Fig. 2. ASE-triggered Ca²⁺ oscillations in enucleated eggs mimic those induced by sperm in eggs. (A) A second series of large enucleated cytoplasts was made. A large bore micropipette was advanced using a hydraulic manipulator to the edge of the egg where the chromatin was positioned (a). Suction was then applied by mouth aspirator such that a portion of the egg started to enter the pipette (b). Suction was stopped as soon as the chromatin had been removed (c), the pipette withdrawn from the egg (d,e), and the fragment expelled under gentle pressure to leave an enucleated egg and a small intact egg fragment containing the chromatin (f).Chromatin (in blue) was stained with Hoechst, and the images overlaid on a series of brightfield images. Bar, 50 µm. (Ba) ASE was injected into these large cytoplasts and the time at which the Ca²⁺ oscillations stopped [23.3±1.46 minutes (mean ± s.e.m.) n=9, three animals] was not significantly different from the mean of the times recorded for control, unmanipulated eggs (23.09±1.42 minutes, P=0.91). (b) Confirmation that these large cytoplasts could oscillate for an extended period of time. The cytoplast was first injected with 90 cyclin B1::GFP protein, and the Ca²⁺ oscillations persisted throughout the entire period they were observed (n=6, two animals).