Immunohistochemical detection and biological activities of CYP17 (P450c17) in the indifferent gonad of the frog Rana rugosa.

Sex steroids play a crucial role in the gonad differentiation in various species of vertebrates. However, little is known regarding the localization and biological activity of steroid-metabolizing enzymes during gonadal sex differentiation in amphibians. In the present study, we showed by real-time RT-PCR analysis that the expression of CYP17, one of the key steroidogenic enzymes, was higher in the indifferent gonad during sex differentiation in male than in female tadpoles of Rana rugosa but that there was no difference detected in the 3betaHSD mRNA level between the male and female gonads. We next examined the localization of CYP17, 3betaHSD and 17betaHSD in the indifferent and differentiating gonads by using three kinds of antibodies specific for CYP17, 3betaHSD and 17betaHSD, respectively. Positive signals for CYP17, 3betaHSD and 17betaHSD were observed in somatic cells of the indifferent gonad of males and in the interstitial cell of the testis. The enzymatic activity of CYP17 was also examined in the gonad during sex differentiation in this species. [(3)H]Progesterone (Prog) was converted to [(3)H]androstenedione (AE) in the indifferent gonad in males and females, but the rate of its conversion was higher in males than in females. Moreover, fluorescence in situ hybridization (FISH) analysis revealed that the CYP17 gene was located on the q arm of chromosome 9, indicating that CYP17 was autosomal in R. rugosa. Taken together, the results demonstrate that the CYP17 protein is synthesized in somatic cells of the indifferent gonad during gonadal sex differentiation in R. rugosa and that it is more active in converting Prog to AE in males than in females. The data suggest that CYP17 may be involved in testicular formation during sex differentiation in this species.

Steroidogenic gene expression during sex determination in the frog Rana rugosa.

Rana rugosa is unique among frog species in that it has two distinct types of sex chromosomes in two separate forms (XX/XY and ZZ/ZW). Treatment with sex steroids can reverse its gender from female to male or male to female. This phenomenon makes it a novel model for studying gonadal differentiation. The physiological role of sex steroids in sex differentiation in amphibians is yet unclear, however. To address this issue, we cloned the cDNAs of 17betaHSD types 8 (17betaHSD8) and 12 (17betaHSD12), 5alpha-reductase type 1 (5alphaRed1), and the steroidogenic acute regulatory protein known as StAR in the steroidogenic pathway. Then, we measured the mRNA levels of these genes during sex differentiation by real-time RT-PCR. The levels of CYP11A1, 3betaHSD, CYP17 and CYP19 mRNA were also measured by real-time RT-PCR. As a result, we detected transcripts of all such genes except for that of 17betaHSD8 in the indifferent gonad before the onset of sex determination. The expression of CYP17 occurred in indifferent gonads in both sexes; and its transcript levels were much higher in the male gonads. By contrast, the levels for CYP19 were much higher in the female gonads. CYP11A1, 3betaHSD, 17betaHSD12, 5alphaRed1 and StAR showed no sexually dimorphic expression during gonadal sex differentiation. Taken together, the results suggest that CYP17 has a major influence on testis development and that CYP19 plays a similar role in ovary development. However, the factors that up-regulate their expression remain to be identified.

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.

Elevated expression of P450c17 (CYP17) during testicular formation in the frog.

Sex steroids play decisive roles in gonadal differentiation in many species of vertebrates. The sex can be changed by sex steroids in some species of amphibians, but the mechanism of the sex-reversal is largely unknown. In this study, we cloned and characterized 3 cDNAs encoding sex steroid-synthesizing enzymes, i.e., CYP11A1, CYP17 and 3beta-HSD from the frog Rana rugosa. RT-PCR analysis showed that the CYP17 expression was much higher in male gonads than in female ones during sex determination in R. rugosa, whereas CYP11A1 and 3beta-HSD showed no sexually dimorphic expression. When testosterone was injected into tadpoles for female-to-male sex reversal, CYP17 expression appeared to be very strong in the gonad at days 16 and 24 after injection of testosterone. CYP11A1 was also transcribed higher at day 16, but its expression was weaker when compared with that of CYP17. The expression of 3beta-HSD did not change during the sex reversal. 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) at days 16 and 24 post testosterone injection, but not in the ovary. Taken together, the results suggest that CYP17 is very involved in testicular differentiation of the gonad in R. rugosa.