothesized to be under complex endocrinological regulation by peptide hormones. To further improve our understanding of the mechanisms underlying this complex regulation, knowledge is needed regarding the hormones not only of the central nervous system (CNS) such as the X-organ/sinus gland (XOSG), brain, and thoracic ganglia, but also the peripheral gonadal tissues. For example, in vertebrates, some gonadal peptide hormones including activin, inhibin, follistatin, and relaxin are known to be involved in the reproductive physiology. Therefore, it is highly likely that some peptide factors from the ovary are serving as the signals among peripheral tissues and central nervous tissues in crustaceans. In this work, we sought to find gonadal peptide hormones and peptide hormone receptors by analyzing the transcriptome of the ovary of the kuruma prawnMarsupenaeus japonicus. The generated ovarian transcriptome data led to the identification of five possible peptide hormones, including bursicon-alpha and -beta, the crustacean hyperglycemic hormone (CHH)-like peptide, insulin-like peptide (ILP), and neuroparsin-like peptide (NPLP). Dominant gene expressions for the bursicons were observed in the thoracic ganglia and the ovary, in the CNS for the CHH-like peptide, in the heart for NPLP, and in the ovary for ILP. Since the gene expressions of CHH-like peptide and NPLP were affected by a CHH (Penaeus japonicussinus gland peptide-I) from XOSG, we produced recombinant peptides for CHH-like peptide and NPLP usingEscherichia coliexpression system to examine their possible peripheral regulation. As a result, we found that the recombinant NPLP increased vitellogenin gene expression in incubated ovarian tissue fragments. Moreover, contigs encoding putative receptors for insulin-like androgenic gland factor, insulin, neuroparsin, and neuropeptide Y/F, as well as several contigs encoding orphan G-protein coupled receptors and receptor-type guanylyl cyclases were also identified in the ovarian transcriptome. These results suggest that reproductive physiology in crustaceans is regulated by various gonadal peptide hormones, akin to vertebrates.
    NIPPON SUISAN GAKKAISHI Japanese Society of Fisheries Science 86 (4) 274 - 287 0021-5392 2020/07 [Refereed]
  • Taketeru Tomita; Masaru Nakamura; Yasuhisa Kobayashi; Atsushi Yoshinaka; Kiyomi Murakumo
    SCIENTIFIC REPORTS NATURE PUBLISHING GROUP 10 (1) 2045-2322 2020/04 [Refereed]
    In viviparous (live-bearing) animals, embryos face an embryo-specific defecation issue: faecal elimination in utero can cause fatal contamination of the embryonic environment. Our data from the viviparous red stingray (Hemitrygon akajei) reveals how viviparous elasmobranchs circumvent this issue. The exit of the embryonic intestine is maintained closed until close to birth, which allows the accumulation of faeces in the embryonic body. Faecal accumulation abilities are increased by (1) the large intestine size (represents about 400-600% of an adult intestine, proportionally), and (2) the modification in the intestinal inner wall structure, specialized to increase water uptake from the faecal matter. According to the literature, faecal accumulation may occur in embryos of the lamniform white shark as well. The reproductive biology of myliobatiform stingrays and lamniform sharks is characterized by the onset of oral feeding before birth (i.e. drinking of uterine milk and eating of sibling eggs, respectively), which is expected to result in the production of large amounts of faeces during gestation. The strong ability of faecal accumulation in these lineages is therefore likely an adaptation to their unique embryonic nutrition mechanism.
  • Yasuhisa Kobayashi; Masaru Nakamura
    Nippon Suisan Gakkaishi (Japanese Edition) Nihon Suisan Gakkai 85 (2) 192  1349-998X 2019
  • Ryo Horiguchi; Ryo Nozu; Toshiaki Hirai; Yasuhisa Kobayashi; Masaru Nakamura
    General and Comparative Endocrinology Academic Press Inc. 257 67 - 73 1095-6840 2018/02 [Refereed]
    The three-spot wrasse, Halichoeres trimaculatus, can change sex from female to male (i.e. protogyny) due to sharp decrease in endogenous estrogen. During the sex change, ovarian tissue degenerates and testicular tissue arises newly. Finally, ovarian tissue disappears completely and replaces into mature testis. In order to predict the molecular mechanisms controlling the processes of sex change, we investigated the expression patterns of four genes (rspo1, figla, sox9b and amh), which have been thought to be associated with ovarian/testicular differentiation in vertebrates. Expression levels of rspo1 and figla, which play important roles for ovarian differentiation in vertebrates, were stable until the middle stage of the sex change, and subsequently down-regulated. Therefore, it was indicated that decrease in rspo1 and figla could result from ovarian degeneration. On the other hand, basis on the expression pattern, it was indicated that sox9b and amh, which are involved in testicular differentiation in vertebrates, were implicated in testicular formation and spermatogenesis during the sex change as well. The present results could be fundamental information for investigating the relationship between these factors and E2 depletion, which is crucial trigger for sex change.
  • Yasuhisa Kobayashi; Ryo Nozu; Ryo Horiguchi; Masaru Nakamura
    Reproductive and Developmental Strategies Springer Japan 321  2509-5536 2018 [Refereed]
  • Kei Tamura; Yasuhisa Kobayashi; Asuka Hirooka; Keiko Takanami; Takumi Oti; Takamichi Jogahara; Sen-ichi Oda; Tatsuya Sakamoto; Hirotaka Sakamoto
    JOURNAL OF COMPARATIVE NEUROLOGY WILEY 525 (7) 1586 - 1598 0021-9967 2017/05 [Refereed]
    Several regions of the brain and spinal cord control male reproductive function. We previously demonstrated that the gastrin-releasing peptide (GRP) system, located in the lumbosacral spinal cord of rats, controls spinal centers to promote penile reflexes during male copulatory behavior. However, little information exists on the male-specific spinal GRP system in animals other than rats. The objective of this study was to examine the functional generality of the spinal GRP system in mammals using the Asian house musk shrew (Suncus murinus; suncus named as the laboratory strain), a specialized placental mammal model. Mice are also used for a representative model of small laboratory animals. We first isolated complementary DNA encoding GRP in suncus. Phylogenetic analysis revealed that suncus preproGRP was clustered to an independent branch. Reverse transcription-PCR showed that GRP and its receptor mRNAs were both expressed in the lumbar spinal cord of suncus and mice. Immunohistochemistry for GRP demonstrated that the sexually dimorphic GRP system and male-specific expression/distribution patterns of GRP in the lumbosacral spinal cord in suncus are similar to those of mice. In suncus, we further found that most GRP-expressing neurons in males also express androgen receptors, suggesting that this male-dominant system in suncus is also androgen-dependent. Taken together, these results indicate that the sexually dimorphic spinal GRP system exists not only in mice but also in suncus, suggesting that this system is a conserved property in mammals. J. Comp. Neurol. 525:1586-1598, 2017. (c) 2016 Wiley Periodicals, Inc.
  • Tomoki Sunobe; Tetsuya Sado; Kiyoshi Hagiwara; Hisaya Manabe; Toshiyuki Suzuki; Yasuhisa Kobayashi; Makoto Sakurai; Shin-ichi Dewa; Midori Matsuoka; Akihiko Shinomiya; Kazuya Fukuda; Masaki Miya
    SCIENCE OF NATURE SPRINGER HEIDELBERG 104 (3-4) 15  0028-1042 2017/04 [Refereed]
    Size-advantage and low-density models have been used to explain how mating systems favor hermaphroditism or gonochorism. However, these models do not indicate historical transitions in sexuality. Here, we investigate the evolution of bidirectional sex change and gonochorism by phylogenetic analysis using the mitochondrial gene of the gobiids Trimma (31 species), Priolepis (eight species), and Trimmatom (two species). Trimma and Priolepis formed a clade within the sister group Trimmatom. Gonadal histology and rearing experiments revealed that Trimma marinae, Trimma nasa, and Trimmatom spp. were gonochoric, whereas all other Trimma and Priolepis spp. were bidirectional sex changers or inferred ones. A maximum-likelihood reconstruction analysis demonstrated that the common ancestor of the three genera was gonochoristic. Bidirectional sex change probably evolved from gonochorism in a common ancestor of Trimma and Priolepis. As the gonads of bidirectional sex changers simultaneously contain mature ovarian and immature testicular components or vice versa, individuals are always potentially capable of functioning as females or males, respectively. Monogamy under low-density conditions may have been the ecological condition for the evolution of bidirectional sex change in a common ancestor. As T. marinae and T. nasa are a monophyletic group, gonochorism should have evolved from bidirectional sex change in a common ancestor.
