Animal eggs and embryos from a variety of organisms including mammals, amphibians and echinoderms exhibit extracellular matrices which play and important role in early development. In the sea urchin, sperm first contact a layer of egg jelly, which contains fucose sulfate-rich glycoconjugates (FSG) that induce the acrosome reaction. Acrosome reacted sperm proceed to bind to receptors in the vitelline layer (VL), an extracellular coat, and then fuse with the egg plasma membrane. Sperm fusion elicits cortical granule exocytosis which releases enzymes and structural proteins that modify the VL to produce a chemically toughened fertilization envelope (FE). Exocytosis also results in polymerization of hyalin forming an extracellular coat responsible for adhesion of blastomeres in the early embryo. Finally, in a later secretory process the blastula secretes a basal lamina which lies at the interface between the blastocoel and embryonic epithelium. In this proposal Dr. Chandler aims to study the structure of egg jelly, its interaction with sperm, and the conversion of the VL to the FE. To accomplish this he will prepare high resolution platinum replicas of eggs that have been quick frozen at functionally distinct stages of early development, then freeze-fractured and deep etched. His aims are 1) to visualize by SEM and TEM of platinum replicas the structure of whole egg jelly, 2) to fractionate egg jelly into constituent glycoconjugates and to determine by rotary-shadowing the architecture of egg jelly macromolecules, 3) to localize FSG within whole egg jelly by immunocytochemistry, and 4) to characterize two assembly domains within the fertilization envelope as to their different complements of assembly enzymes and substrates. In addition, he intends to study the ultrastructure of the hyalin-based matrix at the apical surface and of the basal lamina at the basal surface of the blastular epithelium in regards to 1) secretion and assembly during normal development as well as during reassociation of dissociated embryonic cells, and 2) immunocytochemical localization of vertebrate-like extracellular matrix proteins.
