双酚A对腔前卵泡体外发育的干扰作用

Interfering effect of bisphenol A on the growth of preantral follicles in vitro

目的探讨双酚A(BPA)对腔前卵泡体外发育的干扰作用。方法将分离获取的小鼠腔前卵泡体外培养11d,培养液中分别添加0μmol/L BPA、1μl/ml DMSO、4.5μmol/L BPA和45μmol/L BPA,即为正常组、对照组、4.5μmol/LBPA组和45μmol/LBPA组。观察卵泡、卵母细胞和颗粒细胞的生长发育情况;统计卵泡成腔率、裸卵率和卵母细胞成熟率;采用ELISA法、免疫荧光染色法和Western blotting检测培养液中雌二醇(E2)含量、卵母细胞雌激素受体的表达,以及卵泡中生长分化因子9(GDF-9)和骨形态发生蛋白15(BMP-15)的表达。结果与正常组比较,45μmol/L BPA组腔前卵泡的成腔率(10.1%)、卵母细胞成熟率(60.0%)、卵泡直径和颗粒细胞厚度明显降低(P<0.05),而裸卵率(28.6%)明显升高(P<0.05);且45μmol/LBPA明显降低了D8和D10卵泡培养液中的E2含量、卵母细胞雌激素受体的表达以及卵泡中GDF-9和BMP-15的表达(P<0.05)。4.5μmol/L BPA组的各项指标与正常组比较差异均无统计学意义。结论45μmol/LBPA可抑制腔前卵泡中雌激素的合成和分泌,降低卵母细胞中雌激素受体的表达,影响卵母细胞分泌促生长因子GDF-9和BMP-15,进而干扰卵泡发育、颗粒细胞增殖和卵母细胞成熟。

Objective To investigate the effect of bisphenol A (BPA)on the growth of preantral follicles in vitro.Methods Preantral follicles were harvested from mouse ovaries,cultured in vitro for 11days and randomly divided into 4groups with separately adding 0μmol/L BPA,1μl/ml DMSO,4.5μmol/L BPA and 45μmol/L BPA as to form normal group,control group,4.5μmol/L BPA group and 45μmol/L group.The growth and development of the follicles,oocytes and cumulus cells were observed.The percentages of antral follicles, denuded oocytes and the maturing rate ofoocytes were calculated.The content of estradiol (E2)in the culture medium and the expressions of estrogen receptor (ER),growth differentiation factor 9(GDF-9)and bone morphogenetic protein-15(BMP-15)in oocytes were detected by ELISA,immunofluorescence staining and Western blotting.Results Compared with normal group,45μmol/L BPA significantly reduced the percentage of antral follicles (10.1%),maturation rate of oocytes (60.0%),diameter of follicles and the thickness of the cumulus cell layers, but increased the rate of denuded oocytes (28.6%,P<0.05).The contents orE2in culture media of 45μmol/L BPA group were significantly decreased at the 8th and 10th day of preantral follicle culture (P<0.05).In addition,45μmol/L BPA significantly lowered the expressions of ER,GDF-9and BMP-15in oocytes (P<0.05).For the indexes mentioned above,no significant difference existed between 4.5μmol/L BPA group and normal group.Conclusion 45μmol/L BPA interferes with the growth of preantral follicles,the proliferation of cumulus cells and the maturation of oocytes in vitro through the inhibition on the estrogen synthesis and secretion from preantral follicles,and the reduced expression ofER,GDF-9and BMP-15in oocytes.