title: Effect of histone deacetylase inhibitor valproic acid treatment on donor cell growth characteristics, cell cycle arrest, apoptosis, and handmade cloned bovine embryo production efficiency.
abstract: In this study, we tested the effects of valproic acid (VPA), a known histone deacetylase inhibitor (HDACi), on the growth characteristics, apoptosis, and cell cycle stages distribution of donor cells, as well as cloning efficiency, embryo development, and histone methylation. Our results showed that treatment of donor cells with VPA (2.5 mM, 5.0 mM, 7.5 mM, or 10 mM) for 24 h resulted in altered cell proliferation, extent of apoptosis and necrosis, and cell cycle stage distribution, whereas no changes in cell viability and chromosomal complements were observed. Measurement of relative gene expression using real-time PCR of a few developmentally important genes in treated donor cells showed decreased expression of HDAC1 and increased expression of BAX (p<0.05). No change in relative expression of HDAC2 and Bcl2 was noticed. Treatment of donor cells with VPA for 24 h before electrofusion significantly (p<0.05) increased the blastocyst formation rate of somatic cell nuclear transfer (SCNT) embryos compared to the control embryos. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive nuclei in SCNT blastocysts derived from VPA-treated donor cells were significantly decreased compared to the control blastocysts (p<0.05). Immunolocalization studies revealed that the levels of histone H3 at lysine 9 (H3K9me3) were lower in VPA-treated donor cells derived cloned blastocysts than nontreated cloned embryos, and was at the level of in vitro fertilization (IVF) counterparts, although no effects of treatments were found in donor cells. Our study demonstrates that the use of VPA in SCNT has been beneficial for efficient reprogramming of donor cells. Its effect on histone methylation in cloned embryos correlates with their developmental potential and may be a useful epigenetic marker to predict the efficiency of SCNT.
authors.affiliation: 1 Department of Biomedical Sciences, Ontario Veterinary College , Guelph Canada, N1G 2W1.
authors.forename: Naresh L
authors.initials: NL
authors.lastname: Selokar
authors.forename: Liz
authors.initials: L
authors.lastname: St John
authors.forename: Tamas
authors.initials: T
authors.lastname: Revay
authors.forename: W Allan
authors.initials: WA
authors.lastname: King
authors.forename: Suresh K
authors.initials: SK
authors.lastname: Singla
authors.forename: Pavneesh
authors.initials: P
authors.lastname: Madan
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001483:Base Sequence; D002417:Cattle; D002453:Cell Cycle / Q000187:drug effects*; D002455:Cell Division / Q000187:drug effects*; D002478:Cells, Cultured; D019976:Cloning, Organism; D017931:DNA Primers; D004622:Embryo, Mammalian; D020869:Gene Expression Profiling; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology*; D020287:In Situ Nick-End Labeling; D060888:Real-Time Polymerase Chain Reaction; D014635:Valproic Acid / Q000494:pharmacology*
chemical_list: D017931:DNA Primers; D056572:Histone Deacetylase Inhibitors; D014635:Valproic Acid