SNARE Complex Assembly Is Required for Human Sperm Acrosome Reaction

Developmental Biology 243, 326 –338 (2002) doi:10.1006/dbio.2002.0567, available online at http://www.idealibrary.com on

SNARE Complex Assembly Is Required for Human Sperm Acrosome Reaction Claudia N. Tomes,* ,1 Marcela Michaut,* Gerardo De Blas,* Pablo Visconti,† Ulf Matti,‡ and Luis S. Mayorga* *Laboratorio de Biologı´a Celular y Molecular, Instituto de Histologı´a y Embriologı´a (IHEMCONICET), Facultad de Ciencias Me´dicas, CC 56, Universidad Nacional de Cuyo, 5500 Mendoza, Argentina; †Department of Cell Biology, Center for Recombinant Gamete Contraceptive Vaccinogens, University of Virginia, Room 3-101 Jordan Hall, 1300 Jefferson Park Avenue, Charlottesville, Virginia 22903; and ‡Medizinische Hochschule Hannover, Zentrum Biochemie, Institut fu¨r Physiologische Chemie, OE 4310, Carl-Neuberg-Strasse 1, 30623 Hannover, Germany

Exocytosis of the acrosome (the acrosome reaction) is a terminal morphological alteration that sperm must undergo prior to penetration of the extracellular coat of the egg. Ca 2ⴙ is an essential mediator of this regulated secretory event. Aided by a streptolysin-O permeabilization protocol developed in our laboratory, we have previously demonstrated requirements for Rab3A, NSF, and synaptotagmin VI in the human sperm acrosome reaction. Interestingly, Rab3A elicits an exocytotic response of comparable magnitude to that of Ca 2ⴙ. Here, we report a direct role for the SNARE complex in the acrosome reaction. First, the presence of SNARE proteins is demonstrated by Western blot. Second, the Ca 2ⴙ-triggered acrosome reaction is inhibited by botulinum neurotoxins BoNT/A, -E, -C, and -F. Third, antibody inhibition studies show a requirement for SNAP-25, SNAP-23, syntaxins 1A, 1B, 4, and 6, and VAMP 2. Fourth, addition of bacterially expressed SNAP-25 and SNAP-23 abolishes exocytosis. Acrosome reaction elicited by Rab3-GTP is also inhibited by BoNT/A, -C, and -F. Taken together, these results demonstrate a requirement for members of all SNARE protein families in the Ca 2ⴙ- and Rab3A-triggered acrosome reaction. Furthermore, they indicate that the onset of sperm exocytosis relies on the functional assembly of SNARE complexes. © 2002 Elsevier Science (USA) Key Words: acrosome; calcium; exocytosis; Rab3; SNAP-25; SNARE; sperm; syntaxin; toxin; VAMP.

INTRODUCTION Fertilization is the process whereby individual gametes from the female (the egg) and male (the sperm) unite to produce offspring (Yanagimachi, 1994). Interaction with the egg is restricted to the sperm head, which contains a large secretory vesicle, the acrosome, overlying the sperm’s nucleus (Yanagimachi, 1994). The acrosomal membrane underlying the plasma membrane is referred to as the “outer” acrosomal membrane, and that overlying the nucleus is referred to as the “inner” acrosomal membrane. Exocytosis of the acrosome (the acrosome reaction, AR) 2 is 1

To whom correspondence should be addressed. Fax: 54-261449-4117. E-mail: [email protected]. 2 Abbreviations used: AR, acrosome reaction; BoNT, botulinum neurotoxin; BSA, bovine serum albumin; DTT, dithiothreitol; PBS,

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a terminal morphological alteration that must occur prior to penetration of the extracellular coat of the egg (zona pellucida). As is the case in somatic cells’ regulated exocytosis, Ca 2⫹ is an essential mediator of the AR (Florman et al., 1998). The AR differs strikingly from other known exocytotic events in ways that can be summarized as follows: (1) sperm contain a single secretory vesicle; (2) there are multiple fusion points between the outer acrosomal membrane and

phosphate buffered saline; PVDF, polyvinylidene fluoride; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulphate; SNAP-25/23, synaptosome-associated protein of 25/23 kDa; SLO, streptolysin-O; SNARE, SNAP receptor; TPEN, N,N,N⬘,N⬘tetrakis (2-pyridymethyl) ethylenediamine; VAMP, vesicle-associated membrane protein. 0012-1606/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved.

