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Fig. 4. Role of Ca2+ in mitosis entry after MAPK inactivation in P. lividus eggs. (A) The time course of Ca2+i changes in (a) unfertilized control eggs, (b) fertilized eggs or (c) unfertilized eggs treated with 1 µM U0126. The fluorescence ratio (340 nm to 380 nm) measured using fura-2 dextran indicate relative Ca2+i changes. Each color corresponds to one egg: a, no clear change was recorded in unfertilized control eggs during 3 hours; b, all Ca2+i signals were recorded in eggs showing elevation of the fertilization membrane, whereas absence of Ca2+i signal corresponded to eggs that were not fertilized; c, eggs treated with U0126 showed a slow increase in Ca2+i; calculations of average ratios indicated with an arrow at (1) 62 minutes, (2) 82 minutes and (3) 106 minutes (enlarged in inset) are given in the text and suggest small oscillations of Ca2+i level. (B) Ca-EGTA inhibits entry in mitosis triggered by 1 µM U0126. The injection buffer containing Ca-EGTA and carboxyfluorescein allowed to visualize the injected eggs (left panels). All eggs, including the injected ones, were treated with the MEK inhibitor (UO126) or not treated (Control) and observed 3 hours after treatment. Injected eggs did not show NEB, whereas non-injected eggs entered mitosis and sometimes showed constrictions. (C) Effect of U0126 or PD98059 on egg activation induced by injection of Ins(1,4,5)P3. MEK inhibitors reduced egg activation, as seen by elevation of the fertilization membrane, in eggs injected with less than 10 µM Ins(1,4,5)P3. (D) Effect of the absence of external Ca2+ on U0126-treated eggs. Eggs were treated with 5 µM U0126 (UO126) or not (Control, upper panels) in ASW containing 10 mM Ca2+ (Ca, left panels) or not (0 Ca, right panels) for 3 hours. Entry into mitosis was induced by U0126 even at zero external Ca2+, with deformations of the eggs that were even more severe.
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