Effect of DNMT inhibitor on bovine parthenogenetic embryo development

https://doi.org/10.1016/j.bbrc.2015.09.060Get rights and content

Highlights

  • RG108 inhibited the development of bovine parthenogenetic preimplantation embryos.

  • RG108 decreased the DNA methylation levels of Nanog, α-satellite and satellite I.

  • RG108 increased the expression level of Nanog in bovine parthenogenetic blastocyst.

  • RG108 induced apoptosis in bovine parthenogenetic preimplantation embryos.

Abstract

DNA methylation catalyzed by DNA methyltransferase (DNMT) family plays an important role during mammal preimplanted embryo development. However, the effects of RG108, a DNMT inhibitor (DNMTi), on DNMT in the development of bovine preimplanted embryos are not fully elucidated. In this study, we investigated the role of RG108 on the development, dynamics of gene-specific DNA methylation and transcription of bovine parthenogenetic preimplantation embryos. We found that Dnmt1 and Dnmt3b showed highly transcription in parthenogenetic 2-cell embryos, and then the transcription levels decreased during the following development stages, whereas Dnmt3a was always maintained at a lower transcription level during bovine parthenogenetic preimplantation embryo development. Treatment with RG108 blocked the development of bovine parthenogenetic preimplantation embryos and induced hypomethylation in the embryos. RG108 decreased the methylation level of the Nanog gene promoter region, which caused activation of the Nanog gene in 8-cell embryos and increased the transcription level. RG108 also induced the hypomethylation of the repeat elements (satellite I and α-satellite), which may cause genome instability, increasing the number of apoptotic cells in the blastocysts and also the transcription level of the apoptotic gene Bax. These results indicate that RG108, a DNMT inhibitor (DNMTi), inhibits the development of bovine parthenogenetic preimplantation embryos, suggesting that the DNMT is necessary for bovine parthenogenetic preimplanatation 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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