| As a consequence of the hierarchical nature of gonad development, a single gene or environmental cue operating at the beginning of development can drive the gonad pathway towards one direction or another. However it is still unknown that how environmental override the role of genotype and switch the mechanism to affect the sex determination and differentiation in fish. In this study, the combined effects of temperature and17α-methyltestosterone(MT) on longitudinal expression of sex-determining gene were analyzed by using Box-Behnken design and response surfaced method. The main conclusions showed as that:1. Longitudinal expression change of Sox9 a induced by combination of temperature and MT Sox9, a downstream effector of Sry, is thought to play a key role in differentiation and development of testicular in fish. The results showed that the linear and quadratic effects of temperature and the quadratic effects of inducted duration on the expression of Sox9 a were highly significant(P<0.01), while the linear and quadratic effects of MT and the linear effects of inducted duration were not significant(P>0.05). The interaction between temperature and inducted duration was significant(P<0.05), but the interactions were not significant between MT and temperature or inducted duration(P>0.05). The effect of temperature on Sox9 a expression was greater than that of inducted duration,which in turn was greater than that of MT. With increased temperature, Sox9 a m RNA expression was slightly decreased and then increased. In contrast, Sox9 a m RNA expression increased with the increasing of MT concentration, and then decreased. Through response surface methodology, a model equation of Sox9 a expression on the three factors was established(R2=0.9778, Adj.R2 =0.9493), suggesting that the fitting capability of the model was satisfactory.2. Longitudinal expression change of ARα induced by combination of temperature and MT The biological activity of androgen is mediated by androgen receptors(AR). The results showed that the linear and quadratic effects of temperature, MT and induction duration on ARα were significant(P<0.05), but the interactive effects between any two of three factors was not significant(P>0.05). The effect of temperature on ARα expression was greater than that of inducted duration, which in turn was greater than that of MT. ARαm RNA expression was up-regulated with increasing temperature and duration, and up- and down-regulated with the increasing of MT concentration. Through response surface methodology, a model equation of ARα expression on the three factors was established(R2=0.9972, Adj.R2 =0.9937), suggesting that the fitting capability of the model was satisfactory.3. Longitudinal expression change of GATA4 induced by combination of temperature and MT GATA4, a member of the GATA family, is a well-known transcription factor implicated in the regulation of sex determination and sexual differentiation in mammals,and still little known action of temperature and MT on it. In the present study, the join effects of temperature, MT and induction duration on the expression of GATA4 in O.Niloticus were analyzed by using response surfaced method. The linear and quadratic effects of temperature, MT and induction duration GATA4 expression were significant(P<0.05). The interaction between temperature and induction duration, interactive effect of MT and induction duration were highly significant(P<0.01), but the interaction between MT and temperature was nonsignificant(P>0.05). The effect of temperature on ARαexpression was greater than that of MT, which in turn was greater than that of inducted duration. GATA4 m RNA expression was up-regulated with the rising of temperature, and up- and down-regulated with the increasing of MT concentration and duration. Through response surface methodology, a model equation of GATA4 expression on the three factors was established(R2=0.9918, Adj.R2 =0.9813), suggesting that the fitting capability of the model was satisfactory. |
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