Evidence for the involvement of a pertussis toxin-insensitive G-protein in egg activation of the frog, Xenopus laevis

Abstract

Activation responses of the frog egg at fertilization include the release of calcium from intracellular stores and the opening of calcium-dependent chloride channels, which produce the fertilization potential. To investigate the presence of guanine nucleotide-binding proteins (G-proteins), and their role in initiation of these events in the egg of the frog Xenopus laevis, we assayed for pertussis and cholera toxin substrates, and applied activators and inhibitors of G-proteins. Pertussis toxin catalyzed the [32P]ADP ribosylation of a Mr 40,000 component, but no cholera toxin substrates were demonstrated. Injection of greater than or equal to 25 pmole of guanosine-5'-O-(3-thiotriphosphate) GTP-gamma-S), an activator of G-proteins, produced a change in membrane potential that mimicked the fertilization potential and also caused cortical granule exocytosis and cortical contraction. Injections of up to 600 pmole of guanosine 3':5'-cyclic monophosphate or 9 nmole of guanosine-5'-(beta-gamma-imido)triphosphate did not active eggs. The membrane potential response to GTP-gamma-S injection showed the same peak and chloride dependence as the fertilization potential, although the duration of the GTP-gamma-S response was somewhat greater. GTP-gamma-S did not activate eggs if the calcium rise was prevented by prior injection of the calcium chelator BAPTA. Injection of up to 200 ng of cholera toxin did not activate eggs. However, eggs were activated by applying 1 nM serotonin to eggs that had been injected with a specific mRNA for the serotonin 1c receptor, a member of the class of receptors that act by way of G-proteins. Egg activation in response to either sperm or serotonin was not inhibited by pertussis toxin, under experimental conditions where approximately 80-90% of the toxin substrate was ADP-ribosylated. These results support the hypothesis that sperm activate Xenopus eggs at fertilization by way of a pertussis and cholera toxin-insensitive G-protein.