184 EXPRESSION OF GENES RELATED TO DNA METHYLATION AND GLUCOSE METABOLISM DURING THE PRE-IMPLANTATIONAL STAGE OF BOVINE EMBRYOS

Abstract

During pre-implantation embryonic development in mammals, extensive epigenetic reprogramming occurs; patterns of DNA methylation are erased during the first cell divisions and are restored at the 8- to 16-cell stage (Dean et al. 2001). An important step during this developmental phase, which is controlled by epigenetic factors such as DNA methylation, is inactivation of the X chromosome (XCI). It is established in the morula stage, where the paternal X is inactivated, with immediate reactivation in the blastocyst (Ferreira et al. 2010). The aim of this study was to evaluate the mRNA expression profile of the DNMT3a and DNMT3b genes, which are related to DNA methylation, and the G6PD and PGK1 genes, which are related to glucose metabolism and are subject to XCI. Cumulus–oocyte complexes (COC) were isolated from slaughterhouse ovaries from Bos taurus indicus Nellore cows. Oocytes were matured in vitro and inseminated with X-sorted sperm from a Holstein bull. Embryos of each developmental stage [4-cell (44 hpi), 8- to 16-cell (72 hpi), morula (144 hpi), blastocyst (156 hpi), and expanded blastocyst (168 hpi)] were produced. Three pools of 17 embryos for each stage were frozen at –80°C until RNA extraction. Total RNA was isolated using the Invisorb RNA Spin Cell Mini Kit (Invitek, Berlin, Germany) according to the manufacturer’s protocol. Complementary DNA was done using Oligo dT primers (Invitrogen, São Paulo, Brazil) and SuperScript III reverse transcriptase (Invitrogen). Relative expression of target genes was assessed by quantitative PCR using a Power Sybr^®Green PCR Master Mix (Applied Biosystems, Foster City, CA) in an ABI Prism 7500 Fast Sequence Detection System (Applied Biosystems). The cyclophilin-A gene was used as an endogenous control. To compare gene expression between treatments, we used the ΔΔC_t method. The identity of PCR products was confirmed by the amplicon size in agarose gel and by the melt curve in quantitative PCR. Gene quantification was compared among treatments using one-way ANOVA and Tukey’s test, in the Prophet program, version 5.0 (1996, BBN Systems and Technologies, Cambridge, MA). The 4 genes showed higher expression (P ≤ 0.05) in 4-cell stage embryos than in morulae, blastocysts, or expanded blastocysts. The 8- to 16-cell embryos showed no differences for any gene compared with other embryos, except for the DNMT3b gene, which was not tested in these embryos. We conclude that these genes have a greater quantity of mRNA molecules because of the presence of maternal mRNA and that this stock was being spent during embryo development until the expanded blastocyst stage.