Effects Of 5-AZA-2'-Deoxycytidine (5-AZA-CdR) On The Development Of Preimplantation Mouse Embryos

During the development of preimplantation mouse embryos, DNA methylation is always in the dynamic balance of methylation and demethylation. After fertilization, the male pronucleus firstly undergoes active and rapid genome-wide demethylation prior to DNA replication. And then, a stepwise drop in passive demethylation occurs up to the morula stage following the completion of the first cell cycle due to the absence of Dnmt1, a maintenance methylase. However, by the blastocyst stage, ICM undergoes de novo methylation, while the trophectoderm still maintains the low level of DNA methylation. 5-AZA-CdR is a widely-used agent of demethylation, which is very important in research of development of embryos, cell differentiation and cancer therapy. In our experiments, we used 5-AZA-CdR's demethylation to study the relationship between DNA methylation and mouse preimplantation embryo development. We will understand the relationship between DNA methylation and mouse embryo development more and deeply through our research work. What's more, it is helpful for the study of the mechanism of 5-AZA-CdR and the therapy of cancer.1. 5-AZA-CdR and the developmental competence of preimplantation mouse embryosWe added various concentrations of 5-AZA-CdR (0.2, 1.0, or 5.0μM) from pronuclear embryos, 2-cell embryos and 4-cell embryos, then these embryos were cultured continuously in 5-AZA-CdR until they stop developing in vitro. Fertilized oocytes exposed to CZB containing 5-AZA-CdR at the pronuclear stage were unable to form morulae (0.2 and 1.0μM) or 4-cell embryos (5.0μM), while 2-cell stage embryos exposed to 5-AZA-CdR developed into uncompacted 8-cell (0.2 and 1.0μM) or 3/4-cell (5.0μM) stage embryos. The rate of morula formation was significantly lower in 4-cell embryos cultured in 5-AZA-CdR (1.0, or 5.0μM) than in control embryos (p<0.05), and none of the morulae could develop to blastulae as control. These data indicate that 5-AZA-CdR inhibits the development of mouse preimplantation embryos, especially the development of 8-cell embryos, morulae and blastulae, in a dose-dependent manner.Apoptosis also, was detected by TUNEL and Annexin V assays in our research. We found that 8-cell embryos and early morulae obtained from 5.0μM 5-AZA-CdR underwent apoptosis, while the low concentrations of 5-AZA-CdR did not lead to the apoptosis. This indicated that the development arrest led by 5-AZA-CdR was relative to apoptosis in some degree.2. 5-AZA-CdR and DNA methylation of preimplantation mouse embryosWe detected DNA methylation using immunofluoresence assay and the relative amount of fluorescence intensity was quantified using Image software. After treated with 5-AZA-CdR, DNA methylation of 8-cell embryos and early morulae (compacted 8-cell) decreased significantly (p<0.05), but DNA methylation of 2-cell and 4-cell embryos was on the same level with the control (p>0.05).Because the change of DNA methylation caused by 5-AZA-CdR was mediated by Dnmt1, we detected the expression of Dnmt1o at each stage of embryos. Dnmt1o only expressed in cytoplasm in 2-cell and 4-cell embryos before and after the treatment of 5-AZA-CdR. In 8-cell embryos and morulae, Dnmt1o disappeared from cell nuclei after treated with 5-AZA-CdR. So the decrease of DNA methylation may be related to the absence of Dnmt1o in nuclei. We can give the conclusion that normal DNA methylation is very important for the development of 8-cell embryos, morulae and blastulae, and Dnmt1o is needed to maintain the normal DNA methylation.3. 5-AZA-CdR and the related developmental genes expressionIn this experiment, we studied the relationship of DNA methylation and preimplantation mouse embryos on molecular level. Firstly, we detected genome transcriptional activity by BrUTP immofluorescence assay. The transcriptional activity of 2-cell and 4-cell embryos was almost the same with the embryos in control (p>0.05), while the activity of 8-cell embryos and early morulae was decreased significantly than control (p<0.05).Secondly, we chose Cx31, Cx43, Cx45, Cdh1 and Ctnnb1, which are very close related to the compaction and cavitation, and detected the mRNA expression by real-time quantitative PCR. The results showed that the expression of the five genes did not change before and after 5-AZA-CdR was added. In the 5-7-cell embryos obtained from pronuclear embryos exposed to 5-AZA-CdR, the expression of the five genes almost decreased to zero. And the expression of the five genes decreased sharply in the uncompacted 8-cell embryos obtained from 2-cell embryos exposed to 5-AZA-CdR. When 4-cell embryos exposed to 5-AZA-CdR developed to 8-cell embryos, the expression of the five genes decreased in different degree, and the expression continued to decrease when developed to the early morulae, compared to their respective control. Therefore, these data revealed that the developmental failure of preimplantation mouse embryos induced by hypomethylation was close related with the aberrant expression of the five genes.