DNA甲基化抑制因子5-AzaDc对犬卵母细胞体外培养核成熟的影响

Effect of DNA Methyltransferase Inhibitor 5-AzaDc on Nuclear Maturation of Canine Oocytes in vitro

本实验旨在研究DNA甲基化酶抑制剂5-氮-2-脱氧胞苷(5-Aza Dc)对犬卵母细胞体外成熟的影响。研究比较添加不同浓度5-Aza Dc(1、2μmol/L)处理不同时间(1、2、48、72 h)观察的犬卵母细胞成熟情况,并通过DNA甲基化的免疫荧光染色,观察5-Aza Dc对DNA甲基化在犬卵母细胞中的分布情况及趋势变化。结果表明:添加2μmol/L的5-Aza Dc处理48 h,所有卵母细胞均脱离GV期,并且与对照组相比显著提高进入GVBD-MⅡ期的比率(28.3%vs.87.1%);免疫荧光染色结果显示,成熟卵母细胞的DNA甲基化染色位点主要集中在细胞核内,并未在细胞质中出现,DNA甲基化程度显著降低,并使染色质凝集。综上所述,添加2μmol/L的5-Aza Dc处理48 h可以通过抑制DNA甲基化水平提高犬卵母细胞体外成熟率,短时间的抑制DNA甲基化水平对犬卵母细胞的减数分裂恢复有积极促进作用。

Researches showed that DNA methylation was very important for the maturation of oocytes, however, the effect of DNA methylation on canine oocytes maturation in vitro has not been examined. The objective of this study was to evaluate the effect of DNA methyltransferase inhibitor 5-AzaDc on nuclear maturation of canine oocytes in vitro. We compared the in vitro nuclear maturation of canine oocytes treated with various concentrations （1, 2 μmol/ L） of 5-AzaDc for different durations （1, 2, 48 or 72 h）. Immunodetection of 5-AzaDc treatment was conducted on the distribution and trend of DNA methylation in canine oocytes, the results indicated that the treatment of 2 p, mol/L 5-AzaDc for 48 h reduced the proportion of canine oocytes that blocked in GV stage, and significantly increased the percentage of canine oocytes that reached GVBD-MII in vitro. Immunofluorescence staining of anti-5-methylcytosine showed that the localization of DNA methylation during canine oocyte centralized in nuclear area, but not in cytoplasm, and the treatment with 5-AzaDc significant decreased the DNA methylation level. In conclusion, 2 ~tmol/L 5-AzaDc supplementation during IVM for 48 h improved the maturation of canine oocytes by inhibiting the DNA methylation levels of oocytes, and the inhibition of DNA methylation levels for a short time active effect on meiotic resumption of canine oocytes.