Ryo Horiguchi

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.

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.

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.

Survival of ovarian somatic cells during sex change in the protogynous wrasse, Halichoeres trimaculatus

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.

Immunohistochemical Evidence for 11β-hydroxylase (P45011β) and Androgen Production in the Gonad During Sex Differentiation and in Adults in the Protandrous Anemonefish Amphiprion clarkii

Abstract To obtain basic information on the endocrine mechanisms underlying sex change in the protandrous anemonefish Amphiprion clarkii, we examined the immunolocalization of the steroidogenic enzyme cytochrome 11β-hydroxylase (P45011β), which is involved in 11-ketotestosterone (11-KT) production, and analyzed the ability of gonads to produce steroid hormones throughout the sex differentiation process and at the breeding stage. Immunopositive reactions against P45011β appeared in sexually undifferentiated gonads at 30 days post hatching (dph). The number of immunopositive cells continued to increase during ovarian differentiation (from 60 to 180 dph) and throughout the formation of ambisexual gonads with both ovarian and testicular tissue until 270 dph. In the male phase, strongly immunopositive cells were observed in the cellular interstices of both testicular and ovarian tissues. P45011β was localized only in the theca cells enclosing developed oocytes in the female phase. In-vitro 11-KT production in the gonads gradually increased with testicular differentiation (before, during, and after differentiation). Production of 11-KT in the gonads was higher in the male phase than during testicular differentiation or in the female phase. Our results suggest that androgen is involved in testicular differentiation during sex differentiation and spermatogenesis.

Differentiation of steroid-producing cells during ovarian differentiation in the protogynous Malabar grouper, Epinephelus malabaricus

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.

Expression profile of doublesex/male abnormal-3-related transcription factor-1 during gonadal sex change in the protogynous wrasse, Halichoeres trimaculatus

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. Mol. Reprod. Dev. 82: 859–866, 2015.

Histological observation of doublesex-mab 3-related transcription factor 1 (DMRT1) localization in the adult testis of three-spot wrasse

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.