DNA methylation is essential for embryo development. DNA methyltransferases (DNMTs) are a group of enzymes which catalyse methylation of CpG dinucleotides (5meC). Global DNA methylation levels are similar in the male and female pronucleus and then show little change up to the 8-cell stage of development. After compaction the inner cells show reduced 5meC levels relative to the outer cells and by the blastocyst stage the ICM is markedly hypomethylated compared to the trophectoderm 1. The expression patterns of DNMTs define 5meC levels within cells, yet there is considerable controversy in the literature about the levels and localization of DNMTs within the nucleus at various stages of development. We have found that conventional published fixation and staining methods for DNMTs give artefactual representations of expression. Stable staining of DNMTs in different stage embryos required an acid treatment step (4N HCl for 10 mins) to denatured chromatin prior to immunofluorescence staining. The results give a new unexpected stable pattern of expression summarised as: 1) nuclear expression of DNMT1 from 1-cell, 2-cell, 4-cell, 8-cell, morula and the trophectoderm, but not ICM cells; 2) nuclear expression of DNMT3A in 1-cell through to 8-cell, after which it is mostly lost; 3) nuclear expression of DNMT3B from 8-cell stage, and restricted to the outer cells and trophectoderm, but not present in ICM. The treatment with DNMT 3B morpholino oligonucleotides prevented its upregulation from the 8-cell stage and reduced the differential methylation of TE and ICM in blastocysts. These results show that DNMTs are masked in acid-sensitive manner and show. Differential methylation of the trophectoderm and ICM is caused by differential expression and nuclear localization of DNMT1 and DNMT3B in these lineages.
1 Li, Y., Seah, M. K. Y. & O'Neill, C. Reproduction 151, 83-95, (2016).