AR relies on cAMP synthesized by sAC. A, intact (nonpermeabilized) sperm were exposed to the indicated concentrations of KH7 for 15 min at 37 °C. Acrosomal exocytosis was initiated by adding 10 μm A23187 (left), 15 μm progesterone (Pg, right), or 50 μm 8pCPT-2′-O-Me-cAMP (8pCPT, right) and incubating at 37 °C for a further 15 min. Cells were fixed; acrosomal exocytosis was evaluated by FITC-PSA binding, and data were plotted without normalization (mean ± S.E. of at least three independent experiments); **, p < 0.01 compared with A23187 (left) or progesterone (right). B, SLO-permeabilized human sperm were treated for 15 min at 37 °C with increasing concentrations of KH7. AR was subsequently initiated by adding 0.5 mm CaCl₂ and incubating for 15 min at 37 °C. Exocytosis was evaluated by FITC-PSA binding, and data were normalized as described under “Experimental Procedures.” Plotted results represent the mean ± S.E. of at least three independent experiments.

Epac signaling mobilizes calcium in a PLC-dependent manner. Human sperm (nonpermeabilized) were loaded with 2 μm Fluo-3-AM and 0.02% pluronic acid and incubated for 30 min at 37 °C. At the indicated times (arrows) 50 μm 8-pCPT-2′-O-Me-cAMP (8pCPT) or 4 μm progesterone (Pg) were added with (B) or without (A) previous incubation with 1 μm U73122 or U73343. Maximal [Ca²⁺]_i response was calibrated with 0.1% Triton X-100 (TX-100) at the end of the incubation period. Shown are traces representative of five experiments. The increase in fluorescence is expressed as (F/F₀) − 1 ((maximum fluorescence intensity/initial fluorescence) − 1) versus time in seconds. Bars represent mean ± S.E. of five experiments; data were normalized against the calcium response to 4 μm progesterone (A) or 50 μm 8 pCPT-2′-O-Me-cAMP (B). ***, p < 0.001; *, p < 0.05 compared with 8 pCPT-2′-O-Me-cAMP.

Rab3A-GTP-γ-S requires cAMP, Epac, Rap1, and an active PLC to induce exocytosis. A, SLO-permeabilized human sperm were treated for 15 min at 37 °C with 10 μm KH7, 2 μg/ml PDE, or 10 μm H89 before initiating acrosomal exocytosis with 300 nm GTP-γ-S-bound Rab3A (Rab3A) and incubating at 37 °C for 15 min. Sperm were fixed; the AR was measured by FITC-PSA binding, and the data were normalized as described under “Experimental Procedures.” B, SLO-permeabilized spermatozoa were incubated with 6.7 or 134 nm anti-Epac antibodies, 20 μg/ml anti-Rap antibodies (Santa Cruz Biotechnology), 1 μg/ml Ral-GDS-RBD, or 15 μm U73122 for 15 min at 37 °C. Acrosomal exocytosis was evaluated by FITC-PSA binding after an additional 15 min of incubation at 37 °C in the absence (control) or presence of 0.5 mm CaCl₂ (calcium) or 300 nm GTP-γ-S-bound Rab3A (Rab3A). Plotted results represent the mean ± S.E. of at least three independent experiments.

Epac is not a GEF for Rab3A. The dissociation of the Rab3A-mGDP complex was monitored by a time-dependent decrease in fluorescence intensity. Rab3A (200 nm) was loaded with the fluorescent nucleotide analogue mGDP and incubated in the presence (squares) or absence (circles) of inactive (□) or activated (500 μm cAMP treated; ■) 150 nm Epac1 (left) or Epac2 (center) after the addition (●) or not (○) of a 100-fold excess (20 μm) of unlabeled GDP, as indicated under “Experimental Procedures.” For clarity of presentation and to avoid the overlay of symbols, in the left panel the data points of □ and ■ were shifted by 0.05 and 0.1 relative units, respectively. Likewise, in the center panel data points of ●, □, and ■ were shifted by 0.05, 0.10, and 0.15 units, respectively. Please note that mGDP can be displaced very slowly from Rab3A (note the difference in time scales between the right panel and the rest) by an excess of GDP; thus the Rab protein is properly folded in a native, nucleotide-interacting state. The right panel demonstrates that the Epac1 (■) and Epac2 (▴) preparations were active and exhibited GEF activity toward Rap. Briefly, Rap1 (200 nm) was loaded with the fluorescent nucleotide analogue mGDP and incubated in the absence (○) or presence of activated (100 μm cAMP treated) 150 nm Epac1 (■) or Epac2 (▴) after the addition of a 100-fold excess (20 μm) of unlabeled GDP, as indicated under “Experimental Procedures.” The data represent the mean ± S.E. of at least two independent experiments.

