Effects Of Estrogen And Androgen On Sex Differentiation And Gonadal Gene Expression

The sex of fish was very plastic and easily influenced by environmental factors, especially sex steroids. There have been many studies on the role of estrogen in sex defferentiation, Serum estrogen level was up-regulated during male to female sex reversal in the hermaphroditic and gonochoric fish. Long term treatment of aromatase inhibitor, Fadrozole, even induced secondary sex reversal of the differentiated ovary into functional testis. Further more, estrogen is synthesized earlier in females than androgen in males. Accordingly, estrogen synthesizing enzymes are expressed earlier in ovaries than androgen synthesizing enzymes in testes. In tilapia, several essential steroidogenic enzymes, Cyp11a1, 3β-HSD, Cyp17α1, Cyp19a1 a, were detected in the gonads of XX fry at 5 days after hatching(dah) by immunohistochemistry(IHC). In contrast, Cyp11a1, 3β-HSD, Cyp17α1, and Cyp11b2 were not observed in the testis of XY fish until 30 dah. In a recent study of gonadal transcriptomes from the Nile tilapia, we further confirmed that almost all steroidogenic enzyme genes, including cyp19a1 a but not including cyp11b2, were up-regulated in the XX gonads at 5 dah, while these enzyme genes, including cyp11b2 but not including cyp19a1 a, were detected at 30 dah in the XY gonads. While the role of androgen in sex differentiation is controversial. There has been no report on simultaneous treatment of estrogen and androgen on sex differentiation and gonadal gene expression profile, even though there has been many studies focused on the impact of estrogen or androgen treatment alone. Recently, our group demonstrated that when treated with 17β-estrodiol(E2) and 17α-methyltestrosterone(MT) in the same dose alone or simultaneously, MT could induce female to male sex reversal in XX fish while MT+E2 could not. In contrast, E2 or MT + E2 could induce male to female sex reversal in XY fish. However, it is unclear whether the gonadal gene expression profile of the treated fish is testis-like, ovary-like or disrupted. Recently research has showed that differentiated ovary(even in adult fish) could be induced to change sex to functional testis by long term treatment of aromatase inhibitors. In contrast, sex reversal of differentiated XY fish has been restricted in the period of one month with a low sex reversal rate. In the present study, we performed transcriptome analysis of the gene expression profile of the MT+E2 treated XX and XY gonads. We also treated the one month XY fish with inhibitor of 11β-hydoxylase(Metopirone, MN) +E2 and inhibitor of 3β-hydroxysteroid dehydrogenase(Trilostane, TR) +E2 to induce male to female secondary sex reversal. The results as follows:All-XX and all-XY tilapia were treated with the same dose of MT(50μg/g) and E2(50μg/g) from 0 to 30 dah and then fed with control diet to 90 dah. Gonadal transcriptome analyses indicating that the total gene counts of both MT+E2 treated XX(19979) and XY(19034) gonads were larger than that of XX control(18100), while similar to that of XY control(19790). Transcriptomic cluster analysis revealed that the MT and E2 treated XX and XY gonads clustered into the same branch with the XX control. However, a small fraction of genes, which showed disordered expression profile, may be associated with stress response. These results indicated that estrogen is important in sex differentiation. Simultaneous treatment with estrogen and androgen resulted in disordered gene expression and endocrine disruption in tilapia.TR(150μg/g), MN(150μg/g) and E2(150μg/g) alone or incombination with E2 were used to induce 30 dah XY male fish sex reversal. The XY fish were fed with experimental diet contained drugs for 60 days(till 90 dah) and then fed with control diet. Statistical analysis at 90 dah revealed that, TR and MN treatment alone resulted in no sex reversal, and E2 treatment resulted in 8.3% sex reversal, TR+E2 treatment resulted in 88.8% sex reversal. Interestingly, MN+E2 resulted in no sex reversal at 90 dah, however, when checked at 180 dah, 60.0% of the treated fish sex reversed into female. Serum 11-KT level of TR+E2 treated fish was decreased and E2 level was increased significantly compared with XY control at 90 dah, while 11-KT level of MN+E2 treatment group was decreased and E2 level was increased significantly at 180 dah. IHC analysis revealed that Cyp19a1 a, Cyp11b2 and Dmrt1 were expressed in the gonads of the TR+E2 treatment group at 90 dah, and in the gonads of MN+E2 treatment group at 180 dah. These results indicated that endogenous androgen is critical for the maintainance of male phenotype.In conclution, our results indicate that both XX and XY fish developed as female in the presence of estrogen and androgen, further demonstrating that estrogen is important in sex differentiation. Further more, simultaneous treatment with estrogen and androgen also resulted in disordered gene expression in gonads and endocrine disruption in XX and XY tilapia. Endogenous androgen is critical for the maintainance of male phenotype. Estrogen could induce differentiated the testis of the one month old XY fish reversed into an ovary only when androgen synthesis was inhibited.