  • Expression and Localization of Two Gonadotropin Receptors in Gonads of the Yellowtail Clownfish , Amphiprion Clarki
    Kobayashi Y; Nozu R; Nakamura M
    Journal of Aquaculture & Marine Biology 5 (3) 2017/03 [Refereed]
  • Ryo Nozu; Ryo Horiguchi; Yasuhisa Kobayashi; Masaru Nakamura
    MOLECULAR REPRODUCTION AND DEVELOPMENT WILEY-BLACKWELL 82 (11) 859 - 866 1040-452X 2015/11 [Refereed]
    Sex change in fish involves a dramatic transformation of gonadal tissue and a switch in gametogenesis. Doublesex/male abnormal-3-related transcription factor-1 (DMRT1), encoded by the DMRT1 gene, is involved in testicular differentiation in a wide range of vertebrates as well as in sexual differentiation and gonadal sex change. In the present study, we investigated changes in the expression of dmrt1 during artificial gonadal sex change in the three-spot wrasse, Halichoeres trimaculatus, by real-time quantitative PCR and immunolocalization, using an anti-wrasse-Dmrt1 antibody that we prepared. We found that dmrt1 expression was predominantly observed in the testes, and that Dmrt1 was expressed in Sertoli cells of testes and a few granulosa cells surrounding vitellogenic oocytes of the ovary. Additionally, the upregulation of dmrt1 expression was consistent with an increase in spermatogenic cyst quantity rather than proliferation of presumptive spermatogonia, suggesting that dmrt1 is involved in the progression of spermatogenesis during sex change. Changes in the localization of Dmrt1 during gonadal sex change further implied that Sertoli cells originate from somatic cells adjacent to gonial germ cells during testicular formation in the three-spot wrasse.
  • Kobayashi Y; Mototani T; Murayama F; Sakamoto T
    Zoological letters 1 25  2015/09 [Refereed]
  • Narayan Prasad Pandit; Ramji Kumar Bhandari; Yasuhisa Kobayashi; Masaru Nakamura
    High temperature treatments induce germ cell loss in gonads of vertebrate animals, including fish. It could be a reliable source for induction of sterility if the treatments led to a permanent loss of germ cells. Here we report that heat treatment at 37 degrees C for 45-60 days caused a complete loss of germ cells in female Nile tilapia, Oreochromis niloticus, and that sterility was achieved in fish at all stages of their life cycle. Unlike previous observations, germ cells did not repopulate even after returning them to the water at control conditions suggesting permanent depletion of germ cells. Gonadal somatic cells immunopositive for 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) were clustered at one end of the germ cell depleted gonads close to the blood vessel. Serum level of testosterone, 11-ketotestosterone, and 17 beta-estradiol was significantly decreased in sterile fish compared to control. Body weight of sterile fish was higher than control fish at the end of experiment. Our observations of increased growth and permanent sterilization in the high temperature-treated fish suggest that this method could be an appropriate and eco-friendly tool for inducing sterility in fish with a higher thermal tolerance. (C) 2015 Elsevier Inc. All rights reserved.
  • Ryosuke Murata; Yasuhisa Kobayashi; Hirofumi Karimata; Kazuo Kishimoto; Motofumi Kimura; Masaru Nakamura
    BIOLOGY OF REPRODUCTION SOC STUDY REPRODUCTION 91 (1) 25  0006-3363 2014/07 [Refereed]
    To clarify the cause of sex change recovery after the withdrawal of androgen treatment, immature female Malabar grouper were fed a diet containing 17alpha-methyltestosterone (MT) at 50 mu g/g for 7 mo and then a normal diet for 6 mo. The MT brought about precocious sex change from immature ovaries to mature testes with active spermatogenesis, including the development of spermatozoa, and sex change reversed soon after MT treatment withdrawal. This result indicates that precocious sex change in immature Malabar grouper with oral MT treatment is impermanent. The expression of three steroidogenic enzymes (Cyp11a, Cyp19a1a, and Cyp11b) in the gonads of the Malabar grouper were analyzed immunohistochemically at the end of the 7-mo treatment. No apparent differences were seen in the expression pattern of these enzymes between the mature testes of MT-treated fish and the immature ovaries of control fish. In addition, serum estradiol-17beta and 11-ketotestosterone levels in treated fish were the same as those in control fish. These results indicate that in the case of immature Malabar grouper MT might have little effect on endogenous steroidogenesis during precocious sex change even though it induced active spermatogenesis in the gonads of treated fish. From these results, we also concluded that MT might have little effect on the steroidogenic endocrine pathway, and this is one cause of sex change recovery after treatment withdrawal.
  • Yasuhisa Kobayashi; Ryo Nozu; Ryo Horiguchi; Masaru Nakamura
    International Aquatic Research Springer Verlag 6 (2) 1 - 9 2008-6970 2014 [Refereed]
    Doublesex-mab 3-related transcription factor 1 (DMRT1) has been identified as the first conserved gene involved in the testicular differentiation of vertebrates. However, the precise role of DMRT1 in spermatogenesis has not been made clear. In this study, immunohistochemical method was used to observe DMRT1 protein localization in order to resolve cellular profile of DMRT1 in the adult testis of three-spot wrasse. DMRT1 protein was clearly and specifically localized in the Sertoli cells of all spermatogenic cells and epithelial cells comprising the efferent duct, but not in the germ cells. In addition, adult males were treated with aromatase inhibitor (AI) for investigating the role of estrogen on the transcription of DMRT1. AI treatment caused an increase in the levels of DMRT1 transcripts in the efferent duct region, concomitant with a decrease in spermatogonia and spermatocytes. © 2014 The Author(s).
  • Saori Miura; Yasuhisa Kobayashi; Ramji Kumar Bhandari; Masaru Nakamura
    All undifferentiated gonads of anemonefish first differentiate into ovaries, and then testicular tissue appear among ovarian tissue, and finally form ambisexual gonads with both ovarian and testicular tissues. The role of estradiol-17beta (E2) in differentiation of ovarian cells is well conserved across phyla; however, its role in development of ambisexual gonads is poorly understood. Here we demonstrate that the E2 produced during the differentiation of ovarian cells does not allow testicular cells to differentiate in the prospective ambisexual gonad. We examined the immunolocalization of the steroidogenic enzyme cytochrome aromatase (P450(arom)), which is involved in E2 production. In the gonads, numbers of the P450(arom)-positive cells increased during ovarian differentiation. However, immunopositive cells with weak signal intensity were seen in the interstitial areas among oocytes and between oocytes and testicular tissue undergoing testicular differentiation. In contrast, P450(arom)-positive cells were not found in any testicular tissues of the ambisexual gonads. We also examined changes in E2 production in vitro in the gonads during testicular differentiation. E2 was high in the ovaries before the appearance all of testicular tissue, and decreased accompanying the differentiation of testicular tissue. These results suggest a balance of estrogen/androgen seems to be important during sex differentiation, and then a shift from estrogen to androgen production may induce testicular differentiation in the ovary. Further, exogenous E2 treatment suppressed naturally occurring differentiation of testicular cells forming exclusively ovarian tissues in the gonad in vivo, suggesting the increase of estrogen blocks the differentiation of testicular tissue and the formation of ambisexual gonad. J. Exp. Zool. 319A: 560-568, 2013. (c) 2013 Wiley Periodicals, Inc.
