Tohru Kobayashi

Doublesex- and Mab-3-related transcription factor-1 repression of aromatase transcription, a possible mechanism favoring the male pathway in tilapia.

Doublesex- and Mab-3-related transcription factor-1 (Dmrt1) is an important transcription factor implicated in early testicular differentiation in vertebrates, but its target genes are largely unknown. In the Nile tilapia, estrogen is the natural inducer of ovarian differentiation. Our recent studies have shown that Forkhead-l2 up-regulated transcription of the Cyp19a1a gene (aromatase) in the gonads in a female-specific manner. However, the upstream factor(s) down-regulating Cyp19a1a expression during testicular differentiation remains unclear. In the present study, we used in vitro (promoter analysis) and in vivo (transgenesis and in situ hybridization) approaches to examine whether Dmrt1 inhibits Cyp19a1as transcriptional activity. The in vitro analysis using luciferase assays revealed that Dmrt1 repressed basal as well as Ad4BP/SF-1-activated Cyp19a1a transcription in HEK 293 cells. Luciferase assays with various deletions of Dmrt1 also showed that the Doublesex and Mab-3 domain is essential for the repression. In vitro-translated Dmrt1 and the nuclear extract from tilapia testis could directly bind to the palindrome sequence ACATATGT in the Cyp19a1a promoter, as determined by EMSAs. Transgenic overexpression of Dmrt1 in XX fish resulted in decreased aromatase gene expression, reduced serum estradiol-17beta levels, retardation of the ovarian cavitys development, varying degrees of follicular degeneration, and even a partial to complete sex reversal. Our results indicate that aromatase is one of the targets of Dmrt1. Dmrt1 suppresses the female pathway by repressing aromatase gene transcription and estrogen production in the gonads of tilapia and possibly other vertebrates.

Comparative responsiveness to natural and synthetic estrogens of fish species commonly used in the laboratory and field monitoring

Exposure to estrogenic chemicals discharged into the aquatic environment has been shown to induce feminization in wild freshwater fish and although fish species have been reported to differ in their susceptibility for these effects, empirical studies that directly address this hypothesis are lacking. In this study, in vitro ERα activation assays were applied in a range of fish species used widely in chemical testing (including, zebrafish, fathead minnow, medaka) and/or as environmental monitoring species (including, roach, stickleback, carp) to assess their comparative responsiveness to natural (estrone, estradiol, estriol) and synthetic (17α-ethinylestradiol (EE2), diethylstilbestrol (DES)) estrogens. In vivo exposures to EE2 via the water (nominal 2 and 10 ng/L for 7 days) were also conducted for seven fish species to compare their responsiveness for hepatic vitellogenin (VTG) mRNA induction (an ER mediated response). Of the fish species tested, zebrafish ERα was found to be the most responsive and carp and stickleback ERα the least responsive to natural steroid estrogens. This was also the case for exposure to EE2 with an ERα-mediated response sensitivity order of zebrafish > medaka > roach > fathead minnow > carp > stickleback. For VTG mRNA induction in vivo, the order of species responsiveness was: rainbow trout (not tested in the ERα activation assays) > zebrafish > fathead minnow > medaka > roach > stickleback > carp. Overall, the responses to steroid estrogens in vitro via ERα compared well with those seen in vivo (VTG induction for exposure to EE2) showing in vitro screening of chemicals using fish ERα-mediated responses indicative of estrogenic responses (VTG induction) in vivo.

