| The sex determination and differentiation of fish were influenced by a combination of genetic and environmental factors. Environmental factors, such as temperature, photoperiod, density and steroids, could override the genetic factors and change the fate of the gonad, especially sex steroids. The function of sex steroids for sex determination and their mechanism are controversial. There are two main hypotheses. One is "balance hypothesis". It presented a hypothesis for sex determination based on the ratio of androgen to estrogen in the gonad during sexual differentiation. The sex of an individual was controlled by the dominate steroids. This was confirmed by the wide use of steroids for sex reversal in fish. In general, estrogens feminize genetic males and androgens masculinize genetic females. Another is "deficiency hypothesis". It was argued that the fate of undifferentiated gonads is determined by the presence of estrogens during the critical period of sex determination. The undifferentiated female gonads could synthesize estrogens, in contrast, the undifferentiated male gonads could synthesize neither estrogens nor androgens. The pivotal role of estrogens in sex determination and differentiation was reported in tilapia and rainbow trout. Based on the above-mentioned facts, dose androgen has any effect in sex determination of fish? What would happen to the gonad if XX and XY fish were treated simultaneously with both estrogen and androgen during the critical period of sex determination? Recently research has showed that differentiated ovary (even in adult fish) could be induced to change sex to functional male by long term treatment of aromatase inhibitors. However, sex reversal of XY fish has been restricted in the critical period of sex determination. Surprisingly, there is almost no research showing that XY fish with differentiated gonad could be successly induced into female.To answer these questions, we treated all-female and all-male tilapia fry with estrogen and androgen simultaneously during the critical period of sex determination and tried to induce sex reversal of the sex-differentiated XY male fish to study the effect of the sex steroids in sex determination and differentiation of fish.XY and XX tilapia fry were treated with17β-estradiol (E2) and17a-methyltestosterone (MT) in same dose simultaneously and respectively during the critical period of sex determination (5dah, day after hatching). The5dah XY and XX fish were treated for30days and then fed with control diet to120dah.1) Statistical analysis of gonad revealed that, MT could induce sex reversal of100%XX fish while MT+E2could not. E2could induce sex reversal of62.5%XY fish and MT+E2induce52%.2) Immunohistochemistry (IHC) revealed that, during the sex reversal of XY fish induced by E2, only Dmrtl was expressed at5dat (dat, day after treatment), Dmrt1and Cypl9ala were expressed simultaneously at10dat, and only Cypl9ala was expressed at30dat. While in MT induced the XX fish, only Cypl9ala was expressed at5dat, Cypl9ala and Dmrtl were expressed simultaneously at10dat, and only Dmrtl was expressed at30dat.3) Gonad IHC and the serum E2and11-KT enzyme immunoassay at120dah demonstrated that, Cyp19a1a and Cyp11b2were co-expressed in the interstitial cells of ovaries in both MT+E2treated XX and XY fish. Furthermore, the serum11-KT level of those MT+E2treated female fish were almost the same as that of the XY control fish, and E2level were almost the same as that of the XX control fish. These results demonstrated that, estrogen determined the fate of gonads during the critical period of sex determination. Estrogen could feminize XY fish even in the presence of androgen. The sex steroids induced sex reversal experienced a process of antagonism between the male and female pathway, and simultaneous treatment with both estrogen and androgen up-regulated the androgen synthesis in the ovary.GA and E2were used to induce30dah XY male fish for sex reversal. The30dah XY fish were treated for60days (till90dah) and then fed with control diet to150dah.1) Statistical analysis revealed that, the sex reversal rate of GA and GA+E2+11-KT treatment groups were0, while that for E2treatment group was8.3%and GA+E2treatment group was46.7%.2) Expression level of cyp19ala、cyp11b2、11β-hsd and dmrtl at60dat were analyzed by qPCR. Compared with control group, the expressions of cyp11b2and11β-hsd down-regulated in E2、GA and GA+E2treatment groups. While the expressions of cyp19a1a and dmrt1of GA+E2treatment group were significantly higher than the other groups.3) IHC revealed that Cyp19a1a, Cyp11b2and Dmrt1were expressed in the gonads of the GA+E2treatment group at60dat, while only Cypl9ala and Dmrtl were expressed at150dah.4) At60dat, the serum11-KT level of GA+E2treatment group was significantly lower than that of E2treatment group, while there was no significant difference in serum E2level between the two groups. The results demonstrated that the endogenous11-KT affects the ability of E2to induce sex reversal. Only through decrease of endogenous11-KT level could E2induce sex reversal of the sex-differentiated male gonad. As Dmrtl was continuously expressd during and after sex reversal, this type of sex reversal is incomplete.In conclusion, this study demonstrated that estrogen played a critical role, while androgen has no effect in sex determination of Nile tilapia. These results have supported the "deficiency hypothesis". The sex reversal induced by steroids would experienced a process of antagonism between the male and female pathway, and simultaneous treatment with both estrogen and androgen would up-regulate the androgen synthesis in the ovary. However, once11-KT began synthesis, it would affect the ability of E2to induce sex reversal. Furthermore, even block the synthesis of11-KT, E2could only induce incomplete sex reversal. Moreover, we were unable to induce sex reversal of the XY fish older than30dah with E2right now. These results suggested that androgen plays an important role in testis maintenance. |
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