DNA Methylation at a Bovine Alpha Satellite I Repeat CpG Site during Development following Fertilization and Somatic Cell Nuclear Transfer

Incomplete epigenetic reprogramming is postulated to contribute to the low developmental success following somatic cell nuclear transfer (SCNT). Here, we describe the epigenetic reprogramming of DNA methylation at an alpha satellite I CpG site (αsatI-5) during development of cattle generated either by artificial insemination (AI) or in vitro fertilization (IVF) and SCNT. Quantitative methylation analysis identified that SCNT donor cells were highly methylated at αsatI-5 and resulting SCNT blastocysts showed significantly more methylation than IVF blastocysts. At implantation, no difference in methylation was observed between SCNT and AI in trophoblast tissue at αsatI-5, however, SCNT embryos were significantly hyper-methylated compared to AI controls at this time point. Following implantation, DNA methylation at αsatI-5 decreased in AI but not SCNT placental tissues. In contrast to placenta, the proportion of methylation at αsatI-5 remained high in adrenal, kidney and muscle tissues during development. Differences in the average proportion of methylation were smaller in somatic tissues than placental tissues but, on average, SCNT somatic tissues were hyper-methylated at αsatI-5. Although sperm from all bulls was less methylated than somatic tissues at αsatI-5, on average this site remained hyper-methylated in sperm from cloned bulls compared with control bulls. This developmental time course confirms that epigenetic reprogramming does occur, at least to some extent, following SCNT. However, the elevated methylation levels observed in SCNT blastocysts and cellular derivatives implies that there is either insufficient time or abundance of appropriate reprogramming factors in oocytes to ensure complete reprogramming. Incomplete reprogramming at this CpG site may be a contributing factor to low SCNT success rates, but more likely represents the tip of the iceberg in terms of incompletely reprogramming. Until protocols ensure the epigenetic signature of a differentiated somatic cell is reset to a state resembling totipotency, the efficiency of SCNT is likely to remain low.不完全表观重编程（epigenetic reprogramming）被认为是造成体细胞核移植（somatic cell nuclear transfer, SCNT）后发育成功率偏低的重要原因之一。本研究针对人工授精（artificial insemination, AI）、体外受精（in vitro fertilization, IVF）及体细胞核移植来源的牛胚胎，解析了其发育过程中α卫星I型CpG位点（alpha satellite I CpG site, αsatI-5）的DNA甲基化表观重编程动态。定量甲基化分析结果显示，体细胞核移植供体细胞在αsatI-5位点呈现高度甲基化状态，其所获得的体细胞核移植囊胚的甲基化水平显著高于体外受精囊胚。着床阶段，在αsatI-5位点，体细胞核移植与人工授精的滋养层组织甲基化水平并无显著差异，但该时点体细胞核移植胚胎整体的甲基化水平显著高于人工授精对照组。着床完成后，人工授精组胎盘组织中αsatI-5位点的甲基化水平逐渐下降，而体细胞核移植组未出现该变化趋势。与胎盘组织形成鲜明对比的是，发育过程中肾上腺、肾脏及肌肉组织内αsatI-5位点的甲基化占比始终维持在较高水平。体细胞组织中甲基化平均占比的差异幅度小于胎盘组织，但整体而言体细胞核移植体细胞组织在αsatI-5位点仍呈现高甲基化状态。尽管所有种公牛的精子在αsatI-5位点的甲基化水平均低于体细胞组织，但与对照组种公牛相比，克隆种公牛的精子在该位点仍普遍呈现高甲基化特征。本发育时序分析证实，体细胞核移植后确实会发生表观重编程，至少在一定程度上如此。但体细胞核移植囊胚及其细胞衍生物中检测到的甲基化水平升高现象表明，卵母细胞内可供使用的重编程因子数量不足或作用时长不够，无法确保实现完全的表观重编程。该CpG位点的不完全重编程或许是体细胞核移植成功率偏低的诱因之一，但从表观重编程不完全的整体情况来看，这可能仅仅是冰山一角。唯有通过技术手段将分化体细胞的表观遗传特征重设为类似全能性的状态，体细胞核移植的效率才有望得到显著提升，否则其效率将持续维持在较低水平。