The basic purpose of the proposed 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 biochemical basis of the signaling system. The current emphasis is on the interactive components of the egg (including extracellular matrices or attached cells) and the spermatozoon. The current state of knowledge now allows the following specific aims to be approached: First, the primary structure of the egg peptide receptors will be determine. Oligonucleotide probes corresponding to receptor amino acid sequences will be used to screen testis cDNA libraries. Full length cDNA inserts will then be sequenced and compared from 2 sea urchin species. Based on such information potential """"""""generic"""""""" cDNA probes will be constructed, and the mRNA and genomic DNA of other animals will be analyzed for similar sequences. Second, the cDNA corresponding to the mRNA for resact, the egg peptide from Arbacia punctulata, will be cloned and sequenced. Such information will allow identification of the expected protein precursor of react. The corresponding cDNA for speract will then be cloned and again """"""""generic"""""""" probes will be constructed if areas of the mRNA are conserved. Third, studies will continue to focus on the biochemical basis of the physiological effects of the egg peptides. Special emphasis will be on the mechanisms of regulation of cyclic nucleotide metabolism and the function of G- o/G-i. The basis of sperm chemotaxis will be investigated. In the final specific aim, the effects of Ca2+, cyclic nucleotides, egg conditioned media, ZP-3 (zona pellucidae) and oviductal fluid on endogenous protein phosphorylation of mammalian spermatozoa will be investigated. Factor(s) that alter the phosphoprotein profile will be subsequently characterized.
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