Molecular cloning and gene expression of Foxl2 in the frog Rana rugosa

Foxl2 is a transcription factor that plays a crucial role in the regulation of the early development of the female gonad in mammals and fish. However, little is known regarding its role in ovarian differentiation in amphibians. In this study, we isolated a Foxl2 cDNA from the ovary of the frog Rana rugosa and Xenopuslaevis and examined its expression during gonadal sex differentiation in R. rugosa. Alignment of known Foxl2 sequences from vertebrates showed high identity of the Foxl2 open reading frame and protein sequences, in particular the forkhead domain and C-terminal region, with other vertebrate sequences. Among different adult tissues, Foxl2 was expressed at its highest level in the ovary. Real-time RT-PCR analysis showed that Foxl2 expression was sexually dimorphic during gonadal sex differentiation in R. rugosa. In addition, Foxl2, which was detected immunochemically in somatic cells surrounding oocytes in the ovary, promoted R. rugosaCYP19 transcription in luciferase promoter assays conducted in A6 cells. We also found by FISH analysis that Foxl2 was an autosomal gene. Altogether, these results suggest that Foxl2 probably plays a very important role in ovarian differentiation of R. rugosa by possibly regulating CYP19 expression. The factor that up-regulates Foxl2 expression in female gonads still remains to be identified.

Sex Change in the Gobiid Fish Is Mediated through Rapid Switching of Gonadotropin Receptors from Ovarian to Testicular Portion or Vice Versa

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.

Molecular Aspects of Gonadal Differentiation in a Teleost Fish, the Nile Tilapia

In contrast to many developmental processes, sex-determining mechanisms show no clear evolutionary conservation among phyla. However, recent studies indicate that some downstream products of sex determination genes are functionally similar in diverse species. To date, numerous conserved genes involving gonadal sex differentiation have been examined in the teleost fish Nile tilapia (Oreochromis niloticus). Morphogenesis during gonadal differentiation is also conserved, as is evident in the differentiation and development of parenchyma/medullary cells (testis cord) and follicles. Therefore, it is important to understand the mechanisms of gonadal sex differentiation from the perspective of the relationship between conserved gene expression cascades and morphogenesis during gonadal sex differentiation. This article reviews the expression profiles of male- and female-related genes involved in histogenesis during sex differentiation in tilapia and discusses gene function in gonadal sex differentiation, especially the role of endogenous estrogens for ovarian differentiation.

Gene expression profiles in the testis associated with testis-ova in adult Japanese medaka (Oryzias latipes) exposed to 17α-ethinylestradiol

The occurrence of oocytes in the testis (testis-ova) of several fish species is often associated with exposure of estrogenic chemicals. However, induction mechanisms of the testis-ova remain to be elucidated. To develop marker genes for detecting testis-ova in the testis, adult male medaka were exposed to nominal concentration of 100 ng L(-1) of 17α-ethinylestradiol (EE2) for 3-5 weeks, and 800 ng estradiol benzoate (EB) for 3 weeks (experiment I), and a measured concentration of 20 ng L(-1) EE2 for 1-6 weeks (experiment II). Histological analysis was performed for the testis, and microarray analyses were performed for the testis, liver and brain. Microarray analysis in the estrogen-exposed medaka liver showed vitellogenin and choriogenin as estrogen responsive genes. Testis-ova were induced in the testis after 4 weeks of exposure to 100 ng L(-1) EE2, 3 weeks of exposure to 800 ng EB, and 6 weeks of exposure to 20 ng L(-1) EE2. Microarray analysis of estrogen-exposed testes revealed up-regulation of genes related to zona pellucida (ZP) and the oocytes marker gene, 42Sp50. Using quantitative RT-PCR we confirmed that Zpc5 gene can be used as a marker for the detection of testis-ova in male medaka.

Chromosome Doubling in Early Spermatogonia Produces Diploid Spermatozoa in a Natural Clonal Fish

