The sex-determining gene of amphibians having the XY-type sex-determining system differs from that of ones having the ZW-type sex-determining system

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The aim of this research was to isolate the sex-determining gene in the frog Rana rugosa having two sex-determining systems (the XY and ZW types). We thought that the Dmrt1 gene was the best candidate for the sex-determining gene in R. rugosa having the ZW-type sex-determining system, since this gene was considered to be the sex-determining gene in chickens. However, FISH analysis revealed that Dmrt1 was an autosomal gene. The sex of the ZW type R. rugosa can be reversed from male to female by estrogens, whereas that of the XY type R. rugosa can be changed from female to male by androgens. However, the mechanism of the sex-reversal is largely unknown. To clarify the mechanism of the sex-reversal, we cloned and characterized 3 genes encoding sex steroid-synthesizing enzymes, i.e., CYP11A1, CYP17 and 3βHSD from the frog R. rugosa. RT-PCR analysis showed that the CYP17 expression was higher in male gonads during sex determination in R. rugosa, whereas CYP11A1 and 313-HSD showed no sexually dimorphic expression. When the sex was changed from female to male by injection of testosterone, the level of CYP17 transcripts increased in the sex-reversed gonad, whereas there was no change in the levels of CYP11A1 and 3β-HSD transcripts. In addition, in situ hybridization analysis revealed that CYP17 was expressed in somatic cells of the indifferent male gonad and in those of the testis. Positive signals of CYP17 were also produced in somatic cells of a female to male sex-reversed gonad (testis), but not in the ovary. Taken together, the results suggest that CYP17 is closely involved in testicular differentiation of the gonad in R. rugosa. CYP17 is the key-gene for female-to-male sex-reversal in R. rugosa.