南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (1): 93-99.doi: 10.12122/j.issn.1673-4254.2021.01.13

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双酚A对小鼠卵巢腔前卵泡颗粒细胞凋亡和卵巢发育的影响

梁 猛，周今朝，孙训英，贺超凡，张克甲，胡 柯   

  • 出版日期:2021-01-26 发布日期:2021-01-25

Effects of bisphenol A on apoptosis of ovarian preantral follicular granulosa cells and ovarian development in mice

  • Online:2021-01-26 Published:2021-01-25

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摘要：

目的 探索环境雌激素双酚A（BPA）对小鼠卵巢腔前卵泡颗粒细胞凋亡和卵巢发育的作用及机制。方法 选取10 d ICR雌性小鼠，分离小鼠卵巢腔前卵泡颗粒细胞进行体外培养。予以不同浓度（0、1、10、50、100、150、200和500 μmol/L）BPA处理卵巢腔前卵泡颗粒细胞，CCK-8方法检测细胞增殖活力，流式细胞仪分析细胞周期、凋亡和线粒体膜电位变化，Western blot检测Bcl-2、Bax、
p53和CyclinD1蛋白表达水平。根据对腔前卵泡颗粒细胞效应显著的BPA浓度灌胃处理ICR孕鼠1周，记录孕鼠的体质量变化，子代出生以后分别取10、17、21和42 d子代雌鼠卵巢组织称质量，然后进行卵巢组织切片HE染色及显微镜形态学观察，qPCR检测Bcl-2 mRNA水平。结果 与对照组比较，低浓度BPA（50 μmol/L）通过增加线粒体膜电位来抑制卵巢腔前卵泡颗粒细胞凋亡，进而促进细胞增殖（P<0.05）；高浓度BPA（200 μmol/L）通过减弱线粒体膜电位来促进卵巢腔前卵泡颗粒细胞凋亡，进而抑制细胞增殖（P<0.05）。低浓度BPA（50 μmol/L）和高浓度BPA（200 μmol/L）对卵巢腔前卵泡颗粒细胞中凋亡相关蛋白Bcl-2（P<0.01）、Bax（P<0.05）和p53（P<0.05）表达具有相反效应。体内实验表明，BPA（10 和35 mg/kg）处理导致孕鼠体质量增加较快及子代雌鼠卵巢指数发生变化，Bcl-2 mRNA表达与子代卵巢指数变化趋势相似，并且高浓度BPA（35 mg/kg）能够促使子代雌鼠21 d时卵泡发育加速进入有腔卵泡阶段。结论 BPA对卵巢腔前卵泡颗粒细胞功能调控具有浓度依懒性，并对孕鼠和子代雌鼠早期卵巢发育具有潜在影响。

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Abstract:

Objective To investigate the effect of environmental estrogen bisphenol A (BPA) exposure on apoptosis of mouse ovarian preantral follicular granulosa cells and ovarian development and explore the underlying mechanism. Methods Mouse ovarian preantral follicular granulosa cells were isolated from female ICR mice at postnatal day (PND) 10 and cultured in vitro. The cultured cells were treated with 0, 1, 10, 50, 100, 150, 200 and 500 μmol/L BPA, and the changes in cell proliferation, cell cycle, apoptosis and mitochondrial membrane potential were analyzed with CCK-8 method and flow cytometry. The protein expressions of Bcl-2, Bax, p53 and cyclin D1 in the treated cells were determined with Western blotting. Pregnant ICR mice were treated for a week with BPA at the concentration that produced significant effects on the preantral follicular granulosa cells, and the weight changes of the pregnant mice were recorded. The ovarian tissues of the offspring female mice were weighed at PND 10, 17, 21 and 42 followed by histological observation with HE staining and examination of Bcl-2 mRNA expression level with RT-qPCR. Results Compared with the control cells group, the isolated cells exposed to a low concentration of BPA (50 μmol/L) showed a significantly lowered apoptosis rate, increased mitochondrial membrane potential, and enhanced cellular proliferation (P<0.05). Exposure to a higher BPA concentration at 200 μmol/L obviously enhanced cell apoptosis by reducing the mitochondrial membrane potential and repressed the cell proliferation (P<0.05). BPA exposure at 50 μmol/L and 200 μmol/L produced opposite effects on the protein expressions of Bcl-2 (P<0.01), Bax (P<0.05) and p53 (P<0.05) in mouse ovarian preantral follicular granulosa cells. BPA exposure at the doses of 10 and 35 mg/kg caused rapid weight increment of the pregnant mice and changes in ovarian index of the offspring female mice. In the offspring female mice, the changes in Bcl-2 mRNA expression in the ovarian tissue showed a similar pattern to that of ovarian index. Exposure of the pregnant mice to a high BPA concentration at 35 mg/kg resulted in accelerated follicular development into antral follicular stage in PND 21 offspring female mice. Conclusion BPA can concentration- dependently regulate the function of ovarian preantral follicular granulosa cells in mice and potentially affects both the pregnant mice and the offspring female mice in light of early ovarian development.

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