The co-administration of estradiol/17α-methyltestosterone leads to male fate in the protogynous orange-spotted grouper, Epinephelus coioides.

Estrogen plays a pivotal role in the sex differentiation of teleosts, whereas the precise function of androgens is more controversial. In this study, orange-spotted grouper (Epinephelus coioides) fry were treated with letrozole (an aromatase inhibitor, AI), 17α-methyltestosterone (MT), or MT and 17ß-estradiol (E2) simultaneously, during the period of gonadal formation and sex differentiation. MT feeding at 50 days after hatching resulted in gonadal dysgenesis, which could be rescued by E2 supplementation. Different doses of AI treatment led to different phenotypes: undifferentiated gonads were maintained in the AI group fed a low dose (5 mg/kg diet), whereas female-to-male sex reversal was observed in the AI group fed a high dose (100 mg/kg diet). MT and MT + E2 treatment could induce female-to-male sex reversal during sex differentiation (90 days after hatching). The expression of female pathway genes was suppressed, while the expression of genes in the male pathway was up-regulated in the MT + E2 group. Consistent with the expression of sex-related genes, the serum 11- ketotestosterone level was also upregulated in MT and MT + E2 group. Finally, we examined the expression of male-specific mark (DMRT1) and proliferating cell nuclear antigen in MT and MT + E2 induced sex reversal, and the result indicated that male germ cells and somatic cells may origin from the gonium and proliferative somatic cells surrounding the efferent duct, respectively. Overall, our data suggested that estrogen acts as a natural inducer of female differentiation, and that the co-administration of estrogen and androgen during sex differentiation leads to a male sex fate in the protogynous orange-spotted grouper.

Molecular regulation of sex change induced by methyltestosterone -feeding and methyltestosterone -feeding withdrawal in the protogynous orange-spotted grouper.

The sex identity of fish can be easily manipulated by exogenous hormones. Treatment with 17-methyltestosterone (MT) has been widely used to induce a male fate, but the molecular and cellular processes underlying sex changes induced by MT treatments and the withdrawal of MT are not well studied. In this study, we systematically investigated gonadal histology, gene expression profiles, sex steroid hormone levels, and cellular changes during sex changes induced by MT-feeding and MT-feeding withdrawal in the protogynous orange-spotted grouper, Epinephelus coioides. Based on gonadal histology, we demonstrated that MT-feeding-induced sex reversal can be divided into early and late phases: in the early phase, male and female germ cells coexist, and MT-feeding withdrawal leads to a female fate; in the late phase, only male germ cells are observed, and MT-feeding withdrawal does not reverse the process, leading to a male fate. In both the early and late phases, cytochrome P450 family19 subfamily A member 1 (cyp19a1a) gene expression increased in response to MT-feeding withdrawal. Finally, by tracing doublesex- and Mab-3-related transcription factor 1 (dmrt1)-expressing cells, we found that gonia-like cells in the germinal epithelium might be the major germ cell sources for developing testes during sex reversal. Collectively, our findings provide insights into the molecular and cellular mechanisms underlying sex changes induced by exogenous hormones.

Molecular mechanism of feedback regulation of 17ß-estradiol on two kiss genes in the protogynous orange-spotted grouper (Epinephelus coioides).

Kisspeptins are considered critical regulators in the hypothalamic-pituitary-gonadal axis because they can stimulate secretion of Gonadotropin-releasing hormone in mammals, and may also mediate the feedback regulation of sex steroids in the hypothalamus. Two kiss1 paralogues (kiss1 and kiss2) were identified in teleosts, hinting at their increased complexity of signaling for sex-steroid feedback regulation. In the present study, molecular pathways by which 17ß-estradiol (E2 ) exerted feedback regulation on two kiss genes, via three types of estrogen receptors, were investigated in the protogynous orange-spotted grouper (Epinephelus coioides). kiss2 expression in the brain significantly increased in ovariectomized orange-spotted groupers, while E2 replacement in ovariectomized fish reversed these changes to levels in the sham-surgery group; conversely, kiss1 expression did not change. Dual-label in situ hybridization showed that kiss1 and kiss2 neurons express erα, erß1, and erß2, indicating that E2 may directly regulate kiss1 and kiss2. Indeed, E2 treatment of transiently transfected HEK293T cells decreased the activity of both kiss promoters in the presence of erß1 and erß2 rather than erα. Further deletion and site-directed mutagenesis of the kiss promoters indicated that kiss1 is regulated by E2 via an estrogen-responsive element (ERE)-dependent, classical pathway utilized by Erß1, as well as via an Activator protein 1 (Ap1)-dependent, non-classical pathway utilized by Erß2. kiss2 was also differently regulated by E2 through the Creb transcription factor, utilized by Erß1 as well as a half-ERE-dependent, classical pathway utilized by Erß2. Taken together, multiple signaling pathways in orange-spotted grouper are clearly involved in the feedback regulation of E2 on kiss genes via different estrogen receptors.

