Oral Presentation The Annual Scientific Meeting of the Endocrine Society of Australia and the Society for Reproductive Biology 2014

Formation of the trophectodermal layer in the mouse blastocyst requires increased global CpG methylation of the genome. (#145)

Yan Li 1 , Chris O'Neill 1
  1. Centre for Developmental and Regenerative Medicine, Kolling Institute, Sydney Medical School, University of Sydney, St Leonards, NSW, Australia

Kolling Institute, Sydney Medical School, NSW, 2065

DNA methylation plays an important role during early embryo development. Global levels of 5meC persist throughout zygotic maturation and the first cell-cycles (to the 8-cell stage). By the morula stage, cells encased in an inner position show a ~ 50% reduction in global levels of immuno-detectable 5meC and a further loss occurs in the inner cell mass (ICM) of the blastocyst stage embryo. High levels of 5meC persist in the outer cells of the morula and the resulting trophectoderm (TE) of the blastocyst stage embryo. To test whether this differential in 5meC levels between the totipotent inner cell mass (ICM) and the TE is a requirement for the formation of the TE, the ICM was micro-surgically isolated and then cultured for 24h during which time a new TE formed from the ICM. This was accompanied by global hypermethylation of the new TE as assessed by immunofluorescence. Selectively blocking DNA methyltransferase activity (5-aza-2ยด-deoxycytidine or Nanaomycin) during this regeneration of the TE from the ICM reduced this remethylation and also the formation of a normal TE. When low inhibitor concentrations were used some regeneration of blastocysts still occurred but the cells with TE morphology did not express normal levels of 5meC nor the core TE markers, CDX2 and EOMES. The resulting blastocysts failed to show normal blastocyst outgrowth, indicating aberrant TE function in the absence of remethylation. The results show global hypermethylation of the TE relative to the levels observed in the ICM is required for normal cellular differentiation of the TE from the totipotent ICM in the early embryo.