Estrogen biosynthesis in the gonad of the frog Rana rugosa

In certain species of amphibians gonadal differentiation is influenced by steroid hormones. In the case of the frog Rana rugosa testosterone given to tadpoles reverses sex from female to male, while the opposite reversal - male to female - can be achieved using estradiol-17β. In this study, we investigated whether CYP19 (P450 aromatase), the enzyme responsible for a production of estradiol-17β, was present in the differentiating gonad of R. rugosa. Initially, we immunized rabbits against frog CYP19 peptides and performed immunostaining using specific antibodies purified from that serum. CYP19-reactive signals were observed in gonadal somatic cells of the female, but not male tadpoles at stage (St.) I (the stage prior to phenotypic sex determination in tadpoles of R. rugosa). Immunopositive signals were also produced in ovarian somatic cells froglets at St. XXV (just after the completion of metamorphosis). We also examined the enzymatic activity of CYP19 in the differentiating gonad of R. rugosa. Reverse-phase HPLC (high performance liquid chromatography) analysis revealed that [(3)H]testosterone was converted to [(3)H]estradiol-17β in the gonad of tadpoles at St. I. Interestingly, the rate of conversion was much higher in females than in males. To the best of our knowledge, this is the first report on the biosynthesis of estradiol-17β in the gonad of amphibians, and the co-incident identification of active CYP19 enzyme in the differentiating gonad of R. rugosa. Based on our results, we conclude that estradiol-17β may be involved in ovarian differentiation in this species.