235 THE INVESTIGATION OF mRNA EXPRESSION OF SEVERAL CHROMATIN REMODELLING GENES DURING BOVINE PREIMPLANTATION DEVELOPMENT

Abstract

The efficiency of obtaining live calves from somatic cell nuclear transfer remains quite low. One factor implicated in this failure is inadequate chromatin remodelling of the donor nucleus. Five Polycomb group (PcG) genes were investigated as potential remodelling factors in bovine oocytes and preimplantation embryos. These genes (Cbx6, Eed, Edr1, Yy1, and Zfp144) are involved in transcriptional activation and cell cycle regulation. We hypothesize that inadequate expression may cause the developmental abnormalities seen following cloning. This study is aimed at characterizing normal expression, prior to comparative studies with cloned embryos. Three single abattoir-derived in vitro-matured (IVM) oocytes or in vitro-produced (IVP) embryos from each of the following stages: 2-cell, 4-cell, 8-cell, 16–32 cell, morula, Day 7 blastocyst, and Day 8 hatched blastocyst, were studied. Messenger RNA was isolated from individual samples with Dynabeads (Dynal, Inc., Lake Success, NY, USA) and then cDNA was created and amplified with a SMART cDNA synthesis kit (BD Biosciences Clontech, Palo Alto, CA, USA). Products were diluted 1:10 and used to amplify target genes by PCR. PCR products were sequenced for confirmation of identity. All amplified embryo samples expressed the housekeeping genes Poly(A) polymerase and actin. Similarly, all embryos up to the 8-cell stage expressed GDF9, an oocyte-specific gene, while IFN-tau, involved in maternal recognition of pregnancy, was expressed in one morula and all blastocyst samples. Cbx6 was not expressed at any stage. The other four genes were all expressed fairly consistently throughout the pre-implantation period. Eed transcript was detected in all samples, with the exception of one oocyte, one 4-cell embryo, and one 8-cell embryo. Edr1 transcript was detected in all samples except for two oocytes, one 16–32-cell embryo, and one Day 7 blastocyst. Yy1 was expressed in all but one oocyte, one 2-cell embryo, two 4-cell embryos, and one Day 7 blastocyst. Finally, Zfp144 transcript was detected in one oocyte and in all embryos until the 16–32 stage, and then was not detected until seen in one Day 7 and all Day 8 blastocysts. Cbx6, yet to be fully characterized in any species, is also known as the neuronal pentraxin receptor, and is involved in transport and clearance of synaptic debris. It is known to have the characteristic chromobox domain of the Cbx family, of which several family members play a role during pre-implantation development. Eed, Edr1, Yy1, and Zfp144 are expressed throughout the pre-implantation period, although levels of Zfp144 mRNA appear to drop during the embryonic genome activation and then reappear by the late blastocyst stage. This consistent expression may suggest a role for the proteins in chromatin remodelling or modulating patterns of gene expression in early development. Further studies are required to determine if these factors are expressed incorrectly in cloned embryos, potentially providing a link to the abnormalities observed post nuclear transfer.