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(362) QUANTIFICATION OF HISTONE ACETYLTRANSFERASE TRANSCRIPTS IN EARLY BOVINE EMBRYO DEVELOPMENT. McGraw, Serge1, Robert, Claude1, Massicotte, Lyne1, Sirard, Marc-André1, 1 Centre de Recherche en Biologie de la Reproduction, Québec, CA ABSTRACT- Mammalian oocytes are exceptionally powerful totipotent cells which are capable of removing existing gene expression patterns to impose new ones. This genomic reprogramming involves altering the chromatin structure. These modifications alter the higher order chromatin organization to render the DNA accessible to the transcription machinery. One mechanism known for this action is the acetylation of the nucleosomal histones. By means of real time quantitative RT-PCR, we established mRNA expression profiles for three histone acetyltransferases (HAT1, GCN5 and MORF) and one nucleosomal histone (H2A) throughout early bovine embryo development. Oocytes were harvested from slaughterhouse ovaries and cattle embryos were produced in vitro through standard techniques. The oocytes and embryos were mechanically stripped of their cumulus cells, washed in PBS buffer and stored at −80°C. Triplicates of 20 oocytes (GV and MII) or embryos (2-cell, 8-cell and blastocyst) were used for each of the developmental stages. Total RNA was extracted and treated with DNase. The reverse transcriptions were performed in 20 KEY WORDS: Reprogramming, Chromatin, Acetyltransferase, Oocyte/Embryo |
l total and the cDNA corresponding to 2 oocytes or embryos (1 for H2A) were used for each of the quantitative PCR assays on a Light Cycler apparatus (Roche Diagnostics, Laval, Canada). The values obtained were normalized with the values of H2A. Our results demonstrate that the expression of HAT1 is low and relatively constant during the pre-blastocyst stages, before a major level increase in blastocyst. The patterns observed for GCN5 and MORF are unlike the one for HAT1. Their highest levels are expressed in the MII oocytes and 2-cell embryos, and their lowest levels are in the 8-cell embryo and blastocyst. MORF mRNA transcripts are extremely abundant in every developmental stages verified. Throughout the pre-blastocyst period (GV oocyte to 8-cell), MORF is 500 and 3500 times more abundant than GCN5 and HAT1. In the blastocyst it is 440 and 320 times higher than GCN5 and HAT1. These results indicate that MORF acetyltransferase enzyme maybe more important in early stages of embryonic development, potentially implicated in the regulation of the transcriptional activity. This project was supported by Natural Sciences and Engineering Research Council of Canada.