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 17β-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.