Fig. 1. The correlation between Ca²⁺ transients, Cdk1-cyclin B, MPF and MAP-kinase activities and pronucleus (Pn) formation. (A) Fertilization stimulates a series of Ca²⁺ transients that persist for about 4 hours, stopping close to the time of pronucleus formation. The schematics show the state of the eggs during the time course of the Ca²⁺ transients (A, inset). During the timecourse of the Ca²⁺ oscillations, Cdk1-cyclin B activity was determined by measuring H1-kinase activity and MAP-kinase activity by measuring phosphorylation of myelin basic protein (MBP). Kinase activities were recorded in unfertilized oocytes arrested at MII, in fertilized eggs that had extruded the second polar body (Pb2) within 2 hours of insemination and after Pn formation 4-6 hours after insemination. Data are from two experiments each with two replicates. Data are normalized to 100% activity in unfertilized eggs. The time that the Ca²⁺ oscillations stopped relative to the time of Pn formation is shown in Bi (n=20) and Bii (n=18). The zero time point is defined as the point at which the pronuclei were visible under bright-field observation (Bi) or by accumulation of FITC-NLS-BSA (Bii). (C) Fluorescent images of FITC-NLS-BSA (left column) and bright-field images (right column) illustrating the assessment of Pn formation. The sperm fusion site, or fertilization cone, can be seen in the first bright-field image (arrow). The first evidence of Pn formation is evident in the FITC-NLS-BSA image (arrows, Cii). The first evidence of pronuclei in the bright field optics is some 20 minutes later (arrow, Civ).

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