If not fertilized, synchronous apoptosis is induced in starfish eggs at similar to 11 h after stimulation with the hormone, l-methyladenine. In this study, a membrane-impermeant substrate of caspase-3, acetyl-Asp-Glu-Val-Asp-coumarylamido-4-methanesulfonic acid (Ac-DEVD-CAMS), was synthesized and microinjected into a starfish egg. Caspase-3 activity in unfertilized egg was detected similar to 30 min before blebbing by quantifying the accumulation rate of a membrane-impermeant, fluorogenic product, 7-aminocoumarin-4-methanesulfonic acid (ACMS), using a photomultiplier mounted on a fluorescence microscope. When active recombinant human caspase-3 was microinjected into an egg at 3 h after l-methyladenine treatment, the injected caspase-3 activity decreased and disappeared within 2 h. This decrease is probably due to proteasome-dependent degradation of the enzyme, since the injected caspase-3 was degraded and a proteasome inhibitor blocked its degradation. In contrast, in aged eggs at similar to 10 h after l-methyladenine treatment, no degradation of the injected caspase-3 was observed, suggesting that endogenous caspase-3 may stabilize at this point, therefore, inducing apoptosis. (c) 2006 Elsevier Inc. All rights reserved.