The expression patterns of DNA methylation reprogramming related genes are associated with the developmental competence of cloned embryos after zygotic genome activation in pigs

Abstract

DNA methylation reprogramming, regulated by DNA methylation and demethylation related genes, is essential for early embryo development; however, it is incomplete in cloned embryos, leading to poor cloning efficiency. Previous studies have shown that DNA methylation inhibitor, 5-aza-2'-deoxycytidine (5-aza-dC), could enhance the development of cloned embryos, thus, the genes regulating DNA methylation reprogramming should appropriately express in these embryos. To examine whether there is a correlation between embryo development and the expression patterns of DNA methylation reprogramming related genes, we investigated the developmental progress and transcription levels of candidate genes containing DNA methyltransferases (Dnmt1 and Dnmt3a), ten eleven translocation (Tet) dioxygenases (Tet1, Tet2 and Tet3) and base excision repair related genes including activation induced deamination (Aid), thymine DNA glycosylase (Tdg) and AP endonuclease 1 (Apex1) in porcine early embryos. In this study, our results demonstrated that compared with in vitro fertilized embryos, delayed and reduced development and downregulated transcripts of DNA methylation reprogramming related genes after the 4-cell stage were observed in cloned embryos, showing the significantly (P < 0.05) lower proportions of embryos at the 8-cell, morula and blastocyst stages (19.69% vs 32.64% at 72 h, 16.67% vs 25.49% at 120 h and 19.82% vs 26.29% at 156 h, respectively) and transcription levels of Dnmt3a, Tet1, Tet2, Tet3, Aid, Tdg and Apex1. When cloned embryos were treated with 5-aza-dC, the developmental progress and transcription levels of DNA methylation reprogramming related genes were improved, more similar to those detected in fertilized counterparts. Furthermore, we found that the transcripts of zygotic genome activation and blastocyst quality related genes were also effectively promoted in porcine cloned embryos after 5-aza-dC treatment. In conclusion, our results demonstrated that the disturbed transcripts of DNA methylation reprogramming related genes were observed in porcine cloned embryos, while the enhanced development of porcine cloned embryos induced by 5-aza-dC was accompanied with the improved expression of DNA methylation reprogramming related genes after the 4-cell stage, providing a positive correlation between the expression patterns of DNA methylation reprogramming related genes and the developmental competence of porcine cloned embryos after zygotic genome activation.

Keywords: Cloned embryos; DNA methylation reprogramming; Development; Pig; Transcription; Zygotic genome activation.