  • Nao Kagawa; Yudai Nishiyama; Kanoko Kato; Hideya Takahashi; Yasuhisa Kobayashi; Hirotaka Sakamoto; Tatsuya Sakamoto
    General and Comparative Endocrinology Academic Press Inc. 194 257 - 263 1095-6840 2013/12 [Refereed]
    The hypothalamic hormones, arginine-vasotocin (VT) and isotocin (IT), play central roles in osmoregulation and in the regulation of social behaviors including aggressive behavior in many vertebrates including fish. Here, we examined whether these hormones are associated with aggressive behavior in the mudskipper (Periophthalmus modestus). The mudskipper is an amphibious fish, which lives in the brackish water of river mouths and displays unique aggressive behavior. Upon introduction to each other in an experimental tank with aquatic and terrestrial areas, a pair of males can be classified as aggressive dominant or submissive subordinate based on the frequency of their aggressive acts, which is significantly higher in dominant male. Additionally, the length of stay in terrestrial area of dominant was longer than that of the subordinate. The latter remained in aquatic area almost throughout the period of behavioral observation. The expression of brain VT mRNA was significantly higher in subordinate than in dominant, whereas neither IT mRNA expression nor plasma cortisol level differed between subordinate and dominant male. On the other hand, an intracerebroventricular injection of VT increased aggressive behaviors in mudskippers. In addition to known roles of VT in mediation of aggressive behavior, these results may shed light on the role of endogenous VT toward water migration in submissive mudskippers. The amphibious fish is a valuable experimental model to observe the relationship between effects of central VT on the osmoregulation and social behavioral regulation in vertebrates. © 2013.
  • Ryo Horiguchi; Ryo Nozu; Toshiaki Hirai; Yasuhisa Kobayashi; Yoshitaka Nagahama; Masaru Nakamura
    Developmental Dynamics 242 (4) 388 - 399 1058-8388 2013/04 [Refereed]
    Background: Sex change in fishes provides a good experimental model for understanding the mechanisms and plasticity of sex determination and differentiation. The three-spot wrasse, Halichoeres trimaculatus is a protogynous hermaphrodite. During sex change from female to male, the ovary is replaced by the testis through the degeneration of oocytes and subsequent spermatogenesis. In the present study, we cloned a cDNA-encoding gonadal soma-derived factor (GSDF) from protogynous wrasse and examined its expression pattern in the sexually mature gonads and the sex-changing gonad induced experimentally by aromatase inhibition. Results: Expression of gsdf was predominantly observed in the testis, and it was mainly localized to the supporting cells surrounding the spermatogonia. In the ovary, only slight expression of gsdf was observed in morphologically undifferentiated supporting cells in contact with oogonia. During sex change, strong expression of gsdf appeared first in the supporting cells surrounding the gonial germ cells before the onset of spermatogenesis. Thereafter, the expression of gsdf continually increased in the supporting cells surrounding the proliferating spermatogonia throughout the sex change. Conclusions: These results suggest that gsdf is involved in the proliferation of spermatogonia and subsequent spermatogenesis in both the testis and the gonad in the early stages of sex change. © 2013 Wiley Periodicals, Inc.
  • Physiological and endocrinological mechanisms of sex change in the grouper
    Yasuhisa Kobayashi; Ryosuke Murata; Masaru Nakamura
    Sexual Plasticity and Gametogenesis in Fishes Nova Science Publishers, Inc. 221 - 233 2013 
    Groupers of the genus Epinephelus are one of the most important aquaculture species in the world. The sexuality of groupers is almost protogynous hermaphrodite. Since the gonadal re-constriction occurred in adult, grouper provide a good experiment model to investigate the sexual differentiation in vertebrate. Due to the economical and biological importance of groupers, physiological and endocrinological data are eagerly needed for improvement aquaculture. However, the endocrinological mechanism of sex change is not clearly understood. This review addresses the physiological and endocrinological mechanisms and also explains the process of sex differentiation in groupers. Key regulatory factors underlying sex change has been identified to date include sex steroids and follicle stimulating hormone (FSH). © 2013 Nova Science Publishers, Inc.
  • Y. Kobayashi; Y. Nagahama; M. Nakamura
    SEXUAL DEVELOPMENT KARGER 7 (1-3) 115 - 125 1661-5425 2013 [Refereed]
    Among vertebrates, fishes show an exceptional range of reproductive strategies regarding the expression of their sexuality. Fish sexualities were categorized into gonochorism, synchronous/sequential hermaphrodite, or unisexual reproduction. In gonochoristic fishes, sex is determined genetically or by environmental factors. After sex determination, the gonads are differentiated into ovary or testis, with the sex remaining fixed for the entire life cycle. In contrast, some sequential hermaphrodite fishes can change their sex from male to female (protandrous), female to male (protogynous), or serially (bi-directional sex change) in their life cycle. In many cases, sex change is cued by social factors such as the disappearance of a male or female from a group. This unique diversity in fishes provides an ideal animal model to investigate sex determination and differentiation in vertebrates. This review first discusses genetic-orientated sex determination mechanisms. Then, we address the gonadal sex differentiation process in a gonochoristic fish, using an example of the Nile tilapia. Finally, we discuss various types of sex change that occur in hermaphrodite fishes. Copyright (c) 2012 S. Karger AG, Basel
  • Atsushi Sogabe; Hiromi Takata; Yasuhisa Kobayashi
    ICHTHYOLOGICAL RESEARCH SPRINGER JAPAN KK 60 (1) 85 - 88 1341-8998 2013/01 [Refereed]
    This study investigated ovarian structure and mode of egg production in the seaweed pipefish, Syngnathus schlegeli. The ovary had a rolled sheet-like structure where developing follicles were arranged serially in sequence according to their development, with a single germinal ridge running along the edge of the sheet. Oocytes of various developmental stages were concomitantly present in the ovary, and the number of mature eggs increased continuously over time, indicating that egg production is asynchronous. This would be a physiological basis for multiple spawning within a short time span associated with polygamy in this fish.
  • Ryo Nozu; Ryo Horiguchi; Ryosuke Murata; Yasuhisa Kobayashi; Masaru Nakamura
    Fish Physiology and Biochemistry 39 (1) 47 - 51 0920-1742 2013 [Refereed]
    The three-spot wrasse (Halichoeres trimaculatus), which inhabits the coral reefs of Okinawa, changes sex from female to male. Sex change in this species is controlled by a social system. Oocytes disappear completely from the ovary, and male germ cells and somatic cells comprising testicular tissue arise a new during the sex change process. However, little is known of the fate and origin of the gonadal tissue-forming cells during sex change. In particular, the fate of ovarian somatic cells has not been determined, although the ovarian tissue regresses histologically. To approach this question, we analyzed apoptosis and cell proliferation in the sex-changing gonads. Unexpectedly, we found that few apoptotic somatic cells were present during sex change, suggesting that ovarian somatic cells might survive during the regression of the ovarian tissue. On the other hand, cell proliferation was detected in many granulosa cells surrounding the degenerating oocytes, a few epithelial cells covering ovigerous lamella and a few somatic cells associated with gonial germ cells at an early stage of sex change. Then, we found that proliferative ovarian somatic cells remained in the gonads late in the sex change process. Based on these results, we concluded that some functional somatic cells of the ovary are reused as testicular somatic cells during the gonadal sex change in the three-spot wrasse. © 2012 Springer Science+Business Media B.V.
  • Ryosuke Murata; Yasuhisa Kobayashi; Hirofumi Karimata; Kazuo Kishimoto; Motofumi Kimura; Akio Shimizu; Masaru Nakamura
    The aim of this study was to clarify the roles of 2 gonadotropins (GTHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), on sex differentiation in the protogynous Malabar grouper, Epinephelus malabaricus. To do this, the mRNA expression patterns of GTH subunits (cga, fshb, and lhb) in the fish pituitary throughout gonadal sex differentiation were investigated. Real-time reverse transcriptase (RT)-PCR showed that cga and fshb were present in the undifferentiated and ovarian differentiation stages, and that the expression levels significantly increased after ovarian differentiation (AOD). However, lhb was not expressed before ovarian differentiation (BOD) and was first detected AOD. Next, to investigate the differentiation and distribution of Fshb and Lhb-producing cells in the pituitary of fish throughout gonadal sex differentiation, immunohistochemical analysis was used to detect teleost GTH subunits. Positive immunoreactivity against Fshb and Lhb was not detected in the pituitary BOD; Fshb and Lhb-positive cells first appeared in the pituitary AOD. It therefore seems unlikely that pituitary gonadotropins play a major role in the control of gonadal sex differentiation in the Malabar grouper. (c) 2012 Elsevier Inc. All rights reserved.
