BIOCHEMISTRY OF SPERM-EGG INTERACTION AND FERTILIZATION

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The objective of this research is to gain fundamental knowledge of the cell biology and biochemistry of sperm-egg interaction. Sea urchins and abalone are used because they are nonmammalian, yet exhibit striking similarities to mammalian gamete interaction. The cell biology and biochemistry of sperm-egg interaction is one of the least understood fundamental biological processes. Basic knowledge gained by this research may be used in the future development of novel methods of contraception in mammals. In sea urchins this proposal focuses on the interaction of REJ (the sperm membrane receptor for egg jelly) with ARI (the acrosome reaction inducing glycoprotein purified from egg jelly). 50% of REJ is homologous to the human polycystic kidney disease protein PKD1. Study of how REJ regulates sperm ion channels may aid in the understanding of this significant human disease. In abalone, this project focuses on the egg vitelline envelope ligand for lysin, the sperm protein which dissolves the egg envelope by a nonenzymatic mechanism. This study may aid in understanding the biochemical mechanism mammalian sperm use to penetrate the egg zona pellucida. The specific aims for sea urchin sperm are: 1. To identify sperm membrane proteins associated with the 210kDa receptor for egg jelly (REJ). 2. To clone, sequence and characterize the proteins associated with REJ. 3. To determine the functions of the protein modules of REJ. 4. To characterize the acrosome reaction inducer (ARI) from sea urchin egg jelly which binds sperm REJ. 5. To isolate and characterize the oligosaccharide of ARI which binds to REJ. 6. To use syntaxin and synaptotagmin, which we have cloned from sperm, to identify proteins associated with exocytosis of the acrosomal vesicle. The specific aims for the abalone ligand for lysin are: 7. To study the biochemistry and ultrastructure of the vitelline envelope ligand (VE-L) for lysin. To determine the affinities of homospecific and heterospecific combinations of lysins and VE-Ls. 8. To clone, sequence and characterize the cDNA for the VE-L. 9. To determine if the binding of lysin to VE-L is based on protein or carbohydrate components of the VE-L. 10. To cleave the VE-L into the smallest fragment retaining affinity for lysin. To isolate and characterize this fragment. To cocrystallize lysin complexed with this fragment for x-ray diffraction analysis. 11. To determine which residues of lysin are responsible for its species-specific dissolution of the egg vitellin envelope (VE). 12. To determine the mechanism by which lysin dissolves the VE. Is it based on the stereospecific severing of hydrogen bonds, or hydrophobic interaction?