Degree Type

Dissertation

Date of Award

1997

Degree Name

Doctor of Philosophy

Department

Zoology and Genetics

First Advisor

Sheldon Shen

Abstract

The requirement for both inositol-1,4,5 trisphosphate (InsP3) and ryanodine-sensitive intracellular Ca2+ release mechanisms during fertilization was studied in sea urchin eggs. The postulated pathway of cGMP-dependent protein kinase (PKG) activation of ADP-ribosyl cyclase for production of cADPR to activate the ryanodine receptor Ca2+ channel was tested with a variety of activators and inhibitors. Our observations are consistent with Ca2+ release by cGMP in the egg being dependent on an isoform of PKG that is distinct from the mammalian enzyme. PKG activity in the sea urchin egg was activated by cIMP and was insensitive to cGMP analogs, which are normally an inhibitor and potent activators of mammalian isozymes, respectively. Surprisingly inhibitors of either PKG or ADP-ribosyl cyclase activities did not prevent the transient rise in intracellular Ca2+ activity ((Ca2+] i) in 0.7-1.0 mg/mL heparin-loaded eggs during fertilization. These results suggest the synthesis of cADPR during fertilization is not necessary for regulating the Ca2+ event. The production on inositol 1,4,5-triphosphate (InsP3) to mediate the transient rise in (Ca2+] i in sea urchin eggs during fertilization was studied by inhibiting the hydrolydid of phosphatidylinositol-4,5-bisphosphate to InsP3 and 1,2-diacylglycerol by phospholipase C (PLC). U73122, a PLC inhibitor, eliminated the sperm-induced Ca 2+ release in a dose-dependent manner. It also prevented the accompanying rise intracellular pH (pH i), which is mediated by the activation of the Na+-H+ antiporter. The antiporter is regulated through activation of protein kinase C by 1,2-diacylglycerol. U73122 inhibition was not due to a failure of fertilization, since incorporated sperm pronuclei were evident in U73122-treated eggs. The inhibition of InsP3 production during the first 2 min of fertilization by U73122 was confirmed by InsP3 mass measurements. In addition, U73122 also inhibited the GTP[gamma]S-induced Ca2+ release and pH i rise in unfertilized eggs. These results suggested that the transient rise in Ca2+ in sea urchin during fertilization requires the production of InsP3 via a PLC[beta]-dependent pathway. In summary, the InsP3-mediated pathway is the primarily required mechanism to regulate the rise in (Ca2+] i during fertilization in sea urchin eggs.

DOI

https://doi.org/10.31274/rtd-180813-13280

Publisher

Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/

Copyright Owner

Shyh-Jye Lee

Language

en

Proquest ID

AAI9814661

File Format

application/pdf

File Size

105 pages

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