Pivotal role of cAMP-Epac in governing two parallel but converging signaling pathways during the late steps of the AR. SLO-permeabilized human sperm were treated for 10 min at 37 °C with 2 μg/ml PDE, 10 μm KH7, or anti-Epac (6.7 or 134 nm) antibodies (A) or anti-Rab3A (20 μg/ml), PTP1B D181A (300 nm), or NSF Y83E (186 nm) (B) followed by 0.5 mm CaCl₂ and an additional 15 min of incubation. To rescue the effect of the blockers, we added adenophostin (5 μm) and incubated for a further 10 min at 37 °C (black bars). Controls (gray bars) included the following: background AR in the absence of any stimulation (control), AR stimulated by 0.5 mm CaCl₂ (calcium), and AR inhibition by 2 μg/ml PDE, 10 μm KH7, anti-Epac (6.7 or 134 nm), and anti-Rab3A (20 μg/ml) antibodies, 300 nm PTP1B D181A, and 186 nm NSF Y83E. Sperm were fixed; acrosomal exocytosis was evaluated by FITC-PSA binding, and data were normalized as described under “Experimental Procedures.” C, SLO-permeabilized spermatozoa were loaded with 6.7 nm anti-Epac antibodies for 10 min at 37 °C to selectively block the signaling pathway leading to IP₃-sensitive calcium mobilization. AR was subsequently initiated by adding 0.5 mm CaCl₂. After 10 min of incubation at 37 °C to allow exocytosis to proceed to the Epac-sensitive step, sperm were treated with antibodies that recognize Rab3A (20 μg/ml) or NSF (1:300) and incubated for an additional 10 min at 37 °C. Finally, we added 5 μm adenophostin to rescue the anti-Epac antibody block and incubated as before (black bars). We ran several controls in parallel (gray bars) as follows: background AR in the absence of any stimulation (control), AR stimulated by 0.5 mm CaCl₂ (calcium), and the inhibitory effect of the antibodies when present throughout the experiment (anti-Epac → anti-Rab3A/NSF → calcium → adenophostin). Sperm were fixed, and AR was measured by FITC-PSA binding as described under “Experimental Procedures.” The data represent the mean ± S.E. of at least three independent experiments.

Working model for the biochemical cascade leading to the AR. Calcium enters the cell from the extracellular milieu when channels open in the plasma membrane (PM) in response to progesterone or other physiological inducers, when a calcium ionophore transports it to the cytosol, or through the SLO-generated pores. Cyclic AMP synthesized by sAC (or exogenously added 8-pCPT-2′-O-Me-cAMP) activates Epac, which directly activates Rap1. In turn, active Rap1 stimulates the synthesis of IP₃ through the stimulation of a PLC activity. This IP₃ (or exogenously added adenophostin) elicits the efflux of calcium from an IP₃-sensitive store (likely the acrosome). 2-APB blocks calcium efflux through IP₃-sensitive channels (data not shown). Epac-cAMP also indirectly activates Rab3A, triggering the tethering of the acrosome to the plasma membrane through the assembly of large macromolecular complexes. A reaction taking place during or as a consequence of tethering initiates the activation and/or recruitment of PTP1B, which in turns activates NSF. Next, NSF/α-SNAP render SNARE proteins fusion-competent. Both a local increase in calcium coming from the acrosome through IP₃-sensitive channels and SNAREs converge to accomplish the final steps of membrane fusion. Recombinant Rab3A (rRab3A-GTP) initiates the AR through an alternative pathway (gray) that presumably begins with the recruitment of RIM to achieve tethering. The large quantities of RIM recruited by recombinant Rab3A-GTP-γ-S bind Epac and the intracellular calcium pathway proceeds as described. OAM, outer acrosomal membrane. Solid arrows mean there is one step between the terms connected, and dashed arrows mean that the number of steps is either unknown or not depicted for simplicity.

