Abstract
Developmental defects in somatic cell nuclear transfer (SCNT) embryos are principally attributable to incomplete epigenetic reprogramming. Small-molecule inhibitors such as histone methyltransferase inhibitors (HMTi) and histone deacetylase inhibitors (HDACi) have been used to improve reprogramming efficiency of SCNT embryos. However, their possible synergistic effect on epigenetic reprogramming has not been studied. In this study, we explored whether combined treatment with an HMTi (chaetocin) and an HDACi (trichostatin A; TSA) synergistically enhanced epigenetic reprogramming and the developmental competence of porcine SCNT embryos. Chaetocin, TSA, and the combination significantly increased the cleavage and blastocyst formation rate, hatching/hatched blastocyst rate, and cell numbers and survival rate compared to control embryos. In particular, the combined treatment improved the rate of development to blastocysts more so than chaetocin or TSA alone. TSA and combined chaetocin/TSA significantly reduced the H3K9me3 levels and increased the H3K9ac levels in SCNT embryos, although chaetocin alone significantly reduced only the H3K9me3 levels. Moreover, these inhibitors also decreased global DNA methylation in SCNT embryos. In addition, the expression of zygotic genome activation- and imprinting-related genes was increased by chaetocin or TSA, and more so by the combination, to levels similar to those of in vitro-fertilized embryos. These results suggest that combined chaetocin/TSA have synergistic effects on improving the developmental competences by regulating epigenetic reprogramming and correcting developmental potential-related gene expression in porcine SCNT embryos. Therefore, these strategies may contribute to the generation of transgenic pigs for biomedical research.
Highlights
Somatic cell nuclear transfer (SCNT) is a powerful method that allows reprogramming of terminally differentiated cells to the totipotent state (Gurdon and Wilmut, 2011)
Previous studies have demonstrated that both treatment with 0.5 nM chaetocin and 50 nM trichostatin A (TSA) for 24 h after activation improves the epigenetic reprogramming and developmental competence of porcine somatic cell nuclear transfer (SCNT) embryos (Zhang et al, 2007; Cao et al, 2017; Jeong et al, 2020)
Combined chaetocin/TSA remarkably increased the blastocyst formation rate compared to either inhibitor alone (Figure 1C and Supplementary Table 2); the combination synergistically enhanced the developmental rate of porcine SCNT embryos
Summary
Somatic cell nuclear transfer (SCNT) is a powerful method that allows reprogramming of terminally differentiated cells to the totipotent state (Gurdon and Wilmut, 2011). SCNT has applications in many areas, including animal husbandry, biomedical research, and endangered animal conservation (Yang et al, 2007). Pigs serve as excellent experimental models for biomedical research in areas such as human disease, bioreactors, and xenotransplantation due to anatomic, physiologic and genetic similarities between pigs and humans (Simon and Maibach, 2000; Prather et al, 2003; Schook et al, 2008; Giraldo et al, 2012). Many transgenic pigs have been created, the cloning efficiency remains extremely low due to a low rate of full-term development, and high rates of pregnancy loss and fetal abnormalities (Wilmut et al, 2002; Whitworth and Prather, 2010; Chavatte-Palmer et al, 2012). Increasing evidence suggests that incomplete epigenetic reprogramming of the donor cell (in terms of DNA methylation and histone methylation/acetylation) causes low cloning efficiency (Dean et al, 2001; Matoba and Zhang, 2018; Simmet et al, 2020; Wang et al, 2020)
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