Non-conservation of mammalian preimplantation methylation dynamics

In mammals, active demethylation of sperm pronuclear DNA shortly after fertilisation is thought to be important for reprogramming subsequent embryonic development [1–3]. A further passive loss of methylation has been observed as DNA replicates between 2-cell and morula stages, with somatic cell levels being re-established at or after the blastocyst stage when differentiated lineages are first formed [1,3,4]. We now demonstrate non-conservation in the DNA methylation dynamics of the sheep embryo and suggest this challenges the perceived role of DNA methylation in mammalian preimplantation development. A dramatic loss of cytosine methylation from the male pronucleus has previously been observed in mouse, pig and cow with a 5-methylcytosine antibody [1]. By contrast, we did not observe loss of methylation from either pronucleus in the in vivo-derived ovine zygote (Figure 1A). With the same immunostaining technique, we observed asymmetry in pronuclear methylation in mouse embryos as reported previously, ruling out procedural differences (Figure 1B). Examination of pronuclear stages revealed demethylation in the human zygote (Figure 1C) and no demethylation in the rabbit zygote (Figure 1D). In contrast with a previous study [1], we observed only partial asymmetric demethylation in the bovine zygote (Figure 1E). Collectively, this suggests that demethylation of the paternal genome is not an obligate requirement for early mammalian development. Also in contrast to the mouse and cow [1], we observed no passive demethylation throughout sheep preimplantation development upon purely visual inspection, but rather an apparent increase between the 8-cell and morula stages (Figure 2A–E). Only at the blastocyst stage is demethylation visible in the sheep trophectoderm, whereas the cells of the inner cell mass (ICM) remain methylated (Figure 2F). This might be important as trophectoderm cells are the first differentiated cell type to form during development and trophoblast-specific gene expression is essential for embryonic nutrition and implantation. Quantification of confocal images (Figure 2G), demonstrates a significant decrease in methylation intensity from the 2-cell to the 8-cell stage (43%, p < 0.05). However, nuclear size also decreases between these stages (41%, p < 0.01), thus the ratio of mean methylation intensity to nuclear size was not different. Moreover, quantification revealed that the apparent increase observed between the 8-cell and morula stages is also a visual artefact. A simple explanation is that methylation levels do not increase but nuclear intensities appear higher due to the increased nuclear compaction (55% size reduction from 8-cell to morula (p < 0.001, Figure 2G); we …