title: DNA methylation dynamics in mouse preimplantation embryos revealed by mass spectrometry.
abstract: Following fertilization in mammals, paternal genomic 5-methyl-2'-deoxycytidine (5 mC) content is thought to decrease via oxidation to 5-hydroxymethyl-2'-deoxycytidine (5 hmC). This reciprocal model of demethylation and hydroxymethylation is inferred from indirect, non-quantitative methods. We here report direct quantification of genomic 5 mC and 5 hmC in mouse embryos by small scale liquid chromatographic tandem mass spectrometry (SMM). Profiles of absolute 5 mC levels in embryos produced by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) were almost identical. By 10 h after fertilization, 5 mC levels had declined by ~40%, consistent with active genomic DNA demethylation. Levels of 5 mC in androgenotes (containing only a paternal genome) and parthenogenotes (containing only a maternal genome) underwent active 5 mC loss in the first 6 h, showing that both parental genomes can undergo demethylation independently. We found no evidence for net loss of 5 mC 10-48 h after fertilization, implying that any passive 'demethylation' following DNA replication was balanced by active 5 mC maintenance methylation. However, levels of 5 mC declined during development after 48 h, to 1% (measured as a fraction of G-residues) in blastocysts (~96 h). 5 hmC levels were consistently low (<0.2% of G-residues) throughout development in normal diploid embryos. This work directly quantifies the dynamics of global genomic DNA modification in mouse preimplantation embryos, suggesting that SMM will be applicable to other biomedical situations with limiting sample sizes.
authors.affiliation: Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya 468-8503, Japan.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Okamoto
authors.affiliation: Department of Stem Cell Pathology, Kansai Medical University, 2-5-1 Shinmachi, Hirakata, Osaka 573-1010, Japan.
authors.forename: Naoko
authors.initials: N
authors.lastname: Yoshida
authors.affiliation: Laboratory of Mammalian Molecular Embryology, Department of Biology and Biochemistry, University of Bath, BA2 7AY, England.
authors.forename: Toru
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Tsukuba Primate Research Center (TPRC), National Institute of Biomedical Innovation (NIBIO), 1-1 Hachimandai, Tsukuba, Ibaraki 305-0843, Japan.
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Shimozawa
authors.affiliation: Laboratory of Mammalian Molecular Embryology, Department of Biology and Biochemistry, University of Bath, BA2 7AY, England.
authors.forename: Maki
authors.initials: M
authors.lastname: Asami
authors.affiliation: Research Center for Environmental Quality Management, Kyoto University, 1-2 Yumihama, Otsu, Shiga 520-0811, Japan.
authors.forename: Tomonari
authors.initials: T
authors.lastname: Matsuda
authors.affiliation: Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya 468-8503, Japan.
authors.forename: Nakao
authors.initials: N
authors.lastname: Kojima
authors.affiliation: Laboratory of Mammalian Molecular Embryology, Department of Biology and Biochemistry, University of Bath, BA2 7AY, England.
authors.forename: Anthony C F
authors.initials: AC
authors.lastname: Perry
authors.affiliation: Laboratory of Cell Engineering, Department of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577, Japan.
authors.forename: Tatsuyuki
authors.initials: T
authors.lastname: Takada
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000378:metabolism*; D019175:DNA Methylation; D003841:Deoxycytidine / Q000031:analogs & derivatives; D004625:Embryo, Nonmammalian; D005260:Female; D005307:Fertilization in Vitro; D013058:Mass Spectrometry; D008540:Meiosis / Q000235:genetics; D051379:Mice; D008938:Mitosis / Q000235:genetics; D009865:Oocytes / Q000378:metabolism; D020554:Sperm Injections, Intracytoplasmic
chemical_list: D003841:Deoxycytidine; C016569:5-methyldeoxycytidine