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.

Towards Functional Annotation of the Preimplantation Transcriptome: An RNAi Screen in Mammalian Embryos

“…In summary, GO term analysis offered no striking outliers in functional categories within our screen results and suggests that preimplantation development equally requires all aspects of cellular and molecular function for success. Supporting this notion, previous studies have found essential roles during preimplantation for genes within each of these functional categories (including DNA/RNA/protein binding4546, catalytic activity4748, various transporter activity495051). …”

Oviduct: roles in fertilization and early embryo development

“…For example, 5′ long terminal repeat-containing element is demethylated after fertilization (Smith et al 2014). As embryos develop to the blastocyst stage, repression is slowly reversed in the inner cell mass, and methylation status becomes increased (Smith et al 2012, Guo et al 2014, Okamoto et al 2016.…”

Active DNA demethylation by DNA repair: Facts and uncertainties

“…In the mammalian lifecycle, the most pronounced epigenetic remodeling occurs during two distinct developmental stages, where the acquired epigenetic information has to be erased and reset [16][17][18]. Widespread loss of DNA methylation has been observed in the zygotes immediately following fertilization [42] and again during maturation of the primordial germ cells (PGCs), the progenitors of sperm and oocytes (Fig. 1B) [43].…”