L-半胱氨酸对绵羊卵巢颗粒细胞增殖、凋亡和类固醇激素分泌的影响

doi:10.11843/j.issn.0366-6964.2024.01.018

摘要/Abstract

摘要： 旨在探讨L -半胱氨酸（L-Cys）对体外培养绵羊卵巢颗粒细胞（GCs）增殖、凋亡和类固醇分泌的影响。本研究采集1~1.5岁龄体重相近饲养条件相同的30只健康性成熟小尾寒羊母羊新鲜卵巢，从优势卵泡（2~8 mm）中收集GCs，随机分为 5 组，每组 6 个重复，各组添加L-Cys 浓度分别为0、100、300、500和700 μmol·L^-1，培养48 h 后用CCK-8试剂盒进行细胞增殖活力测定，Annexin V-FITC/PI流式细胞仪检测GCs的凋亡情况，采用 ELISA 检测GCs培养液上清中孕酮和雌二醇的分泌水平并采用实时荧光定量PCR和 Western blot 分析L-Cys对绵羊颗粒细胞增殖相关基因（PCNA、CCND2、CCNB1）、凋亡相关基因（BAX、Caspase-3、Bcl-2）和类固醇分泌相关基因的（STAR、3β-HSD、CYP11A1、CYP19A1）mRNA和蛋白表达的影响。结果显示，100、300和500 μmol·L^-1组的L-Cys均可显著促进绵羊GCs的增殖活力（P<0.05），并显著抑制其凋亡率（P<0.05）。添加300 μmol·L^-1的L-Cys显著抑制了孕酮（P₄， P<0.05）的分泌。进一步研究发现，300 μmol·L^-1和500 μmol·L^-1组的L-Cys 可以上调增殖相关基因（PCNA、CCND2、CCNB1）mRNA表达水平，其中300 μmol·L^-1组的增殖蛋白（PCNA、CCND2、CCNB1）表达量显著上升（P<0.05）；100 μmol·L^-1和300 μmol·L^-1组显著下调凋亡相关基因（BAX、Caspase-3）mRNA（P<0.05）及其蛋白表达水平（P<0.05）。添加300 μmol·L^-1 L-Cys 显著降低了类固醇相关基因（STAR、3β-HSD）mRNA及蛋白表达水平（P<0.05），添加100 μmol·L^-1和300 μmol·L^-1 L-Cys 显著升高了类固醇相关基因（CYP11A1、CYP19A1）mRNA（P<0.05）及其蛋白表达水平（P<0.05）。综上，L-Cys 通过上调基因PCNA、CCNB1、CCND2、Bcl-2 mRNA及其蛋白表达、下调基因BAX、Caspase-3 mRNA及其蛋白表达，促进绵羊GCs增殖，抑制其凋亡；L-Cys通过下调基因STAR、3β-HSD mRNA及其蛋白表达以及上调基因CYP11A1 mRNA及其蛋白表达抑制P₄ 的分泌。

关键词: 绵羊, 卵巢颗粒细胞, L-半胱氨酸, 增殖, 凋亡, 类固醇激素生成

Abstract: This study was conducted to investigate the effects of L-cysteine (L-Cys) on proliferation, apoptosis and the secretion of steroid hormones (progesterone and estrogen, P₄ and E₂) in ovine ovarian granulosa cells (GCs). Thirty sexually mature ewes aged between 1-1.5 years with the similar body weight and the same feeding condition were selected, and the fresh ovaries were collected. Then, GCs were collected from the dominant follicles (2-8 mm), and the GCs were randomly divided into 5 groups with 6 replicates per group. GCs were treated with 0, 100, 300, 500 and 700 μmol· L^-1 L-Cys for 48 h. The cell proliferation, apoptosis assay and the secretion of steroid hormones were measured using the CCK-8 kit, Annexin V-FITC/PI flow cytometry and ELISA, respectively. Real-time quantitative PCR and Western blot were used to analyze the mRNA and protein expression of the cell proliferation related genes (PCNA, CCND2, CCNB1), apoptosis-related genes (BAX, Caspase-3, Bcl-2) and steroid-secretion-related genes (STAR, 3β-HSD, CYP11A1, CYP19A1). The results showed that 100, 300 and 500 μmol·L^-1 L-Cys significantly promoted the cell viability (P<0.05) and significantly inhibited the apoptotic rate (P<0.05) of GCs. The addition of 300 μmol·L^-1 L-Cys significantly inhibited the secretion of P₄ (P<0.05). The mRNA expression of proliferation-related genes (PCNA, CCND2, CCNB1) were upregulated in the 300 μmol·L^-1 and 500 μmol·L^-1 L-Cys groups, while the protein expression of proliferation-related proteins (PCNA, CCND2, CCNB1) were increased in the 300 μmol·L^-1 L-Cys group (P<0.05). The mRNA and protein expressions of apoptosis-related genes (BAX, Caspase-3) were significantly downregulated in 100 μmol·L^-1 and 300 μmol·L^-1 L-Cys groups (P<0.05). The mRNA and protein expressions of the steroid-related genes (STAR, 3β-HSD) were significantly decreased in 300 μmol·L^-1 L-Cys group (P<0.05), but the mRNA and protein expressions of the steroid-related genes (CYP11A1, CYP19A1) were significantly increased in 100 μmol·L^-1 and 300 μmol·L^-1 L-Cys groups (P<0.05). In summary, L-Cys promoted cell proliferation and inhibited the apoptosis of ovine ovarian GCs by upregulating the mRNA and protein expressions of PCNA, CCNB1, CCND2, Bcl-2 and downregulating the mRNA and protein expressions of BAX, Caspase-3; L-Cys inhibited secretion of P₄ by downregulating the mRNA and protein expressions of STAR, 3β-HSD and upregulating the mRNA and protein expressions of CYP11A1.

Key words: sheep, ovarian granulosa cells, L-cysteine, proliferation, apoptosis, steroidogenesis

中图分类号:

S826.3

段香茹, 康佳, 杨若晨, 单新雨, 李太春, 赵雯, 张英杰, 刘月琴. L-半胱氨酸对绵羊卵巢颗粒细胞增殖、凋亡和类固醇激素分泌的影响[J]. 畜牧兽医学报, 2024, 55(1): 179-191.

DUAN Xiangru, KANG Jia, YANG Ruochen, SHAN Xinyu, LI Taichun, ZHAO Wen, ZHANG Yingjie, LIU Yueqin. Effect of L-cysteine on Proliferation, Apoptosis and the Secretion of Steroid Hormone in Ovine Ovarian Granulosa Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(1): 179-191.

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