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.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD036022-05
Application #
6693387
Study Section
Reproductive Biology Study Section (REB)
Program Officer
Rankin, Tracy L
Project Start
2000-02-01
Project End
2005-01-31
Budget Start
2004-02-01
Budget End
2005-01-31
Support Year
5
Fiscal Year
2004
Total Cost
$159,647
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Pharmacology
Type
Schools of Medicine
DUNS #
800771545
City
Dallas
State
TX
Country
United States
Zip Code
75390
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Chapman, Karen M; Powell, Heather M; Chaudhary, Jaideep et al. (2013) Linking spermatid ribonucleic acid (RNA) binding protein and retrogene diversity to reproductive success. Mol Cell Proteomics 12:3221-36
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Izsvak, Zsuzsanna; Frohlich, Janine; Grabundzija, Ivana et al. (2010) Generating knockout rats by transposon mutagenesis in spermatogonial stem cells. Nat Methods 7:443-5
Hamra, F Kent (2010) Gene targeting: Enter the rat. Nature 467:161-3
Carlson, Anne E; Burnett, Lindsey A; del Camino, Donato et al. (2009) Pharmacological targeting of native CatSper channels reveals a required role in maintenance of sperm hyperactivation. PLoS One 4:e6844
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Wang, Dan; Hu, Jie; Bobulescu, I Alexandru et al. (2007) A sperm-specific Na+/H+ exchanger (sNHE) is critical for expression and in vivo bicarbonate regulation of the soluble adenylyl cyclase (sAC). Proc Natl Acad Sci U S A 104:9325-30

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