| The sex of fish was very plastic and sex determination and differentiation of fish were influenced by combination of genetic and environmental factors.Among environmental factors,temperature can affect the fate of gonadal differentiation and change the sex ratio of fish during the period of sex differentiation.During the same period,in teleost,endogenous estrogens are synthesized in the ovary and act as the natural inducer of ovarian differentiation.In addition,the change of endogenous steroid hormones can even cover genetic factors and change the direction of gonadal differentiation.Nile tilapia is a kind of fish whose sex differentiation direction is influenced by both genes and temperature.That is to say,in the period of sex differentiation,temperature can play a guiding role in the direction of gender differentiation and even reverse the process of gender differentiation.How does temperature affect the sex differentiation.At high temperatures,does endogenous estrogen undertake the task of changing the direction of gender differentiation through changes in its level? What is the level of estrogen change? The molecular mechanisms governing this process remain a mystery.We develop three all-female families by hybridization of wild females and sex reversed males.Because Letrozole(Le)could specifically inhibit aromatase activity and decrease the levels of E2,in order to explore the effects of high temperature induced on the sex steroid hormone level and the male ratio in the early stage of tilapia Gonadal differentiation,and the corresponding relationship,the female control group(XX),the male control group(XY),the high temperature treatment group(36?,XX+HT)and Le different concentrations group were set.In this way,the ability of high temperature and Le to down-regulate E2 levels in the early stage of sex differentiation was compared and analyzed.Although the levels of E2 and 11-KT in the gonads of tilapia did not change during the early stage of high temperature treatment(13dpf).However,at 21 dpf,high temperature significantly reduced E2 levels tonearly half of the control females' E2 levels and close to XX+L1.5(4?g/L)E2 levels.In the Le group,the E2 level gradually decreased as the amount of Le was added.The analysis of male ratio showed that the male ratio of XX+HT group(70%)was even higher than that of XX+L4.5 group(65.9%).It is worth noting that the level of E2 in gonad of XX+HT group was very close to that of XX+L1.5 group.Experiments have shown that high temperature induced under the condition of the same lower estrogen levels,still maintained a high rate of male,we speculate that the high temperature in addition to the cut way of E2,may also through a variety of other regulatory pathways involved in regulating the Nile tilapia females sex reversal.However,the E2 pathway down-regulated by high temperature may be one of the most important pathways.The gonad aromatase,Cyp19a1 a,is a steroid-producing enzyme that is responsible for the catalytic production of estrogen.In the experiment,in situ hybridization,qrt-pcr,immunohistochemistry and western blot were used to detect and analyze the mRNA expression and protein expression of Cyp19a1 a,a gonad aromatase of Nile tilapia.The results showed that high temperature significantly decreased the expression of Cyp19a1 a in the gonad at 21 dpf.On this basis,the activity of aromatase was detected by radioimmunoassay.Similarly,the activity of aromatase in the gonad of XX+HT group was significantly decreased.Based on the research on reptiles,in order to further verify the down-regulation mechanism of Cyp19a1 a,we conducted bisulfite sequencing on the promoter region of Cyp19a1 a.At 21 dpf,the methylation level of Cyp19a1 a promoter region in Nile tilapia was significantly increased by high temperature induction,which also showed the same results in sea bass and turtles.It indicates that high temperature has down-regulated the expression of Cyp19a1 a mRNA by influencing the methylation degree of Cyp19a1 a promoter in tilapia at 21 dpf,which might be the main reason for the down-regulation of E2 level.In addition,the expression of Dmrt1 mRNA and protein was analyzed.The results showed that Dmrt1,a key gene of male sex differentiation,had been significantly up-regulated at high temperature,indicating that the male pathway had been opened during the critical period of gender differentiation,which further demonstrated that E2 played a key role in the sexual differentiation period of Nile tilapia.In conclusion,the results showed that high temperature down-regulation of E2 was themain pathway to induce female sex reversal of Nile tilapia during the period of sex differentiation.High temperature can reduce the expression of Cyp19a1 a mRNA and protein by affecting the methylation degree of Cyp19a1 a promoter,and then regulate the down-regulation of E2 level,which may be the key reason for the sexual reversal induced by high temperature in Nile Tilapia. |
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