Epigenetic modification of fetal fibroblasts improves developmental competency and gene expression in porcine cloned embryos.
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.
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Gyeongsang National University, 501, Jinju-daero, Jinju, 660-701, Republic of Korea.
B Mohana
BM
Kumar
Geun-Ho
GH
Maeng
Yeon-Mi
YM
Lee
Jeong-Hyeon
JH
Lee
Byeong-Gyun
BG
Jeon
Sun-A
SA
Ock
Taeyoung
T
Kang
Gyu-Jin
GJ
Rho
D000107:Acetylation; D000818:Animals; D019976:Cloning, Organism / Q000662:veterinary; D019175:DNA Methylation; D015257:DNA-Cytosine Methylases / Q000235:genetics / Q000378:metabolism; D004622:Embryo, Mammalian / Q000196:embryology*; D044127:Epigenesis, Genetic; D005307:Fertilization in Vitro / Q000662:veterinary; D005347:Fibroblasts / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental; D051548:Histone Acetyltransferases / Q000235:genetics / Q000378:metabolism; D006655:Histone Deacetylases / Q000235:genetics / Q000378:metabolism; D006657:Histones / Q000235:genetics / Q000378:metabolism; D053652:Nuclear Transfer Techniques / Q000662:veterinary; D034421:Sus scrofa / Q000196:embryology*
D006657:Histones; D015257:DNA-Cytosine Methylases; D051548:Histone Acetyltransferases; D006655:Histone Deacetylases