Fig. 3. Ca²⁺ buffers inhibit actin nucleation and DNA replication, but not pronuclear motion, in F. serratus zygotes. (A) The speeds at which sperm pronuclei progress towards egg pronuclei did not differ significantly between FITC-loaded and BAPTA dextran-loaded zygotes. Data are mean±s.e.m. (B) Pronuclear fusion in zygotes loaded with the Ca²⁺ buffer BAPTA dextran (0.4 mM). In these representative images, the sperm pronucleus ('sp') was labelled with 100 µM Hoechst 33342 and visualized using 2-photon microscopy. The left-hand image, taken 120 minutes after fertilization, shows the sperm pronucleus as a bright blue dot sitting next to the egg pronucleus ('ep') against a green FITC background (co-loaded with BAPTA dextran). In the right-hand image, taken 20 minutes later, the sperm pronucleus is less bright and has started to decondense, indicating that pronuclear fusion has started. (C) Untreated polarized 24-hour-old zygotes stained with Texas Red phalloidin to show actin nucleation in a cone at the germinating rhizoid apex. (D) Zygotes loaded biolistically with the Ca²⁺ buffer BAPTA dextran (0.4 mM) 30 minutes after fertilization and stained with Texas Red phalloidin 24 hours after fertilization. Neither zygotic polarization nor actin nucleation was observed (n=8). (E) Zygotes treated continuously with the CDK inhibitor olomoucine (100 µM) did not display zygotic polarization, but did show non-polarized cortical actin nucleation when stained with Texas Red phalloidin 24 hours after fertilization. (F) The Ca²⁺ buffer BAPTA dextran (0.4-0.8 mM) inhibited S-phase progression when loaded into zygotes 30 minutes after fertilization. Zygotes were biolistically loaded with GFP-PCNA, Texas Red dextran and BAPTA dextran and nuclear GFP-PCNA/Texas Red ratios (R) were plotted relative to their starting values (R₀) for control (white circles) and BAPTA dextran-treated zygotes (black circles). Control zygotes displayed significant nuclear GFP-PCNA increases between 3 and 5 hours after fertilization (see also Fig. 1B), but these increases were completely abolished following treatment with BAPTA dextran. Data are mean±s.e.m.; ^*P<0.05.