The Molecular Mechanism Of Follicle-Stimulating Hormone Regulating Sex Differentiation In The Orange-Spotted Grouper (Epinephelus Coioides)

Fish,as the largest taxa of vertebrate,exhibit extremely diverse and plastic patterns of sex determination mechanisms,including genotypic sex determination(GSD)and environmental sex determination(ESD).Furthermore,the sex ratio is determined by a sex determining mechanism,can be influenced during the process of sex differentiation by various environmental factors.And it is now clearly established that sex steroid hormones play a critical role in the sex differentiation of teleosts,especially the estrogen.Moreover,the synthesis of endogenous estrogen is controlled by its synthetic ratelimiting enzyme,the aromatase(code by cyp19a1a).In addition,the expression of cyp19a1 a is regulated by gonadotropin(GtH)and a large number of researches indicated that the follicle-stimulating hormone(FSH,one of GtH)is involing in the sex differentiation of teleost.The orange-spotted grouper(Epinephelus coioides),a protogynous hermaphroditic fish,develops ovaries at a young age and therefore it is considered as an excellent model to investigate the molecular mechanisms underlaying sex differentiation.In our earlier studies,we found that fsh? and its receptor fshr were highly expressed during sex differentiation and these suggested that they may be involved in gonadal sex differentiation of the orange-spotted grouper.However,the precise function and molecular mechanism of follicle-stimulating hormone regulating sex differentiation in the orange-spotted grouper is largely unknown.This study aims to slove these problems,and results of this study can be summarized as follows:1.Long-term FSH treatment initially promoted the formation of ovaries but subsequently induced a male fate.The expression of female pathway genes was initially increased but then decreased,whereas the expression of male pathway genes was upregulated only during long-term FSH treatment.The genes related to the synthesis of sex steroid hormones,as well as serum 11-ketotestosterone and estradiol,were also upregulated during long-term FSH treatment.Short-term FSH treatment activated genes in the female pathway(especially cyp19a1a)at low doses but caused inhibition at high doses.Genes in the male pathway were up-regulated by high concentrations of FSH over the short term.Finally,we found that low,but not high,concentrations of FSH treatment activated cyp19a1 a promoter activities in human embryonic kidney(HEK)293 cells.Overall,our data suggested that FSH may induce ovarian differentiation or a change to a male sex fate in the protogynous orange-spotted grouper,and that these processes occurred in an FSH concentration-dependent manner.2.MT-feeding at 50 days after hatching(DAH)resulted in gonadal dysgenesis,which could be rescued by E2 supplementation.MT,and MT+E2,treatment could induce female to male sex reversal during sex differentiation(90-DAH 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 up-regulated in MT and MT+E2 group.Finally,we examined the expression of male specific mark(DMRT1),and proliferating cell nuclear antigen(PCNA)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.3.Different doses of aromatase inhibitor(AI)treatment during sex differentiation led to different phenotypes: undifferentiated gonads were maintained in the AI group fed a low dose whereas female to male sex reversal was observed in the AI group fed a high dose.Interestingly,AI and AI+E2,treatment could induce female to male sex reversal,while AI-feeding withdrawal and AI+E2-feeding withdrawal led to a female sex fate.The expression of female pathway genes was suppressed while the expression of genes in the male pathway was up-regulated in the AI and AI+E2 group.However,the suppression effect of female pathway genes and activation effect of male pathway genes were disappeared respond to AI and AI+E2 withdrawal.Consistent with the expression of sex-related genes,the serum 11-ketotestosterone level was up-regulated in AI and AI+E2+E2 group and down-regulated in AI and AI+E2 withdrawal group,while serum E2 level exhibit the opposite result.Finally,we examined the proliferating cell nuclear antigen(PCNA)and cell apoptosis,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 the upregulated of cyp19a1 a promote ovarian differentiation and the down-regulation of cyp19a1 a expression is likely to be responsible for testicular differentiation.4.Short-term MT,E2 and MT+E2 injection activate the expression of fsh? in pituitary of the orange-spotted grouper.The genome sequence of the 5' flanking regions of fsh? was analyzed and the binding regions of two estrogen response element(ERE)and four androgen response element(ARE)were predicted.The promoter region was cloned,inserted into luciferase reporter gene vectors and transfected into HEK-293 T cells.We found that E2 could up-regulate the activty of fsh? promoter through era and 11-KT could up-regulate the activty of fsh? promoter through ara and arb.