Kaneda Masahiro, Akagi Satoshi, Watanabe Shinya, Nagai Takashi
Reproductive Biology and Technology Research Team, National Institute of Livestock and Grassland Science (NILGS), National Agriculture and Food Research Organization (NARO), 2 Ikenodai, Tsukuba, Ibaraki 305-0901, Japan.
BMC Proc. 2011 Jun 3;5 Suppl 4(Suppl 4):S3. doi: 10.1186/1753-6561-5-S4-S3.
DNA methylation of cytosine residues in CpG dinucleotide controls gene expression and dramatically changes during development. Its pattern is disrupted in cloned animals suggesting incomplete reprogramming during somatic cell nuclear transfer (the first reprogramming). However, the second reprogramming occurs in the germ cells and epigenetic errors in somatic cells of cloned animals should be erased. To analyze the DNA methylation changes on the spermatogenesis of bulls, we measured DNA methylation levels of three repetitive elements in blastocysts, blood and sperm.
DNA from PBLs (peripheral blood leukocytes), sperm and individual IVF (in vitro fertilized) and parthenogenetic blastocysts was isolated and bisulfite converted. Three repetitive elements; Satellite I, Satellite II and art2 sequences were amplified by PCR with specific pairs of primers. The PCR product was then cut by restriction enzymes and analyzed by agarose gel electrophoresis for determining the DNA methylation levels.
Both Satellite I and Satellite II sequences were highly methylated in PBLs, whereas hypo-methylated in sperm and blastocysts. The art2 sequence was half methylated both in PBLs and sperm but less methylated in blastocysts. There was no difference in DNA methylation levels between IVF and parthenogenetic blastocysts.
These results suggest that there is a dynamic change of DNA methylation during embryonic development and spermatogenesis in cattle. Satellite I and Satellite II regions are methylated during embryogenesis and then de-methylated during spermatogenesis. However, art2 sequences are not de-methylated during spermatogenesis, suggesting that this region is not reprogrammed during germ cell development. These results show dynamic changes of DNA methylation levels during bovine embryogenesis, especially genome-wide reprogramming in germ cells.
CpG二核苷酸中胞嘧啶残基的DNA甲基化控制基因表达，并在发育过程中发生显著变化。其模式在克隆动物中被破坏，这表明在体细胞核移植（第一次重编程）过程中重编程不完全。然而，第二次重编程发生在生殖细胞中，克隆动物体细胞中的表观遗传错误应该被消除。为了分析DNA甲基化对公牛精子发生的影响，我们测量了囊胚、血液和精子中三个重复元件的DNA甲基化水平。
从外周血白细胞（PBLs）、精子以及单个体外受精（IVF）和孤雌生殖囊胚中分离DNA，并进行亚硫酸氢盐转化。通过使用特定引物对进行PCR扩增三个重复元件；卫星I、卫星II和art2序列。然后用限制性内切酶切割PCR产物，并通过琼脂糖凝胶电泳进行分析，以确定DNA甲基化水平。
卫星I和卫星II序列在PBLs中高度甲基化，而在精子和囊胚中低甲基化。art2序列在PBLs和精子中均为半甲基化，但在囊胚中甲基化程度较低。IVF和孤雌生殖囊胚之间的DNA甲基化水平没有差异。
这些结果表明，牛胚胎发育和精子发生过程中存在DNA甲基化的动态变化。卫星I和卫星II区域在胚胎发生过程中甲基化，然后在精子发生过程中去甲基化。然而，art2序列在精子发生过程中没有去甲基化，这表明该区域在生殖细胞发育过程中没有重编程。这些结果显示了牛胚胎发生过程中DNA甲基化水平的动态变化，特别是生殖细胞中的全基因组重编程。
