Modification of the Egg Extracellular Matrix at Fertilization

点击翻译按钮获取中文摘要

0315657WesselSuccess at fertilization requires both sperm-egg fusion and blockage of excess sperm fusions, or polyspermy. Polyspermy is lethal in most animals, and oocytes have evolved mechanisms to minimize its occurrence. The most conserved mechanism throughout phylogeny involves a rapid remodeling of the egg extracellular matrix following fertilization by secretion of cortical granules, which are stimulus-dependent secretory vesicles unique to eggs and oocytes. Cortical granule contents modify the nascent egg extracellular matrix to form both a permanent block to polyspermy and to provide protection for early embryonic development. The general question addressed in this application is: How do eggs so rapidly and efficiently construct their new extracellular matrix? We will use sea urchin eggs for this project because: 1) we can obtain, on average, 106 eggs per female, enabling large-scale biochemical approaches; 2) each of the constituents of this process - the cortical granules, the fertilization envelope, and the vitelline layer, can be isolated en masse in a functional form; 3) cDNA clones have been isolated that represent each major functional protein of this process; and 4) sea urchin eggs undergo one of the most extreme modifications at fertilization. They invest 6% of their protein mass and nearly 20% of their mRNA to this transformation of the nascent extracellular, vitelline layer into an impervious fertilization envelope within 60 seconds.The broader implications of this project are that undergraduate and graduate students will be trained in diverse contemporary methods of cell, molecular, and developmental biology, and that general principles of fertilization and extracellular matrix biology will be integrated in a classically described, tractable system for educational purposes. In addition, project results and other educational material for K-16 will be continually updated on the Educational Resources page of our lab web site (http://www.brown.edu/Research/Wessel_Lab/).Three Specific Research Aims are proposed:1. Identify the vitelline layer proteins essential for fertilization envelope construction. 2. Determine the domains in cortical granule proteins required for construction of the extracellular matrix and block to polyspermy at fertilization. 3. Identify the peroxide-generating mechanism essential for envelope cross-linking.