EGCG对水牛卵母细胞体外成熟的影响

Effect of EGCG on oocytes in vitro maturation of buffalo(Bubalus bubalis)

【目的】探讨表没食子儿茶素没食子酸酯（EGCG）对水牛卵母细胞体外成熟质量与早期胚胎发育能力的影响,为优化水牛体外胚胎生产体系及提高其体外胚胎生产效率提供参考。【方法】在水牛卵母细胞体外成熟液中添加不同浓度的EGCG,以排出第一极体为细胞核成熟标志,观察卵母细胞核成熟情况,检测MII期卵母细胞内活性氧（ROS）和谷胱甘肽（GSH）含量,并统计经孤雌激活及体外受精后早期胚胎的发育情况。【结果】在体外成熟液中添加10~20μmol/L EGCG有利于水牛卵母细胞体外成熟过程中卵丘扩展,卵丘扩展指数（CEI）分别为2.49%和2.42%,卵母细胞成熟率（59.31%和50.98%）显著高于未添加EGCG的CK组（P〈0.05,下同）,MII期卵母细胞的GSH含量（3.21和3.25 pmol/枚）也显著高于CK组;添加20~30μmol/L EGCG可显著降低水牛MII期卵母细胞的ROS水平。以体外成熟培养获得的水牛MII期卵母细胞分别进行孤雌激活及体外受精,结果发现以在体外成熟液中添加10μmol/L EGCG的效果最佳,其对应的卵裂率和囊胚发育率均显著高于CK组。【结论】EGCG可用于优化水牛卵母细胞体外成熟体系,能有效提高卵母细胞的成熟率和体外发育潜力,且以水牛体外成熟液中添加10μmol/L EGCG的效果最佳。

【Objective】In order to provide reference for improving efficiency of embryo in vitro culture, the present experiment was conducted to investigate effect of epi-gallocatechin-3-gallate（EGCG） on buffalo oocytes in vitro maturation and early embryo viability. 【Method】The buffalo oocytes was cultured in vitro in the maturation medium adding to different concentrations of EGCG, with discharge of first polar body as nucleus maturity mark, the maturation degree of buffalo oocyte nucleus was observed. Then the contents of glutathione（GSH） and reactive oxygen（ROS） in oocytes at MII phase were determined, and the early embryo development after parthenogenetic activation and in vitro fertilization were analyzed. 【Result】The results showed that, adding 10-20 μmol/L EGCG to maturation medium had positive effects on expansion of cumulus in process of oocyte in vitro maturation, the corresponding cumulus expansion indexes（CEI） were2.49% and 2.42%, respectively, and the maturation rates of oocytes were 59.31% and 50.98%, respectively, which were significantly higher than that of oocytes in CK group（maturation medium without EGCG）（P 0.05, the same below）, moreover, the contents of GSH in oocytes at MII phase（3.21 and 3.25 pmol/oocyte） were higher than that of oocytes in CK group. In addition, adding 20-30 μmol/L EGCG to maturation medium could significantly reduce ROS level of oocyte at MII phase. Furthermore, it was found that adding 10 μmol/L EGCG to maturation medium showed the best effect on oocytes after parthenogenetic activation and in vitro fertilization, and the corresponding cleavage rate and blastocyst developmental rate were significantly higher than those of CK group. 【Conclusion】EGCG can be widely used to optimize oocyte in vitro maturation system, so as to effectively improve buffalo＇s oocytes maturation rate and embryo in vitro development. Moreover, the effect is optimum when adding 10 μmol/L EGCG to maturation medium.