Fig. 5. Ca²⁺ buffers inhibit cell cycle progression and reveal differential effects on zygotic polarization. (A) The effect of Ca²⁺ buffers on cell cycle progression. Zygotes were treated 1 hour after fertilization (white circles) or 7 hours after fertilization (black circles) with one of four treatments: biolistic FITC loading, biolistic FITC+BAPTA dextran loading, biolistic FITC+Br₂BAPTA loading or superfusion with TPEN. The effect of Ca²⁺ buffers on cell cycle progression was quantified by counting the percentage of 24-hour-old zygotes that were binucleate and had clearly undergone mitosis. Buffer concentrations were lognormally distributed so the x-axis is logarithmic. Data are means±s.e.m. with n>5 for each point. (B) The effect of Ca²⁺ buffers on zygotic polarization when applied 1 hour after fertilization. The logistic regressions of A were used to calculate cell cycle rates for each zygote and predicted length/width ratios were subtracted from measured length/width ratios to give length/width residuals (circles). Linear regressions are shown as thick black lines, with 99% confidence limits as broken black lines. The FITC dextran and Br₂BAPTA regressions do not differ significantly from the null model (dotted horizontal line), but those for BAPTA dextran and TPEN are significantly lower than the null model. (C) The effect of Ca²⁺ buffers on zygotic polarization when applied 7 hours after fertilization. Data are presented as in B. The regression lines for FITC, BAPTA dextran and Br₂BAPTA do not differ significantly from the null model. However, the regression line for TPEN is significantly lower than the null model. (D) Representative images to show the maximum inhibition of polarization seen in binucleate zygotes. Cell cycle progression and polarization may be uncoupled either by BAPTA dextran treatment 1 hour, but not 7 hours, after fertilization or by TPEN treatment either 1 or 7 hours after fertilization.