玻璃化冷冻后小鼠卵母细胞及其早期胚胎中DNA甲基转移酶的表达模式

Dynamic Regulation of DNA Methyltransferases in Vitrified Mouse Oocytes and Their in vitro Fertilized Early Embryos

卵母细胞玻璃化冷冻导致的胚胎发育阻滞与其DNA甲基化模式的异常密切相关。本研究以小鼠为模型,旨在探讨玻璃化冷冻对卵母细胞及其形成的早期胚胎中DNA甲基转移酶(DNMTs)表达模式的影响。采用免疫荧光染色结合激光共聚焦显微成像技术检测了DNMT1、DNMT3A和DNMT3B在新鲜和冷冻卵母细胞及其形成的各阶段早期胚胎中的表达与分布,结果表明:在卵母细胞及受精后的合子中冷冻组3种DNMTs的表达均出现了异常。采用实时定量PCR技术检测了新鲜和冷冻卵母细胞及其形成的囊胚中,Dnmt1、Dnmt3a和Dnmt3b的m RNA表达水平,显示在冷冻卵母细胞中3种Dnmts的表达水平均显著下降,至囊胚阶段Dnmt3b的表达水平仍然很低(P<0.05)。研究结果表明卵母细胞玻璃化冷冻导致了小鼠早期胚胎发育过程中DNMTs表达模式的异常,这可能是DNA甲基化异常的重要原因之一。

The embryo developmental retardation caused by oocyte vitrification is closely related to abnormal DNA methylation pattern. The purpose of this study is investigate the expression pattern of DNA methyltransferases （DNMTs） during early embryo development and the effect of oocyte vitrification on it. The expression and distribution characteristics of DNMTI, DNMT3A and DNMT3B were detected by immunostaining and laser confocal scanning in oocytes and resultant early embryos, it showed the abnormal expression of DNMTs in vitrified oocytes and subsequent zygotes. The mRNA expression levels of Dnmtl, Dnmt3a and Dnmt3b were examined using real-time quantitative PCR in oocytes and resultant blastocysts, it revealed that the relative mRNA expression levels of the three Dnmts were declined remarkably in vitrified oocytes and the relative mRNA expression level of Dnmt3b was still significantly lower than the fresh group in blastocysts （P〈0.05）. The result indicated that ooeyte vitrification caused abnormal expression pattern of DNMTs during early embryo development, which could be one of the core reasons for lhe abnormal DNA methylation.