Yan Li, Chris O’Neill

Covalent modification of cytosine nucleotides within the genome encode essential epigenetic information, with methylation (5meC) and hydroxymethylation (5hmC) having received most attention. It has been proposed that the formation of 5hmC is an intermediate in the active demethylation of 5meC. Some reports show that global loss of 5meC in the newly fertilised embryo is accompanied by increased 5hmC, but others have failed to confirm this finding. These analyses have relied on immuno-localization of these modifications. In this study we have established the conditions required for equilibrium binding of antibodies to 5meC and 5hmC in zygotes. Simultaneous detection of these antigens required denaturation of chromatin by acid treatment followed by antigen retrieval by tryptic digestion. Equilibrium binding then required incubation at 4°C for greater than 6 h. These are more demanding conditions than generally reported and resulted in the consistent detection of 5meC and 5hmC in both male and female pronuclei throughout zygotic maturation. No dynamic reciprocal change in the level of 5meC relative to 5hmC was observed. Both 5meC and 5hmC accumulated within the peri-nucleolar regions and this was more pronounced in the male pronucleus. Staining of 5meC was relatively more intense within the cortical and 5hmC in the central regions of pronuclei. The results are not consistent with a role for 5hmC in global demethylation in the zygote. The persistence of both modifications throughout zygotic maturation, and their differing patterns of localization and solvent exposure infer each modification provides its own epigenetic information to the early embryo.