The long-term goal of this grant is to provide a basis for intervention at the level of sperm surface receptors for the practical purpose of increasing or decreasing fertilization rates. The potential positive effects on human health and welfare could be dramatic, possibly helping childless couples with infertility problems while at the same time offering a means to control the rapidly increasing human population. Positive effects on fertilization rates in Agriculture could also increase the World's food supply. In addition, the research fostered by this grant has now been shown to directly impact on diseases such as hypertension and diarrhea. The basic purpose of this research is to determine the structure of those molecules that specifically interact with spermatozoan receptors, to identify the receptor molecules, to define the physiological events that occur in response to receptor occupation, and to determine the molecular basis of the signaling system. There are three specific aims entailed within this application. The first is to define the functional roles of adenylyl cyclase and of cyclic AMP in spermatozoa. Within the context of this application, the major goal will be to clone and express the mRNA for the sperm adenylyl cyclase, and to define its regulatory properties. The second specific aim is to define the functional roles of guanylyl cyclase and of cyclic GMP in spermatozoa. The sites of phosphorylation of the sea urchin sperm guanylyl cyclase will be determined. The protein kinase(s) and protein phosphatase(s) responsible for regulation of the phosphorylation state of guanylyl cyclase will be identified and their regulatory properties defined. Preliminary work will be initiated to determine the molecular sites of action of cyclic GMP where both PCR technology and low stringency hybridization will be brought to bear on the question. The third specific aim is to identify the G-protein coupled receptors of spermatozoa. Spermatozoa contain various G-proteins and considerable research suggests that the ZP-3 receptor couples to G- proteins. PCR will be used to search for such receptors. Sperm-specific sequences will be subsequently used to obtain full length cDNA clones which will be expressed in cultured cells. One sperm-specific receptor, most similar to known adenosine receptors, is now being studied. Ligands for each receptor will be identified, and the G-protein alpha-subunit that is activated will be identified.
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