Urokinase Receptor Gene Expression Inhibited by siRNA Cocktails in Co-culture of Spermatogenic Cells with Sertoli Cells from 3-Week-Old Rat

ObjectiveTo study the functions of urokinase receptor (uPAR) during spermatogenesis by adding targeted siRNA cocktails to the co-culture of Sertoli cell with spermatogenic cells.MethodsThe seminiferous tubule samples from SD rats aged 20-23 d were digested with collagenase for 15-20 min, then were cut into small fragments. Tubular fragments were digested with collagenase again for 5-10 min, then gently resuspended in F12/DMEM supplemented with vitamins and hormones. Cell samples were seeded in plate and cultured at 32°C for 2 weeks. After 48 h, siRNA cocktails at a concentration of 15 nmol/L or 30 nmol/L were introduced into this cocultured Sertoli/spermatogenic cells. After then for 48 h, expression of uPAR mRNA was analyzed by real-time RT-PCR.ResultsSome spermatogenic cells took place morphologic changes and generated an obvious flagellum. The expression levels of uPAR mRNA silencing with siRNA cocktails at a concentration of 15 nmol/L or 30 nmol/L were significantly lower than those in nontransfected group (P<0.05); the inhibition rate was 63.5% or 76.7%, respectively.ConclusionThe meiosis and spermiogenesis could take place in this culture system, and the siRNA cocktails can effectively inhibit the gene expression of uPAR in co-cultured Sertoli/spermatogenic cells.