Faculty of Life and Environmental Sciences, University of Yamanashi, Yamanashi, 400-8510, Japan.
Advanced Biotechnology Center, University of Yamanashi, Yamanashi, 400-8510, Japan.
Development. 2020 Aug 7;147(15):dev190777. doi: 10.1242/dev.190777.
The reason for the poor development of cloned embryos is not yet clear. Several reports have suggested that some nuclear remodeling/reprogramming factors (RRFs) are removed from oocytes at the time of enucleation, which might cause the low success rate of animal cloning. However, there is currently no method to manipulate the amount of RRFs in oocytes. Here, we describe techniques we have developed to gradually reduce RRFs in mouse oocytes by injecting somatic cell nuclei into oocytes. These injected nuclei were remodeled and reprogrammed using RRFs, and then RRFs were removed by subsequent deletion of somatic nuclei from oocytes. The size of the metaphase II spindle reduced immediately, but did recover when transferred into fresh oocytes. Though affected, the full-term developmental potential of these RRF-reduced oocytes with MII-spindle shrinkage was not lost after fertilization. When somatic cell nuclear transfer was performed, the successful generation of cloned mice was somewhat improved and abnormalities were reduced when oocytes with slightly reduced RRF levels were used. These results suggest that a change in RRFs in oocytes, as achieved by the method described in this paper or by enucleation, is important but not the main reason for the incomplete reprogramming of somatic cell nuclei.
克隆胚胎发育不良的原因尚不清楚。有几项报告表明，在去核时，一些核重编程因子（RRFs）从卵母细胞中被去除，这可能导致动物克隆的成功率低。然而，目前还没有方法可以操纵卵母细胞中 RRFs 的数量。在这里，我们描述了我们开发的技术，通过将体细胞核注射到卵母细胞中，逐渐减少卵母细胞中的 RRFs。这些注射的核被 RRFs 重塑和重编程，然后通过随后从卵母细胞中删除体细胞核来去除 RRFs。中期 II 纺锤体的大小立即减小，但在转移到新鲜卵母细胞中时恢复。尽管受到影响，但在受精后，具有 MII-纺锤体收缩的 RRF 减少的卵母细胞的完整发育潜能并未丧失。当进行体细胞核移植时，使用 RRF 水平略有降低的卵母细胞可以略微提高克隆小鼠的成功生成率，并减少异常。这些结果表明，卵母细胞中 RRF 的变化，如本文所述的方法或去核所实现的变化，是重要的，但不是体细胞核不完全重编程的主要原因。
