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Treatment of somatic cells with DNA methylation and histone deacetylation inhibitors has been hypothesized to improve the potential reprogramming after nuclear transfer (NT). The objective of this study was to investigate the developmental competence and gene expression during the porcine preimplantation development of in vitro fertilized (IVF) and embryos cloned with porcine fetal fibroblasts (pFF) (pFF-NT), and pFF treated by 0.5 μM 5-azacytidine (5-azaC) (pFF+5-azaC-NT) or 1.0 mM sodium butyrate (NaB) (pFF+NaB-NT). IVF embryos had significantly (P < 0.05) higher blastocyst rates (27.7 ± 2.6 %) and total cell numbers (46.7 ± 3.9). However, NT embryos from pFF+5-azaC and pFF+NaB showed enhanced developmental potential with significantly (P < 0.05) higher rates of blastocysts (21.3 ± 2.9 % and 22.4 ± 1.7 %, respectively) than those from pFF (15.1 ± 2.5 %). Further, NT embryos from pFF+5-azaC and pFF+NaB (33.8 ± 4.1 and 35.7 ± 5.2, respectively) had significantly (P < 0.05) higher total cell numbers than those from pFF (24.6 ± 3.5). Differential expression pattern of genes involved in DNA methylation (DNA methyltransferases- DNMT1, DNMT2, DNMT3A and DNMT3B), histone acetylation (histone acetyltransferase 1- HAT1) and histone deacetylation (histone deacetylases- HDAC1, HDAC2 and HDAC3) were observed in NT embryos when compared to IVF counterparts. However, the relative expressions of genes in pFF+5-azaC-NT and pFF+NaB-NT groups were largely comparable to those of IVF embryos than pFF-NT embryos. In conclusion, modification of the epigenetic status by reducing DNA methylation or enhancing histone acetylation levels in pFF improved the developmental rates, total cell number and the transcription profile of porcine NT embryos. Thus, somatic cells with relatively hypomethylated or hyperacetylated genome may enhance reprogramming efficiency in porcine NT.

The purpose of this study was to examine the telomerase activity, population doubling time (PDT), morphological alterations, and the cell cycle status with activity of senescence-associated-ß-galactosidase in porcine mesenchymal stem cells (MSCs) and fibroblasts during an extended in vitro culture. MSCs and fibroblasts were isolated from bone marrow and ear skin of a miniature pig, respectively, and cultured up to 20 passages. The analysis was carried out in MSCs and fibroblasts at 1, 5, 10, 15, and 20 passages. Relative telomerase activity (RTA) levels were significantly (P < 0.05) higher in MSCs than in fibroblasts at all the passages. The PDT and cellular size slightly increased in MSCs at later passages. In contrast, fibroblasts had significantly (P < 0.05) increased PDT and cellular size, and the morphology revealed senescent-like abnormal type after passage 10. Further, the high incidence of ß-galactosidase stained cells was observed in fibroblasts compared to that of MSCs at passage 15, and cell cycle stage at G0 / G1 phase was significantly (P < 0.05) increased in the fibroblasts at 15 and 20 passages compared to that of MSCs. Based on these observations, we concluded that porcine MSCs possessed more tolerance against senescence and aging compared to fibroblasts following prolonged in vitro culture.

The poor outcome of somatic cell nuclear transfer (SCNT) is thought to be a consequence of incomplete reprogramming of the donor cell. The objective of this study was to investigate the effects of treatment with S-adenosylhomocysteine (SAH) a DNA demethylation agent, on DNA methylation levels and X-chromosome inactivation status of bovine female fibroblast donor cells and the subsequent impact on developmental potential after SCNT. Compared with non-treated controls, the cells treated with SAH revealed (i) significantly (P<0.05) reduced global DNA methylation, (ii) significantly (approximately 1.5-fold) increased telomerase activity, (iii) diminished distribution signals of methylated histones H3-3mK9 and H3-3mK27 on the presumptive inactive X-chromosome (Xi), (iv) alteration in the replication pattern of the Xi, and (v) elevation of transcript levels for X-chromosome linked genes, ANT3, MECP2, XIAP, XIST, and HPRT. SCNT embryos produced with SAH-treated donor cells compared with those derived from untreated donor cells revealed (i) similar cleavage frequencies, (ii) significant elevation in the frequencies of development of cleaved embryos to hatched blastocyst stage, and (iii) 1.5-fold increase in telomerase activity. We concluded that SAH induces global DNA demethylation that partially reactivates the Xi, and that a hypomethylated genome may facilitate the nuclear reprogramming process.