The overall objective of this study is to characterize the components of the cell surface of the sea urchin egg and to elucidate the role of these components in the initial events in fertilization. 1) We will continue studies on the components of a cell surface complex isolated from eggs that consists of the plasma membrane, coated on the outer side with peripheral proteins of the vitelline layer and on the inner side with cortical vesicles. Following further subfractionation the vitelline components, plasma membrane, cortical vesicle membrane, and the contents of the cortical vesicles will be isolated, and analyzed comparatively for polypeptides, lipids, and marker enzymes. To obtain a clearer picture of the fate of these components following fertilization, they will be compared with known components of the hyaline layer and the fertilization envelope. 2) The ion permeability of vesicles prepared from the plasma membrane, the cortical vesicle membrane and a mosaic membrane consisting of both of them will be measured to determine if one of these membranes is responsible for the enhanced influx of Ca++ and Na+ into the egg observed upon fertilization. 3) We will investigate the possibility that Ca++ dependent breakdown and arachidonic acid release. The cell surface complex may be an excellent model to study the kinetic and functional relationships between secretion and arachidonic acid production and metabolism. 4) The structure of the sulfated polysaccharides in egg jelly coat will be studied to define the structural differences that result in species specific induction of the acrosomal reaction in sperm. Using jelly coat as an affinity ligand we will undertake the isolation of the protein on the surface of the sperm that functions as a receptor of jelly coat. The properties and mode of action of the isolated receptor in inducing the acrosomal reaction will be studied. 5) Using binding, or antibody prepared towards a glycopeptide fragment of the receptor as tools, we will isolate the receptor for sperm from solubilized egg cell surface membranes. The purified receptor will be characterized and the structural components of it responsible for both sperm adhesion and species specificity will be defined.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD018590-10
Application #
3315661
Study Section
Reproductive Biology Study Section (REB)
Project Start
1989-08-01
Project End
1992-05-31
Budget Start
1991-06-01
Budget End
1992-05-31
Support Year
10
Fiscal Year
1991
Total Cost
Indirect Cost
Name
State University New York Stony Brook
Department
Type
Schools of Arts and Sciences
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
Ohlendieck, Kay (2004) Purification of membrane proteins. Methods Mol Biol 244:301-8
Hirohashi, N; Lennarz, W J (2001) Role of a vitelline layer-associated 350 kDa glycoprotein in controlling species-specific gamete interaction in the sea urchin. Dev Growth Differ 43:247-55
Huggins, L G; Lennarz, W J (2001) Inhibitors of procollagen C-terminal proteinase block gastrulation and spicule elongation in the sea urchin embryo. Dev Growth Differ 43:415-24
Susan, J M; Just, M L; Lennarz, W J (2000) Cloning and characterization of alphaP integrin in embryos of the sea urchin Strongylocentrotus purpuratus. Biochem Biophys Res Commun 272:929-35
Ohta, K; Sato, C; Matsuda, T et al. (2000) Co-localization of receptor and transducer proteins in the glycosphingolipid-enriched, low density, detergent-insoluble membrane fraction of sea urchin sperm. Glycoconj J 17:205-14
Ohta, K; Sato, C; Matsuda, T et al. (1999) Isolation and characterization of low density detergent-insoluble membrane (LD-DIM) fraction from sea urchin sperm. Biochem Biophys Res Commun 258:616-23
Hirohashi, N; Lennarz, W J (1998) The 350-kDa sea urchin egg receptor for sperm is localized in the vitelline layer. Dev Biol 204:305-15
Hirohashi, N; Lennarz, W J (1998) Sperm-egg binding in the sea urchin: a high level of intracellular ATP stabilizes sperm attachment to the egg receptor. Dev Biol 201:270-9
Tian, J; Gong, H; Thomsen, G H et al. (1997) Xenopus laevis sperm-egg adhesion is regulated by modifications in the sperm receptor and the egg vitelline envelope. Dev Biol 187:143-53
Just, M L; Lennarz, W J (1997) Reexamination of the sequence of the sea urchin egg receptor for sperm: implications with respect to its properties. Dev Biol 184:25-30

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