Extended Exposure to Trichostatin A after Activation Alters the Expression of Genes Important for Early Development in Nuclear Transfer Murine Embryos

Abstract

ABSTRACT. The low viability of embryos reconstructed by somatic cell nuclear transfer (SCNT) is believed to be associated with epigenetic modification errors, and reduction of those errors may improve the viability of SCNT embryos. The present study shows the effect of trichostatin A (TSA), a strong inhibitor of histone deacetylase, on the development of murine SCNT embryos. After enucleation and nuclear injection, reconstructed murine oocytes were activated with or without TSA for 6 hr (TSA-6 hr). After activation, TSA treatment was extended to 3 hr (TSA-9 hr), 5 hr (TSA-11 hr) and 18 hr (TSA-24 hr) during culture. As a result, the SCNT embryos in the TSA-11 hr group showed a remarkably higher blastocyst rate (21.1%) when compared with the nontreated embryos (3.4%), while the concentration of TSA did not significantly affect embryonic development. The expressions of histone deacetylase (HDAC1 and HDAC2) and DNA methylation (DNMT3a and DNMT3b) genes decreased in the TSA-11 hr and TSA-24 hr groups, while there was an increase in the expression of histone acetyltransferase (P300 and CBP), pluripotency (OCT4 and NANOG) and embryonic growth/trophectoderm formation (FGF4)-related genes in the same groups. The expression of CDX2, a critical gene for trophectoderm formation was upregulated only in the TSA-24 hr group. Our results show that TSA treatment during the peri-and postactivation period improves the development of reconstructed murine embryos, and this observation may be explained by enhanced epigenetic modification of somatic cells caused by TSA-induced hyperacetylation, demethylation and upregulation of pluripotency and embryonic growth after SCNT. Advances in cloning techniques [4] using somatic cell nuclear transfer (SCNT) started with the birth of the first mammalian clone, Dolly the sheep, in 1997 [55]. SCNT is a powerful tool for the study of cell reprogramming as well as animal cloning. The applications of SCNT technology include the preservation of endangered species, pet or domestic animal cloning and cell therapy using autologous embryonic stem cells [49][50][51]54]. However, like other species, mouse cloning by SCNT has demonstrated very low success rates since the first cloned mouse was reported in 1998 [52]. Although a noticeable number of SCNT murine embryos reach the blastocyst stage in vitro when produced using published SCNT protocols, the postimplantation development is very limited. The factors affecting the development of SCNT embryos include oocyte activation [19], timing of enucleation and injection of the somatic cell nucleus [48], donor cell pretreatment before nuclear transfer [47] and supplementation of various factors in culture medium [18,35]. In addition to high abortion and fetal death rates, surviving cloned mice often show a variety of abnormalities including obesity, large placenta and abnormal expression of genes important for development [30,40,42,53]. Operational defects of epigenetic factors may cause these anomalies [33]. In recent studies, molecular analyses of cloned emb...

Histone Demethylase KDM4D Could Improve the Developmental Competence of Buffalo (Bubalus Bubalis) Somatic Cell Nuclear Transfer (SCNT) Embryos

“…It is generally believed that compared with the IVF embryos, the expression pattern of pluripotent-related genes in SCNT embryos tend to be lower, so these genes are thought as critical indicators of the blastocyst quality (Inoue et al, 2002; Aston et al, 2010; Kang & Roh, 2011). Thus, in this study, to determine if overexpression KDM4D could affect the blastocyst quality of buffalo SCNT embryos, the expression level of pluripotency-related genes were detected through RT-qPCR.…”

Trichostatin A Improves Preimplantation Development of Bovine Cloned Embryos and Alters Expression of Epigenetic and Pluripotency Genes in Cloned Blastocysts

“…Although the difference in Nanog expression between the long-term and short-term groups cannot be exactly explained, short-term treatment with TSA might not be the optimal exposure duration of TSA for effective modification of Nanog gene. In ridents, extended exposure to TSA after activation altered the expression of genes related to pluripotency (OCT4, NANOG) and early embryonic development (FGF4, CDX2) in cloned embryos [17]. In addition, it was reported that nuclear reorganization of centromeric/pericentromeric sequences is often abnormal in SCNT embryos and is improved by TSA treatment [2525].…”

Generation of Induced Pluripotent Stem (iPS) Cells by Nuclear Reprogramming

“…Compatibility of mitochondrial DNA haplotypes between donor cells and host oocytes improves reprogramming efficiency possibly by epigenetic modification. To increase the SCNT viability, treatment with chemicals that causes chromatin modification like Trichostatin-A (TSA) decreases expression of histone deacetylase (HDAC1 and HDAC2) and DNA methylation (DNMT3a and DNMT3b) while increases the expression of histone acetylation (P300 and CBP), pluripotency (OCT4 and NANOG) genes [55]. Histone deacetylase inhibitor valproic acid (VPA) enabled reprogramming of human fibroblasts with only Oct4 and SOX2 [56].…”

“…TSA-treated embryo showed decreased expression of histone deacetylase (both HDAC1 and HDAC2) and DNA methylation genes (DNMT3a and DNMT3b) where expression levels increased for expression of histone acetyltransferase (P300 and CBP) and pluripotency markers (OCT4 and NANOG). Thus chemicals like TSA can be used to compensate for the shortcomings of iPS [55]. Epigenetic changes happen when differentiation takes place from pluripotency and vice versa [69].…”