Functional Identification Of Sox9 Gene In The Early Male Differentiation Of Pelodiscus Sinensis

In mammals,Sox9 gene is directly activated by the sex-determining gene Sry on the Y chromosome,which promotes the development of undifferentiated gonads toward the testis and is located upstream of the molecular pathway of male development.Compared with mammals,the specific function of Sox9 gene in reptilian sex differentiation and its position in developmental pathway remain unclear.The Chinese soft-shelled turtle(Pelodiscus sinensis)is one of the famous,special and excellent aquatic products in China.The male has the advantages of fast growth,large size,wide calipash and high unit price,so it has higher breeding benefits than the female.The molecular mechanism of sexual differentiation in P.sinensis is still unclear,which hinders the development of high-male(all-male)breeding technology.In this study,the Sox9,male differentiation conserved gene in vertebrates,was taken as the research object.The expression distribution of Sox9 gene in ZZ and ZW embryonic gonads of different developmental stages were detected,and the biological function of Sox9 gene in early male differentiation of P.sinensis was identified by constructing the embryonic models of Sox9 gene interference and over-expression.The research contents and results are as follows:(1)Spatio-temporal expression analysis of Sox9 gene in embryonic gonads of P.sinensisThe RNA-Seq,q RT-PCR and immunofluorescence staining were used to detect the expression distribution of Sox9 gene at m RNA and protein levels in the embryonic gonads of both sexes at different developmental stages of P.sinensis.The results showed that Sox9 m RNA had begun to expressed in the embryonic gonads of stage 14(St.14,the early stage of sex determination),and the expression level in ZZ gonads was significantly higher than that in ZW gonads.The expression level reached the highest level in ZZ gonads at St.15.Subsequently,the high expression pattern of Sox9 in males accompanied the whole period of sexual differentiation(St.15-St.21).SOX9 protein had been widely expressed in ZZ gonads of St.15,mainly located in the nucleus of Sertoli precursor cells.With the differentiation and development of gonads,the detected fluorescence signal of SOX9 protein gradually increased.By the end of embryonic development(St.27),an obvious sex-cord structure surrounding germ cells had formed in ZZ gonads,and SOX9 protein was mainly distributed in the nucleus of supporting cells on the sex cords at this time,while almost no expression signal of SOX9 protein was detected in ZW gonads during embryogenesis.During the critical stages of sex determination and sexual differentiation(St.14-St.21),Sox9 maintained highly specific expression in male gonads,suggesting that this gene plays an important role in early male differentiation of P.sinensis.(2)Knockdown of Sox9 in ZZ gonads resulted in male-to-female sex reversalThe expression level of Sox9 in ZZ gonads was down-regulated by RNA interference at St.14,constructing the ZZ embryonic models with Sox9 knockdown.When the embryos developed to St.27,sex reversal analysis was performed on the knockdown embryos.The observation results of gonadal histology showed that ZZ gonads with Sox9 knockdown showed typical female characteristics,exhibiting the male-to-female sex reversal: gonads changed from short and thick cylindrical shape to long and thin flat shape;cortical area was highly developed,forming multi-layer cell structure,and medullary area was seriously degraded,and obvious lacunae structure appeared.In addition,the distribution pattern of germ cells was similar to female cortical distribution.Molecular level detection results showed that the expression levels of male-specific genes Dmrt1 and Amh were significantly decreased,while the expression levels of female-specific gene Foxl2 was remarkably increased in ZZ gonads with Sox9 knockdown.DMRT1 protein expression almost disappeared,while FOXL2 protein expression was induced.The results showed that after Sox9 knockdown,the ZZ gonads underwent a male-to-female sex reversal,with a reversal rate of 87.88%(29/33),suggesting that Sox9 gene is a necessary key regulatory factor in the early male differentiation of P.sinensis.(3)Over-expression of Sox9 in ZW gonads led to in female-to-male sex reversalThe expression level of Sox9 in ZW gonads was up-regulated by RNA overexpression at St.15,constructing the ZW embryonic models over-expressing Sox9.When the embryos developed to st27,the sex reversal analysis of the over-expressed embryos was carried out through gonadal histology observation and molecular level detection.The results showed that after over-expression of Sox9,ZW gonads differentiated into males,but the sex reversal was not complete,presenting an intersex state: there were a few male-like sex-cords in gonads;Dmrt1 and Amh expressions were up-regulated,and Foxl2 expressions were downregulated.Fluorescence signals of DMRT1 and FOXL2 protein expression were detected in the same gonad.In addition,this study found that in ZW gonads over-expressing Sox9,the expression of Dmrt1 and Amh,the two key genes of male differentiation,did not change significantly at St.16 before the onset of sexual differentiation.However,our research group previously found that the over-expression of Dmrt1 and Amh in ZW gonads,respectively,could rapidly up-regulate Sox9,and then induce the complete female-to-male sex reversal.These results suggest that the ectopic expression of Sox9 can induce ZW gonads differentiate to males(ovotestis),and this gene is regulated by upstream genes Dmrt1 and Amh.In conclusion,Sox9 is a necessary key gene in early male differentiation,and its deletion or over-expression can lead to varying degrees of sex reversal,locating downstream of Dmrt1 and Amh in molecular pathway of male development in P.sinensis.This study laid a theoretical foundation for elucidating the molecular regulatory network of sex determination and sexual differentiation and establishing the all-male breeding technology of P.sinensis.