The overall goal of the proposed research is to investigate the role of reactive oxygen species durign fertilization, both beneficial and deleterious, using a system where these species are clearly playing a physiologic role. When sea urchin eggs are fertilized, high concentrations of hydrogen peroxide are formed and used as substrate by ovoperoxidase to modify the extracellular matrix of the egg. The mechanism of hydrogen peroxide formation by eggs and the possible involvement of species such as superoxide will be investigated. The role of ovothiol, a novel thiol containing histidine derivative recently isolated from eggs, in hydrogen peroxide formation and protection from oxidant stress will be characterized. Intracellular ovothiol levels during fertilization and cell division will be measured using 1H spin echo NMR or by derivatization with (14C) iodoacetic acid. The effect of depletion of egg ovothiol on fertilization envelope elevation, cell division, hydrogen peroxide generation and egg susceptibility to oxidant damage will be investigated. Evidence for specific forms of oxidant-mediated damage, including protein cleavage, lipid peroxidation, and fragmentation of DNA, will be investigated and specific mechanisms of damage characterized.
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