Scientia Agricultura Sinica ›› 2025, Vol. 58 ›› Issue (11): 2265-2274.doi: 10.3864/j.issn.0578-1752.2025.11.014

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles    

Effects of β-mercaptoethanol on Rapamycin Induced Autophagy, Apoptosis and Steroid Hormone Synthesis in Buffalo Granulosa Cells

XU YuanYuan1,2(), HUANG LiQing1, LU XingRong1,2, FENG Chao1,2, SHANG JiangHua1,2()   

  1. 1 Guangxi Buffalo Research Institute/Guangxi Key Laboratory of Buffalo Genetics, Breeding and Reproduction, Nanning 530001
    2 Key Laboratory of Buffalo Genetics, Breeding and Reproduction Technology Ministry of Agriculture and Rural Affairs, Nanning 530001
  • Received:2024-12-04 Accepted:2025-04-03 Online:2025-06-01 Published:2025-06-09
  • Contact: SHANG JiangHua

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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

Table 1

Primer information"

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

Fig. 1

Effects of 2-ME and Rapamycin on cell viability of granulosa cells Compared with the control group (0 µmol·L-1), *, significant difference (P<0.05); **, extremely significant difference (P<0.01); ns, No significant difference (P>0.05). The same as below"

Fig. 2

Effects of 2-ME on the expression of autophagy and apoptosis related genes in granulosa cells"

Fig. 3

Effects of 2-ME on the secretion of estradiol and progesterone in granulosa cells"

Fig. 4

Effects of 2-ME on the expression of autophagy and apoptosis related proteins in granulosa cells"

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