The Demethylating Agent 5-Aza-2′-Deoxycytidine (5-AZA-CdR) Inhibits The Development of Preimplantation Mouse Embryos

DNA methylation is crucial for mammalian development, and DNA methylation is always in the dynamic status during preimplantation mouse embryos development. The effects of 5-A-ZA-CdR on the development of preimplantation mouse embryos were evaluated. Preimplantation mouse embryos created by in vitro fertilization were cultured continuously in 5-AZA-CdR (0.2, 1.0, or 5.0 mu mol/L). Fertilized oocytes exposed to CZB containing 5-A-ZA-CdR at the pronuclear stage were unable to form morulae (0.2 and 1.0 mu mol/L) or 4-cell embryos (5.0 mu mol/L), while 2-cell stage embryos exposed to 5-AZA-CdR developed into uncompacted 8-cell (0.2 and 1.0 mu mol/L) or 3/4-cell (5.0 mu mol/L) stage embryos. The rate of morula formation was significantly lower in 4-cell embryos cultured in 5-AZA-CdR (1.0 or 5.0 mu mol/L) than that in control embryos (P < 0.05). These data indicate that 5-A-ZA-CdR inhibits the development of mouse preimplantation embryos. Apoptosis, DNA methylation, and transcriptional activity were analyzed to determine the reason for these developmental defects. An annexin V-PI assay revealed that high doses of 5-AZA-CdR led to apoptosis. Compared to the controls, DNA methylation was significantly reduced in uncompacted 8-cell embryos and morulae (P < 0.05) in a dose-dependent manner, whereas no significant change was detected in 2- or 4-cell embryos (P > 0.05). The observed changes in transcriptional activity, determined by measuring the incorporation of BrUTP, were similar to the observed alterations in DNA methylation. Therefore, the developmental defects induced by 5-AZA-CdR appear to be mediated by alterations in DNA methylation and transcriptional activity in preimplantation mouse embryos.