Zebularine significantly improves the preimplantation development of ovine somatic cell nuclear transfer embryos

Abstract

Aberrant DNA methylation reduces the developmental competence of mammalian somatic cell nuclear transfer (SCNT) embryos. Thus, hypomethylation-associated drugs are beneficial for improving reprogramming efficiency. Therefore, in the present study we investigated the effect of zebularine, a relatively novel DNA methyltransferase inhibitor, on the developmental potential of ovine SCNT embryos. First, reduced overall DNA methylation patterns and gene-specific DNA methylation levels at the promoter regions of pluripotency genes (octamer-binding transcription factor 4 (Oct4), SRY (sex determining region Y)-box 2 (Sox2) and Nanog) were found in zebularine-treated cumulus cells. In addition, the DNA methylation levels in SCNT embryos derived from zebularine-treated cumulus cells were significantly reduced at the 2-, 4-, 8-cell, and blastocyst stages compared with their corresponding controls (P < 0.05). The blastocyst rate was significantly improved in SCNT embryos reconstructed by the cumulus donor cells treated with 5 nM zebularine for 12 h compared with the control group (25.4±1.6 vs 11.8±1.7%, P < 0.05). Moreover, the abundance of Oct4 and Sox2 mRNA was significantly increased during the preimplantation stages after zebularine treatment (P < 0.05). In conclusion, the results indicate that, in an ovine model, zebularine decreases overall DNA methylation levels in donor cumulus cells and reconstructed embryos, downregulates the DNA methylation profile in the promoter region of pluripotency genes in donor cells and ultimately elevates the expression of pluripotency genes in the reconstructed embryos, which can lead to improved development of SCNT embryos.

Enhancement of epigenetic reprogramming status of porcine cloned embryos with zebularine, a DNA methyltransferase inhibitor

“…In most cancer cells, Zb shows a consistent pattern of growth inhibition and demethylation effects with little evidence of toxicity (Cheng et al, ; Cheng et al, ). In embryo production studies, Zb has been applied to modify the epigenetic status of donor fibroblast cells, and it could enhance the subsequent in vitro developmental competence and quality of SCNT embryos of pigs (Diao et al, ), yaks (Xiong et al, ), and sheep (Cao et al, ; Fang et al, ). In the present study, we first investigated the effects of Zb treatment in a porcine IVP system, and we found that supplementation with Zb during IVC could affect the subsequent embryo development of SCNT embryos in different ways according to its concentration and treatment time.…”

Optimizing treatment of DNA methyltransferase inhibitor RG108 on porcine fibroblasts for somatic cell nuclear transfer

“…Generally, there are two approaches to facilitate reprogramming process of DNA methylation, either on donor cells to decrease the methylation status before SCNT (Cao et al, ; Sun et al, ) or during early cultivation of cloned embryos to mimic the demethylation process as in vivo embryos (Zhai et al, ). Several DNA methyltransferase (DNMT) inhibitors, such as 5‐azacytidine, zebularine and RG108, have been applied to assist somatic nucleus to mimic DNA methylation to an optimal status for further reprogramming process (Cao et al, ; Huangfu et al, ; Zhai et al, ).…”

Reproduction of Sheep through Nuclear Transfer of Somatic Cells: A Bibliometric Approach

“…Cluster two in bright green consists of 13 words referring to the development efficiency of cloned embryos at the blastocyst stage using different types of cells as donor of genetic information, most commonly cumulus cells and fibroblasts. The cells were treated with egg extracts [51], zebularine [47], chaetocin [52] and histone demethylase enzymes [53] to promote the nuclear reprogramming of somatic cells and improve the epigenetic status of ovine cloned embryos.…”