Effects of Downregulating DNA Methyltransferase 1 Transcript by RNA Interference on DNA Methylation Status of the Satellite I Region and In Vitro Development of Bovine Somatic Cell Nuclear Transfer Embryos

Abstract

Abstract. For the successful production of cloned animals by somatic cell nuclear transfer (NT), the epigenetic status of the differentiated donor cell is reversed to an embryonic totipotent status. However, in NT embryos, this process is aberrant, with genomic hypermethylation consistently observed. Here, we investigated the effects of silencing DNA methyltransferase 1 (DNMT1) mRNA by small interfering RNA (siRNA) on the DNA methylation status of the satellite I region and in vitro development of bovine NT embryos. First, the levels of DNMT1 expression were analyzed at 0, 24, 48, 72, 120 and 192 h after in vitro culture. Real-time PCR and western blotting analyses detected a significant decrease in DNMT1 mRNA in the siRNA-injected NT (siRNA-NT) group up to 72 h after in vitro culture. Next, the levels of DNA methylation of the satellite I region were analyzed at several time points after in vitro culture. The level of DNA methylation detected in siRNA-NT embryos was significantly less than those in NT embryos throughout in vitro development. Moreover, the developmental rate of embryos to blastocysts in the siRNA-NT group was significantly higher than that of NT embryos. Our data suggest that knockdown of DNMT1 mRNA in NT embryos can induce DNA demethylation, which may enhance reprogramming efficiency. Key words: DNA methylation, DNA methyltransferase, Embryo development, Nuclear transfer, RNA interference (J. Reprod. Dev. 57: [393][394][395][396][397][398][399][400][401][402] 2011) hile there have been many attempts to improve the developmental competence of somatic cell nuclear transfer (NT) embryos, the success rate of producing viable offspring is still very low, with less than 5% of embryos transferred to surrogate females [1]. In bovine cloning, high rates of embryonic, fetal, neonatal and postnatal abnormalities have been consistently observed [2][3][4]. To achieve developmental competence of NT embryos, the donor nucleus of the differentiated cell needs to undergo epigenetic reprogramming during preimplantation development. However, this process needed for a differentiated donor nucleus to achieve embryonic status is delayed and incomplete in NT embryos [5]. In fact, many studies have demonstrated that epigenetic statuses such as DNA methylation and histone acetylation, are abnormally established in NT embryos during preimplantation development [6][7][8][9][10][11][12]. As a result, abnormal gene expression including imprinted genes [10,[13][14][15] can prevent normal development of NT embryos [16].DNA methylation of cytosine residues in CpG dinucleotides is one of the epigenetic modifications mediated by three DNA methyltransferases (DNMTs), DNMT1, DNMT3a and DNMT3b [17]. DNA methylation patterns are dynamic, yet tightly regulated, during mammalian development [18]. In normal embryos, both maternal and paternal gametes undergo extensive demethylation after fertilization, with the paternal genome undergoing demethylation within hours of fertilization [19]. In contrast, the maternal genome is passively...

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

“…However, in cloned embryos, continuous expression of Dnmt1s leads to aberrant demethylation (Chung et al, 2003;Yamanaka et al, 2011), and our results also showed that Dnmt1 transcripts were higher in porcine cloned embryos before the 8-cell stage. Additionally, Dnmt1 and Dnmt3a were not effectively activated after ZGA, possibly explaining the cause of incomplete DNA methylation reprogramming and low development of cloned embryos (Huan et al, 2014;Yamanaka et al, 2011). Furthermore, the expression patterns of Dnmt1 and Dnmt3a and the development of cloned embryos in IVF and NA embryos also reveal that effective silence and activation of Dnmts are essential for normal development of early embryos.…”

“…Before ZGA, a passive demethylation by a DNA replication dependent mechanism is observed in IVF embryos, probably as Dnmt1 is present in the cytoplasm (Rodriguez-Osorio et al, 2010). However, in cloned embryos, continuous expression of Dnmt1s leads to aberrant demethylation (Chung et al, 2003;Yamanaka et al, 2011), and our results also showed that Dnmt1 transcripts were higher in porcine cloned embryos before the 8-cell stage. Additionally, Dnmt1 and Dnmt3a were not effectively activated after ZGA, possibly explaining the cause of incomplete DNA methylation reprogramming and low development of cloned embryos (Huan et al, 2014;Yamanaka et al, 2011).…”

Epigenetic Modification of Cloned Embryos ImprovesNanogReprogramming in Pigs

“…After SCNT, the methylation levels of the Nanog promoter and 5¢-UTR were higher than those in IVF embryos, and the methylation of the first exon was also reprogrammed inefficiently, suggesting that incomplete Nanog methylation reprogramming could be the cause of the poor development of cloned embryos. As for the reason for incomplete Nanog methylation reprogramming, it is possible that there is a mechanism that preserves the methylation pattern of donor cells against reprogramming by oocyte factors (Yamanaka et al, 2011).…”

Epigenetic Modification Agents Improve Gene-Specific Methylation Reprogramming in Porcine Cloned Embryos

“…However, in this study, gene-specific methylation reprogramming was incomplete in porcine cloned embryos, which probably leads to poor development of cloned embryos. As for the reason for incomplete methylation reprogramming of Oct4 and Thy1 in cloned embryos, it is possible that there is a potential mechanism which preserves the tissue specific methylation pattern of donor cells against reprogramming by oocyte factors [13, 22, 23]. …”