Fig. 3. Degradation of securin and cyclin B1 is initiated simultaneously. Eggs were microinjected with the Ca²⁺ reporter fura2-dextran and cRNA to either securin::GFP (A,B) or cyclin B1::GFP (C). Sperm were added at the beginning of recording and following gamete fusion a series of Ca²⁺ spikes were observed that were responsible for initiating exit from MII arrest. Degradation of cyclin B1 and securin occurred at the same time: approximately 10-20 minutes after the initiation of Ca²⁺ spiking. (A) Representative securin::GFP images captured from an inseminated egg every 2 hours at the times indicated (in hours); time is relative to the addition of sperm, and in this egg polar body extrusion occurred at 2.3 hours and pronucleus formation at 6.5 hours. Scale bar: 20 µm. (B,C) Sperm induced a series of Ca²⁺ spikes that lasted for several hours (black trace), simultaneous imaging of eggs for GFP showed that degradation of both GFP constructs were complete by second polar body formation. Levels increased again as the pronuclei formed in the 1-cell embryo. A single representative recording from 16 eggs is shown for both constructs. GFP levels are represented as the mean egg average fluorescence (in arbitrary units) from a region-of-interest defined on the Metafluor program.