Figure 6

Extracellular calcium elicits calcium release from the acrosomal store through IP ₃ -gated channels in a cAMP/Epac and Rab3-dependent manner. SLO-permeabilized human sperm were treated with 100 nM botulinum toxin B to prevent exocytosis, loaded with the fluorescent calcium indicator Fluo3-AM (2 μM) and incubated with 100 μM 2-APB (D), 10 μM KH7 (E), 6.7 nM anti-Epac (F) or 70 nM anti-Rab3A (G) antibodies, 300 nM PTP1BD181A (H). Samples were stimulated with 0.5 mM CaCl₂ (calcium, B, D-H) or 5 μM adenophostin A (AdA, C) and the fluorescence intensity visualized as described in the Methods section. Images at top are pseudo-coloured to show intensity of fluorescence. Scale is shown on right (“warm” colours show high [Ca²⁺]). Changes in fluorescence are localised to the acrosome; fluorescence in the mid piece was invariant. Scale bars = 5 μM. Each line graph shows the recording of [Ca²⁺] changes in the cell shown above. Plotted are the normalized Fluo3 fluorescence variations in the acrosome (ac, red) and the mid piece (mp, green) in response to the application of CaCl₂ or adenophostin A (indicated by vertical arrows) vs. time. Bar graphs illustrate the population response to each treatment (mean ± SEM; 17-50 cells in three experiments). Bar graphs show the relative fluorescence from the acrosomal (ac, red) and mid piece (mp, green) regions at the beginning (0’sec, assigned 100%) and 300’seconds after the initiation of the recording. Asterisks indicate significant difference (**P < 0.01, ***P < 0.001) from the initial value (single group analysis, 99.9% confidence interval), ns indicates that statistical differences between bars were non-significant (P > 0.05). Note: around 80% cells responded to CaCl₂ in all experiments included in the analysis.