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Updated: Jul 27, 2025

RhoC GTPase Activation Assay
Published on: August 22, 2010
Evidence for the involvement of the Rho GTP-binding protein in egg activation of the ascidian Halocynthia roretzi
Satoshi Toratani1, Hideyoshi Yokosawa1
1Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060, Japan.
Abstract:
Eggs of the ascidian Halocynthia roretzi are activated by insemination and by treatment with calcium ionophore, leading to elevation of the vitelline coat. Here we describe the effects on egg activation of microinjection of guanosine 5'-(γ thio) triphosphate (GTPγS, a non-hydrolyzable GTP analog), heparin (an antagonist of the inositol 1,4,5-trisphosphate receptor) and a monoclonal antibody to the Rho GTP-binding protein. Microinjected GTPγS induced elevation of the vitelline coat, but not when it was co-injected with EGTA or heparin. Pre-injected heparin or the anti-Rho monoclonal antibody blocked subsequent sperm-induced elevation of the vitelline coat, but not calcium ionophore-induced elevation. We also demonstrated that the amount of cytosolic inositol 1,4,5-trisphosphate was increased by insemination. These results strongly suggest that the Rho GTP-binding protein functions prior to the heparin-blocked inositol 1,4,5-trisphosphate receptor-mediated Ca2+ release in the sperm induced activation process of H. roretzi eggs.
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