中国农业科学2025，Vol.58Issue(11)：2265-2274,10.DOI:10.3864/j.issn.0578-1752.2025.11.014
β-巯基乙醇对雷帕霉素诱导的水牛颗粒细胞自噬、凋亡和类固醇激素分泌的影响
Effects of β-mercaptoethanol on Rapamycin Induced Autophagy,Apoptosis and Steroid Hormone Synthesis in Buffalo Granulosa Cells
摘要
Abstract
[Objective]The development of mammalian follicles involves a series of complex processes,which is regulated by various factors,among which granulosa cells(GCs)play an important role.The proliferation and differentiation of granulosa cells directly affect the development of follicles.Autophagy is a crucial biological process in the development of granulosa cells,and β-Mercaptoethanol(2-ME)is often used as an antioxidant to optimize the in vitro maturation culture system of domestic animal oocytes,but there is currently a lack of systematic research on its effects on the growth and development of buffalo follicular granulosa cells.In view of this,the aim of this study was to investigate the effects of 2-ME on rapamycin induced autophagy,apoptosis,and steroid hormone secretion in buffalo follicular granulosa cells.[Method]Buffalo ovaries were collected,and GCs were isolated and cultured in vitro.After the cells were passaged,the different concentrations of 2-ME and Rapamycin were added exogenously to measure the cell viability by CCK8 method.And then,the optimal concentrations of 2-ME and Rapamycin were selected to treat GCs.Real-time fluorescence quantitative PCR was used to detect the effects of 2-ME on the gene expression of Rapamycin induced autophagy,apoptosis and steroid hormone secretion in buffalo granulosa cells.In addition,contents of estradiol(E2)and progesterone(P4)were detected by ELISA.And the expression levels of autophagy and apoptosis related proteins in granulosa cells under different treatments were detected by Western blotting.[Result]The optimal treatment concentrations for 2-ME and Rapamycin were 50 and 25 µmol·L-1,respectively.Overactivation of autophagy could extremely significantly upregulate the expression of autophagy related genes ATG5,LC3,and Beclin1(P<0.01),while the expression of all three genes was down regulated after the addition of 2-ME and Rapamycin simultaneously.Overactivation of autophagy could extremely significantly upregulate the expression of Bax and Caspase3(P<0.01)and significantly upregulate the expression of Bcl2(P<0.05).However,the addition of 2-ME and Rapamycin at the same time restored the expression of Bax and Caspase3 genes.Overactivation of autophagy could reduce the expression of CYP19A1 and CYP11A1 genes,while adding 2-ME and Rapamycin simultaneously increased the expression levels of CYP19A1 and CYP11A1 genes.Overactivation of autophagy could significantly reduce the secretion levels of E2 and P4 in granulosa cells(P<0.05),while adding 2-ME after activating autophagy can increase the secretion levels of E2 and P4.Autophagy could upregulate the expression levels of LC3,Beclin1 and Caspase3 proteins.However,the expression levels of all three proteins decreased to varying degrees after the addition of 2-ME and Rapamycin simultaneously.[Conclusion]Autophagy could promote the apoptosis of granulosa cells in buffalo follicles and inhibit their ability to secrete E2 and P4.However,2-ME could alleviate cell apoptosis caused by autophagy in granulosa cells and restore the ability of granulosa cells to secrete E2 and P4 reduced by autophagy.In conclusion,2-ME played an important role in maintaining autophagy homeostasis of buffalo granulosa cells.关键词
水牛/β-巯基乙醇/颗粒细胞/自噬/类固醇生成Key words
buffalo/β-mercaptoethanol/granulosa cells/autophagy/steroidogenesis引用本文复制引用
徐媛媛,黄丽清,陆杏蓉,冯超,尚江华..β-巯基乙醇对雷帕霉素诱导的水牛颗粒细胞自噬、凋亡和类固醇激素分泌的影响[J].中国农业科学,2025,58(11):2265-2274,10.基金项目
广西科技重大专项(桂科AA24263009)、中央引导地方科技发展资金项目(桂科ZY21195007)、国家现代农业产业技术体系广西奶水牛产业创新团队项目(nycytxgxcxtd-2021-21-01)、广西农业科技自筹经费项目(Z2024041) （桂科AA24263009）
