KNOCKDOWN OF Dnmt1 AND Dnmt3a ENZYMES DISRUPTS PREIMPLANTATION EMBRYO DEVELOPMENT BY AFFECTING GLOBAL DNA METHYLATION

Abstract

DNA methylation is one of the epigenetic mechanisms that play critical roles in preimplantation embryo development executed by DNA methyltransferase (Dnmt) enzymes. Dnmt1, responsible for the maintenance of global DNA methylation and Dnmt3a for the de novo methylation, are gradually erased from zygote to blastocyst stage by a demethylation process, and then reestablished by de novo methylation around blastocyst stage. This important differentiation mechanism indicates pivotal roles of global DNA methylation during the course of preimplantation embryo development. So, a fundamental question which also served as the aim of the current study was to address the spatio-temporal expression of Dnmt1 and Dnmt3a and thus the establishment of DNA methylation could affect the embryo development. We set up four groups as control, Dnmt1 siRNA, Dnmt3a siRNA, and non-targeted siRNA. In vivo developed Balb/c mouse zygotes were collected, divided into groups and then cultured for 96 h to reach blastocyst stage. Zygotes were transfected with 50 nM Dnmt1 and Dnmt3a-spesific small interfering RNA (siRNA) duplexes vs non-targeting control siRNA duplexes for 96 h using DharmaFECT as a transfection reagent. Nontargeting siRNA duplex served as the negative control. Knockdown of Dnmt genes using siRNA was confirmed by analyzing the proteins using immunofluorescence and Western blot techniques after 96 h in culture. Following knockdown of Dnmt1 and Dnmt3a, we analyzed the developmental competence of embryos. We also evaluated global DNA methylation levels by 5 methylcytosine staining in blastocyst stage. We found that both Dnmt genes were successfully knocked-down (approximately 80%) in 2-4 cell embryos. In Dnmt1 siRNA group, embryo arrest rates were 29.5% in 2-cell, 15.7 in %4-cell, and 9.8% in morula stages. Only 16.7% embryos reached to blastocyst stage in this group. In Dnmt3a siRNA group, embryos arrested in 26.2% 2-cell, 21.9% 4-cell, 9.2% morula and 29.5% embryos reached to blastocyst stage. Degenerated embryo rates were 28.2% in Dnmt1 siRNA, and 13.1% in Dnmt3a siRNA group. Interestingly, we noted that global DNA methylation level significantly decreased in Dnmt1 knockdown group while increased in Dnmt3a knockdown group (P<0.001). These results suggested that temporal Dnmt enzymes could contribute to the process of early embryonic development and thus play vital roles in the mouse embryos. Therefore it seems evident that these enzymes should also be elucidated in human to check whether they play any major role in the human preimplantation embryo development.