title: Treating cloned embryos, but not donor cells, with 5-aza-2'-deoxycytidine enhances the developmental competence of porcine cloned embryos.
abstract: The efficiency of cloning by somatic cell nuclear transfer (SCNT) has remained low. In most cloned embryos, epigenetic reprogramming is incomplete, and usually the genome is hypermethylated. The DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) could improve the developmental competence of cow, pig, cat and human SCNT embryos in previous studies. However, the parameters of 5-aza-dC treatment among species are different, and whether 5-aza-dC could enhance the developmental competence of porcine cloned embryos has still not been well studied. Therefore, in this study, we treated porcine fetal fibroblasts (PFF) that then were used as donor nuclei for nuclear transfer or fibroblast-derived reconstructed embryos with 5-aza-dC, and the concentration- and time-dependent effects of 5-aza-dC on porcine cloned embryos were investigated by assessing pseudo-pronucleus formation, developmental potential and pluripotent gene expression of these reconstructed embryos. Our results showed that 5-aza-dC significantly reduced the DNA methylation level in PFF (0 nM vs. 10 nM vs. 25 nM vs. 50 nM, 58.70% vs. 37.37% vs. 45.43% vs. 39.53%, P<0.05), but did not improve the blastocyst rate of cloned embryos derived from these cells. Treating cloned embryos with 25 nM 5-aza-dC for 24 h significantly enhanced the blastocyst rate compared with that of the untreated group. Furthermore, treating cloned embryos, but not donor cells, significantly promoted pseudo-pronucleus formation at 4 h post activation (51% for cloned embryos treated, 34% for donor cells treated and 36% for control, respectively, P<0.05) and enhanced the expression levels of pluripotent genes (Oct4, Nanog and Sox2) up to those of in vitro fertilized embryos during embryo development. In conclusion, treating cloned embryos, but not donor cells, with 5-aza-dC enhanced the developmental competence of porcine cloned embryos by promotion of pseudo-pronucleus formation and improvement of pluripotent gene expression.
authors.affiliation: College of Life Science, Northeast Agricultural University, Haerbin 150030, China.
authors.forename: Yan Jun
authors.initials: YJ
authors.lastname: Huan
authors.forename: Jiang
authors.initials: J
authors.lastname: Zhu
authors.forename: Bing Teng
authors.initials: BT
authors.lastname: Xie
authors.forename: Jian Yu
authors.initials: JY
authors.lastname: Wang
authors.forename: Shi Chao
authors.initials: SC
authors.lastname: Liu
authors.forename: Yang
authors.initials: Y
authors.lastname: Zhou
authors.forename: Qing Ran
authors.initials: QR
authors.lastname: Kong
authors.forename: Hong Bin
authors.initials: HB
authors.lastname: He
authors.forename: Zhong Hua
authors.initials: ZH
authors.lastname: Liu
mesh_terms: D000003:Abattoirs; D000818:Animals; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001755:Blastocyst / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D001757:Blastomeres / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D002478:Cells, Cultured; D019976:Cloning, Organism / Q000662:veterinary*; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects*; D000077209:Decitabine; D015536:Down-Regulation / Q000187:drug effects; D004477:Ectogenesis / Q000187:drug effects*; D046149:Embryo Culture Techniques / Q000662:veterinary; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D044127:Epigenesis, Genetic / Q000187:drug effects; D005260:Female; D005333:Fetus / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D059471:In Vitro Oocyte Maturation Techniques / Q000662:veterinary; D008297:Male; D053652:Nuclear Transfer Techniques / Q000662:veterinary; D012333:RNA, Messenger / Q000378:metabolism; D034421:Sus scrofa
chemical_list: D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D000077209:Decitabine; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D001374:Azacitidine