| Granulosa cells are one of the functional cells in follicles,which play an important role in the regulation of follicles development and ovulation.When granulosa cells function is abnormal,it will causes follicle atresia and abnormal ovulation.As a member of the Sirtuins family,Sirt2 is an important histone deacetylase that relies on NAD+activity to function.It can catalyze the deacetylation of different substrates and participate in the regulation of microtubule dynamics,chromosomes arrangement,oxidative stress and energy metabolism and other physiological processes.However,the roles and regulatory mechanisms of Sirt2 in cells cycle,proliferation,apoptosis,antioxidant and steroid hormones secretion of sheep ovarian granulosa cells have not been reported.In this study,sheep ovarian granulosa cells were used as experimental materials,si-Sirt2 transfection was used to interfere the expression of Sirt2 in granulosa cells.Co-culture with sheep bare eggs to explore the effects of interfering the expression of Sirt2 in granulosa cells on oocytes maturation under co-culture system.siRNA interference,immunofluorescence staining,MTT assay,Annexin V-FITC/PI double staining and ELISA were used to investigate the effects of Sirt2 on cells cycle,proliferation,apoptosis,antioxidant and steroids secretion of sheep ovarian granulosa cells and the related regulatory mechanisms.The results showed that:(1)si-Sirt2 transfection can successfully knock down the expression of Sirt2 in sheep ovarian granulosa cells.In sheep ovarian granulosa cells,Sirt2 was expressed in both cytoplasm and nucleus,but mainly in cytoplasm;(2)When sheep ovarian granulosa cells were co-cultured with bare eggs,the maturation rate of the co-cultured bare eggs after interfering with the expression of Sirt2 was 33.60%,which was significantly lower than the 47.98%maturation rate of the control group(P<0.05);(3)In terms of the effect of Sirt2 on sheep ovarian granulosa cells cycle,after transfection of granulosa cells for 24 h,compared with the control group,the proportion of G0/G1 phase cells was significantly increased,while the proportion of S phase and G2/M phase cells were significantly decreased(P<0.05);(4)In terms of granulosa cells proliferation,after transfection of granulosa cells at 24 h,48 h and 72 h,there was no significant difference in cells proliferation activity compared with the control group(P>0.05);(5)In terms of granulosa cells apoptosis,24 h after transfection,the apoptotic rate was significantly higher than that of the control group(P<0.05),and the expression of anti-apoptotic gene Bcl-2 was significantly decreased(P<0.05),the expression levels of pro-apoptotic genes Bax and Caspase-3 were significantly increased(P<0.05);(6)In terms of antioxidant function of granulosa cells,ROS level was significantly increased after transfection for 24 h(P<0.05),and the expressions of FoxO3a,SOD2,CAT and GPX4 were significantly decreased(P<0.05);(7)Interfering Sirt2 expression in sheep ovarian granulosa cells could promote the secretion of P4 by increasing the genes expression of StAR and CYP11A1,and inhibiting the genes expressions of CYP17A1,CYP19A1,3β-HSD and 17β-HSD(P<0.05),but had no significant effects on the secretion of E2 and T(P>0.05);Moreover,interference with Sirt2 expression could activate the ERK1/2 signaling pathway,thereby increasing the secretion of P4.In conclusion,interference with Sirt2 expression in sheep ovarian granulosa cells could block the cells cycles,promote cells apoptosis,weaken the antioxidant capacity of cells,induce the synthesis and secretion of P4,and thus inhibit oocytes maturation under the co-culture system.Moreover,Sirt2 could regulate the secretion of P4 through the ERK1/2 signaling pathway. |
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