Activation of Epac by 8-pCPT-2′-O-Me-cAMP stimulates a GEF for Rab3A during sperm exocytosis. A, permeabilized spermatozoa were incubated for 10 min at 37 °C with 4 μm GDI (in all the experiments using GDI, the buffer was supplemented with 0.7 mm MgCl₂ and 0.2 mm GTP) followed or not by 300 nm Rab3A-GDP and further incubation for 10 min at 37 °C. Acrosomal exocytosis was initiated by adding 50 μm 8-pCPT-2′-O-Me-cAMP and incubating for an additional 10 min at 37 °C (black bars). Controls (gray bars) included the following: background AR in the absence of any stimulation (control), AR stimulated by 0.5 mm CaCl₂ (calcium) and 50 μm 8-pCPT-2′-O-Me-cAMP (8pCPT), and lack of effect on the AR of 300 nm Rab3A-GDP and 4 μm GDI plus 300 nm Rab3A-GDP. Sperm were fixed; acrosomal exocytosis was evaluated by FITC-PSA binding, and data were normalized (mean ± S.E.) as described under “Experimental Procedures.” B, SLO-permeabilized spermatozoa were incubated for 10 min at 37 °C with 5 μg/ml RIM-(11–398) followed or not by 300 nm Rab3A-GDP and further incubation for 10 min at 37 °C. Acrosomal exocytosis was initiated by adding 0.5 mm CaCl₂ or 50 μm 8-pCPT-2′-O-Me-cAMP (8pCPT) and incubating for an additional 10 min at 37 °C (black bars). Controls (gray bars) included the following background AR in the absence of any stimulation (control), AR stimulated by 0.5 mm CaCl₂ (calcium) and 50 μm 8-pCPT-2′-O-Me-cAMP (8pCPT), and lack of effect on the AR of 300 nm Rab3A-GDP. Samples were processed, and the AR was scored as in A. C, capacitated human sperm were permeabilized with SLO, and the onset of the AR was prevented by 100 μm 2-APB. Sperm suspensions were subsequently incubated with (right) or without 20 μg/ml anti-Rap1 antibodies or with 1 μg of His₆-Rab3A (top left). AR inducers 50 μm 8-pCPT-2′-O-Me-cAMP (top left and right) and 0.5 mm CaCl₂ (bottom left) were added and incubations proceeded as described under “Experimental Procedures.” Whole sperm lysates were subjected to pulldown assay using GST-RIM-Sepharose, and the levels of GTP-bound Rab3A were determined by Western blot with an anti-Rab3A antibody. Shown are experiments representative of three repetitions; right, quantification (carried out with Image J, freeware from National Institutes of Health) is depicted above the immunoblot as mean ± S.E. from all replicates; different letters indicate statistical significance (p < 0.05).

Rap1 participates in a pathway that mobilizes calcium from an IP₃-sensitive store. A, permeabilized spermatozoa were loaded with 10 μm NP-EGTA-AM (NP) for 10 min at 37 °C to chelate intra-acrosomal calcium. The AR was subsequently initiated by adding 0.5 mm CaCl₂. After a further 15-min incubation at 37 °C to allow exocytosis to proceed up to the intra-acrosomal calcium-sensitive step, sperm were treated for 15 min at 37 °C with 20 μg/ml anti-Rap antibody (Santa Cruz Biotechnology) or 1 μg/ml Ral-GDS-RBD. All these procedures were carried out in the dark. UV flash photolysis of the chelator was induced at the end of the incubation period (hv), and the samples were incubated for 5 min (NP → calcium → inhibitor → hv; black bars). Several controls were run (gray bars) as follows: background AR in the absence of any stimulation (control); AR stimulated by 0.5 mm CaCl₂ (calcium), inhibitory effect of NP-EGTA-AM in the dark (NP → calcium → dark), and the recovery upon illumination (NP → calcium → hv); and the effect of the inhibitors when present throughout the experiment (NP → inhibitor → calcium → hv). Sperm were fixed; the AR was measured by FITC-PSA binding, and the data were normalized as described under “Experimental Procedures.” B, permeabilized spermatozoa were incubated with 15 μm U73343 or U73122 for 15 min at 37 °C. Acrosomal exocytosis was then initiated by adding 0.5 mm CaCl₂ (calcium) or 50 μm 8-pCPT-2′-O-Me-cAMP (8pCPT) and a further 15-min incubation at 37 °C (black bars). Sperm were stained, and the AR scored as in A. C, permeabilized spermatozoa were incubated with 20 μg/ml anti-Rap antibodies (Santa Cruz Biotechnology), 1 μg/ml Ral-GDS-RBD, or 15 μm U73122 for 10 min at 37 °C. Exocytosis was initiated by adding 0.5 mm CaCl₂. To demonstrate that Rap1-GTP and an active PLC were located upstream of the intracellular calcium efflux, 5 μm adenophostin was added to mobilize calcium from IP₃-sensitive stores, and incubations proceeded for an additional 10 min at 37 °C (black bars). Controls (gray bars) included the following: background AR in the absence of any stimulation (control), AR stimulated by 0.5 mm CaCl₂ (calcium), AR inhibition by 20 μg/ml anti-Rap antibodies, 1 μg/ml Ral-GDS-RBD, or 15 μm U73122, and AR unaffected by 5 μm adenophostin. Sperm were stained, and the AR scored as in A. Plotted results represent the mean ± S.E. of at least three independent experiments.

