Persistence of Cytosine Methylation of DNA following Fertilisation in the Mouse

Abstract

Normal development of the mammalian embryo requires epigenetic reprogramming of the genome. The level of cytosine methylation of CpG-rich (5meC) regions of the genome is a major epigenetic regulator and active global demethylation of 5meC throughout the genome is reported to occur within the first cell-cycle following fertilization. An enzyme or mechanism capable of catalysing such rapid global demethylation has not been identified. The mouse is a widely used model for studying developmental epigenetics. We have reassessed the evidence for this phenomenon of genome-wide demethylation following fertilisation in the mouse. We found when using conventional methods of immunolocalization that 5meC showed a progressive acid-resistant antigenic masking during zygotic maturation which gave the appearance of demethylation. Changing the unmasking strategy by also performing tryptic digestion revealed a persistence of a methylated state. Analysis of methyl binding domain 1 protein (MBD1) binding confirmed that the genome remained methylated following fertilisation. The maintenance of this methylated state over the first several cell-cycles required the actions of DNA methyltransferase activity. The study shows that any 5meC remodelling that occurs during early development is not explained by a global active loss of 5meC staining during the cleavage stage of development and global loss of methylation following fertilization is not a major component of epigenetic reprogramming in the mouse zygote.

Epigenetics and developmental programming of welfare and production traits in farm animals

“…Some groups (e.g. Santos et al 2010) have used immunofluorescent techniques to visually quantify global 5-methyl-cytidine staining to assess effects of ART procedures on DNA methylation in embryos, but this method lacks sensitivity, cannot identify locus-specific changes in methylation and possesses other methodological limitations (Li and O'Neill 2012). However, in a microarray analysis of 1536 CpG sites in just over 700 genes, Katari et al (2009) found that imprinted loci were no more or less likely to be differentially methylated than nonimprinted loci.…”

Repeats as global DNA methylation marker in bovine preimplantation embryos

“…First of all, changes in the staining protocol may lead to different results. Li and O'Neill (2012) used a step of trypsin digestion in methylation staining and a different result of paternal demethylation during mouse zygote maturation was found. Besides the staining method itself, the lack of standard quantification for staining may contribute to different results as well.…”

“…However, conflicting results were found among these studies, for example as to whether methylation is more intensive in inner cell mass (ICM) or trophectoderm (TE) cells in IVF bovine blastocysts, and the staining result can be affected by the protocol used (Li and O'Neill 2012). Therefore, another simple and direct method is needed for determining the global DNA methylation in preimplantation embryos.…”

Lessons from zebrafish on reprogramming the epigenetic code after fertilisation

“…6,7 This masking was caused by changes in nuclear proteins and could be reversed by brief tryptic digestion of fixed zygotes. When 5meC staining was performed under conditions favouring antibody binding under thermodynamic equilibrium and after antigen retrieval by a combination of acid-induced denaturation and tryptic digestion of chromatin, 5meC (and 5hmC) were found to persist throughout zygotic maturation and there was no evidence of a preferential loss of 5meC staining from the paternally inherited genome compared to that inherited from the oocyte.…”

“…When 5meC staining was performed under conditions favouring antibody binding under thermodynamic equilibrium and after antigen retrieval by a combination of acid-induced denaturation and tryptic digestion of chromatin, 5meC (and 5hmC) were found to persist throughout zygotic maturation and there was no evidence of a preferential loss of 5meC staining from the paternally inherited genome compared to that inherited from the oocyte. 6,7 In the mouse zygote, 5meC tends to accumulate at heterochromatic regions and the differing organisation of heterochromatin in the paternally-and maternallyderived genomes, 8 giving the appearance of differing patterns of methylation.…”