ABSTRACT
Exogenous factors such as temperature, social behavior, and salinity play a crucial role during the critical sensitive period of sex differentiation in many vertebrates. In fishes, amphibians, and reptiles temperature treatment is known to induce all-male (or female) individuals, and genes related to sex differentiation have been studied. The Japanese pufferfish, Takifugu rubripes, possesses the most compact genome among vertebrates and has immense potential for studies focusing on comparative genome analysis. In this study, we describe gonadal morphology and vasa (germ cell marker) and dmrt1 (Sertoli cell marker) expression on a molecular level in relation to the development of temperature-treated pufferfish. To investigate the relationship between temperature and gonadal development, pufferfish were exposed to high-temperature conditions (32 degrees C) during early gonadal development. Morphological observations showed that this high-temperature treatment did not influence sexual differentiation as determined by ovarian cavity characteristics; however, high-temperature treatment induces gonadal degeneration that is devoid of germ cells. RT-PCR results revealed no vasa expression within germ cell-degenerated gonads. In situ hybridization results showed that dmrt1 was expressed in somatic cells of germ cell-degenerated ovaries. These results suggest that high-temperature treatment during early gonadal development induces germ cell degeneration and masculinization of ovarian somatic cells in pufferfish.
Subject(s)
Germ Cells/cytology , Takifugu/growth & development , Temperature , Animals , Body Weight , Cell Death , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Female , Gene Expression Regulation, Developmental , Germ Cells/enzymology , Gonads/cytology , Gonads/enzymology , Gonads/growth & development , In Situ Hybridization , Male , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
In order to assess the involvement of aromatase CYP19 isoforms and endogenous sex steroids in gonadal sex differentiation and development of the Japanese fugu (Takifugu rubripes), an aromatase inhibitor (AI, fadrozole) was administered to developing fishes from the 'first feeding' till the 100th day after hatching. It was observed that ovarian cavity formation was inhibited by fadrozole at doses of 500 and 1000 microg/g diet, which was followed by testicular differentiation in all treated fugu. In the non-treated fugu, CYP19A was predominantly expressed in the ovary and CYP19B in the brain (in both sexes), although both were expressed interchangeably at low levels. An exceptionally high expression of CYP19B was also evident in testis throughout the study period. Both forms of CYP19 mRNA showed low levels of expression in brain and gonad with no significant differences between the two AI treatments. AI treatment inhibited CYP19A mRNA in trunk during the crucial period of ovarian cavity formation and CYP19B in gonad and brain by the end of gonadal sex differentiation. An elevation of testosterone and 11-ketotestosterone was observed which can be associated with the down-regulation of the circulating 17beta-estradiol production during the AI treatment period. After stopping AI treatment, both circulating estrogen and androgen were normalized. The current results suggest that suppression of CYP19A before and during morphological sex differentiation inhibits ovarian cavity formation in fugu. Furthermore, non-detectable limits of 17beta-estradiol and high testosterone levels by the end of the gonadal differentiation period can be ascribed to inhibition of CYP19B, suggesting that conversion of 17beta-estradiol from testosterone is plausibly regulated by CYP19B, and that this factor (CYP19B) may play an important role in AI-induced testicular development after gonadal sex differentiation through regulation of the testosterone-17beta-estradiol balance in fugu.