title: Comparison of DNA methylation levels of repetitive loci during bovine development.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: 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. taku@affrc.go.jp.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Kaneda
authors.forename: Satoshi
authors.initials: S
authors.lastname: Akagi
authors.forename: Shinya
authors.initials: S
authors.lastname: Watanabe
authors.forename: Takashi
authors.initials: T
authors.lastname: Nagai