Calcium and 8-pCPT-2′-O-Me-cAMP activate Rap1 during sperm exocytosis. A, proteins from whole sperm homogenates (100 × 10⁶ cells) were partitioned in Triton X-114 and precipitated with organic solvents as described under “Experimental Procedures.” Proteins from the aqueous and detergent phases (normalized by initial cell counts) were resolved in 10% Tricine/SDS-polyacrylamide gels and immunoblotted with the custom-made anti-Rap1 antibody. Molecular mass markers are indicated on the left. B, top, SLO-permeabilized human sperm were treated with 20 μg/ml of an anti-Rap1 polyclonal antibody (Santa Cruz Biotechnology) for 15 min at 37 °C. Acrosomal exocytosis was subsequently initiated by adding 50 μm 8-pCPT-2′-O-Me-cAMP (8pCPT, black bar) and incubating for a further 15 min at 37 °C. Bottom, SLO-permeabilized human sperm were incubated for 15 min at 37 °C with 20 μg/ml of the commercial or the custom-made (CM) anti-Rap antibodies (black bars). Asterisks indicate that the antibodies had been pretreated with 205 μg/ml recombinant Rap (anti-Rap* → calcium) or 453 μg/ml Rap peptide (CM-anti-Rap* → calcium). Sperm were challenged with 0.5 mm CaCl₂ and incubated for an additional 15 min at 37 °C. Control condition (gray bars) include the following: background AR in the absence of any stimulation (control), AR induced with 0.5 mm CaCl₂ (calcium), or 50 μm 8-pCPT-2′-O-Me-cAMP (8pCPT); lack of effect on the AR of recombinant Rap1 and 200 μm 6-Bnz-cAMP alone; and unaffected exocytosis by 20 μg/ml anti-Rab11 and nonimmune antibodies (anti-Rab11/IgG → calcium). Cells were fixed; acrosomal exocytosis was evaluated by FITC-PSA binding, and data were normalized as described under “Experimental Procedures.” C, sperm suspensions (20 × 10⁶ cells) were treated (+) or not (−) with 50 μm 8-pCPT-2′-O-Me-cAMP for 10 min at 37 °C in the presence of 100 μm 2-APB to prevent protein loss due to exocytosis. Cells were disrupted; whole cell lysates were subjected to pulldown assays using GST-Ral-GDS-RBD-Sepharose, and the levels of GTP-bound Rap1 were determined as described under “Experimental Procedures.” Shown is a blot representative of three repetitions. D, permeabilized spermatozoa were incubated with 1 μg/ml Ral-GDS-RBD or 1 μg/ml GST for 15 min at 37 °C. Acrosomal exocytosis was then initiated by adding 0.5 mm CaCl₂ (calcium) or 50 μm 8-pCPT-2′-O-Me-cAMP (8pCPT) and a further 15-min incubation at 37 °C. Sperm were stained and the AR scored as in B. The data represent the mean ± S.E. of at least three independent experiments.