| As a lower vertebrate,the sex determination and differentiation of fish are not only controlled by genetic factors,but also influenced by environmental factors.Environmental factors include light,temperature,pH and steroid hormones among which temperature and steroid hormones are the two most important environmental factors.A large number of experiments have proved that temperature changes sex by changing the level of cortisol in the body.Cortisol as the major glucocorticoid in fish,most studies focus on osmotic pressure regulation,inflammatory response,and immune regulation.Little research has been done on the relationship between cortisol and fish sex.So far,studies linking cortisol to sex differentiation in fish have been linked to changes in sex through high temperature mostly.In Japanese eels,Rainbow trout and Fathead minnow,high temperatures can increase cortisol levels,which can lead to masculinization.In medaka,high temperatures or direct exposure to exogenous cortisol can lead to masculinization or even sexual reverse.Above studies show that cortisol does play a role in sex determination and differentiation of fish,but the mechanism is still unclear.so,we designed this experiment to explore how cortisol affects the sex of fish,and Nile tilapia as model.The main findings as follows:1,Cortisol works through Gr.In this study,we have isolated two gr genes,gr1 and gr2,which are similar in structure.In phylogenetic and synteny analyses,we found that most of fish had two gr genes,but little has one gr.we speculate that it is loss in evolution.Both of the gr in Nile tilapia were copolymerized with the gr in medaka.The upstream and downstream conserved genes kctd16,fgf1 a,ndfip1,and gnpda1 of gr1 in Nile tilapia showed synteny in teleosts.GRXCR2,KCTD16 b,YIPF5,ARHGAP26 and ndfip1,the upstream and downstream conserved genes of gr2 in Nile tilapia,which also showed synteny in teleosts.Gr is a extensive expressed gene in Nile tilapia.Gr1 is most highly expressed in the testis and head kidney,while gr2 is highly expressed in the brain,liver,testis and head kidney.Moreover,the expression level of gr1 is higher than gr2 in both of the testis and ovaries.Gr1 is highly expressed in ovaries about 30 dah.In the testis,the expression was highest at 90 dah,and then continued high expression.Gr2 is highly expressed in ovary at 5 dah,and is low expressed in other stages.In the testis,gr2 is high expressed from 30 dah to 180 dah.The result of in situ hybridization showed that gr1 was mainly expressed in leydig cells in the testis.In ovary,gr1 is expressed in follicular cells and interstitial cells mainly.However,no gr2 signal was detected in the testis and ovaries.2,The serum cortisol levels of male and female Nile tilapia were detected at 90 dah,120 dah,150 dah and 180 dah,and the cortisol levels of male tilapia were always significantly higher than that of female tilapia.The expression levels of related genes in the cortisol synthesis pathway in head kidneys of female and male fish at 90 dah and 180 dah were detected.It was found that the expressions level of 3?hsdI and StARI in head kidney of male fish were significantly higher than those in female fish.Therefore,it is speculated that the difference in cortisol concentration between females and males is caused by the different expression of these two genes.3,?)The genetic females were continuously treated with cortisol until 60 dah.Histological observation and qRT-PCR analysis were performed on the gonads of the treatment group and the control group respectively.The results showed that oocytes of I-II phase were found in the control group,but no oocytes were found in the treatment group.Immunohistochemical results showed that both the control group and the treatment group could detect the positive signal of germ cell marker gene vasa,but the positive signal of the treatment group was significantly weaker than that of the control group.In the control group,the expression level of cyp19a1 a,a key gene for estrogen synthesis,was clearly observed,but its signal was not detected in the treatment group.Gsdf(Gonadal soma-derived factor)with high expression in the testis(low expression in the ovaries)was significantly increased after cortisol treatment compared to the control group.At the same time,we detected the expression of relevant genes about steroid synthesis in gonads through Real time PCR,and found that,the expression levels of germ cell marker gene vasa,meiosis related gene sycp3,ovarian specific expression gene cyp19a1 a and anti-apoptosis gene bcl-2 were significantly reduced.The expression levels of cyp11b2(androgen synthesis gene),gsdf and bax(apoptosis-promoting gene)were significantly increased.II)Cortisol treatment with estrogen.Histological observation was performed on the gonads of 60 dah and 180 dah respectively.At 60 dah,it was found that estrogen can rescue oocyte development retardation caused by cortisol to some extent.A small number of oocytes were observed in the rescue group,but no oocytes were observed in cortisol treatment group,and the number of oocytes in rescued ovaries was significantly lower than that in the normal control group.At the same time,we found that treatment with cortisol alone increased the mortality of juvenile fish,but if treated with estrogen,the mortality could be reduced.In 180 dah,we found that the female fish showed a sexual reversal and became a testis.III)If the genetic female larvae were treated with cortisol,the treatment was stopped at 30 dah,and then they were fed with normally feed until to 90 dah.Immunohistochemistry and Real time PCR showed that the marker gene Vasa of germ cells was slightly expressed in the treatment group,but significantly lower than the control group.The expression level of Cyp19a1 a was significantly decreased,and immunohistochemical results showed that there was no positive signal in the treatment group.The expression level of gsdf was significantly increased.The results of Real time PCR showed that the expression levels of meiosis related gene sycp3 and anti-apoptosis related gene bcl-2 were significantly reduced.Meanwhile,the expression levels of cyp11b2(a gene specifically expressed in the testis),and bax(a gene that promotes apoptosis),were significantly increased.IV)When normal feeding to 40 dah,and then cortisol treatment to 90 dah.From the histological observation of gonad,it was found that there was no obvious difference in gonad between the treatment and control female fish.Immunohistochemical results showed that the marker gene 3?hsd II(ovarian specific expression gene)and the key gene for estrogen synthesis cyp19a1 a were expressed in both treatment and control groups.Real time PCR showed that no significant difference between the two groups in 3?hsdII,but cyp19a1 a was significantly lower in treatment group than control group.In conclusion,gr1 may be the main cortisol receptor that plays a major role in sex determination and differentiation of Nile tilapia.The expression of gr1 has a sex dimorphism,and the expression level in the testis is higher than that in the ovaries,If female are treated with cortisol alone before meiosis,it will cause irreversible damage to the development of female sex.Estrogen can recued it in the early stage,but with the extension of treatment time,it will still cause sexual reversal.Cortisol treatment alone after meiosis had no significant effect on female gonadal development.Therefore,it is speculated that the effect of exogenous cortisol on the sex of female fish in early stage of sex development. |
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