246 EVIDENCE OF FOCAL ADHESION ASSEMBLY IN BOVINE OOCYTES

Abstract

Integrins are located on the plasma membrane of bovine oocytes and have been implicated in bovine fertilization and activation (Campbell et al. 2000; Sessions et al. 2006). In response to signals from the extracellular matrix, activated integrins cluster at focal adhesions and interact with the cytoskeleton via signalling molecules, including focal adhesion kinase (FAK), α-actinin, phosphotyrosine, and talin. Immunohistochemistry was used to illustrate the formation of focal adhesions at the site of sperm binding in bovine oocytes during fertilization. To confirm antibody specificity, proteins were recovered from bovine oocyte lysate, separated by electrophoreses, and exposed to Western blotting. Specificity was confirmed based on antibody binding to the correctly sized bovine protein in the blot. Bovine oocytes were recovered, matured in vitro, and exposed to spermatozoa as described previously. Antibodies were then used to directly identify co-localization of FAK, α-actinin, phophotyrosine, and talin with each other in addition to localization with β-integrin isotypes within the bovine oocyte plasma membrane. Appropriate controls were performed in which all steps were duplicated with the elimination of the primary antibodies, to confirm that immunohistochemistry was specific for the proteins of interest. The results of immunohistochemistry indicate a co-localization of FAK protein clusters that involves specific oocyte membrane integrin proteins during fertilization. These findings illustrate the formation of focal adhesions via colocalization of signalling molecules with integrin β subunits in bovine oocytes and further support the hypothesis that induction of signalling pathways is initiated with sperm–oocyte binding in the bovine.