Gene discovery in the pre-implantation embryo

Progression through the interval of pre-implantation development is one of the biggest challenges a mammalian embryo faces. Understanding the events of pre-implantation development is important to gaining insight into the basic biology of development and is integral to improving the efficacy and safety of assisted reproductive technologies (ART) applied in both agricultural species and humans. The overall objective of this thesis was to advance our understanding of the molecular mechanisms regulating development to the blastocyst stage. To address this objective, I employed differential display-reverse transcription-polymerase chain reaction (DD-RT-PCR) to assess patterns of messenger RNA expression at different stages of bovine pre-attachment development. My experiments demonstrated that mRNA expression patterns varied according to embryo stage but that a specific pattern was established at the 8--16 cell stage that was largely unchanged through to the blastocyst stage. Treatment of embryos with alpha-amanitin in coordination with DD-RT-PCR demonstrated minor levels of transcriptional activity in early cleavage-stage embryos, supporting findings by other investigators. In addition, several stage-specific and alpha-amanitin-sensitive cDNAs were isolated and provided a focus for subsequent studies. The first study confirmed and characterized the expression of IQGAPs and members of the small Rho-GTPases family of proteins, rac-1 and cdc42 throughout murine pre-implantation development. Based on our characterization of the sub-cellular localization of IQGAP-1 and rac-1 and the role of IQGAP proteins in mediating epithelial cell-cell adhesion, we proposed a hypothesis that interactions between rac-1 and IQGAP-1 proteins regulate the onset of E-cadherin-mediated cell-to-cell adhesion at the onset of compaction. The second study confirmed the expression of p38 regulated/activated kinase (PRAK) and members of the p38 MAPK signaling pathway throughout murine pre-implantation development. Pharmacological studies in which embryos were treated with SB220025 to specifically inhibit p38 MAPK activity demonstrated that p38 MAPK is required at the 8-cell stage to promote compaction and also subsequent morula and blastocyst formation. These studies have expanded our understanding of gene expression that supports pre-implantation development and may ultimately be applied to improve the efficacy and safety of assisted reproductive technologies applied in both livestock and humans.