The production of bovine embryos through in vitro maturation and fertilization is an important tool of the genomic revolution in dairy cattle. Gene expression analysis of these embryos revealed differences according to the culture conditions or oocyte donor's pubertal status compared to in vivo derived embryos. We hypothesized that some of the methylation patterns in oocytes are acquired in the last step of folliculogenesis and could be influenced by the environment created in the follicles containing these oocytes. These altered patterns may not be erased during the first week of embryonic development in culture or may be sensitive to the conditions during that time. To quantify the changes related to culture conditions, an in vivo control group consisting of embryos (Day 12 post fertilization for all groups) obtained from superovulated and artificially inseminated cows was compared to in vitro produced (IVP) embryos cultured with or without Fetal Bovine Serum (FBS). To measure the effect of the oocytes donor's age, we also compared a fourth group consisting of IVP embryos produced with oocytes collected following ovarian stimulation of pre-pubertal animals. Embryonic disk and trophoblast cells were processed separately and the methylation status of ten imprinted genes (H19, MEST, KCNQ1, SNRPN, PEG3, NNAT, GNASXL, IGF2R, PEG10, and PLAGL1) was assessed by pyrosequencing. Next, ten Day 7 blastocysts were produced following the same methodology as for the D12 embryos (four groups) to observe the most interesting genes (KCNQ1, SNRPN, IGF2R and PLAGL1) at an earlier developmental stage. For all samples, we observed overall lower methylation levels and greater variability in the three in vitro groups compared to the in vivo group. The individual embryo analysis indicated that some embryos were deviant from the others and some were not affected. We concluded that IGF2R, SNRPN, and PEG10 were particularly sensitive to culture conditions and the presence of FBS, while KCNQ1 and PLAGL1 were more affected in embryos derived from pre-pubertal donors. This work provides markers at the single imprinted control region (ICR) resolution to assess the culture environment required to minimize epigenetic perturbations in bovine embryos generated by assisted reproduction techniques, thus laying the groundwork for a better comprehension of the complex interplay between in vitro conditions and imprinted genes.

中文翻译：

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在授精后第 7 天和第 12 天评估的牛胚胎中与卵母细胞供体年龄和培养条件相关的特定印记基因去甲基化

通过体外成熟和受精生产牛胚胎是奶牛基因组革命的重要工具。这些胚胎的基因表达分析显示，与体内衍生胚胎相比，根据培养条件或卵母细胞供体的青春期状态存在差异。我们假设卵母细胞中的一些甲基化模式是在卵泡发生的最后一步获得的，并且可能受到包含这些卵母细胞的卵泡中产生的环境的影响。在胚胎发育的第一周内，这些改变的模式可能不会被消除，或者可能对这段时间的条件敏感。为了量化与培养条件相关的变化，由从超排卵和人工授精的奶牛获得的胚胎（所有组受精后第 12 天）组成的体内对照组与体外产生的 (IVP) 胚胎进行比较，这些胚胎在有或没有胎牛血清 (FBS) 的情况下培养。为了测量卵母细胞供体年龄的影响，我们还比较了第四组，该组由在青春期前动物的卵巢刺激后收集的卵母细胞产生的 IVP 胚胎组成。分别处理胚胎盘和滋养层细胞，并通过焦磷酸测序评估 10 个印迹基因（H19、MEST、KCNQ1、SNRPN、PEG3、NNAT、GNASXL、IGF2R、PEG10 和 PLAGL1）的甲基化状态。接下来，按照与 D12 胚胎（四组）相同的方法产生 10 个第 7 天的囊胚，以观察最有趣的基因（KCNQ1、SNRPN、IGF2R 和 PLAGL1) 处于早期发育阶段。对于所有样品，与体内组相比，我们观察到三个体外组的总体甲基化水平更低，变异性更大。单个胚胎分析表明，一些胚胎与其他胚胎有偏差，而有些没有受到影响。我们得出结论，IGF2R、SNRPN 和 PEG10 对培养条件和 FBS 的存在特别敏感，而 KCNQ1 和 PLAGL1 在来自青春期前供体的胚胎中受影响更大。这项工作提供了单一印记控制区 (ICR) 分辨率的标记，以评估最小化辅助生殖技术产生的牛胚胎表观遗传扰动所需的培养环境，