Gonadal sex differentiation of rice field frog hoplobatrachus rugulosus (wiegmann, 1834)

The rice field frog Hoplobatrachus rugulosus, a species widely distributed in Thailand, has potential to be a model in many fields of research. A preliminary study demonstrated that the gonadal differentiation in this frog showed tendency to be an undifferentiated type since all individuals had ovaries at complete metamorphosis. However, the information was still unclear. Therefore, this study aimed to investigate the chronology and pattern of gonadal sex differentiation as well as steroidogenic potential of gonadal tissues during development. The frog embryos were obtained by stimulating fertilization in laboratory under natural light and temperature conditions. Their developmental stage was identified using Gosner (1960) system. The time period during each stage of development was also recorded. H. rugulosus spent 25-33 days for development to complete metamorphosis. The gonad was firstly observed by stereomicroscope at Gosner stage 33 (9-12 days post hatch, dph), while the testis and ovary were distinguishable by morphology from 3–4 weeks after metamorphosis. Histological analysis showed that genital ridge formation began at stage 25 (1-2 dph) and ovarian differentiation began at stage 36 (13-17 dph). The developing ovary appeared with numerous primary oogonia which developed into oocytes, while the medulla regressed to form an ovarian cavity. During metamorphosis, only an ovary was observed. Testicular differentiation began later, during the first week after metamorphosis, and occurred via an intersex condition. The intersex gonads contained developing testicular tissue with both normal and atretic oocytes. The fully developed testis was firstly identified at 6 weeks after metamorphosis. Thus, the pattern of gonadal sex differentiation in H. rugulosus is an undifferentiated type, in which only female gonads are observed during metamorphosis and intersex and male gonads are observed later. For steroidogenic potential of gonad, the expression level of mRNA encoding cytochrome P450 17-hydroxylase/C17–20 lyase (CYP17) and cytochrome P450 aromatase (CYP19) were observed by quantitative real-time RT-PCR and localization of CYP17 mRNA in the tissues was observed by in situ hybridization. The results showed that CYP17 mRNA levels in male group at 4-11 weeks after metamorphosis showed a higher level than those of other groups. This result corresponded to their localization in the gonadal tissues that the CYP17 signals were specifically detected in Leydig cells of testicular tissue in both testes and intersex gonads at 4-16 weeks after metamorphosis but undetectable in all ovary samples. The CYP19 mRNA levels in female group at 4-11 weeks after metamorphosis was higher than those of other groups which corresponded with the histological results that the number of oocytes and follicular cells were increased, indicating potential steroidogenic function of the ovary. In intersex group at 4-11 weeks after metamorphosis, the level of CYP17 mRNA was high, whereas the level of CYP19 mRNA was relatively lower. It could be suggested that the testicular tissue of intersex gonad has developed and had steroidogenic function, while the ovarian tissue of intersex gonad may not function. Based on the present results, the role of CYP17 and CYP19 mRNA in sex differentiation in H. rugulosus may occur after the gonadal sex differentiated and the steroidogenic potential of the gonads exhibited sexual dimorphic pattern. These results provide a crucial basis for further research on developmental biology in anuran species.