  • Yasuhisa Kobayashi; Takeshi Usami; Tomoki Sunobe; Hisaya Manabe; Yoshitaka Nagahama; Masaru Nakamura
    ZOOLOGICAL SCIENCE ZOOLOGICAL SOC JAPAN 29 (2) 121 - 126 0289-0003 2012/02 [Refereed]
    The gobiid fish Trimma okinawae changes its sex bi-directionally according to its social status. Morphological changes in the urinogenital papillae (UGP) of this fish have been reported during sex change. However, there have been no detailed observations of such changes. Here, we histologically examined the UGP structure of male-and female-phase fish. UGPs of fish in female and male phase contained both oviducts and sperm ducts. Both ducts were coalesced into one duct within the posterior region of the UGP. Female-phase fish had many longitudinal folds in the hypertrophied tunica mucosa of the oviduct, which was found to be responsible for the transport of eggs and the removal of follicular cells from the oocyte. In contrast, male-phase fish had an immature oviduct and a mature sperm duct in the UGP. In the male-phase fish, the co-existence of spermatozoa and fibrillar secretions was observed in the sperm duct during spermiation.
  • Atsushi Sogabe; Ryota Kawanishi; Hiromi Takata; Yasuhisa Kobayashi
    ICHTHYOLOGICAL RESEARCH SPRINGER JAPAN KK 59 (1) 77 - 82 1341-8998 2012/01 [Refereed]
    The mating behaviour, fecundity characteristics and egg production process were investigated in the barbed pipefish Urocampus nanus under aquarium conditions. The mating behaviour consisted of five motor patterns, some of which have been generally reported in other syngnathids. Neither the number nor size of newborns related to the size of parents, although there was a negative correlation between the number and size of newborns. The ovary consisted of two germinal ridges, and mature eggs were produced synchronously with multiple ovulations. This type of egg production has thus far been reported only in a polyandrous and sex-role-reversed pipefish.
  • Sabina Ruksana; Mohammad Ashraful Alam; Yasuhisa Kobayashi; Masaru Nakamura
    ZOOLOGICAL SCIENCE ZOOLOGICAL SOC JAPAN 28 (11) 845 - 852 0289-0003 2011/11 [Refereed]
    Differentiation and development of steroid-producing cells (SPCs) and folliculogenesis during ovarian differentiation in the Nile tilapia Oreochromis niloticus were immunohistochemically and ultrastructurally examined. Clusters of immunopositive cells (IPCs) against antibodies (ABs) of cholesterol side-chain cleavage cytochrome P450 (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), and cytochrome P450aromatase (P450arom) only appeared in the area near blood vessels in the fish ovaries at 50-60 days after hatching (dah). Ultrastructural results showed that differentiation and development of SPCs from undifferentiated to maturation occurred in the area near blood vessels, indicating that it would be the original site of SPCs. At 70-80 dah, IPC clusters invaded the interstices among oocytes at the perinucleolar stage from the area near the blood vessels. IPCs increased in number in the interstices among the previtellogenic oocytes, and some clusters began to enclose the outer thecal layer of the previtellogenic oocytes at 90 dah. The process of folliculogenesis was ultrastructurally observed. SPCs enclosed by fibroblastic cells invaded the interstitial areas among oocytes and some reached the surfaces of oocytes. The upper portions of these elongations opened and began to enclose the outer surfaces of developed oocytes to become thecal layer. Later, newly migrated SPCs reach the thecal layer to become thecal cells. These results indicate that steroid-producing thecal cells originate from the SPCs in the area near blood vessels. After thecal layer formation, an immunopositive reaction against P450arom AB, but not against P450scc or 3 beta-HSD ABs, appeared first in the granulosa cells enclosing the vitellogenic oocytes at 100 dah. At this time, estrogen production in serum levels rapidly increased. Thus, folliculogenesis could be essential for active production of estrogen in the ovary.
  • Hirokuni Kobayashi; Takashi Iwamatsu; Yasushi Shibata; Michio Ishihara; Yasuhisa Kobayashi
    ZOOLOGICAL SCIENCE ZOOLOGICAL SOC JAPAN 28 (5) 355 - 359 0289-0003 2011/05 [Refereed]
    To investigate how estrogen and androgen affect each other in inducing sex reversal in the medaka, O. latipes, 17 beta-estradiol (E2) and 17 alpha-methyldihydrotestosterone (MDHT) were co-administered by a convenient method for hormonal treatment, in which freshly fertilized eggs were immersed for 24 h in saline containing either or both of the two sex steroids in different concentrations and/or ratios. The minimal concentrations of E2 and MDHT sufficient to induce the maximal rate of sex reversal from male to female and from female to male were 500 ng/ml and 2.5 ng/ml, respectively, both of which were referred to as the most efficacious dose (MED), and each equivalent for the inducing potency in sex reversal. E2 and MDHT, when simultaneously administered at MED, greatly suppressed each other to induce each corresponding sex reversal. Thus, the present experimental results indicate that E2 and DMHT are antagonists that induce corresponding sex reversal, and suggest that genotypic sex in the medaka might be modified through an unknown factor of common affinity to both sex steroids, by which the pathway of differentiation of either sex could be switched at the early stages of development far before gonadal sex differentiation.
  • Yasuhisa Kobayashi; Ryo Nozu; Masaru Nakamura
    DEVELOPMENTAL DYNAMICS WILEY-BLACKWELL 240 (1) 116 - 121 1058-8388 2011/01 [Refereed]
    The three-spot wrasse, Halichoeres trimaculatus, is a protogynous hermaphrodite. Under appropriate social conditions, female fish can become male. Previous studies indicated that estrogens are important regulators of sex change in this fish. However, the role of estrogen in the male is not known. To clarify the involvement of estrogen in spermatogenesis in hermaphrodite fish, we treated initial phase (IP) males for 10 weeks with exemestane, an aromatase inhibitor (AI), to block estrogen synthesis. Fish treated with AI exhibited decreases in gonadal weight, plasma estrogen levels, and spermatogonial proliferation in the testis, together with increases in androgen levels. Additionally, we confirmed that exogenous estrogen treatments stimulated the renewal and proliferation of spermatogonia in the testis of IP males. These results indicate that estrogens play an important role in regulating spermatogenesis in this fish. Developmental Dynamics 240: 116-121, 2011. (C) 2010 Wiley-Liss, Inc.
  • Ryosuke Murata; Hirofumi Karimata; Yasuhisa Kobayashi; Ryo Horiguchi; Kazuo Kishimoto; Motofumi Kimura; Tohru Kobayashi; Kiyoshi Soyano; Masaru Nakamura
    INTERNATIONAL JOURNAL OF DEVELOPMENTAL BIOLOGY U B C PRESS 55 (6) 619 - 625 0214-6282 2011 [Refereed]
    To understand the mechanism of sex differentiation in the protogynous Malabar grouper Epinephelus malabaricus, we performed an immunohistochemical investigation of the expression of three steroidogenic enzymes, cholesterol-side-chain-cleavage enzyme (CYP11a), aromatase (CYP19a1a), and cytochrome P45011beta-hydroxylase (CYP11b), in the gonads during ovarian differentiation. Strong positive immunoreactivity against CYP11a, the key enzyme of steroidogenesis, and CYP19a1a which is essential for estrogen (17beta-estradiol) production, appeared first in the somatic cells surrounding gonial germ cells in undifferentiated gonads and throughout ovarian differentiation. However, positive immunoreactivity against CYP11b, which is important for androgen (11-ketotestosterone) production, first appeared in the cluster of somatic cells in the ovary tunica near the dorsal blood vessel after differentiation. CYP19a1a and CYP11b did not co-localize in any cells. These results indicate that there are two types of steroid-producing cells, estrogen-producing cells and androgen-producing cells, in the gonads of this fish, and they are distributed differently, suggesting that these cells are derived from different somatic cells. Estrogen-producing cells appeared prior to ovarian differentiation, while androgen-producing cells were first detected after ovarian differentiation. These results suggest that endogenous estrogen is involved in ovarian differentiation.