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the overlying plasma membrane; (3) exocytosis does not lead to a single fused membrane, but to vesiculation and actual membrane loss; and (4) the AR is a singular event, with no known membrane recycling taking place following exocytosis. Although several aspects of the AR have been studied extensively, the intrinsic mechanism of outer acrosomal to plasma membrane fusion is still unclear. In many exocytotic cell types, of which neurons and neuroendocrine cells are the most studied, membrane fusion is governed by conserved sets of proteins that have evolved into a few protein families. These proteins include GTPases of the rab family (Novick and Zerial, 1997; Zerial and McBride, 2001), the sec1p/munc-18 protein family (Pevsner, 1996), and SNARE proteins (Gerst, 1999; Chen and Scheller, 2001). The SNARE protein superfamily contains the syntaxin, SNAP-25, and synaptobrevin/VAMP protein families. Syntaxins and SNAP-25-like proteins were originally designated as t-SNAREs due to their localization on target membranes, whereas the vesicular VAMPs were termed v-SNAREs. More recently, these proteins have been reclassified as R-SNAREs (arginine-containing SNAREs) or Q-SNAREs (glutamine-containing SNAREs), based on the identity of a highly conserved residue (Fasshauer et al., 1998). Syntaxin and SNAP-25 are Q-SNAREs, whereas VAMP is an R-SNARE. The Q- and R-SNAREs form tight heterotrimeric complexes during the fusion process. These ternary complexes are highly resistant to denaturing reagents, but undergo cyclic assembly and disassembly with the assistance of chaperone-like molecules known as NSF and SNAPs (Sollner et al., 1993a,b). It is thought that the energy released during complex formation is transfered to the membranes via the proteins membrane anchors and used to initiate membrane fusion by pulling the membranes together. In vitro, isolated sets of cognate SNARE proteins are able to drive liposome fusion, identifying SNAREs as part of the minimal machinery for membrane fusion (Weber et al., 1998). Syntaxin and VAMP are anchored to the lipid bilayer by transmembrane regions found at their carboxy termini, whereas SNAP-25, which lacks a transmembrane region, is thought to be anchored by palmitoylation of a cysteine-rich sequence in the center of the molecule. Interestingly, initial tethering of newly synthesized SNAP-25 to membranes of PC12 cells is a consequence of its association with syntaxin and does not require palmitoylation (Vogel et al., 2000; Gonelle-Gispert et al., 2000). Once the SNAP-25/ syntaxin heterodimers reach the cell surface, palmitoylation consolidates membrane anchorage. In addition to interacting with SNARE proteins, SNAP-25 binds to synaptotagmin (Gerona et al., 2000) and to calcium channels (Rettig et al., 1996; Wiser et al., 1996). SNAP-23, an ubiquitously expressed isoform of SNAP-25, also contains a central cysteine-rich region and binds tightly to multiple syntaxins and VAMPs (Ravichandran et al., 1996). The notion that VAMP, syntaxin, and SNAP-25 have a direct function in synaptic vesicle exocytosis has received strong support from the identification of these proteins as the targets of clostridial neurotoxins. Tetanus toxin and

seven structurally related botulinal neurotoxins (BoNT/A, -B, -C1, -D, -E, -F, and -G) are potent inhibitors of neurotransmitter release due to their highly specific, zincdependent proteolytic cleavage of SNARE proteins. BoNT/A and -E cleave SNAP-25, and BoNT/C cleaves syntaxin and, albeit with lower efficiency, SNAP-25. The remaining BoNTs, as well as tetanus toxin, are specific for VAMP (Pellizzari et al., 1999). SNARE proteins are only vulnerable to neurotoxins when not assembled in tight ternary complexes (Hayashi et al., 1994). Despite all the advances made in unveiling the biochemistry behind membrane fusion events, a still unresolved issue is the mechanism whereby Ca 2⫹ triggers regulated exocytosis. Progress in this field has been hindered by the limited number of membrane fusion systems that can be readily manipulated to dissect the process. We have developed a streptolysin O (SLO)-permeabilized sperm model capable of undergoing acrosome content release upon stimulation with Ca 2⫹ (Diaz et al., 1996; Yunes et al., 2000). Taking advantage of this approach, we have demonstrated the involvement of Rab3A (Yunes et al., 2000), NSF (Michaut et al., 2000), and synaptotagmin VI (Michaut et al., 2001) in the Ca 2⫹-dependent AR of human sperm. Here, we report the requirement of the SNARE complex in the Ca 2⫹ and Rab3A-triggered AR.

