Biochemical and Biophysical Research Communications
Effect of DNMT inhibitor on bovine parthenogenetic embryo development
Introduction
DNA methylation plays critical roles in the transcriptional silencing of genes and retrotransposons, gene imprinting and X chromosome inactivation [1]. The DNMT family includes three main members, DNMT1, DNMT3a and DNMT3b. DNMT1 is largely responsible for maintaining methylation patterns through DNA replication, whereas both DNMT3a and DNMT3b are de novo methyltransferases [2], and act to transfer methyl groups to previously unmethylated CpG dinucleotides within the genome [3]. In mice, the paternal genome is actively demethylated within 6–8 h post fertilization, before the onset of DNA replication, whereas the maternal genome is gradually demethylated until the blastocyst stage [4]. However, recent studies found that both maternal and paternal genomes undergo widespread active and passive demethylation in zygotes before the first mitotic division in mice [5].
The typical pattern of dynamic change in DNA methylation in early mammalian embryos has been demonstrated with anti-5-methylcytosine (5 mC) immunofluorescence staining [6], although the majority of 5 mC immunofluorescence signals are predicted to correspond to multiple-copy repetitive regions [7]. A regulatory and genomic locus-specific DNA methylation reprogramming pattern during mammalian preimplantation development has been detected using genome-wide DNA methylation studies [8], [9], [10], [11]. Accordingly, some differentially methylated regions (DMRs) at imprinted loci are resistant to this wave of active paternal and passive maternal DNA demethylation in the zygote and early preimplantation embryos [12]. Similarly, some repeat sequences, such as intracisternal A particle (IAPs) elements, are also exempted from complete DNA demethylation [13]. In addition, a number of promoter regions in non-imprinted genes also escape the global DNA methylation reprogramming in mouse preimplantation embryos [9].
RG108, a novel DNMT inhibitor, lacks cytotoxic or genotoxic effects compared to five other DNMT inhibitors, 5-aza-CR, 5-aza-CdR, zebularine, procaine and epigallocatechin-3-gallate, in human cell lines [14], [15]. In mice, cloned embryos treated with 500 μM RG108 from the 2-cell to morula/blastocyst stages, show higher POU5F1 expression and increase number of inner cell mass (ICM) cells.
Collectively, these studies suggest that although DNA demethylation plays an important role during mammalian early embryo development, locus-specific DNA methylation maintenance is also necessary for the normal development of mammalian early embryos. DNMT is responsible for the maintenance of locus-specific DNA methylation [16], however, the role of DNMT in the development of bovine preimplanted embryos is not fully elucidated. In this study, we evaluated a novel DNMT inhibitor (DNMTi), RG108, and investigated its effects on the development, dynamics of gene-specific DNA methylation and transcription of bovine parthenogenetic embryos.
Section snippets
Materials and methods
Unless described elsewhere, all chemicals and reagents were purchased from Sigma (St. Louis, MO, USA).
Expressions of the Dnmt gene family in bovine parthenogenetic preimplantation embryos
The quantitative real-time PCR (qRT-PCR) results revealed that transcripts of Dnmt1, Dnmt3a and Dnmt3b were differentially expressed in bovine parthenogenetic preimplantation embryos. Dnmt1 and Dnmt3b showed high expression in the parthenogenetic 2-cell stage embryo, then, the expression levels of Dnmt1 and Dnmt3b decreased during the following developmental stages (Fig. 1). The Dnmt3a showed lower expression compared to Dnmt1 and Dnmt3b during bovine parthenogenetic preimplantation embryo
Discussion
In mammals, DNA methylation plays an essential role in maintaining genomic imprinting, X-chromosome inactivation, transcriptional regulation and suppression of transposable elements during normal development [20]. The paternal and maternal genomes undergo vast reprogramming after fertilization, including alterations in DNA methylation and histone acetylation [21], [22]. The beneficial effect of histone deacetylase inhibitor (HADCi) and DNMTi were observed when cloned embryos were treated after
Author contributions
Ziyi Li designed the study; Sheng Zhang and Bo Tang performed experiments; Congli and Liuxin Shi collected and analysed data; Xueming Zhang and Liguang Sun wrote the manuscript. Ziyi Li edited the manuscript.
Conflict of interest
The authors have no conflicts of interest to declare.
Acknowledgment
This work was supported by grants from National Science and Technology Major Projects, Grant Number 2014ZX-08-007-002-008, Natural Science Foundation of China, Grant Number 31472093 and PCSIRT, Grant Number IRT1248.
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These authors contributed equally to this work.