The long term objective is to determine the molecular alterations that produce ultrastructural and fertilization related functional changes in egg envelopes. Structural changes in the glycoproteins composing the egg envelope will be correlated with functional changes in sperm binding, induction of the sperm acrosome reaction, and sperm penetration of the envelope. The animal being used is the frog Xenopus laevis.
Specific aims i nclude structure-function studies of the coelomic to vitelline envelope conversion which takes place in the pars recta oviduct and the vitelline envelope to fertilization envelope conversion which occurs at fertilization. A pars recta protease responsible for the coelomic to vitelline envelope conversion will be isolated and characterized as a protein and an enzyme. The gene for this protease will be cloned using recombinant DNA methodology to assist in determining protein structure. The chemical action of the enzyme on its glycoprotein envelope substrate will be determined by comparing the primary structure of the 43K substrate and the 41K product. Alteration of the sperm binding properties caused by limited hydrolysis will be investigated. At fertilization, the vitelline envelope is modified by the addition of a fertilization (F) layer and limited hydrolysis of 69K and 64K envelope glycoproteins. The F layer is produced by a cortical granule lectinprefertilization layer ligand reaction. We will determine the primary structure of the glycoprotein lectin and isolate and structurally characterize its ligand. Recombinant DNA methodology will be used to assist in determining protein structure. The ligand will be analyzed for its acrosome reaction inducing activity to test the hypothesis that formation of the F layer blocks sperm penetration because lectin binding inhibits the acrosome reaction inducing activity of the ligand. Evidence will be sought for a role of the 69K, 64K glycoproteins in binding the F layer to the envelope. These structure function studies will increase our understanding of the role of these molecules in fertilization and also our fundamental knowledge of lectin-ligand interactions.
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