| Fish are lower vertebrates and their determining mechanisms include genotypic sex determination(GSD),environmentally-dependent sex determination(ESD),and genetic-environment dependent sex determination(GSD+ESD).Environmental factors include temperature,light,p H,oxygen content,population density,exogenous steroid hormones,etc.The study found that temperature is one of the most important environmental factors in fish sex determination called temperature-dependent sex determination(TSD).The sex determination of some fish is controlled by both genetics and temperature,and has a sex chromosome,called a mixed type of genetic sex determination and temperature-dependent sex determination(GSD+TSD).For example,Nile tilapia is a kind of GSD+TSD fish.Their males fish grows more than 30% faster than females,increasing the breeding of male Nile tilapia can effectively improve economic efficiency.High temperature induction during the critical period of sex differentiation of Nile tilapia can significantly increase the proportion of males,that is,some hereditary females are reversed to physiological males.In this study,Nile tilapia was used as the research object,and the culture was carried out at the normal culture temperature(28 ?)and high temperature treatment temperature(36 ?).By analyzing the differences in the timing of germline cell types,the expression levels of sex-related genes and the differences in expression positions,the similarities and differences between the high temperature-induced female and normal female and male fish gonad development were revealed.The research results are as follows:1.In this study,the gonadal tissues were analyzed by HE staining and Vasa immunohistochemistry.It was found that the gonad development was similar in the 21 dpf,high temperature treatment group and the control female group,and the results showed that the gonad was ovary-like.In this study,the gonadal tissue was analyzed by HE staining.It was found that the gonadal developmental morphology,gonad cell type and quantity were similar in the 21 dpf,high temperature treatment group,control female fish group and control male fish group.At 39 dpf,it was observed that the control female fish group had clearly differentiated into oogonia cells,and the spermatogonia cells were differentiated in the periphery of the gonads of the control male fish group,and the gonads were not differentiated,and the gonads appeared in the center of the gonads.However,the trend of differentiation of gonadal cells tends to be compared with the male group,and the results indicate that the gonads at this time are testis-like.At 99 dpf,the high temperature treatment group and the control male fish group developed in the same state,differentiated spermatogonia,primary spermatocytes and a small number of sperm,the results showed that the gonads have been transformed into complete testis,but in the high temperature treatment group,there are a large number of gonads Honeycomb-like structures may affect the function of the gonads.These results indicate that high temperature treatment delays the differentiation of germ cells in the gonads and affects the integrity of gonadal structure development,but the germ cell type of the final gonadal development is consistent with the control male fish group.2.In this study,the expression levels and expression positions of three groups of fish gonad Cyp19a1 a protein at different developmental stages were detected by immunohistochemistry.The expression of Cyp19a1 a in the gonad of the Nile tilapia high temperature treatment group was detected by immunohistochemistry in 21 dpf.Compared with the control female fish group,the expression level and expression position were similar,and they were located in the gonad mesenchymal cells.No expression of Cyp19a1 a was observed in the control male fish group.It was observed that Cyp19a1 a was only detected in the control female group in 39 dpf,and neither the high temperature treatment group nor the control male fish group was detected.It indicated that the expression level of Cyp19a1 a protein in the high temperature treatment group was significantly down-regulated due to the high temperature,which was consistent with the control male fish group.The gonads of male and female tilapia have been clearly differentiated in 99 dpf,and the gonad cell types can be clearly distinguished.It has been observed that the high temperature treatment group has been successfully reversed.3?The expression of Dmrt1 and Gsdf proteins of male sex-differentiation genes in three groups of Nile tilapia was detected by immunohistochemistry at different stages.There was no significant difference in Dmrt1 and Gsdf protein expression between the high temperature treatment group,the control female group and the control male group at 21 dpf.At 39 dpf,almost no expression of Dmrt1 and Gsdf was observed in the control female group,high temperature treatment group and control male fish.The expression of Dmrt1 and Gsdf was highly expressed in the group,indicating that the high temperature treatment group tends to develop into male.The Vasa immunohistochemistry results in this period indicate that the expression of sex-determining genes in Nile tilapia which is reversible after high temperature treatment is earlier than the sex.Differentiation of gland cells.In the gonadal development to99 dpf,the gonadal cell types in the high temperature treatment group and the control male fish group can be clearly observed,and the spermatogonial cells,spermatocytes and sperm are clearly distinguished,and the Dmrt1 protein expression is localized in the spermatogonial cells of the gonads.In spermatocytes,Gsdf protein expression was mainly localized in supporting cells,and the results further confirmed that the gonads of the high temperature treatment group had been transformed into complete testis.In summary,under high temperature treatment,the gonad development process of female Nile tilapia females undergoes a sexual reversal from female to male.First,the expression pattern of genes related to sex differentiation changed,which was similar to that of the control females,and gradually changed to similar to the control males.Second,the cell type in the gonads changed,which was also consistent with the control females,and gradually changed to be consistent with the control males.However,the specific transformation process of cell types in the gonads requires further study. |
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