  • Mohammad Ashraful Alam; Yasuhisa Kobayashi; Toshiaki Hirai; Masaru Nakamura
    Follicle-stimulating hormone (FSH) and its receptor (FSHR) play important roles in spermatogenesis. We cloned and characterized the honeycomb grouper Epinephelus metro FSHR (EmFSHR) to elucidate its role in the protogynous sex change in groupers. Reverse transcription-polymerase chain reaction (RT-PCR) analysis suggested that EmFSHR was expressed exclusively in the gonads. In situ hybridization showed the distribution of EmFSHR in the granulosa cells of previtellogenic oocytes and Leydig cells in the testis. Quantitative reverse transcription PCR (RT-qPCR) analysis of gonadal EmFSHR transcripts during the process of sex change indicated that the lowest levels were found in the female phase before sex change. EmFSHR transcripts increased during the early transitional phase, when oocytes began to degenerate in parallel with the initiation of gonial germ cell differentiation into spermatogonia. A dramatic increase in EmFSHR transcription occurred during the late transitional phase, when the gonad contained numerous proliferating male germ cells and many degenerated oocytes. EmFSHR expression remained high until the transformation from ovary to testis was complete. The data reveal that female to male sex change is associated with the upregulation of EmFSHR transcripts, and that this upregulation may be responsible for the development of testicular tissue and the progression of spermatogenesis. Furthermore, how the upregulation of EmFSHR is controlled in the initiation of sex change remains to be elucidated. (C) 2010 Elsevier Inc. All rights reserved.
  • Yasuhisa Kobayashi; Mohammad Ashraful Alam; Ryo Horiguchi; Akio Shimizu; Masaru Nakamura
    BIOLOGY OF REPRODUCTION SOC STUDY REPRODUCTION 82 (6) 1030 - 1036 0006-3363 2010/06 [Refereed]
    Recent studies have suggested that the hypothalamic-pituitary-gonadal axis is involved in gonadal sex change in sex-changing teleosts. However, its underlying mechanism remains largely unknown. In this study, we focused on the distinct roles of two gonadotropins (GTHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), in the protogynous hermaphrodite teleost, honeycomb grouper (Epinephelus merra). First, we investigated the expression pattern of mRNAs for GTH subunits (cga, fshb, and lhb) in the pituitaries from fish at the different sexual phases. Real-time RT-PCR analyses showed that fhsb mRNA levels in the female pituitary were low. However, fshb transcripts increased dramatically in association with testis development. In contrast, levels of cga and lhb mRNAs did not significantly vary during sex change. In addition, immunohistochemical observations of Fshb- and Lhb-producing cells in the pituitary, through the use of specific antibodies for detections of teleost GTH subunits, were consistent with sexually dimorphic expression of Fshb. In order to identify the role of GTH in gonad of honeycomb grouper, we treated females with bovine FSH (50 or 500 ng/fish) or LH (500 ng/fish) in vivo. After 3 wk, FSH treatments induced female-to-male sex change and up-regulated endogenous androgen levels and fshb transcripts, whereas LH treatment had no effect on sex change. These results suggest that FSH may trigger the female-to-male sex change in honeycomb grouper.
  • Yasuhisa Kobayashi; Ryo Horiguchi; Saori Miura; Masaru Nakamura
    To investigate the role of estrogen in the gonad of yellowtail clownfish Amphiprion clarkii, we isolated cDNA encoding cytochrome P450 aromatase (Cyp19a1a) from the adult ovary. The full-length cDNA of clownfish cyp19a1a is 1928-bp long and encodes 520 amino acids. Real-time quantitative RT-PCR analysis showed that cyp19a1a was expressed mainly in the ovary of female-phase fish. In situ hybridization and immunohistochemical observations showed that positive signals were restricted to the ovarian follicle of the female-phase fish. In contrast, Cyp19a1a signal was not detected in the ambisexual gonad of the male-phase fish. These findings suggest that Cyp19a1a is involved in oogenesis in the female-phase fish, but not in the ambisexuall gonad of male-phase fish. (C) 2009 Elsevier Inc. All rights reserved.
  • Yasuhisa Kobayashi; Ryo Horiguchi; Ryo Nozu; Masaru Nakamura
    Biology of Sex Differences 1 (1) 3  2042-6410 2010 [Refereed]
    Background: Three-spot wrasse, Halichoeres trimaculatus, is a marine protogynous hermaphrodite fish. Individuals mature either as initial phase (IP) males or females. Appropriate social cues induce the sex change from IP female to terminal phase (TP) male. However, the molecular mechanisms behind such a sex change remain largely unknown. Recently, the forkhead transcription factor 2 (Foxl2) was identified as an essential regulator of vertebrate ovarian development/function/phenotype. Inspired by this information, we characterized the expression patterns of Foxl2 in the protogynous wrasse assuming Foxl2 as the female-specific marker in this species. Methods: First, we clonedFoxl2 cDNA from ovary by reverse transcription polymerase chain reaction (RT-PCR) followed by rapid amplification of cDNA ends (RACE). Next, we analysed expression pattern of Foxl2 messenger RNA (mRNA) and protein in gonads of different sexual phases by real time quantitative PCR assay and flour fluorescence immunohistochemical method, respectively. Additionally, we studied the changes in Foxl2 expression pattern during aromatase inhibitor (AI)-induced sex change. Results: The amino acid sequence (306 AA) of wrasse Foxl2, especially the forkhead domain, shows high identity with that of other reported teleost Foxl2s. Quite unexpectedly, no sexual dimorphism was observable between the testes and ovary in the expression pattern of Foxl2. In female phase fish, signals for Foxl2 protein were detectable in the granulosa cells, but not the theca cells. Transcript levels of Foxl2 in the testes of IP and TP males were identical to that in the ovaries of females and, further, Foxl2 protein was found to be localized in the interstitial cells including tubules and Leydig cells. Treatment with AI induced sex change in male gonads and an up-regulation was seen in the expression of Foxl2 in these gonads. Conclusions: Unlike in other vertebrates, including teleosts, Foxl2 may have a different role in the naturally sex changing fishes. © 2010 Kobayashi et al licensee BioMed Central Ltd.
  • Yasuhisa Kobayashi; Masaru Nakamura; Tomoki Sunobe; Takeshi Usami; Tohru Kobayashi; Hisaya Manabe; Bindhu Paul-Prasanth; Norio Suzuki; Yoshitaka Nagahama
    ENDOCRINOLOGY ENDOCRINE SOC 150 (3) 1503 - 1511 0013-7227 2009/03 [Refereed]
    Sex-changing fish Trimma okinawae can change its sex back and forth from male to female and then to male serially, depending on the social status in the harem. T. okinawae is well equipped to respond to its social status by possessing both ovarian and testicular tissues even though only one gonad remains active at one time. Here we investigated the involvement of gonadotropins in sex change by determining the changes in gonadotropin receptor (GtHR) gene expression during the onset of sex change from female to male and male to female. The expression of the GtHR was found to be confined to the active gonad of the corresponding sexual phase. During the sex-change from female to male, initially the ovary had high levels of FSHR and LHR, which eventually went up in the testicular tissue if the fish was bigger. Changing of the gonads started with switching of GtHR expression discernible within 8-12 h of the visual cue. Further in vitro culture of the transitional gonads with a supply of exogenous gonadotropin (human chorionic gonadotropin) revealed that the to-be-active gonad acquired the ability to produce the corresponding sex hormone within 1 d of the activation of GtHR. Conversely, the to-be-regressed gonad did not respond to the exogenous gonadotropin. Our findings show that the gonads of successive sex-changing fish possess the intrinsic mechanism to respond to the social cue differentially. Additionally, this location switching of GtHR expression also could substantiate the importance of the hypothalamo-pituitary-gonadotropic axis. (Endocrinology 150: 1503-1511, 2009)
  • Ryo Horiguchi; Yasuhisa Kobayashi; Masaru Nakamura
    CYBIUM SOC FRANCAISE D ICHTYOLOGIE 32 (2) 88 - 89 0399-0974 2008/07 [Refereed]
    Estrogens are essential for sex change and behavior in wrasses. To understand the molecular mechanisms of estrogen action in the reproduction of the protogynous wrasse, Halichoeres trimacidatus, we isolated cDNA encoding three estrogen receptor (ER) subtypes from ovary by 5' and 3' rapid amplification cDNA ends (RACE) methods. The deduced amino acid sequence of ER alpha, ER beta 1 and ER beta 2 showed high identity to the red seabream ER alpha (81%), largemouth bass ER beta (82%) and largemouth bass ER gamma (81%), respectively. Real-time polymerase chain reaction revealed some distinctions in tissue expression of ERs, suggesting different functions for each ER subtype in protogynous wrasses.
