The broad objective of this proposal is to understand the molecular basis of fertilization, the principle emphasis being on the mechanisms by which the egg communicates and interacts with the sperm cell. Based on success of intervention at the level of receptors and adhesion/recognition proteins in other cellular systems, it is probably the same will be true with respect to gametes, thereby leading to potential prevention or enhancement of fertilization Two major specific aims are proposed: 1. Define the Function of Newly Discovered Sperm Membrane Proteins.
The specific aim will concentrate on zonadhesin, a mosaic protein containing MAM, D-, and EGF-domains. The experimental approaches to define the function of zonadhesin will include gene disruption, determination of the site(s) during sperm development where the MAM and mucin domains are removed, expression of various domains in cultured mammalian cells followed by binding studies, production of specific antibodies to the MAM, D-, and EGF-domains, determination of whether carbohydrate on D-domains plays a significant role in binding to the zona pellucida, and determination of which components of the zone pellucida bind zonadhesin. 2. Define the Unique Proteins of the Sperm Plasma Membrane and Determine Function. We will utilize the powerful method of Signal Peptide Trapping to define the proteins present on sperm membranes. We have now established a signal of PCR, and the use of partial length cDNAs as probes, the sperm-specific transcripts will be identified These specific cDNAs will then be cloned and based on conserved functional domains, as well as predicted topology, function will be predicted. The priority for a membrane protein to be studied in detail will be sperm-specific expression, and of those fitting the first criterion, the presence of domains or a topology consistent with known functions in cell recognition (adhesion) or cell signaling molecules. We already possess testis-specific transcripts, 2 of which have been previously identified (sp56, TPX-1), 1 which appears to represent a calcium T-type channel, and 3 unique compared to the database. The channel may represent the protein ultimately responsible for induction of acrosome reaction. It is used as an example of our line of inquiry following the discovery of a sperm-specific membrane protein which function can be predicted based on database searches.
Showing the most recent 10 out of 19 publications
