Figure 4.

Active Rab3A triggers cortical granule exocytosis. A) MII eggs were collected and cortical granule exocytosis was not triggered (control) or triggered by in vitro fertilization (IVF), by calcium ionophore (A23187), and SrCl₂ (SrCl₂). After removing the zona pellucida, cells were fixed and mounted for cortical granules’ quantification. Shown are representative images for each condition. Cortical granules were stained with LCA conjugated with FITC (LCA-FITC) and DNA was stained with Hoechst 33342 (DIC-Hoechst). B) Quantification of cortical granules density (CG/100 μm²) in MII eggs activated by in vitro fertilization (IVF), calcium ionophore(A23187) and SrCl₂(SrCl₂). The graph shows raw data for each condition (not normalized). Data represent the means ± SEM of at least three independent experiments. The asterisks indicate significant differences from control condition (***, p< 0.001). C) Quantification of cortical granules density (% CG/100 μm²) in MII eggs activated by SrCl₂ (SrCl₂) and after microinjection of purified and prenylated GST-Rab3A loaded with GTPγS (GTP) or GDPβS (GDP). As negative controls, non-prenylated GST-Rab3A (none), mock preparations without recombinant Rab3A (mock) and glutathione-S-transferase (GST) were tested in the assay. Data were normalized as explained in Materials and Methods and represent the means ± SEM of at least three independent experiments. The asterisks indicate significant differences from control condition (***, p<0.001). D) Quantification of cortical granules density (% CG/100 μm²) in MII eggs activated by SrCl₂ (SrCl₂) and active Rab3A (microinjected Rab3A-GTP) in CZB prepared without (−Ca²⁺) or with calcium (+Ca²⁺). Data represent the means ± SEM of at least three independent experiments. The asterisks indicate significant differences from control condition (***, p< 0.001).