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.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Modified Research Career Development Award (K04)
Project #
1K04HD000619-01
Application #
3073183
Study Section
Reproductive Biology Study Section (REB)
Project Start
1985-01-01
Project End
1989-12-31
Budget Start
1985-01-01
Budget End
1985-12-31
Support Year
1
Fiscal Year
1985
Total Cost
Indirect Cost
Name
Arizona State University-Tempe Campus
Department
Type
Schools of Arts and Sciences
DUNS #
188435911
City
Tempe
State
AZ
Country
United States
Zip Code
85287
Chandler, D E (1991) Multiple intracellular signals coordinate structural dynamics in the sea urchin egg cortex at fertilization. J Electron Microsc Tech 17:266-93
Larabell, C; Chandler, D E (1991) Fertilization-induced changes in the vitelline envelope of echinoderm and amphibian eggs: self-assembly of an extracellular matrix. J Electron Microsc Tech 17:294-318
Mozingo, N M; Chandler, D E (1990) The fluorescent probe BCECF has a heterogeneous distribution in sea urchin eggs. Cell Biol Int Rep 14:689-99
Larabell, C A; Chandler, D E (1990) Stepwise transformation of the vitelline envelope of Xenopus eggs at activation: a quick-freeze, deep-etch analysis. Dev Biol 139:263-8
Larabell, C A; Chandler, D E (1989) Quick-freeze, deep-etch, rotary-shadow views of the extracellular matrix and cortical cytoskeleton of Xenopus laevis eggs. J Electron Microsc Tech 13:228-43
Merkle, C J; Chandler, D E (1989) Hyperosmolality inhibits exocytosis in sea urchin eggs by formation of a granule-free zone and arrest of pore widening. J Membr Biol 112:223-32
Larabell, C A; Chandler, D E (1989) The coelomic envelope of Xenopus laevis eggs: a quick-freeze, deep-etch analysis. Dev Biol 131:126-35
Chandler, D E; Whitaker, M; Zimmerberg, J (1989) High molecular weight polymers block cortical granule exocytosis in sea urchin eggs at the level of granule matrix disassembly. J Cell Biol 109:1269-78
Kazilek, C J; Merkle, C J; Chandler, D E (1988) Hyperosmotic inhibition of calcium signals and exocytosis in rabbit neutrophils. Am J Physiol 254:C709-18
Larabell, C A; Chandler, D E (1988) Freeze-fracture analysis of structural reorganization during meiotic maturation in oocytes of Xenopus laevis. Cell Tissue Res 251:129-36

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