The long-term objective of this application is to provide a molecular understanding of the roles of egg cortical granule (CG) components in the block to polyspermy reaction. This block is critical to survival of the embryo since it insures that the diploid state of the zygote is maintained. Following fertilization, CG material is released by the egg to eliminate sperm binding to the egg envelope, induce egg envelope hardening, and form new egg extracellular matrices important to early embryonic cell-cell interactions. These reactions are conserved among animals, indicating their importance to development. Progress in characterizing the CG components has been slow, especially in mammals due to the very small amounts of egg CG protein available for biochemical analyses. In model systems, such as the frog Xenopus laevis, gametes are much more abundant, but still the progress has been slow due to the labor and cost of purifying proteins and of traditional amino acid sequencing methods. However, recent technological advances have led to the new field of proteomics, which identifies the proteins in a given cellular state, as a starting point for determining the functions of the proteins.
The specific aim of the proposed research is to characterize the cortical granule contents of Xenopus laevis eggs using a proteomics approach. This will entail separating CG components by high resolution two-dimensional gel electrophoresis, and determining amino acid sequences of peptide digests using sensitive and rapid mass spectrometry techniques. The peptide information will be used to: 1) search the now vast cDNA/protein database for matches with known proteins or partially sequenced cDNAs (ESTs), or 2) clone unique proteins using PCR/cDNA cloning methods. This approach will identify potential molecules important in egg extracellular matrix modifications, which will be the subject of future investigations.
