The Joint Annual Scientific Meetings of the Endocrine Society of Australia and the Society for Reproductive Biology 2018

Immunolocalisation of DNA Methylation and DNA methyltransferases In The Post-Implantation Mouse Embryo. (#300)

Romanthi J Madawala 1 , Yan Li 1 , Chris O'Neill 1
  1. Kolling Institute, The University of Sydney, Sydney, NSW, Australia

Dynamic reprogramming of DNA methylation (5meC) is an essential feature of early embryonic development. It is a highly regulated process, with three conserved DNA methyltransferase enzymes responsible for maintenance (DNMT1) and de novo (DNMT3A and DNMT3B) methylation. The complex structure of chromatin within nuclei variably masks 5meC and DNMTs from antibody detection. Immunolocalization techniques that use both acid and tryptic digestion are required to unmask 5meC in the pre-implantation embryo. This method showed that there was a marked loss of immune-detectable global 5meC from the inner cell mass of the preimplantation embryo (1). In this study we used our validated immunolocalization approaches to assess the pattern of global immune-detectable 5meC and the DNMTs in each of the lineages that arise in the early post-implantation embryo.

Changes in 5meC and DNMT enzymes within serial cryo-sections of the post-implantation embryo (E5.5 & E6.5) were mapped. At both these embryonic stages, acid and tryptic digestion was required to retrieve the 5meC epitope while acid digestion was required for full antigenic retrieval of the DNMTs. E5.5 and E6.5 embryos showed similar global levels and patterns of nuclear 5meC staining in all cells of the placental cone, trophectoderm, the hypoblast and epiblast. DNMT1 showed a similar staining profile to 5meC. DNMT3A and DNMT3B were also localised to nuclei of all lineages in the embryo, however, both were strongly upregulated in the epiblast of the post-implantation embryo.

This study shows that the low levels of immuno-detectable 5meC in the inner cell mass of the preimplantation blastocyst was reversed in the post-implantation epiblast and this is associated with a marked upregulation of DNMT3A and DNMT3B within this lineage.  All other lineages showed similar levels of global 5meC and DNMTs.


  1. 1) Li Y, Seah MK, O’Neill C. (2016). Reproduction 151(2):83-95.