Differential display analysis of gene expression in female-to-male sex-reversing gonads of the frog Rana rugosa

Sex steroids play pivotal roles in gonadal differentiation in many species of vertebrates. The sex can be reversed from female to male by testosterone in the Japanese wrinkled frog Rana rugosa, but it is still unclear what genes are up- or down-regulated during the XX sex-reversal in this species. To search the genes for the female-to-male sex-reversal, we employed differential display and 5′/3′-RACE. Consequently, we isolated from the gonads at day 8 after testosterone injection 24 different cDNA fragments showing a testosterone treatment-related change and then obtained three full-length cDNAs, which we termed Zfp64, Zfp112, and Rrp54. The former two cDNAs encoded different proteins with zinc-finger domains, whereas the latter cDNA encoded an unknown protein. Transcripts of the three genes were hardly detectable in the sex-reversing gonads at day 24 after the injection; at this time few growing oocytes were observed in the sex-reversing gonad. Besides, in situ hybridization analysis showed positive signals of the three genes in the cytoplasm of growing oocytes of an ovary when testosterone was injected into a tadpole. Thus, the decrease in expression of these three genes was probably due to the disappearance of growing oocytes and not to their direct involvement in the testis formation. To find the key-gene for testis formation, it will be necessary to analyze, by the differential display method, more genes showing a change in expression pattern during sex reversal.