238 RNA BINDING PROTEIN EXPRESSION IN CULTURED EARLY BOVINE EMBRYOS

Abstract

The control of gene expression in the early embryo requires a highly regulated turnover of specific mRNA, particularly those of maternal origin, as the embryo becomes transcriptionally autonomous. In cattle, the period during which maternal transcripts persist can last 72 to 96 h or longer, suggesting a dynamic, regulated interplay between factors that protect transcripts before this point and those that subsequently facilitate decay. Some decay pathways for specific embryonic transcripts are now known, but many are not. In somatic cells, mRNA decay is often mediated by interactions between defined sequence elements (ARE) in the 3′ untranslated region of important target genes and specific RNA-binding proteins (AUBP) that promote or inhibit decay of the associated transcript. These have not been extensively characterized in embryos. We hypothesized that changes in the pattern of expression of one or several AUBP in the developing bovine embryo would support a role for these proteins in mRNA turnover and the control of gene expression. We, therefore, evaluated the expression of different AUBP (HuR, AUF1, TTP) in bovine oocytes and early embryos in vitro. Bovine oocytes obtained at slaughter were matured, fertilized, and cultured using standard protocols. Oocytes and embryos from different stages were either placed in Trizol for subsequent RNA isolation and RT-PCR analysis or fixed in 4% paraformaldehyde and subsequently processed for immunohistochemical detection of AUBP. Analysis by RT-PCR revealed that AUF1, an mRNA destabilizing protein, was expressed at all stages examined (immature oocyte, mature oocyte, 2 to 4 cells, 8 to 16 cells, morulae, and blastocyst) except in morulae. Another mRNA destabilizing protein, TTP, was expressed at the morula stage only. An mRNA stabilizing factor, HuR, was expressed at all stages except the morula. Immunohistochemical analysis revealed that the pattern of protein expression for AUF1 and TTP essentially mirrored that observed at the RNA level as detected by RT-PCR. Together, these results show that AUBP expression in the early bovine embryo is dynamic, with RNA-binding proteins present at all times during development and changes in expression evident at the morula stage. This suggests that modification of presynthesized (i.e. maternal) AUBP is likely to control mRNA decay during the maternal to embryonic transition (8-cell stage) and that the expression of TTP at the morula stage might mark the onset of embryonic control of mRNA stability. Research was supported by NSERC, OMAFRA, and the Canada Research Chairs Program.