Effects Of BMAL1 On Apoptosis And Testosterone Secretion In Mouse Leydig Cells

Leydig cells are distributed between the seminiferous tubules of the testis,which can synthesize and secrete androgen,thereby promoting spermatogenesis and reproductive organ development in male animals.The level of apoptosis of Leydig cells is closely related to the function of testis.Studies have shown that the circadian clock system plays an important role in maintaining mammalian reproductive performance.BMAL1 is a core circadian clock gene.Studies have shown that it can participate in apoptosis of various cells.The effects of BMAL1 on apoptosis and testosterone secretion of Leydig cells have not been reported.The aim of this study was to investigate the effects of BMAL1 on apoptosis,testosterone secretion and PI3K(Phosphatidylinositol 3-kinase)/AKT(Protein kinase B)signaling pathway in mouse Leydig cells.Using mouse Leydig cell line TM3 as a research object,small interfering RNA(siRNA)was used to inhibit the expression of BMAL1.Flow cytometry,qRT-PCR,and western blot were used to detect the effect of BMAL1 on apoptosis of mouse Leydig cells;ELISA was used to detect the testosterone secretion.qRT-PCR and western blot were used to detect the mRNA and protein expression levels of testosterone synthesis related genes: STAR,CYP11A1 and 3β-HSD;Western Blot was used to detect the phosphorylation level of PI3K/AKT signaling pathway.The results are as follows:1.Transfection of three siRNAs for 24 or 48 hours to detect gene and protein expression,and qRT-PCR was used to detect and screen the best interference efficiency.The results showed that the mixed siRNA interference efficiency was 50 %-60 %.Flow cytometry results showed that knockdown of BMAL1 gene caused a significant increase of the apoptosis in mouse Leydig cells(P<0.05);The results of qRT-PCR and western blot showed that knockdown of BMAL1 gene significantly up-regulated the mRNA and protein expression of the pro-apoptotic gene BAX(P<0.05),and down-regulated the mRNA and protein expression of the anti-apoptotic gene BCL-2(P<0.05).2.Mixed siRNA transfection for 24 or 48 h,the effects of knockdown of BMAL1 expression on mRNA and protein expression of testosterone synthesis related genes STAR,CYP11A1 and 3β-HSD were detected by qRT-PCR and western blot.The results showed that compared with NC(Negative Control)group,testosterone synthesis related genes STAR,CYP11A1 and 3β-HSD mRNA and protein expression levels of the siRNA interference group were significantly reduced(P<0.05).3.The expression levels of PI3 K,p-PI3 K,AKT and p-AKT protein were detected by western blot after knockdown of BMAL1 gene expression in mouse Leydig cells.It was found that the decreased expression of BMAL1 resulted in significant decrease of phosphorylation of PI3 K and AKT.(P<0.05).The results of this study indicate that knockdown of BMAL1 may inhibit the activity of PI3K/AKT signaling pathway,leading to the increase of apoptosis and downregulation of testosterone secretion and key synthetic gene expression in mouse Leydig cells.