Yasuhisa Kobayashi

Diversity and Plasticity of Sex Determination and Differentiation in Fishes

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.

Molecular cloning and quantitative expression of sexually dimorphic markers Dmrt1 and Foxl2 during female-to-male sex change in Epinephelus merra.

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.

Characterization of Two Types of Cytochrome P450 Aromatase in the Serial-sex Changing Gobiid Fish, Trimma okinawae

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-17β. 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.

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.

Sexually Dimorphic Expression of Gonadotropin Subunits in the Pituitary of Protogynous Honeycomb Grouper (Epinephelus merra): Evidence That Follicle-Stimulating Hormone (FSH) Induces Gonadal Sex Change

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.

Sex change in coral reef fish.

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.

Characterization of gonadal soma-derived factor expression during sex change in the protogynous wrasse, Halichoeres trimaculatus

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. Developmental Dynamics 242:388–399, 2013.

Expression and localization of forkhead transcriptional factor 2 (Foxl2) in the gonads of protogynous wrasse, Halichoeres trimaculatus

BackgroundThree-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.MethodsFirst, 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.ResultsThe 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.ConclusionsUnlike in other vertebrates, including teleosts, Foxl2 may have a different role in the naturally sex changing fishes.

Sex- and tissue-specific expression of P450 aromatase (cyp19a1a) in the yellowtail clownfish, Amphiprion clarkii.

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 ambisexual gonad of male-phase fish.

Sex change of adult initial-phase male wrasse, Halichoeres trimaculatus by estradiol-17β treatment

Sex steroids are considered major regulators of sex change processes in fish. Estrogen depletion is shown to be crucial for female-male sex change initiation; however, its role in male-female sex change is largely unknown. In the present study, we examined the effects of estradiol-17 beta (E2) treatments on testes of initial-phase (IP) males of the three-spot wrasse (Halichoeres trimaculatus), which naturally do not undergo male-female sex change. Sexually mature IP males were fed a diet containing E2 (low, 20 microg/g feed; high, 200 microg/g feed) for 6 or 12 weeks, and changes in gonadal structures were examined. Percentage of sex change varied with the dosage of E2 and the duration of treatment. All individuals treated with high-dose E2 for 6 weeks had ovaries with many immature oocytes; whereas 75% of individuals treated with low-dose of E2 for 6 weeks and sampled on the 12th week had ovaries with yolky oocytes and an ovarian cavity indicating a typical mature ovary. No testicular tissue was observed in sex-reversed gonads in both treatment groups. Contrary to the previous assumptions, present results suggest that IP male wrasses have the potential to undergo male-female sex change in response to exogenous estrogen. How the presence or absence of estrogen creates sexual plasticity in gonadal germ and somatic cells remains to be clarified.