Identification and Characterization of Signaling Complexes Required for Egg Activation at Fertilization

University Of California-Santa Barbara, Santa Barbara CA

Investigators

Abstract

0110790 Foltz Fertilization initiates the start of the development of a new organism. Sperm binding and fusion launch the egg from a quiescent state into an active state, committed to cell cycle entry. How does the meeting of these two cells signal the activation of development? In particular, the release of Ca2+ from internal egg stores is a hallmark of egg activation and has been the focus of intensive study. Studies using echinoderm (sea urchin and sea star) gametes have suggested a model for a signal transduction pathway that leads to initiation of Ca2+ release. The working model is that sperm-egg interaction activates a Src family tyrosine kinase(s) (SFK) that in turn (directly or indirectly) activates PLCg, causing the production of IP3 and subsequent release of Ca2+ from internal stores in the egg. This research will test the current signaling pathway model by identifying and characterizing the endogenous components in the egg, evaluating their interactions, and investigating their mode of regulation. The results of the proposed experiments should provide insight into how signaling components are organized in the sea urchin egg and interact with one another to trigger egg activation. There are two specific aims. First, the Src-type egg tyrosine kinase(s) that is activated at fertilization will be identified and characterized. cDNAs encoding egg SFKs are being cloned and antibodies will be raised against the proteins. The necessity of the SFK(s) for Ca2+ release will be tested by inhibiting its function using a dominant-interfering approach. The antibodies will be used to characterize the protein's activity at fertilization, localization in the egg, and potential interaction with PLCg and other egg signaling or cytoskeletal components. The second aim will investigate the mechanism of PLCg activation. The interaction of an egg SFK(s) with egg PLCg will be investigated, as will the localization and tyrosine phosphorylation state of PLCg. Together, the results from the proposed work should provide insight into how sperm-egg interaction results in one of the most important and universal events of egg activation, the release of Ca2+ in the egg.