Immunolocalization of Methylcytosine Indicates Dynamic Global Reprogramming of this Modification During the First Two Cell-Cycles of Mouse Embryo Development.

Abstract

The purpose of this study was to re-examine the pattern of cytosine methylation (5MeC) in mouse preimplantation stage embryos. It is currently considered that progressive demethylation of the genome occurs throughout the preimplantation stage and new methylation occurs at or soon after implantation. Given the essential requirement for DNA methylation for genomic stability this prolonged relative demethylation creates a biological paradox. We have therefore undertaken a reassessment of this phenomenon. The immunofluorescence staining method was used to test the 5MeC in different stages of preimplantation stage embryos. We found that global demethylation of both the paternal and maternal genome progressively occurs throughout the zygote stage and is complete by syngamy. This demethylation can be perturbed after IVF or prolonged culture. By the 2-cell stage, embryos collected direct from the oviduct showed some remethylation high levels of cytosine methylation, and interphase nuclei 4-cell and 8-cell embryos stained extensively for 5meC. The remethylation during the early 2-cell stage was inhibited by the selective DNMT inhibitor, RG108 (5 µM) - late stage zygotes where incubated for 16h with or without RG108 and the resulting 2-cell embryos stained for 5meC. The embryos that developed in the presence of the DNA methyltransferase inhibitor showed significantly less methylcytosine staining than the embryos in the untreated culture conditions (p<0.001). Treatment of embryos during this period with RG108 significantly reduced their capacity to develop to normal blastocysts, indicating that this early DNA re-methylation reaction was important for the normal development of the embryo. These results indicate that the current paradigms of epigenetic reprogramming in the early embryo require reassessment. (poster)