20 ALTERED GENE EXPRESSION IN BOVINE SOMATIC CELL NUCLEAR-TRANSFERRED EMBRYOS AFTER TRICHOSTATIN A TREATMENT

Abstract

Trichostatin A is a histone deacetylase inhibitor that improves histone acetylation and chromatin remodeling of somatic cell nuclear-transferred embryos (Iager et al. 2008 Cloning Stem Cells 10, 371–379; Maalouf et al. 2009 BMC Dev. Biol. 9, 11). We have previously observed that it also improves quality of bovine cloned embryos, which may increase pregnancy rates. This study aimed to evaluate the effect of trichostatin A treatment of zygotes on relative abundance of 9 transcripts in bovine nuclear-transferred blastocysts. In vitro matured oocytes were enucleated, fused to somatic cells and activated with ionomycin (Camargo et al. 2011 Reprod. Fertil. Dev. 23, 122). After activation, putative zygotes were randomly separated into 2 groups: NT-TRICHO, zygotes were cultured for 4 h in 6-DMAP followed by 7 h in CR₂ aa medium plus with 2.5% fetal calf serum (FCS; Nutricell, Campinas, Brazil), both supplemented with 50 nM trichostatin A (Sigma); NT-CONT, zygotes were cultured in the same described conditions without thichostatin A supplementation. In vitro-fertilized embryos (IVF group) were used as a calibrator for relative transcript quantification. Embryos from the 3 groups were cultured in CR₂ aa supplemented with 2.5% FCS under 5% CO₂, 5% O₂ and 90% N₂ at 38.5°C. At 168 h postactivation, the embryos were rapidly frozen in liquid nitrogen. Pools of 10 blastocysts for each group were subject to RNA extraction and reverse transcription, in which cDNA was amplified by real-time PCR using the β-actin and GAPDH genes as endogenous references. The transcripts analysed encode high mobility group N1 (HMGN1), peroxiredoxin 1 (PRDX1), octamer-binding protein 4 (OCT4), insulin-like growth factor 1 and 2 receptors (IGF1r and IGF2r), glucose transporter 1 and 5 (GLUT1 and GLUT5), histone acetyltransferase (HAT) and heat shock protein 70.1 (HSP70) genes. Results were analysed by a pair-wise fixed reallocation randomization test using the REST software v.2. Data from NT-TRICHO and NT-CONT were compared with the IVF group and between themselves. The relative abundance of HSP70, PRDX1, IGF2r and HMGN1 transcripts was higher (P < 0.05) in NT-TRICHO compared with the IVF group and no difference was detected for the other transcripts. In the NT-CONT group, the relative abundance of IGF2r and HAT was higher (P < 0.05), whereas IGF1r and OCT4 were lower (P < 0.05) compared with IVF embryos. When data from NT-TRICHO and NT-CONT were compared, a higher amount (P < 0.05) of stress-associated transcripts (HSP70 and PRDX1) were found in NT-TRICO blastocysts. These results suggest that although trichostatin A may improve chromatin remodeling, alterations on gene expression still persist in bovine somatic cell nuclear-transferred blastocysts in comparison with IVF embryos.

Financial support: Embrapa Project 01.07.01.002, CNPq 403019/2008–7 and Fapemig.