A Preliminary Study On Differential Proteomics Of Gonads In Chicken Embryos

Chicken is an agricultural animal and its gender characteristics have a significant effect on production.During the developmental process of the chicken embryo,the bilateral gonads of male chicken embryos develop symmetrically,but the left and right gonads of female chicken embryos develop asymmetrically.The left side develops into ovary normally while the right side gradually degenerated and atrophied.The regulation mechanism of gonads development in the chicken embryo is not yet clear.In current study,we illustrated the regulatory mechanism of gonadal development and sex differentiation in chicken embryos at the molecular level that might be a foundation for the analysis of sex determination and differentiation in chicken.The factors related to gonadal differentiation between male and female gonads in chicken embryos were analyzed by the protein expression level,and the mechanism of asymmetric development of gonad of the female chicken embryo was evaluated.The results of our findings are as follows:1.Differential proteomic analysis between male and female gonads in chicken embryos.We used the label free tandem mass spectrometry proteomics technique.The protein expression level of the left female gonad and the right female gonad along with left and right male gonads of the chicken embryo day 6.5 were analyzed.The results demonstrated that a total of 7726 proteins were identified and 6858 proteins could be quantified.In this study,with the female gonads as the control group and the male gonads as the experimental group,a total of 454 differentially expressed proteins were accomplished(including 265 upregulated and 189downregulated).Through bioinformatics analysis of DEPs and Gene Ontology(GO)enrichment analysis showed that the molecular function of DEPs in female and male gonads was associated with RNA binding protein.KEGG pathways analysis exhibited that the steroid hormone biosynthesis pathway was significantly enriched,and the metabolic pathways of DEPs were mainly concentrated in some pathways related to metabolic pathways like energy metabolism,fatty acid and amino acid metabolism.2.Differential proteomic analysis between left and right gonads in female chicken embryos.To investigate the molecular regulatory mechanism of asymmetric gonadal development in the female chickens,using the female right gonad as the control group and the left gonad as the experimental group,a total of 94 DEPs were obtained(52 up-regulated and 42 down-regulated).The metabolic pathways of DEPs were mainly implicated in apoptosis,piRNA metabolic process,gonadal development regulation,steroid regulation of sodium reabsorption and cell adhesion molecule CAMs and other metabolic processes.Western Blot confirmed that the expression changes of CVH,DMRT1 and DAZL proteins were consistent with the results of quantitative proteomic analysis.3.A preliminary study on the inhibition of TBX1 in chicken Sertoli cells.In order to investigae the relationship between the DEPs TBX1 and DMRT1,we used RNA interference to inhibite the expression of the TBX1 gene in chicken testicular Sertoli cells.The results showed that knocking down TBX1 caused down-regulation of DMRT1 expression,which confirmed that si RNA interference affected the expression of DMRT1,indicating that the expression of the DMRT1 gene in chicken testicular Sertoli cells was related to the TBX1 gene.Overall,the protein expression profile of chicken embryo gonads were constructed,and the DEPs of female and male gonads and the DEPs of left and right female gonads were obtained.It was confirmed that TBX1 might have significant role on the expression of the sex-determining gene DMRT1 by RNA interference.The results of this study laid a foundation for further studies of sex determination and differentiation of chicken and asymmetric development of female gonads in chicken.