  • Takashi Todo; Mana Sato; Misa Ashida; Naohiro Yamaguchi; Yasuhisa Kobayashi; Shinji Adachi; Kohei Yamauchi; Masaru Nakamura
    CYBIUM SOC FRANCAISE D ICHTYOLOGIE 32 (2) 106 - 106 0399-0974 2008/07 [Refereed]
    Recently, we succeeded in inducing in vitro spermatogenesis in a protogynous hermaphrodite fish, three-spotted wrasse (Halichoeres trimaculatus), during organ culture of ovary with a serum-supplemented medium. In the present study, we further optimized and used this culture system to determine effective steroid doses to induce sex change, and to investigate the importance of apoptosis during sex change in vitro. The results show that the restructuring from ovary to testis in the wrasse could be induced in vitro with a serum-free medium. Gonadal sex change can be triggered during basic in vitro culture due to a lack of endogenous factors (especially, estrogens) for female sexuality, and can be accelerated by androgens. Furthermore, oocyte apoptosis may be an important mechanism effecting gonadal sex change.
  • Mohammad Ashraful Alam; Yasuhisa Kobayashi; Ryo Horiguchi; Toshiaki Hirai; Masaru Nakamura
    The honeycomb grouper (Epinephelus merra) is one of the smallest members of the Serranidae family and is often used to study protogynous sex change. To determine the role of the male-determining gene Dmrt1 and the ovarian-specific gene Foxl2 in sex change, we cloned these two markers from E. merra gonads by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Two isoforms, Dmrt1a and Dmrt1b, resulted from alternative splicing in the coding region, causing the insertion of one glutamine residue in Dmrt1b. RT-PCR revealed that Dmrt1 was expressed only in the gonads, with higher levels in the testis than in the ovary. cDNA encoding Foxl2 was isolated from the ovary; Foxl2 was expressed extensively in the brain, pituitary, gonads, and gill, with its highest level in the ovary, indicating a potential role for Foxl2 in the brain-pituitary-gonad axis. Real-time quantitative RT-PCR analyses showed that Foxl2 mRNA expression was significantly downregulated from the late transitional phase to the completion of sex change. Conversely, Dmrt1 expression increased with the progression of spermatogenesis and continued until the formation of the testis. The expression profiles of these two sex-specific marker genes corresponded closely with the histological process of sex change. The down-regulation of Foxl2 most likely facilitates oocyte degeneration, whereas the up-regulation of Dmrt1 causes the proliferation of gonial germ cells into spermatogina and initiates sex change. (C) 2008 Elsevier Inc. All rights reserved.
  • Saori Miura; Shigeo Nakamura; Yasuhisa Kobayashi; Francesc Piferrer; Masaru Nakamura
    To clarify the relationship between steroid hormones and sex differentiation of the protandrous anemonefish Amphiprion clarkii, we histologically examined its gonadal differentiation. From hatching to 30 days post hatching (dph), all of the gonads surveyed were sexually undifferentiated. The gonads of all fish first differentiated into ovaries at 60 dph, and the oocytes gradually developed and increased in number as the ovaries grew up until 183 dph. Some cysts of differentiated spermatogenic germ cells appeared in the ovaries at 214 dph, and ambisexual gonads with both ovarian and testicular tissues formed by 273 dph. Using immunohistochemistry, we then investigated the expression of cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), during gonadal sex differentiation. P450scc-immunopositive reactions first appeared in sexually undifferentiated gonads at 30 dph. Beginning at 60 dph, the number of strongly positive cells increased throughout the differentiation of the ovaries and continued to increase during the testicular differentiation until 210 dph. Immunopositive cells were observed more frequently in ovarian tissue than in testicular tissue in the ambisexual gonads at 270 dph. These results suggest that endogenous steroid hormones are important for the sex differentiation, including the primary sex differentiation and subsequent testicular differentiation, of the anemonefish. (C) 2007 Elsevier Inc. All rights reserved.
  • Masaru Nakamura; Mohammad Ashraful Alam; Yasuhisa Kobayashi; Ramji Kumar Bhandari
    Groupers are commercially important coral reef fishes because of their excellent flavor and high price. Due to their sex changing nature, commercial aquaculture operations have not yet been developed for groupers. Sex steroids play a critical role in the initiation of sex change, but their exact function and mechanism of action are unknown. We have investigated the role of sex steroids in protogynous sex change using the honeycomb grouper, Epinephelus merra, for a number of years. Our studies have shown that sex steroids play a major role in sex change. In this review, we integrate our findings to better understand the mechanism of sex change in groupers.
  • Toru Komatsu; Ramji K. Bhandari; Yasuhisa Kobayashi; Shigeo Nakamura; Masaru Nakamura
    AQUACULTURE ELSEVIER SCIENCE BV 261 (2) 823 - 824 0044-8486 2006/11 [Refereed]
  • T Komatsu; RK Bhandari; Y Kobayashi; S Nakamura; M Nakamura
    AQUACULTURE ELSEVIER SCIENCE BV 257 (1-4) 558 - 565 0044-8486 2006/06 [Refereed]
    Golden rabbitfish, Siganus guttatus, do not attain spermiation in the first spawning season after hatching in northern Okinawa, Japan. Nevertheless, underyearling males possess spermatogenic germ cells at the late stages of development, including spermatozoa. The fate of those germ cells is still unknown. This study examined whether prespawning treatment with gonadotropin-releasing hormone agonist (GnRHa) may induce further development and/or maturation of those germ cells in an attempt to obtain expressible milt from underyearling golden rabbitfish. Cholesterol pellets containing three different doses of GnRHa (0 (control), 100 and 200 mu g kg(-1) body weight) were implanted intramuscularly in underyearling rabbitfish. The implanted fish were examined 2.5 months later. At the end of experiment, mean gonadosomatic index (GSI) was significantly higher in the 200 mu g kg(-1) group than in the control group. While the testicular histology of about 40% of the control males showed regression from the initial condition, 70-80% of males in both GnRHa-treated groups demonstrated advanced maturation stages, including spermiation. Mean plasma 11-ketotestosterone (11-KT) and testosterone (T) levels in the GnRHa-treated group were significantly higher than in the control group, while no significant changes were shown in 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (DHP) levels. In conclusion, GnRHa treatment induced the development and maturation of spermatogenic germ cells, leading to spermiation in underyearling male testes, demonstrating that GnRHa treatment could serve as a useful way to induce precocious spermiation in male golden rabbitfish. (c) 2006 Elsevier B.V. All rights reserved.
