Bovine imprinted gene analysis in assisted reproduction techniques

通过体外成熟和受精生产牛胚胎正在成为奶牛基因组革命的重要工具。根据父亲和母亲的基因型选择胚胎的能力，以及在怀孕开始前在囊胚水平上进行这种选择的可能性，正在改变动物生产和选择的方式。这些胚胎的分子分析表明，根据父母的年龄和根据培养或激素环境，这些胚胎存在一些差异。这种影响可能是暂时的，但在某些情况下，它可能会影响产后表型，这表明需要更多的研究来了解所观察到的变化。这个项目是基于这样的假设：一些甲基化模式是在卵泡形成的最后阶段获得的，可能会受到产生这些卵母细胞的环境的影响。这些差异可能不会在培养的第一周内消除，也不会在此期间对条件敏感。为了验证这一假设，将从青春期前供体(8)获得的32个胚胎（第12天）与从成人(8)获得的胚胎进行比较，并在体外(8)或不含血清(8)或体内（对照）(8)生产。将从这些胚胎中提取DNA并对其进行亚硫酸盐化处理，以显示甲基化标记。然后，对10个印迹基因的分析将在不同的治疗中进行比较，以评估模式是否被改变，是所有胚胎都受到影响，还是只有一小部分受到影响。第二部分将评估48个囊胚和活组织中4种最佳印迹指标的甲基化水平，以开发一种快速的第7天测试，允许监测任何对培养条件或卵巢刺激的修改。这项工作将产生标记物来评估所需的激素和培养环境，以尽量减少ART产生的牛胚胎的表观遗传扰动。

The production of bovine embryos through in vitro maturation and fertilization is becoming an important tool of the genomic revolution in dairy cows. The capacity to select embryos based on the father and the mother genotypes and the possibility to do that at the blastocyst level before a pregnancy is initiated is changing the way animals are produced and selected. The molecular analysis of these embryos indicate some differences according to the age of the parent and according to the culture or hormonal environment. Such influences may be temporary but in some case, it may influence the post-natal phenotype suggesting that more research is required to understand the changes observed. This project is based on the hypothesis that some of the methylation patterns are acquired in the last portion of folliculogenesis and could be influenced by the environment created to generate these oocytes. Such differences may not be erased during the first week of culture or sensitive to the conditions during this time. To test this hypothesis, 32 embryos (day 12) obtained from pre-puber donors (8)will be compared to embryos obtained from adults and produced in vitro with (8) or without serum (8) or in vivo (control)(8). The DNA from these embryos will be extracted and bisulfited to reveal methylation marks. The analysis of 10 imprinted genes will then be compared across treatments to assess if the pattern is modified and if all embryos are affected or only a subset. The second part will assess the methylation level of the 4 best imprinted indicator in 48 blastocysts and biopsies to develop a rapid day 7 test permitting the monitoring of any modification to the culture conditions or ovarian stimulation. This work will generate markers to assess the required hormonal and culture environment to minimize epigenetic perturbation in bovine embryos generated by ART.