期刊
SCIENTIFIC REPORTS
卷 6, 期 -, 页码 -出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/srep19134
关键词
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资金
- Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan [25550036, 20510063, 21028021, 23651248, 15K14440]
- Ritsumeikan Global Innovation Research Organization (R-GIRO)
- Center of Innovation Program from Japan Science and Technology Agency, JST
- Medical Research Council, UK [G1000839]
- EU Reintegration Grant [PIRG06-GA-2009-256408]
- MRC [MR/N000080/1, G1000839] Funding Source: UKRI
- Grants-in-Aid for Scientific Research [25550036, 23221006, 20510063, 23651248, 21028021, 15K14440] Funding Source: KAKEN
- Medical Research Council [MR/N000080/1, G1000839] Funding Source: researchfish
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 similar to 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 (similar to 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.
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