Molecular cloning, characterization and functional analysis of QRFP in orange-spotted grouper (Epinephelus coioides).

The peptide QRFP plays an important role in the regulation of vertebrate feeding behavior. In this study, we cloned the full length cDNA of a QRFP precursor in a teleost fish, the orange-spotted grouper (Epinephelus coioides). Sequence analysis has shown that the functional regions of QRFP in other vertebrates (QRFP-25 and QRFP-7) are conserved in orange-spotted grouper. RT-PCR demonstrated that the pre-processed mRNA of QRFP is widely expressed in orange-spotted grouper. Three days of food deprivation did not change the hypothalamic pre-processed QRFP expression. However, QRFP expression significantly increased when the fish were reefed after three days of fasting. Intraperitoneal injection of QRFP-25 peptide to orange-spotted grouper suppressed expression of orexin, but elevated expression of pro-opiomelanocortin (POMC) in the hypothalamus. We also investigated the effects of QRFP-25 on the expression of reproductive genes. The peptide suppressed the expression of seabream-type gonadotropin-releasing hormones (sbGnRH), luteinizing hormone beta subunit (LHß) and follicle-stimulating hormone beta subunit (FSHß) in vivo, as well as inhibited the expression of LHß and FSHß in pituitary cells in primary culture. Our results indicate that QRFP may play an inhibitory role in the regulation of feeding behavior and reproduction in orange-spotted grouper.

Expression profiles of gonadotropins and their receptors during 17α-methyltestosterone implantation-induced sex change in the orange-spotted grouper (Epinephelus coioides).

It is known that the hypothalamic-pituitary-gonadal axis participates in the sex change of hermaphrodite teleosts, and gonadal steroid hormones mediate this physiological process. The secretion of gonadal steroids is directly regulated by signaling pathways involving gonadotropins (GtHs) and gonadotropin receptors (GtHRs) in teleosts. To gain insight into the involvement of GtH/GtHR systems in the sex change process, cDNAs encoding follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) were firstly isolated from gonads of orange-spotted grouper (Epinephelus coioides), a protogynous hermaphrodite fish. Reverse transcription-PCR (RT-PCR) analysis demonstrated that the expression of the FSHR was confined to the brain, pituitary gland, ovary, and testis, while the LHR was expressed only in the brain, ovary, and testis. Furthermore, the expression profiles of GtH subunits (FSHß and LHß) and their receptors were analyzed in parallel with the serum levels of estradiol-17ß (E(2) ), testosterone (T), and 11-ketotestosterone (11-KT) during 17α-methyltestosterone (MT)-induced sex change. Quantitative real-time PCR determined that the abundances of FSHß and FSHR were significantly inhibited after MT treatment for 2 and 4 weeks, but subsequently returned to the control level after 6 weeks. In contrast, the mRNA levels of LHß and LHR were significantly elevated throughout the sex change process. During MT-induced sex change, serum concentrations of E(2) remained constant while T and 11-KT levels were significantly increased. Taken together, our results suggest that GtH/GtHR systems are involved in MT-induced sex change, and two signaling pathways may have distinct roles in modulating the variations of the corresponding steroid hormones in the orange-spotted grouper.

Molecular cloning, characterization and expression profiles of three estrogen receptors in protogynous hermaphroditic orange-spotted grouper (Epinephelus coioides).

Estrogen plays key roles in vertebrate reproductive system via estrogen receptors (ERs) as mediating pathways. In the present study, three full-length ERs cDNA sequences were isolated from a protogynous teleost, the orange-spotted grouper (Epinephelus coioides), and were 2235bp for gERα, 1967bp for gERß1 and 2158bp for gERß2, respectively. Phylogenetic and amino acid alignment analyses showed that each gER was clustered in the corresponding taxonomic groups of the perciformes and exhibited high evolutional conservation in functional domains. RT-PCR revealed that gERs expressed at different levels in all the obtained tissues. gERα highly expressed in mature ovaries, gERß1 mainly expressed in immature ovaries and gERß2 varied greatly during ovarian development. During female to male sex reversal induced by 17α-methyltestosterone (MT) implantation, gERα decreased gradually, gERß1 increased gradually, and gERß2 decreased firstly and recovered subsequently in male stage. The present study speculated the potential roles of gERs during female maturation and female to male sex reversal induced by MT in the protogynous grouper E. coioides.