MATERIALS AND METHODS Reagents Streptolysin O (SLO) was obtained from Murex (Dartford, UK). Gamete Preparation Media (GPM; Serono, Madrid, Spain) was used as spermatozoa culture medium. Anti-syntaxin 4 and 6 antibodies (mouse monoclonals, purified IgG 1, clones 49 and 30, respectively) were from Transduction Laboratories (Lexington, KY). Anti-VAMP and anti-VAMP-2 (mouse monoclonals, isotype IgG 1, clones 10.1 and 69.1), anti-syntaxin 1A and 1B (rabbit polyclonals, whole serum), and anti-SNAP-25 (mouse monoclonal, isotype IgG 1, clone 71.1) were from Synaptic Systems (Go¨ ttingen, Germany). An antimembrin antibody (mouse monoclonal, purified IgG 1) was from StressGen (Victoria, BC, Canada). An anti-SNAP-23 antibody (rabbit polyclonal, purified IgG) was a kind gift from Dr. Sidney Whiteheart (University of Kentucky, Lexington, KY). Affinitypurified rabbit anti-mouse IgG (whole molecule), horseradish peroxidase-conjugated goat anti-rabbit IgG (whole molecule and Fc-specific), and horseradish peroxidase-conjugated goat antimouse IgG (whole molecule and Fc-specific) were purchased from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA). ImmunoPure Plus Immobilized Protein A was from Pierce (Rockford, IL). BoNT/A (Hall strain), F (160 strain), and C (London strain) holotoxins were a kind gift from Ms. Laura de Jong and Dr. Rafael Ferna´ ndez (Universidad Nacional de Cuyo, Mendoza, Argentina). Prestained molecular weight standards were from BRL/Life Technologies, Inc. (Gaithersburg, MD). Nickel-nitrilotriacetic acid agarose was from Qiagen (Hilden, Germany). All other reagents were from Sigma (St. Louis, MO).

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Recombinant Proteins The expression plasmid pGEX2T containing the cDNAencoding human Rab3A was generously provided by Drs. M. I. Colombo and P. D. Stahl (Washington University, St. Louis, MO). An expression plasmid encoding human SNAP-23 (pET-His 6hSNAP-23) was a kind gift from Dr. T. Galli (Institute Curie, Paris, France). Plasmids encoding the light chain of BoNT/E (BoNT/E-LC) and pBN10 coding for SNAP-25-His 6 were generously provided by Dr. T. Binz (Medizinische Hochschule Hannover, Hannover, Germany). GST-Rab3A was expressed in Escherichia coli strain XL1blue (Stratagene, La Jolla, CA), purified, prenylated, and activated as previously described (Yunes et al., 2000). SNAP-23 and SNAP 25 were expressed and purified as described in E. coli strains BLR(DE3) (Novagen, Madison, WI) and M15pRep4 (Qiagen), respectively (Binz et al., 1994). His 6-tagged BoNT/E-LC was expressed and purified as in Vaidyanathan et al. (1999).

SLO Permeabilization and AR Assay Human semen samples were obtained from normal healthy donors. Highly motile sperm were recovered following a swim-up separation for 1 h in GPM medium at 37°C in an atmosphere of 5% CO 2/95% air. Concentration was then adjusted to 5–10 ⫻ 10 6 ml, and incubation proceeded for at least 2 h under conditions that support capacitation (GPM medium, 37°C, 5% CO 2/95% air). Permeabilization was accomplished as described (Yunes et al., 2000). Briefly, washed spermatozoa were resuspended in cold PBS containing 0.4 U/ml SLO for 15 min at 4°C. Cells were washed once with PBS, resuspended in ice-cold sucrose buffer (250 mM sucrose, 20 mM Hepes-K 0.5 mM EGTA, pH7) containing 2 mM DTT, and inhibitors were added when indicated and further incubated for 15 min at 37°C. BoNTs were preactivated by incubation for 15 min at 37°C with 5 mM DTT in the presence or absence of 5 mM TPEN. After addition of stimulants to the sperm suspensions, incubation proceeded at 37°C for 15 min. A total of 10 ␮l of each reaction mixture was spotted on eight-well slides, air dried, and fixed/permeabilized in ice-cold methanol for 30 s. Acrosomal status was evaluated by staining with fluorescein isothiocyanatecoupled Pisum sativum according to Mendoza et al. (1992). At least 200 cells were scored by using a Nikon microscope equipped with epifluorescence optics. Negative (no stimulation) and positive (10 ␮M calcium) controls were included in all experiments. For each experiment, the data were normalized by subtracting the number of reacted spermatozoa in the negative control from all values and expressing the result as a percentage of the AR observed in the positive control.

