Epigenetic Profile of Developmentally Important Genes in Bovine Oocytes
Epigenetic Profile of Developmentally Important Genes in Bovine Oocytes
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DOI：
10.1002/mrd.21281
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发表时间：
2011-03-01
影响因子：
2.5
通讯作者：
Niemann, H.
中科院分区：
文献类型：
--
作者：
Heinzmann, J.;Hansmann, T.;Niemann, H.
辅助生殖技术与表观遗传畸变的发生率增加有关，特别是在印记基因中。在这里，我们使用牛卵母细胞作为模型，以确定推定的表观遗传突变在三个印记基因位点所造成的类型的成熟，无论是在体外成熟（IVM）在组织培养基199（TCM）或改良的合成输卵管液（mSOF）培养基，或在体内成熟。我们应用有限稀释法和直接亚硫酸氢盐测序来分析H19/IGF 2、PEG 3和SNRPN的单个等位基因（DNA分子）的甲基化谱，这些等位基因各自与人类和/或小鼠模型中的印迹缺陷相关，并且已知在牛胚胎中差异甲基化。总的来说，我们从未成熟卵母细胞中获得了203个等位基因（含4，512个CpG位点）的甲基化模式，从TCM成熟卵母细胞中获得了213个等位基因（含4，779个CpG位点）的甲基化模式，在mSOFC成熟卵母细胞中获得了215个等位基因/4，725个CpG位点，从体内成熟卵母细胞中获得了78个等位基因/1，672个CpG位点。个体CpG和整个等位基因甲基化错误的总比率在两个IVM组和体内组之间没有显著差异，表明目前的IVM方案对这些关键的表观遗传标记没有影响或仅有轻微影响。此外，三个印迹基因和一个面板的八个其他基因指示卵母细胞的能力的mRNA表达谱进行了测定，通过定量实时PCR。我们发现体内成熟卵母细胞与体外成熟卵母细胞之间存在不同的mRNA表达谱，这表明对DNA甲基化以外的调控机制的影响。
Assisted reproductive technologies are associated with an increased incidence of epigenetic aberrations, specifically in imprinted genes. Here, we used the bovine oocyte as a model to determine putative epigenetic mutations at three imprinted gene loci caused by the type of maturation, either in vitro maturation (IVM) in Tissue Culture Medium 199 (TCM) or modified synthetic oviduct fluid (mSOF) medium, or in vivo maturation. We applied a limiting dilution approach and direct bisulfite sequencing to analyze the methylation profiles of individual alleles (DNA molecules) for H19/IGF2, PEG3, and SNRPN, which are each associated with imprinting defects in humans and/or the mouse model, and are known to be differentially methylated in bovine embryos. Altogether, we obtained the methylation patterns of 203 alleles containing 4,512 CpG sites from immature oocytes, 213 alleles with 4,779 CpG sites from TCM-matured oocytes, 215 alleles/4,725 CpGs in mSOF-matured oocytes, and 78 alleles/1,672 CpGs from in vivo-matured oocytes. The total rate of individual CpGs and entire allele methylation errors did not differ significantly between the two IVM and the in vivo group, indicating that current IVM protocols have no or only marginal effects on these critical epigenetic marks. Furthermore, the mRNA expression profiles of the three imprinted genes and a panel of eight other genes indicative of oocyte competence were determined by quantitative real-time PCR. We found different mRNA expression profiles between in vivo-matured oocytes versus their in vitro-matured counterparts, suggesting an influence on regulatory mechanisms other than DNA methylation.