Activation of newt eggs in the absence of Ca2+ activity by treatment with cycloheximide or D2 O.

Abstract

Unfertilized eggs of urodeles that exhibit physiological polyspermy are difficult to activate by ordinary egg-activating agents, such as pricking and Ca2+ ionophores, that easily activate monospermic anuran eggs. Therefore, we have tested the effects of other agents that cause egg activation in non-amphibian species in order to investigate the mechanism of egg activation in urodeles. We have found that cycloheximide (a protein synthesis inhibitor), D2 O (that induces microtubule polymerization) and 6-DMAP (a protein kinase inhibitor) caused activation of unfertilized eggs of the newt, Cynops pyrrhogaster. The cell cycle, arrested at meiotic metaphase II, was resumed to form the second polar body accompanied by a loss of maturation promoting factor and cytostatic factor activity. The treated eggs underwent abnormal cleavage. These results indicate that protein synthesis followed by protein phosphorylation is necessary to maintain M phase in unfertilized Cynops eggs. Unfertilized eggs failed to be activated by pricking, but were activated by the ionophore A23187, but only at a concentration 30 times higher than that required to activate Xenopus eggs. Eggs whose intracellular Ca2+ ions had been chelated by BAPTA could also be activated by either cycloheximide or D2 O. Cycloheximide- as well as 6-DMAP-induced egg activations were not inhibited by nocodazole, a microtubule-depolymerizing agent. These results suggest that the inhibition of synthesis and phosphorylation of short-lived proteins acts as an egg activation mechanism, downstream of the site of Ca2+ action and independently of microtubule polymerization.