  • Mohammad Ashraful Alam; Ramji Kumar Bhandari; Yasuhisa Kobayashi; Kiyoshi Soyano; Masaru Nakamura
    Aquaculture 255 (1-4) 532 - 535 0044-8486 2006/05 [Refereed]
    Artificial sex change was induced within two full moons by an aromatase inhibitor (AI 1 mg/fish) during the breeding season to establish the quickest method of sex change and natural spawning of the honeycomb grouper (Epinephelus merra). The sex change from female (ovary) to male (testis) occurred during the time between the two full moons (3 weeks) following AI implantation, and the efferent ducts of sex-changed males were filled with sperm. To examine sperm fertility, sex-changed males were mated with natural, normal females and produced fertilized eggs. Most of the hatched larvae grew normally without any morphological deformities. Therefore, the use of this method which is the quickest known sex change method using AI, may contribute to quality sperm production for grouper aquaculture. © 2006 Elsevier B.V. All rights reserved.
  • MA Alam; RK Bhandari; Y Kobayashi; S Nakamura; K Soyano; M Nakamura
    MOLECULAR REPRODUCTION AND DEVELOPMENT WILEY-LISS 73 (2) 206 - 214 1040-452X 2006/02 [Refereed]
    11-ketotestosterone (11-KT), a potent male-specific androgen in fish, has important roles on spermatogenesis, male behavior, and nuptial coloration. The site of 11-KT synthesis and its role on male germ cell development during protogynous sex change is not clearly understood. We examined the dynamics of steroidogenic enzymes immunolocalization, viz cholesterol side-chain cleavage (P450scc), biomarker of steroids and cytochrome P45011 beta-hydroxylase (P45011 beta), downstream to 11-KT production, throughout the process of sex change in honeycomb grouper (Epinephelus merra). In female, P450scc immunoreactivity (-ir) was observed in the theca layer and tunica near blood vessels (BV). During the onset of sex change, P450scc reactive cells were observed in the remaining follicle layer of degenerated oocyte of the ovo-testis in early transitional (ET) and late transitional (LT). In male, P450scc-ir was localized in the interstitial Legdig cells of testis. P45011 beta reactive cells were observed in the tunica near BV in female but not in theca layer. In ET and LT phases gonads P45011 beta localized in remaining follicle layer of degenerated oocyte and tunica near BV. On the other hand, in male, both interstices and tunica near BV showed strong signals against P45011 beta. Moreover, in vivo and in vitro levels of 11-KT related with the changes in the nuclei diameter of P45011 beta-positive cells in both tunica near BV and remaining follicle layer of degenerated oocyte to interstices during the progress of sex change. The present results suggest that 11-KT produced in the tunica near BV may provide the stimulus for female to degenerate oocytes and initiate sex change. However, 11-KT produced both in tunica near BV and remaining follicle layer of degenerated oocyte possibly plays critical role during testicular differentiation as well as gonadal restructuring at mid to late phases (ET to LT) of sex change in honeycomb grouper.
  • Y Kobayashi; T Sunobe; T Kobayashi; M Nakamura; N Suzuki; Y Nagahama
    involvement of Ad4BP/SF-1 was investigated using the serial sex changing goby Trimma okinawae. First, a cDNA encoding Ad4BP/SF-1 was cloned from ovarian follicles. The open reading frame of goby Ad4BP/SF-1 encodes a protein of 489 amino acids. Ad4BP/SF-1 was expressed in gonadal tissues, brain, and kidney. Second, transcript levels of Ad4BP/SF-1 were measured in the ovary and testis of the same individuals. Expressions were related to sexual phases. Moreover, ovarian expression of Ad4BP/SF-1 increased during vitellogenesis and declined sharply during the post-vitellogenic period. Finally, the expression profile of Ad4BP/SF-1 was measured during serial sex change in the ovary. Ad4BP/SF-1 increased in parallel with the onset of the female-phase and decreased as female became male. Therefore, Ad4BP/SF-1 probably acts as an important indirect regulator of oocyte growth and maturation at female-phase of serial sex changing gobiid fish T okinawae. (c) 2005 Elsevier Inc. All rights reserved.
  • T Sunobe; M Nakamura; Y Kobayashi; T Kobayashi; Y Nagahama
    The role of aromatase (Arom) in the process of bi-directional sex change in the gobiid fish Trimma okinawae was investigated by immunohistochemical methods. Irrespective of sexual phase, gonads comprised both ovarian and testicular tissues. In each sexual phase of females, the 2nd (2DF-M) and 4th (4DF-M) days after initiation of sex change to male, males, and the 2nd (2DM-F), 4th (4DM-F) and 6th (6DM-F) days after the initiation of reversion from male to female, ovarian and testicular histological observations were made. During the female, 2DF-M, 4DF-M and 6DM-F phases, the ovary contained vitellogenic and previtellogenic oocytes, compared with previtellogenic oocytes in the other phases. Although sperm was found in the testis in every phase, sperm ducts were apparent in the male phase, but not the female phase. Arom immunoreactivity was detected in the interstitial cells between the oocytes in all phases. On the other hand, it was localized in the thecal and granulosa cells of the follicular layer enclosing the oocytes in the female, 2DF-M, 4DF-M and 6DM-F phases. Activity of Arom in the thecal and granulosa cells is thought to be important for the development of oocytes and subsequent sex change. (c) 2005 Elsevier Inc. All rights reserved.
  • Masaru Nakamura; Yasuhisa Kobayashi
    FISH PHYSIOLOGY AND BIOCHEMISTRY SPRINGER 31 (2-3) 117 - 122 0920-1742 2005/04 [Refereed]
    Gonadal differentiation can take many forms in fish, ranging from gonochorism, where individuals directly develop as male or female and finally possess only testis or ovaries at sexual maturation, to hermaphroditism where the same individuals can produce mature male and female gametes at some time in their lives. Hermaphrodite fish are, thus, an excellent model for studying the plasticity of sex determination and differentiation in vertebrates. We have shown that sex steroids play a principal role in sex differentiation and sex change in fish. Our laboratory implements several fish models that undergo sex change from female to male or male to female or in both directions. In this review, we will briefly discuss recent advances in our understanding of the mechanism of sex change in coral reef fish.
  • Yasuhisa Kobayashi; Tomoki Sunobe; Tohru Kobayashi; Yoshitaka Nagahama; Masaru Nakamura
    FISH PHYSIOLOGY AND BIOCHEMISTRY SPRINGER 31 (2-3) 123 - 127 0920-1742 2005/04 [Refereed]
    Despite numerous endocrine studies on sex change in teleost, no general mechanism that mediates sex change has emerged. The gobiid fish, Trimma okinawae, can change sex in both directions repeatedly. This phenomenon of sex change in goby assigns it as an excellent animal model to elucidate the understanding mechanisms of sex change. In hermaphrodite fishes, estrogen plays a particularly important role in natural and experimentally induced sex change. To investigate the role of estrogen in the serial-sex changing fish T. okinawae, we cloned and analyzed the 5'-flanking regions of P450arom genes from goby genome DNA. Both regions have consensus sequences of TATA, CRE and ERE. Ad4 binding site was restricted in the region of P450aromA. These findings indicate that different regulators control the expression of the two P450arom genes.
  • T Sunobe; M Nakamura; Y Kobayashi; T Kobayashi; Y Nagahama
    ICHTHYOLOGICAL RESEARCH SPRINGER TOKYO 52 (1) 27 - 32 1341-8998 2005/02 [Refereed]
    The process of sex change in the gobiid fish Trimma okinawae was investigated by gonad histology and immunohistochemistry of two steroidogenic enzymes, P450 cholesterol-side-chain-cleavage ( P450scc) and 3beta- hydroxysteroid dehydrogenase (3beta- HSD). Irrespective of sexual phase, gonads comprised both ovarian and testicular tissues. Females changed sex to male within 7 days, reverting again to female over an 11-day period. In each sexual phase of the females, the 2nd (2DF-M) and 4th ( 4DF- M) day after the initiation of sex change to male, the males, and 2nd ( 2DM- F), 4th ( 4DMF), and 6th ( 6DM- F) days after the initiation of reversion from male to female, histological observations were made. In the ovary during the female, 2DF- M, 4DF- M, and 6DM- F phases, both vitellogenic and previtellogenic oocytes were present, but only previtellogenic oocytes were found in the other phases. The testis contained sperm in all phases, but sperm ducts were not visible in the female phase. In the ovary, P450scc immunoreactivity of interstitial cells was strongly or moderately detected, although weak in the male phase. In contrast, P450scc immunoreactivity in thecal cells was found in all but the male and 2DM- F phases. 3beta- HSD immunoreactive interstitial cells were detected in all phases, but only weakly so in the male and 2DM- F phases. 3beta- HSD immunoreactive thecal cells were observed in all stages without the male and 2DM- F and 4DM-F phases. In the testis, moderate P450scc and 3beta-HSD immunoreactivity was regularly found in the Leydig cells in all the phases. These results suggest that functional steroids including testosterone are produced in any sexual phases.
