Title

Signal transduction during fertilization of starfish eggs

Date of Completion

January 1996

Keywords

Biology, Molecular|Biology, Cell|Biology, Animal Physiology

Degree

Ph.D.

Abstract

One of the most dramatic events of egg activation during fertilization is the rise in intracellular free calcium. Currently, the signal transduction events responsible for egg activation and the rise in intracellular free calcium are unknown. This dissertation investigates the mechanism linking sperm-egg interaction and egg activation.^ Starfish eggs were challenged with the proteases trypsin, chymotrypsin, and pronase to determine if they could be activated by an action confined to the cell surface. These enzymes elicited fertilization-like responses, including cortical granule exocytosis, elevation of a fertilization membrane, an increase in intracellular free calcium, and initiation of DNA synthesis. The egg activation event did not occur if the proteases were preincubated with inhibitors, nor if an inactive form of the enzyme was used. Reducing the amount of free calcium in the surrounding seawater to levels that would make any calcium influx physiologically insignificant did not inhibit exocytosis in response to trypsin or chymotrypsin.^ RNAs for the serotonin 2c receptor and a PDGF/FGF chimeric receptor were prepared by in vitro translation and microinjected into starfish oocytes. After a culture period, the oocytes were matured and challenged with either serotonin or PDGF, depending upon which RNA had been injected. Stimulation of either receptor was sufficient to cause a fertilization-like response, including cortical granule exocytosis, elevation of a fertilization membrane, an increase in intracellular free calcium, initiation of DNA synthesis, and in some eggs, cleavage.^ Microinjection of a fusion protein including the N- and C-terminal SH2 domains of phosphoinositide-specific phospholipase C$\sb\gamma$ inhibited the initiation of calcium release at fertilization. This inhibition could be bypassed by microinjection of IP$\sb3$, or by stimulation of exogenously expressed serotonin 2c receptor. Microinjection of a mutated form of this fusion protein had no effect on calcium release during fertilization.^ These results indicate that starfish eggs possess a cell surface protein capable of initiating egg activation and that calcium release during fertilization is initiated by SH2 domain-dependent activation of PLC$\sb\gamma$. ^

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