吉林大学学报(医学版) ›› 2023, Vol. 49 ›› Issue (5): 1154-1160.doi: 10.13481/j.1671-587X.20230507

• 基础研究 • 上一篇    

睾酮在体外对人卵巢颗粒细胞SVOG凋亡的影响及其内质网应激机制

仝晓丽1,2,范明慧1,孟娇1,朱继红3,盛敏佳1()   

  1. 1.吉林大学中日联谊医院生殖医学中心,吉林 长春 130033
    2.山西省临汾市中心医院生殖健康 与不孕不育科,山西 临汾 041000
    3.吉林大学第一医院生殖医学?产前诊断中心, 吉林 长春 130021
  • 收稿日期:2022-11-30 出版日期:2023-09-28 发布日期:2023-10-26
  • 通讯作者: 盛敏佳 E-mail:shengmj@jlu.edu.com
  • 作者简介:仝晓丽(1993-),女,山西省大同市人,医师,医学硕士,主要从事辅助生殖临床方面的研究。
  • 基金资助:
    吴阶平医学基金会专项项目(320.6750.2021-19-1);吉林省教育厅科学技术研究项目一般项目(JJKH20211200KJ);吉林省财政厅科研项目(JLSWSRCZX2020-062)

Effect of testosterone on apoptosis of human ovarian granulosa cells SVOG in vitro and its endoplasmic reticulum stress mechanism

Xiaoli TONG1,2,Minghui FAN1,Jia MENG1,Jihong ZHU3,Minjia SHENG1()   

  1. 1.Center of Reproductive Medicine,China-Japan Union Hospital,Jilin University,Changchun 130033,China
    2.Department of Reproductive Health and Infertility,Central Hospital,Linfen City,Shanxi Province,Linfen 041000,China
    3.Reproductive Medicine Center,Prenatal Diagnosis Center,First Hospital,Jilin University,Changchun 130021,China
  • Received:2022-11-30 Online:2023-09-28 Published:2023-10-26
  • Contact: Minjia SHENG E-mail:shengmj@jlu.edu.com

摘要:

目的 探讨睾酮在体外对颗粒细胞凋亡的诱导作用,并阐明其作用机制。 方法 噻唑蓝(MTT)比色法检测人卵巢颗粒细胞SVOG的细胞增殖抑制率,筛选最佳药物作用浓度后,将SVOG 细胞分为对照组、睾酮组(1.0×10-5 mol·L-1睾酮)、睾酮+氟他胺组(1.0×10-5 mol·L-1睾酮+1.0×10-5 mol·L-1氟他胺)、睾酮+牛磺熊去氧胆酸(TUDCA)组(1.0×10-5 mol·L-1睾酮+1.5 g·L-1 TUDCA)。采用实时荧光定量PCR(RT-qPCR)法检测各组 SVOG 细胞中剪切型X盒结合蛋白1 (XBP1s)、激活转录因子4(ATF4)、转录因子C/EBP同源蛋白(CHOP)和死亡受体5(DR5) mRNA表达水平,流式细胞术检测各组 SVOG 细胞凋亡率。 结果 与对照组比较,睾酮组SVOG细胞形态改变,异型细胞增多,胞质中出现较多颗粒,培养基中细胞碎片增多,SVOG细胞增殖抑制率升高(P<0.05);与睾酮组比较,睾酮+氟他胺组和睾酮+TUDCA组SVOG细胞形态变化减少,细胞增殖抑制率降低(P<0.05);流式细胞术,与对照组比较,睾酮组SVOG细胞凋亡率升高(P<0.05);与睾酮组比较,睾酮+氟他胺组和睾酮+TUDCA组SVOG细胞凋亡率降低(P<0.05)。RT-qPCR法,作用48 h后,与对照组比较,睾酮组SVOG细胞中XBP1s、ATF4、CHOP和DR5 mRNA表达水平均明显升高(P<0.05);与睾酮组比较,睾酮+氟他胺组和睾酮+TUDCA组SVOG细胞中XBP1s、ATF4、CHOP和DR5 mRNA表达水平降低(P<0.05)。 结论 睾酮可诱导人卵巢颗粒细胞凋亡并引起卵泡闭锁,其作用机制可能是雄激素与雄激素受体(AR)结合激活CHOP-DR5通路,诱导内质网应激(ERS)发生,促进颗粒细胞凋亡。

关键词: 睾酮, 颗粒细胞, 内质网应激, 雄激素受体, 转录因子C/EBP同源蛋白, 死亡受体5

Abstract:

Objective To discuss the inductive effect of testosterone on the apoptosis of the granulosa cells in vitro,and to clarify its mechanism. Methods The inhibitory rates of proliferation of human ovarian granulosa cells SVOG were detected by methyl thiazolyl tetrazolium(MTT) assay. After screening the optimal concentrations of the drugs, the SVOG cells were divided into control group, testosterone group (1.0×10-5 mol·L-1 testosterone),testosterone+fluorothiamine group(1.0×10-5 mol·L-1 testosterone+ 1.0×10-5 mol·L-1 fluorothiamine),and testosterone+tauroursodeoxycholic acid (TUDCA)group (1.0×10-5 mol·L-1 testosterone+1.5 g·L-1 TUDCA). Real-time fluorescence quantitative PCR (RT-qPCR) method was used to detect the expression levels of spliced X-box binding protein 1 (XBP1s), activating transcription factor 4 (ATF4), transcription factor C/EBP homologous protein (CHOP), and death receptor 5 (DR5) mRNA in the SVOG cells in various groups; flow cytometry was used to detect the apoptotic rates of the SVOG cells in various groups. Results Compared with control group, the SVOG cells in testosterone group showed morphological changes, the number of atypical cells was increased, the granularity in the cytoplasm was increased, the cell fragments in the culture medium was increased, and the inhibitory rate of proliferation of the SVOG cells was increased (P<0.05). Compared with testosterone group, the morphological changes of the cells in testosterone +fluorothiamine group and testosterone+TUDCA group were decreased and inhibitory rates of proliferation of the cells were decreased(P<0.05). The flow cytometry results showed that compared with control group, the apoptotic rate of the SVOG cells in testosterone group was significantly increased (P<0.05). Compared with testosterone group, the apoptotic rates of the SVOG cells in testosterone + TUDCA group and testosterone + fluorothiamine group were significantly decreased (P<0.05). The RT-qPCR analysis results showed that after treated for 48 h, compared with control group, the expression levels of XBP1s, ATF4, CHOP, and DR5 mRNA in the SVOG cells in testosterone group were increased (P<0.05). Compared with testosterone group, the expression levels of XBP1s,ATF4,CHOP, and DR5 mRNA in the SVOG cells in testosterone+TUDCA group and testosterone+fluorothiamine group were decreased (P<0.05). Conclusion Testosterone can induce the apoptosis of human ovarian granulosa cells and cause follicular atresia,and its mechanism may be that androgen combined with adrogen receptor(AR) activates the CHOP-DR5 pathway, induces endoplasmic reticulum stress(ERS), and promotes the apoptosis of the granulosa cells.

Key words: Testosterone, Granulosa cells, Endoplasmic reticulum stress, Androgen receptor, Transcription factor C/EBP homologous protein, Death receptor 5

中图分类号: 

  • R711.75