Beaujean Nathalie, Taylor Jane, Gardner John, Wilmut Ian, Meehan Richard, Young Lorraine
Division of Gene Expression and Development, Roslin Institute, Roslin EH25 9PS, United Kingdom.
Biol Reprod. 2004 Jul;71(1):185-93. doi: 10.1095/biolreprod.103.026559. Epub 2004 Mar 3.
Active demethylation of cytosine residues in the sperm genome before forming a functional zygotic nucleus is thought to be an important function of the oocyte cytoplasm for subsequent embryonic development in the mouse. Conversely, this event does not occur in the sheep or rabbit zygote and occurs only partially in the cow. The aim of this study was to investigate the effect of limited methylation reprogramming in the normal sheep embryo on reprogramming somatic nuclei. Sheep fibroblast somatic nuclei were partially demethylated after electrofusion with recipient sheep oocytes and undergo a stepwise passive loss of DNA methylation during early development, as determined by 5-methylcytosine immunostaining on interphase embryonic nuclei. A similar decrease takes place with in vivo-derived sheep embryos up to the eight-cell stage, although nuclear transfer embryos exhibit a consistently higher level of methylation at each stage. Between the eight-cell and blastocyst stages, DNA methylation levels in nuclear transfer embryos are comparable with those derived in vivo, but the distribution of methylated DNA is abnormal in a high proportion. By correlating DNA methylation with developmental potential at individual stages, our results suggest that somatic nuclei that do not undergo rapid reorganization of their DNA before the first mitosis fail to develop within two to three cell cycles and that the observed methylation defects in early cleavage stages more likely occur as a direct consequence of failed nuclear reorganization than in failed demethylation capacity. However, because only embryos with reorganized chromatin appear to survive the 16-cell and morula stages, failure to demethylate the trophectoderm cells of the blastocyst is likely to directly impact on developmental potential by altering programmed patterns of gene expression in extra-embryonic tissues. Thus, both remodeling of DNA and epigenetic reprogramming appear critical for development of both fertilized and nuclear transfer embryos.
在小鼠中，精子基因组中胞嘧啶残基在形成功能性合子核之前的主动去甲基化被认为是卵母细胞细胞质对后续胚胎发育的一项重要功能。相反，这种现象在绵羊或兔的合子中不会发生，在牛中仅部分发生。本研究的目的是调查正常绵羊胚胎中有限的甲基化重编程对重编程体细胞核的影响。绵羊成纤维体细胞经电融合到受体绵羊卵母细胞后，其细胞核会部分去甲基化，并在早期发育过程中经历DNA甲基化的逐步被动丢失，这是通过对间期胚胎细胞核进行5-甲基胞嘧啶免疫染色确定的。体内来源的绵羊胚胎在八细胞阶段之前也会发生类似的下降，尽管核移植胚胎在每个阶段的甲基化水平始终较高。在八细胞和囊胚阶段之间，核移植胚胎中的DNA甲基化水平与体内来源的胚胎相当，但甲基化DNA的分布在很大比例上是异常的。通过将DNA甲基化与各个阶段的发育潜力相关联，我们的结果表明，在第一次有丝分裂之前未经历DNA快速重组的体细胞核在两到三个细胞周期内无法发育，并且在早期卵裂阶段观察到的甲基化缺陷更可能是核重组失败的直接后果，而不是去甲基化能力失败。然而，由于只有染色质重组的胚胎似乎才能在16细胞和桑椹胚阶段存活，囊胚滋养外胚层细胞未能去甲基化可能会通过改变胚外组织中基因表达的程序化模式直接影响发育潜力。因此，DNA重塑和表观遗传重编程对于受精胚胎和核移植胚胎的发育似乎都至关重要。
