INRAE, UR1037 LPGP, Fish Physiology Ad Genomics, Campus de Beaulieu, 35000, Rennes, France.
Sci Rep. 2021 Feb 17;11(1):3945. doi: 10.1038/s41598-021-83033-2.
Reducing the variability in nuclear transfer outcome requires a better understanding of its cellular and epigenetic determinants, in order to ensure safer fish regeneration from cryobanked somatic material. In this work, clones from goldfish were obtained using cryopreserved fin cells as donor and non-enucleated oocytes as recipients. We showed that the high variability of clones survival was not correlated to spawn quality. Clones were then characterized for their first cleavages pattern in relation to their developmental fate up to hatching. The first cell cycle duration was increased in clones with abnormal first cleavage, and symmetric first two cleavages increased clone probability to reach later on 24 h- and hatching-stages. At 24 h-stage, 24% of the clones were diploids and from donor genetic origin only. However, ploidy and genetic origin did not determine clones morphological quality. DNA methylation reprogramming in the promoter region of pou2, nanog, and notail marker genes was highly variable, but clones with the nicest morphologies displayed the best DNA methylation reprogramming. To conclude, non-enucleated oocytes did allow authentic clones production. The first two cell cycles were a critical determinant of the clone ability to reach hatching-stage, and DNA methylation reprogramming significantly influenced clones morphological quality.
为了确保从低温保存的体细胞材料更安全地再生鱼类，需要更好地了解核转移的细胞和表观遗传决定因素，以降低核转移结果的变异性。在这项工作中，使用冷冻保存的鳍细胞作为供体和未去核的卵母细胞作为受体获得了金鱼的克隆。我们表明，克隆存活率的高度变异性与产卵质量无关。然后，根据其发育命运特征对克隆进行了第一次卵裂模式的分析，直到孵化。第一次细胞周期持续时间在第一次卵裂异常的克隆中增加，而第一次两个卵裂对称增加了克隆达到 24 小时和孵化阶段的概率。在 24 小时阶段，24%的克隆是二倍体，仅来自供体遗传起源。然而，ploidy 和遗传起源并不能决定克隆的形态质量。在 pou2、nanog 和 notail 标记基因启动子区域的 DNA 甲基化重编程高度可变，但形态最好的克隆显示出最好的 DNA 甲基化重编程。总之，未去核的卵母细胞确实允许生产真正的克隆。前两个细胞周期是克隆达到孵化阶段能力的关键决定因素，而 DNA 甲基化重编程显著影响了克隆的形态质量。
