Metaphase II-arrested mouse eggs are stimulated to complete meiosis by sperm-induced Ca2+ spiking. The Ca2+ signal causes activation of the E3 ligase anaphase-promoting complex /cyclosome (APC), leading to the destruction of key proteins necessary for meiotic exit. We show, using western blots of mouse eggs, the presence of both APC activators cdc20 and cdh1, which target D-box and D-box/KEN-box substrates, respectively, for proteolysis. We decided to examine the temporal activation of ApCcdc20 and APCcdh1 by coupling APC substrates to GFP and examining their destruction in real-time following release from second meiotic division arrest. D-box substrates were degraded quickly after the initiation of sperm-induced Ca2+ spiking, such that their degradation was complete by the time of second polar body extrusion. By contrast, KEN-box-containing substrates were degraded when CDK1 activity was low, during the period between polar body extrusion and pronucleus formation. This observation of apparent APCcdh1 activity in meiosis II based on destruction of exogenous GFP-coupled substrates was then confirmed by observing destruction of endogenous APCcdh1 substrates. These data are consistent with a model of initial APCcdc20 activation on sperm-induced activation, followed by APCcdh1 activation after second polar body extrusion. Interestingly, therefore, we propose that mammalian eggs undergo meiosis II with both APCcdc20 and APCcdh1, whereas eggs of other species so far described have APCcdc20 activity only.
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