Lafontaine Simon, Labrecque Rémi, Palomino J Manuel, Blondin Patrick, Sirard Marc-André
Centre de Recherche en Reproduction, Développement et Santé Intergénérationnelle (CRDSI), Département des Sciences Animals, Faculté des Sciences de l'agriculture et de l'alimentation, Université Laval, Québec, Canada.
SEMEX Boviteq, 3450 Rue Sicotte, Saint-Hyacinthe, QC J2S, Canada.
Theriogenology. 2020 Dec;158:321-330. doi: 10.1016/j.theriogenology.2020.09.027. Epub 2020 Sep 24.
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.
通过体外成熟和受精生产牛胚胎是奶牛基因组革命的一项重要工具。对这些胚胎的基因表达分析显示，与体内来源的胚胎相比，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阶段获得的，并且可能受到含有这些卵母细胞的卵泡中所创造环境的影响。这些改变的模式在培养的胚胎发育的第一周可能不会被消除，或者可能对那段时间的条件敏感。为了量化与培养条件相关的变化，将一个由从超排和人工授精母牛获得（所有组均为受精后第12天）的胚胎组成的体内对照组，与在有或没有胎牛血清（FBS）的情况下培养的体外生产（IVP）胚胎进行比较。为了测量卵母细胞供体年龄的影响，我们还比较了第四组，该组由用青春期前动物卵巢刺激后收集的卵母细胞生产的IVP胚胎组成。分别处理胚盘和滋养层细胞，并通过焦磷酸测序评估十个印记基因（H19、MEST、KCNQ1、SNRPN、PEG3、NNAT、GNASXL、IGF2R、PEG10和PLAGL1）的甲基化状态。接下来，按照与第12天胚胎（四组）相同的方法生产十个第7天的囊胚，以便在更早的发育阶段观察最感兴趣的基因（KCNQ1、SNRPN、IGF2R和PLAGL1）。对于所有样本，我们观察到与体内组相比，三个体外组的总体甲基化水平较低且变异性更大。个体胚胎分析表明，一些胚胎与其他胚胎不同，而一些胚胎则未受影响。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。这项工作提供了单印记控制区域（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，从而为更好地理解体外条件与印记基因之间的复杂相互作用奠定基础。