  • Y Kobayashi; T Sunobe; T Kobayashi; Y Nagahama; M Nakamura
    In order to obtain basic information about the role played by endogenous sex hormones in bringing about sex changes in the serial-sex changing gobiid fish Trimma okinawae, the gonadal structure of male and female phases were observed histologically. Steroid-producing cells (SPC; Leydig cells in a testis) were observed ultrastructurally in the ovaries and testes of both female-phase and male-phase fish. In addition, gonadal expression of P450 cholesterol side-chain-cleavage (scc) was examined immunohistochemically. Gonads of fish in female and male phases were observed to have both ovaries,and testes simultaneously. Female-phase fish had matured with many developed vitellogenic oocytes, while male-phase individuals had immature ovaries with many numbers of previtellogenic oocytes at the perinucleolus stage. Testes of fish in different sexual phases had active spermatogenic germ cells. Organellae of SPC in the ovaries of female-phase fish had active structures of steroid production. In contrast, SPC in the ovaries of male-phase fish did not show active structures of steroid production. Immunopositive reactions against the scc antibody in the ovaries of female-phase fish were very strong, but immunoreactions in the ovaries of male-phase fish were very weak. In the testis, moderate immunopositive signals were obtained from dual-phase male/females.
  • Y Kobayashi; T Kobayashi; M Nakamura; T Sunobe; CE Morrey; N Suzuki; Y Nagahama
    ZOOLOGICAL SCIENCE ZOOLOGICAL SOC JAPAN 21 (4) 417 - 425 0289-0003 2004/04 [Refereed]
    To investigate the role of estrogen in the serial-sex changing fish Trimma okinawae, we isolated complementary DNAs encoding two distinct cytochrome P450 aromatase isoforms from adult ovary and brain (termed P450aromA and P450aromB, respectively). Sequence and phylogenic analyses showed that the goby P450arom forms belong to two separate CYP19 subfamilies. Transient expression of these cDNAs in HEK293 cells caused conversion of exogenous testosterone to estradiol-17beta. RT-PCR showed that P450aromA was expressed in the brain, spleen, testis and ovary. P450aromB was expressed in the brain, liver, testis and ovary. In situ hybridization studies showed that P450aromA mRNA, but not P450aromB mRNA, was present in both ovary and testis. Positive signals were restricted to granulosa cells of vitellogenic follicles and interstitial cells of mature testis. Ovarian expression of both P450arom genes during the spawning cycle was examined by quantitative real-time RT-PCR. P450aromA transcripts increased during vitellogenesis and decreased prior to spawning. In contrast, P450aromB transcripts were barely detectable and did not correlate with ovarian development. These findings suggest that P450aromA, but not P450aromB, is involved in regulating ovarian vitellogenesis in goby.


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Research Themes

  • 日本学術振興会:科学研究費助成事業 基盤研究(C)
    Date (from‐to) : 2023/04 -2026/03 
    Author : 小林 靖尚
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research
    Date (from‐to) : 2019/04 -2022/03 
    Author : Kobayashi Yasuhisa
    The red stingray (Hemitrygon akajei), relatively easy to collect among cartilaginous fishes, have an ovoviviparous reproductive system. In this stingray, the gonadal sex determination and differentiation of the embryo exposed to maternal estrogen have occurred in the uterus. In this study, we investigated the process of gonadal sex differentiation of red stingray embryos. Histological observations revealed that morphological sex difference of the gonads occurs in the embryo, which is about 2 cm in total length. These findings help to understand the reproduction of cartilaginous fish.
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research
    Date (from‐to) : 2018/04 -2022/03 
    Author : Kobayashi Tohru
    The sex differentiation cascade of Oryzias sakaizumii is very similar to that of Nile tilapia, a useful fisheries species for aquaculture, unlike O. latipes. This study is conducting research using this species as a new model by comparing it to sex differentiation and masculinization in tilapia and masculinization in Grouper. In this study, we examined the molecular mechanisms of gonadal differentiation and sex change from sex determination. Thus we demonstrate the existence of an unknown testis differentiation pathway as the primary pathway for sex transdifferentiation to the testis.
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C)
    Date (from‐to) : 2016/04 -2019/03 
    Author : Kobayashi Yasuhisa; YAMAMOTO Masayuki
    Groupers of the genus Epinephelus are one of the most important aquaculture species in the world. The sexuality of groupers is almost protogynous hermaphrodite. Therefore, in general, males of a species are larger than females. Larger males in the wild have decreased remarkably in numbers, owing to overfishing so, although both males and females are essential for offspring production, collecting larger male groupers from the field is very difficult. In this study, we developed sex control method of groupers. Androgen treatment easily induced normal sex change in Longtooth grouper. However, it was not possible to induce reverse sex change (male-to-female) in Redspotted grouper. Therefore, it was considered that the sexual plasticity of grouper decreased after sex change.
  • Japan Society for the Promotion of Science:Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B)
    Date (from‐to) : 2014/04 -2016/03 
    Author : Kobayashi Yasuhisa
    Groupers of the genus Epinephelus are one of the most important aquaculture species in the world. Due to the economical and biological importance of groupers, physiological and endocrinological data are eagerly needed for improvement aquaculture. However, the endocrinological mechanism of sex change is not clearly understood. In this research, we focused on the pituitary. First, we had carried out RNA-seq by using grouper pituitary. Based on the data, we successfully isolated several genes of hormones. Second, we quantified hormone gene expressions during sex change in grouper. Finally, we has been identified that key regulatory factor underlying sex change is FSH.
  • 日本学術振興会:科学研究費助成事業 特別研究員奨励費
    Date (from‐to) : 2007 -2009 
    Author : 小林 靖尚
    これまで両方向性転換魚オキナワベニハゼを用いた研究から魚類の性転換には、脳-脳下垂体-生殖腺の情報伝達系が重要な働き巻果たすことを明らかにしてきた。今年度は、雌かう雄へ性転換するハタ科魚類の一種であるカンモンハタを用いて脳下垂体で産出される2種類の生殖腺刺激ホルモン(GTH;FSH,LH)の性転換に果たす役割を調べた。 最初に、GTHを構成する各サブユニット(GPα,FSHβ,LHβ)をクローニングした。つぎに異なる性のステージの下垂体における各サブユニットのmRNA発現量を調べた。その結果、GPαとLHβの遺伝子発現量に性差は見られなかった。しかしFSHβは雌では低発現であったにもかかわらず、雄で非常に高く発現していた。特にFSHβは性転換初期の個体で非常に高く発現していた。これら遺伝子発現の結果はGTH特異抗体を用いた脳下垂体の免疫染色によっても確かめられた。 加えて性転換におけるFSHの具体的な役割を調べるため、ウシのFSH精製品を雌にin vivo投与し生殖腺の変化を観察した。その結果FSH投与群の個体の生殖腺内では卵巣組織の崩壊と精巣組織の増殖が観察され性転換が誘導されていた。以上の結果からカンモンハタの生殖腺の性転換には、脳下垂体から放出されるFSHが引き金的役割を果たすことを明らかにした。ハタ科魚類は水産重要種の一つであり、近年、日本各地でハタ科魚類の種苗生産が試みられている。現在、得られたハタの性転換に関する知見および性のコントロール技術を、実際の養殖現場にて応用しようとしているところである。