Abstract The natural clonal loach Misgurnus anguillicaudatus (Teleostei: Cobitidae) is diploid (2n = 50) and produces genetically identical unreduced eggs, which develop into diploid individuals without any genetic contribution from sperm. Artificially sex-reversed clones created by the administration of 17alpha-methyltestosterone produce clonal diploid sperm. In metaphase spreads from testicular cells of the sex-reversed clones, spermatocytes had twice the normal number of chromosomes (50 bivalents) compared with those of normal diploids (25 bivalents). Thus, the production of unreduced diploid spermatozoa is initiated by premeiotic endomitosis (or endoreduplication), chromosome doubling before meiosis, and is followed by two quasinormal divisions. Larger nuclei in the germ cells were observed in all stages of type B spermatogonia in the testes of the sex-reversed clones. In contrast, besides having larger type A spermatogonia, the sex-reversed clones also had the type A spermatogonia that were the same size as those of normal diploids. It follows that chromosome duplication causing unreduced spermatogenesis occurred in the type A spermatogonia. The presence of tetraploid type A and early type B spermatogonia, identified by labeling with antispermatogonia-specific antigen 1, was verified using DNA content flow cytometry. These results support the conclusion that chromosome doubling occurs at the type A spermatogonial stage in diploid spermatogenesis in the clonal fish.

Understanding the molecular basis for differences in responses of fish estrogen receptor subtypes to environmental estrogens.

Exposure to endocrine disrupting chemicals (EDCs) can elicit adverse effects on development, sexual differentiation, and reproduction in fish. Teleost species exhibit at least three subtypes of estrogen receptor (ESR), ESR1, ESR2a, and ESR2b; thus, estrogenic signaling pathways are complex. We applied in vitro reporter gene assays for ESRs in five fish species to investigate the ESR subtype-specificity for better understanding the signaling pathway of estrogenic EDCs. Responses to bisphenol A, 4-nonylphenol, and o,p-DDT varied among ESR subtypes, and the response pattern of ESRs was basically common among the different fish species. Using a computational in silico docking model and through assays quantifying transactivation of the LBD (using GAL-LBD fusion proteins and chimera proteins for the ESR2s), we found that the LBD of the different ESR subtypes generally plays a key role in conferring responsiveness of the ESR subtypes to EDCs. These results also indicate that responses of ESR2s to EDCs cannot necessarily be predicted from the LBD sequence alone, and an additional region is required for full transactivation of these receptors. Our data thus provide advancing understanding on receptor functioning for both basic and applied research.

Molecular cloning of two isoforms of 11β-hydroxylase and their expressions in the Nile tilapia, Oreochromis niloticus

P450 11beta-hydroxylase, encoded by P450(11beta) gene, is a key mitochondrial enzyme to produce 11beta-hydroxy testosterone, substrate for the production of 11-ketotestosterone (11-KT), which has been shown to be potent androgen in several fish species. In the present work, two alternative splicing isoforms i.e. P450(11beta)-1 and P450(11beta)-2 cDNAs were cloned from the Nile tilapia, Oreochromis niloticus. They were 1614 and 1227bp in length with open reading frames encoding proteins of 537 and 408 amino acids, respectively. In contrast to P450(11beta)-1, which derived from 9 exons of the P450(11beta) gene, the 7th and 8th exons were absent in P450(11beta)-2. Tilapia P450(11beta)-1 shares the highest homology with that of medaka, Oryzias latipes. Expressions of P450(11beta)-1 and -2 were detected in the kidney and head kidney of both sexes, and in the testis but not in the ovary, with P450(11beta)-2 lower than P450(11beta)-1. Ontogenic expressions of both isoforms were detected in testis from 50dah onwards. P450(11beta)-1 and -2 were strongly expressed in sex reversed XX testis after fadrozole and tamoxifen treatment, but completely inhibited in 17beta-estradiol induced XY ovary. The existence of two alternatively spliced isoforms and the sexual dimorphic expression of P450(11beta)s were further confirmed by Northern blot. Strong expression signals in Leydig cells and weak signals in spermatogonia were detected by in situ hybridization and immunohistochemistry. Taken together, our data suggest a role for P450(11beta) in the spermatogenesis of tilapia through the production of 11-KT in testis, in addition to cortisol production in head kidney.

Gonadal soma-derived factor (gsdf), a TGF-beta superfamily gene, induces testis differentiation in the teleost fish Oreochromis niloticus.

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 XX 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.