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

doi:10.3864/j.issn.0578-1752.2025.11.014

摘要/Abstract

摘要：

【目的】哺乳动物的卵泡发育包含一系列复杂的过程，而卵泡发育过程受多种因素的调节，其中颗粒细胞在这一过程中起着重要作用。颗粒细胞的增殖和分化会直接影响卵泡的发育。自噬是颗粒细胞发育过程中至关重要的生物过程，β-巯基乙醇（2-mercaptoethanol，2-ME）常被用作一种抗氧化剂来优化家畜卵母细胞体外成熟培养体系，但目前尚缺乏其对水牛卵泡颗粒细胞生长发育的系统研究。本研究的目的是探究2-ME对雷帕霉素（Rapamycin）诱导的水牛卵泡颗粒细胞自噬、凋亡和类固醇激素分泌的影响。【方法】收集水牛卵巢组织，分离培养卵泡颗粒细胞，CCK8法检测细胞活力，筛选2-ME和Rapamycin的最佳处理浓度；选择最佳作用浓度处理卵泡颗粒细胞，采用实时荧光定量PCR检测2-ME对Rapamycin诱导的水牛颗粒细胞自噬、凋亡和类固醇激素分泌相关基因表达的影响；通过ELISA法分析2-ME对颗粒细胞类固醇激素分泌的影响；并采用Western blotting方法检测不同处理下颗粒细胞自噬和凋亡相关蛋白表达水平。【结果】 2-ME的最佳处理浓度为50 µmol·L^-1，Rapamycin的最佳的处理浓度为25 µmol·L^-1；自噬过度激活能够极显著上调自噬相关基因ATG5、LC3和Beclin1的表达（P<0.01），而同时加入2-ME和Rapamycin后3种基因的表达均下调；自噬过度激活能够极显著上调Bax和Caspase3的表达（P<0.01），显著上调Bcl2的表达（P<0.05），而同时加入2-ME和Rapamycin后，Bax和Caspase3的表达有所回复；自噬过度激活能够降低CYP19A1和CYP11A1的表达，而同时加入2-ME和Rapamycin后CYP19A1和CYP11A1的表达量升高，但差异不显著；自噬过度激活能够显著降低颗粒细胞E2和P4的分泌水平（P<0.05），而激活自噬后添加2-ME能够增加E2和P4的分泌水平；自噬能够上调LC3和Beclin1、Caspase3蛋白的表达水平，而同时加入2-ME和Rapamycin后3种蛋白的表达水平均有不同程度的降低。【结论】自噬能够促进水牛卵泡颗粒细胞的凋亡，抑制颗粒细胞分泌E2和P4的能力；而2-ME能够缓解颗粒细胞自噬引起的细胞凋亡，恢复由自噬降低的颗粒细胞分泌E2和P4的能力。综上所述，2-ME在维持水牛卵泡颗粒细胞自噬稳态方面发挥重要作用。

关键词: 水牛, β-巯基乙醇, 颗粒细胞, 自噬, 类固醇生成

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.

Key words: buffalo, β-mercaptoethanol, granulosa cells, autophagy, steroidogenesis

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.

XU YuanYuan, HUANG LiQing, LU XingRong, FENG Chao, SHANG JiangHua. Effects of β-mercaptoethanol on Rapamycin Induced Autophagy, Apoptosis and Steroid Hormone Synthesis in Buffalo Granulosa Cells[J]. Scientia Agricultura Sinica, 2025, 58(11): 2265-2274.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2025.11.014

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Gene	Primer sequence (5'-3')	Product size (bp)	Annealing temperature (℃)	Accession No.
Atg5	F: ACAAGCAACTCTGGATGGGT R: AGGCCGTTCAGTTGTTGTCT	143	60	XM_045161962
LC3	F: CCGACTTATCCGAGAGCAGC R: ACATGATCAGGCACCAGAAACT	114	60	XM_006046130
Beclin1	F: GGACACTCAGCTCAACGTCA R: GGATCAGCCTCTCCTCCTCT	143	60	XM_044938934
Bcl2	F: ATGACTTCTCTCGGCGCTAC R: CTGAAGAGCTCCTCCACCAC	112	60	XM_025273636
Bax	F: AAGGTGCCCGAGTTGATCAG R: AAAGTAGGAGAGGAGGCCGT	114	60	XM_025269476
Caspase3	F: TGGTGCTGAGGATGACATGG R: CCAGGATCCGTACTTTGCGT	115	60	XM_025280224
Cyp19A1	F: TGGTGACCATCTGTGCTGAT R: CCCAGGAAGAGCACGTTAGA	136	60	NM_001290963
Cyp11A1	F: TGGAGGATGTCAAGGCCAAT R: CGTGCCATCTCGTACAAGTG	94	60	XM_025271874
β-Actin	F: GGACCTGACGGACTACCTCA R: CCATCTCCTGCTCGAAGTCC	134	60	NM_001290932