Use of the sea star oocyte as a quantitative bioassay system and characterization of endogenous Src family kinases and investigation of their role in echinoderm fertilization

The process of fertilization has been under scientific scrutiny for the better part of mans' written history, yet the mechanisms controlling this common phenomenon remain unclear. Echinoderms, such as sea urchins and sea stars, have been invaluable model organisms for investigating the underlying principles of fertilization. In addition, echinoderm oocytes have also proven a valuable in vivo assay system for characterizing the physiological function of novel proteins and metabolites. Chapter 2 of this dissertation describes the novel use of sea star oocytes to test the quantitative effects of vertebrate tau, sirtuins, and the novel metabolite, O-acetylated ADP-ribose, on meiosis, mitosis, and other microtubule mediated events. The question of how a sperm activates the developmental regimen of an egg remains unanswered. One approach to determine how the sperm accomplishes this task is to identify what proteins function in egg activation. Egg activation in echinoderms is characterized by a series of temporally invariant events, including inositol triphosphate (IP3) mediated calcium release from the egg's endoplasmic reticulum. This increase in IP3 levels appears to be mediated in echinoderms by phospholipase C-gamma (PLC-gamma), and PLC-gamma activity is most commonly regulated via tyrosine phosphorylation by either a trans-membrane or membrane associated tyrosine kinase, such as the Src Family tyrosine Kinases (SFKs). Current data using hetero-specific reagents implicates a role for both PLC-gamma and a SFK in the mechanism controlling calcium release during echinoderm fertilization. In order to establish whether endogenous SFKs were playing a role in the calcium release pathway, we identified one SFK from sea urchin (Chapter 3), and three SFKs from sea star (Chapter 4), and tested whether they met the criteria for a bona fide member of the calcium release vi pathway. The data presented in this dissertation support a role for SpSFK1 in egg activation in sea urchins, and a role for both AmSFK1 and AmSFK3 in sea star egg activation. These data represent the first identification of an endogenous member of the calcium release pathway from any system and pave the way for identifying interacting proteins, which compose the signaling complex responsible for egg activation.