Immunoprecipitation Sperm incubated and washed as described were lysed in cold 50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 5 ␮g/ml trypsin inhibitor, 5 ␮g/ml pepstatin, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM benzamidine, 1% NP 40, 1% deoxycholate, and 0.1% SDS (RIPA). After sonicating two times for 15 s and extraction for 30 – 60 min at 4°C, the sperm extracts were clarified by centrifugation at 12,000g for 5 min and used immediately or stored at ⫺20°C. A total of 100 ␮l of sperm lysate (5 ⫻ 10 7 cells) was incubated with 3 ␮l of a mouse monoclonal anti-SNAP-25 antibody overnight at 4°C. A purified rabbit anti-mouse IgG (13 ␮g) was immobilized on 10 ␮l (50% slurry) protein A agarose by incubation overnight at 4°C before immunocomplexes recovery by incubation for 1 h at

room temperature with constant inversion. Beads were washed (three times) with washing buffer (20 mM Tris–HCl, pH 7.2, 150 mM NaCl, 0.2% NP 40, 2 mM EDTA, and the same protease inhibitors described above) and then boiled twice in sample buffer. Extracted proteins were recovered by centrifugation and then subjected to SDS-PAGE. Immunoprecipitation with a rabbit polyclonal anti-SNAP-23 antibody was carried out as described, except that the rabbit anti-mouse antibody was omitted.

SDS-PAGE and Immunoblot Analysis Proteins were separated on polyacrylamide slab gels according to Laemmli (1970) and were Coomassie blue-stained or transferred to 0.2-␮m PVDF membranes (Pierce) (Towbin et al., 1979). Nonspecific reactivity was blocked by incubation for 1 h at room temperature with 0.5% BSA/2% horse serum (SNAP-23/25) or 5% non-fat dry milk (syntaxin 1B/4/6) dissolved in washing buffer (50 mM Tris–HCl, pH 7.6, 100 mM NaCl, 0.1% Tween 20, 0.2% gelatin). Blots were incubated with the primary antibodies (1:10,000 antiSNAP-25; 1:20,000 anti-SNAP-23; 1: 5,000 anti-syntaxins 1B, 4, and 6) for 60 min at room temperature. Horseradish peroxidaseconjugated goat anti-mouse and goat anti-rabbit IgGs were used as secondary antibodies with 45-min incubations. Excess first and second antibodies were removed by washing five times for 10 min in washing buffer. Detection was accomplished with an enhanced chemiluminescence system (SuperSignal West Pico Chemiluminescent Substrate; Pierce) and subsequent exposure to Kodak XAR film (Eastman Kodak, Rochester, NY) for 5–30 s.

Statistical Analysis Differences between experimental and control conditions were tested by a two-way ANOVA and Fisher’s protected least significant difference tests. Percentages (not normalized) were transformed to the arc-sine before analysis. Only significant differences (P ⬍ 0.05) between experimental groups are discussed in the text.

RESULTS SNAP-25 and SNAP-23 Are Present in Human Sperm SNAP-25, a major component of the neuronal SNARE complex, is involved in the final step of the regulated exocytosis of neurotransmitters and hormones (Robinson and Martin, 1998). An ubiquitously expressed isoform, SNAP-23, was first identified in nonneuronal cells (Ravichandran et al., 1996). To further characterize the molecular mechanisms of sperm secretion, we focused on the SNARE proteins, based on our previous findings that active NSF is required for the human sperm AR (Michaut et al., 2000). We initially investigated whether SNAP-25 and SNAP-23 were present in human sperm and had a role in the AR. Specific antibodies were used to immunoprecipitate SNAP-23 (Chen et al., 1999) and SNAP-25 (Bruns et al., 1997) from human sperm extracts. Proteins with an apparent molecular mass of 28 kDa corresponding to SNAP-25 and SNAP-23 (Fig. 1, “sperm”) were subsequently detected by immunoblot using the respective antibodies. Rat brain SNAP-25 (Fig. 1, “brain”) comigrated with sperm SNAP-25,

