Shi Wei, Dirim Fatma, Wolf Eckhard, Zakhartchenko Valeri, Haaf Thomas
Department of Molecular Animal Breeding and Biotechnology, University of Munich, 85764 Oberschleissheim, Germany.
Biol Reprod. 2004 Jul;71(1):340-7. doi: 10.1095/biolreprod.103.024554. Epub 2004 Mar 17.
Active demethylation of the paternal genome but not of the maternal genome occurs in fertilized mouse, rat, pig, and bovine zygotes. To study whether this early demethylation wave is important for embryonic development, we have analyzed the global methylation patterns of both in vivo-fertilized and cloned rabbit embryos. Anti-5-methylcytosine immunofluorescence of in vivo-fertilized rabbit embryos revealed that the equally high methylation levels of the paternal and maternal genomes are largely maintained from the zygote up to the 16-cell stage. The lack of detectable methylation changes in rabbit preimplantation embryos suggests that genome-wide demethylation is not an obligatory requirement for epigenetic reprogramming. The methylation patterns of embryos derived from fibroblast and cumulus cell nuclear transfer were similar to those of in vivo-fertilized rabbit embryos. Fluorescence in situ hybridization with chromosome-specific BACs demonstrated significantly increased chromosomal aneuploidy rates in cumulus cell nuclear transfer rabbit embryos and embryos derived from nuclear transfer of rabbit fibroblasts into bovine oocytes compared with in vivo-fertilized rabbit embryos. The incidence of chromosomal abnormalities was correlated with subsequent developmental failure. We propose that postzygotic mitotic errors are one important explanation of why mammalian cloning often fails.
在受精的小鼠、大鼠、猪和牛的受精卵中，父本基因组会发生主动去甲基化，而母本基因组则不会。为了研究这种早期去甲基化浪潮对胚胎发育是否重要，我们分析了体内受精和克隆兔胚胎的整体甲基化模式。对体内受精兔胚胎进行的抗5-甲基胞嘧啶免疫荧光分析显示，从受精卵到16细胞阶段，父本和母本基因组同样高的甲基化水平在很大程度上得以维持。兔植入前胚胎中未检测到甲基化变化，这表明全基因组去甲基化并非表观遗传重编程的必要条件。来自成纤维细胞和卵丘细胞核移植的胚胎的甲基化模式与体内受精兔胚胎相似。与体内受精兔胚胎相比，用染色体特异性细菌人工染色体（BAC）进行荧光原位杂交显示，卵丘细胞核移植兔胚胎以及将兔成纤维细胞核移植到牛卵母细胞中获得的胚胎的染色体非整倍体率显著增加。染色体异常的发生率与随后的发育失败相关。我们认为合子后有丝分裂错误是哺乳动物克隆常常失败的一个重要原因。
