Expression Of Sox9 Gene And Its Role In Porcine Immature Sertoli Cell

The Sox9 gene is the major target gene of the mammalian testis determining gene SRY and is located on the male Y chromosome.In mammals,Sox9 has been shown to play a key role in male sex determination,testicular development,and porcine sertoi cell differentiation.Porcine sertoi cell as unique somatic cells within the vas deferens,with multiple functions,including providing structural support for developmental germ cells and controlling self-renewal and survival of spermatogonia.They also produce an impermeable immune barrier,the blood testis barrier(BTB),and participate in germ cell activity through spermatogenic epithelial cells.In this study,si RNA technology,real-time quantitative PCR(qRT-PCR),flow cytometry,EDU cell proliferation assay,Cell Counting Kit-8(CCK-8)were used to study the interference of Sox9 gene on the proliferation of pig porcine immature sertoi cell.The role of death and analysis of the expression and immunohistochemistry of Sox9 gene in different developmental stages of porcine testis tissue,the results are as follows:1.Three Sox9 siRNA oligos were constructed and transiently transfected into testicular immature support cells by liposome lip2000.The expression of Sox9 gene was detected by real-time PCR(qRT-PCR).Sox9 gene expression was significantly reduced(interference efficiency was 74%).Therefore,subsequent experiments can be continued;2.After transient transfection of si Sox9 and siSox9 NC,the ability of cell growth,proliferation and apoptosis was observed by flow cytometry,CCK-8 assay,and EdU cell proliferation assay;and three apoptosis-related genes(Bax,Bcl-2,Caspase3)were passed.qRT-PCR assay to determine the biological function of Sox9 gene in immature support cells of porcine testis.The results of cell cycle assay by flow cytometry showed that the proportion of cells in G1 phase increased significantly(P<0.01),while the proportion of cells in S phase decreased significantly(P<0.01),indicating that the cell cycle was arrested in G1 phase.It indicates that the interference with Sox9 will inhibit the proliferation of sertoi celland promote its apoptosis.Meanwhile,CCK-8 and EDU experiments also show that the interference with Sox9 can inhibit the proliferation of sertoi celland promote their apoptosis;qRT-PCR results of apoptosis-related genes It was shown that after Sox9 interference,theexpression of Bax and Caspase3,which are related to apoptosis-promoting,increased,while the expression of Bcl-2,which is related to apoptosis-inhibiting,decreased,which was consistent with the results of western blot.The above results show that the interference of Sox9 gene can inhibit the proliferation of porcine sertoi cell and promote their apoptosis;3.Immunohistochemical analysis of 1 and 180 days of pig testicular tissue showed that the positive expression level of Sox9 protein was higher in D1 stage and more expressed on supporting cells;the positive expression level of D180 stage protein was lower,and a small amount of sertoi cell were formed Immunopositive reaction;4.The expression levels of Sox9 mRNA in pig testis tissues of D1,D30,D60,D90,D120 and D150 were analyzed.The results showed that the overall expression level of Sox9 m RNA increased first and then decreased,and the highest expression at D30.After that,the expression level showed a steady downward trend;In summary,interference of the Sox9 gene inhibits proliferation and promotes apoptosis in porcine immature sertoi cell,and demonstrates that the Sox9 gene acts primarily on porcine sertoi cell.