A major problem in fertilization is how sperm recognize and bind to the egg surface. In animals as diverse as echinoderms and mammals this problem of cell-cell recognition is solved by the presence of an extracellular coat near the egg surface that contains receptors for sperm. This, coat (the vitelline layer (VE) of the sea urchin egg and the zona pellucida (ZP) of the mammalian egg) is the structure to which sperm first bind, one sperm actually going on to fuse with and fertilize the egg. After fertilization, this coat is modified to prevent further sperm binding. Modifications are carried out by enzymes and structural proteins released from the egg by exocytosis just after fertilization. A major goal of this study is to visualize the fiberous structure of the VE and ZP using the quick-freezing method developed by Heuser. This in combination with freeze fracture, deep etching, and rotary shadowing, provides a 3-dimensional replica of the egg surface and its extracellular coats. In both the VE and the ZP we hope to characterize the nature of sperm-coat binding as well as the nature of the post-fertilization modifications to these layers. The modifications that we will be looking for include a) coat degradation by a protease that hydrolyzes sperm receptors; b) crosslinking of the fibers in these layers by ovoperoxidase, and c) coating of the fiberous network by structural proteins, e.g. the 'paracrystalline' protein of the sea urchin egg. Sperm receptor hydrolase, ovoperoxidase, and coating proteins are all contained in cortical granules (CG) and released by exocytosis.
Our second aim i s to study CG exocytosis in both sea urchin and mammalian eggs. In sea urchin eggs, the intracellular reservoir of calcium used to trigger exocytosis will be localized at the electron microscope level using freeze substitution and x-ray microanalysis, and the organelle characterized as to its ATP-dependent 45Ca2+ sequestering ability. In isolated egg cortices, fusion of CG and plasma membranes will be studied using quick freezing and freeze fracture. Finally, in mammalian eggs, conventional freeze fracture techniques will be used to study the temporal and spacial kinetics of cortical granule exocytosis. These data can then be compared to the well known kinetics of CG exocytosis in the sea urchin egg.
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