The Role Of Soxs In The Process Of Sex Differentiation And Artificial Sex Reversal In Pelodiscus Sinensis

Pelodiscus sinensis,also known as the Chinese soft-shelled turtle,is widely distributed in freshwater rivers and lakes in East and Southeast Asia.P.sinensis shows obvious sexual dimorphism.The economic and nutrition value of male individuals are significantly higher than those of female individuals,which are deeply loved by breeders and consumers.Therefore,it has become a hot field to improve the male rate of filial generation by using sex control technology in the all-male breeding of P.sinensis,which has important research and application significance.Pseudo-females,which have been reversed from male to female,have been successfully obtained by estrogen induction,laying the foundation for all-male breeding.Here,comparative transcriptome analyses of female,male,and pseudo-female gonads were performed,and differentially expressed genes were identified in the pairwise comparison of three groups.The Sox genes involved in gonadal development and sex differentiation were selected as the research object.RT-q PCR was performed to analyze their expression changes during sex reversal induced by E₂in P.sinensis.The full-length c DNA of Sox3 was cloned,and the Sox3 lentivirus interference vector was designed.The dynamic expressions of Sox3 in different tissue,embryo development stage,exogenous estrogen induction and lentivirus interference were analyzed to explore the role of Sox3 gene in the process of sex differentiation and sex reversal of P.sinensis.The main results are indicated below:（1）Comparative analysis of gonad transcriptome in P.sinensisIn this study,transcriptome comparative analysis was performed on the gonads of female,male and pseudo-female P.sinensis.A total of 14,430 differentially expressed genes（DEGs）were identified in the three pairwise comparison groups,and3,017 and 975 DEGs were specifically expressed in male and female,respectively.In addition,147 DEGs were only expressed in pseudo-females,and they were not expressed in both females and males.GO and KEGG analysis of DEGs showed sex-related DEGs were mainly concentrated in steroid hormone biosynthesis pathway,such as steroid hormone biosynthesis（ko00140）,ovarian steroidogenesis pathway（ko04913）and progesterone-mediated oocyte maturation（ko04914）.Some candidate genes related to sex differentiation and gonadal development were screened:Cyp19a1,Cyp11a,Hsd3b,Hsd11b2,Hsd17b7,Wnt family genes,R-spondin family genes and Sox family genes.（2）Identification and screening of Sox genes related to sex reversal in P.sinensisAmong the 17 Sox family genes obtained from gonad transcriptome data,10 sex-related Sox genes were differentially expressed in male vs.female,male vs.pseudo-female,and female vs.pseudo-female.The differential expressions of Sox genes in gonads were analyzed by RT-q PCR,and it was found that Sox1,Sox3,Sox6,Sox11and Sox17 showed female-specific expression,while Sox9,Sox12 and Sox30 showed male-specific.Sox8 is highly expressed only in pseudo-females.The expression levels of 6 Sox genes in pseudo-female gonads were significantly different from those in female and male gonads.RT-q PCR was used to analyze their expression changes in the process of estradiol（E₂）induced embryonic sex reversal.The results showed that exogenous estrogen could activate the expression of Sox3 and inhibit Sox8 and Sox9.Sox11,Sox17 and Sox30 were not affected.Therefore,it can be speculated that Sox11,Sox17 and Sox30 do not participate in sex differentiation,but only participate in gonadal development and maintain gonadal function.Sox3 may promote male to female reversal,while Sox8 and Sox9 are inhibited in this process.In other words,Sox3 regulates female sex differentiation through estrogen pathway in embryo sex differentiation of P.sinensis,while Sox8 and Sox9 prevent this process.Sox family genes affect both female and male pathways in the process of sex reversal,which provides a new insight for the all-male breeding of P.sinensis.（3）Expression characteristics of Sox3 in ovarian differentiation of P.sinensisIn this experiment,it was found that Sox3 was only expressed in brain and gonad.Sox3 had no gender difference in the brain,but showed female specificity in the ovary.During embryonic development,Sox3 was highly expressed only at stage 9～13（stage12 is the initiation of ovarian differentiation）,and lowly expressed in other stages and male embryonic development.This may indicate that Sox3 is involved in the initiation of female sex differentiation and ovarian development.The results of exogenous E₂ induced experiment showed that E₂ could significantly enhance the m RNA signal of Sox3 during sex differentiation.The peak time of Sox3 expression in female embryos（12 h after treatment）was significantly earlier than that in male embryos（72 h after treatment）.This may be that Sox3 mainly plays a role in the initiation period of female sex differentiation of P.sinensis.Sox3lentiviral interference experiments found that in females（ZW+RNAi）,the expression of the male sex-determining gene Sox9 was up-regulated,while the expression of the key gene Ctnnb1 in the female pathway was down-regulated.It proved the importance of Sox3 for the initiation of female gonadal differentiation in the early stage of P.sinensis.Sox3 may be one of the early initiation genes of female sex differentiation in P.sinensis,promoting sex differentiation to ovary,and inhibiting the male sex differentiation pathway promoted by Sox9 in this process.