Institute of Farm Animal Genetics (Friedrich-Loeffler-Institut, FLI), Mariensee, Neustadt, Germany.
Mol Reprod Dev. 2011 Mar;78(3):188-201. doi: 10.1002/mrd.21281. Epub 2011 Feb 2.
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.
辅助生殖技术与表观遗传异常的发生率增加有关，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，以确定三种印记基因座中由成熟类型引起的潜在表观遗传突变，分别是在组织培养 199 培养基（TCM）或改良合成输卵管液（mSOF）培养基中的体外成熟（IVM）或体内成熟。我们应用有限稀释方法和直接亚硫酸氢盐测序来分析 H19/IGF2、PEG3 和 SNRPN 的个体等位基因（DNA 分子）的甲基化谱，这些基因都与人类和/或小鼠模型中的印迹缺陷有关，并且已知在牛胚胎中存在差异甲基化。总共从不成熟卵母细胞中获得了包含 4,512 个 CpG 位点的 203 个等位基因的甲基化模式，从 TCM 成熟卵母细胞中获得了包含 4,779 个 CpG 位点的 213 个等位基因，从 mSOF 成熟卵母细胞中获得了包含 4,725 个 CpG 位点的 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包含 1,672 个 CpG 位点的 78 个等位基因/1,672 个 CpG 位点。两个 IVM 组和体内组之间单个 CpG 和整个等位基因甲基化错误的总发生率没有显著差异，这表明当前的 IVM 方案对这些关键的表观遗传标记没有或只有轻微的影响。此外，通过定量实时 PCR 测定了三个印记基因和一组八个其他指示卵母细胞能力的基因的 mRNA 表达谱。我们发现体内成熟卵母细胞与体外成熟卵母细胞之间的 mRNA 表达谱不同，这表明对除 DNA 甲基化以外的调控机制有影响。