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Human Sperm Acrosome Reaction Requires SNARE Proteins

FIG. 1. SNAP-25 and SNAP 23 are present in human sperm. Protein samples were run on 15% gels and analyzed by Western blot using anti-SNAP-25 (left) and anti-SNAP-23 (right) antibodies as probes, followed by horseradish peroxidase-conjugated secondary antibodies (Fc-specific). (Left) lane “brain”, 1 ␮g of a postnuclear membrane pellet from rat brain; lane “rec SNAP-23”, 1.8 ␮g His 6-SNAP-23; lane “rec SNAP-25”, 0.9 ␮g SNAP-25-His 6; lane “sperm (IP)”, SNAP-25 immunoprecipitated from RIPA extracts (5.5 ⫻ 10 7 lysed cells). (Right) lane “rec SNAP-25”, 0.9 ␮g SNAP-25-His 6; lane “rec SNAP-23”, 1.8 ␮g His 6-SNAP-23; lane “sperm (IP)”, SNAP-23 immunoprecipitated from RIPA extracts (5.5 ⫻ 10 7 lysed cells). Electrophoretic migration of SNAP-23/25 and heavy chain of immunoglobulin are indicated with an arrow and an arrowhead, respectively. The molecular weight standards are indicated to the left.

suggesting that related forms are present in these tissues. The specificity of these antibodies was corroborated by immunoblot of bacterially expressed SNAP-23 and SNAP25. Anti SNAP-25 monoclonal antibody recognized recombinant SNAP-25 but not SNAP-23 (Fig. 1). Conversely, the polyclonal anti-SNAP-23 recognized recombinant SNAP-23 but not SNAP-25 (Fig. 1). These results indicate that SNAP-23 and SNAP-25 are present in human sperm.

SNAP-23 Is Required for Calcium-Triggered, Human Sperm AR Because of its widespread distribution, SNAP-23 had been thought to function in the process of membrane fusion in the constitutive secretory pathway. More recently, however, SNAP-23 has been shown to participate in regulated exocytotic processes (Lemons et al., 2000; Flaumenhaft et al., 1999; Chen et al., 2000; Sadoul et al., 1997). A widespread methodology in the study of exocytosis is the use of SLO permeabilization, which we have recently applied to the human sperm AR (Yunes et al., 2000). Permeabilization allows penetration into the cell of exogenous proteins, such as neutralizing antibodies and neurotoxins, that usually are not able to penetrate the plasma membrane and can be used to block the function of target proteins. Furthermore, the SLO-permeabilized human sperm model is suitable for addition of recombinant proteins to replace and/or compete

with the endogenous isoforms. In the present report, we use a combination of these strategies to analyze the role of SNARE proteins in the human sperm AR. To determine the involvement of SNAP-23 in the AR, an antibody was introduced into SLO-permeabilized human sperm before challenging with Ca 2⫹. Ca 2⫹-induced AR was completely inhibited by the addition of a specific antibody against SNAP-23 but not by a nonimmune IgG (Fig. 2), suggesting that SNAP-23 is involved in this exocytotic process. In addition, permeabilized sperm cells were incubated with recombinant SNAP-23 prior to Ca 2⫹ stimulation. The results in Fig. 2 show a typical dose-response curve where exocytosis was completely abrogated by 30 ␮g/ml of bacterially expressed SNAP-23. Taken together, these data demonstrate that SNAP-23 is not only present but also has an essential role in human sperm acrosomal exocytosis.

Calcium-Triggered, Human Sperm AR Requires SNAP-25 to Be Assembled in a Stable SNARE Complex Despite the reported almost exclusive localization of SNAP-25 to neurons and endocrine cells, we were able to detect this protein in human sperm (Fig. 1). Since SNAP-25 mediates fusion between exocytotic vesicles and the plasma membrane, we set out to investigate its possible role in acrosomal exocytosis using several different ap-

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FIG. 2. Anti-SNAP-23 and bacterially expressed, full-length SNAP-23 inhibit Ca 2⫹-triggered acrosomal exocytosis. Permeabilized spermatozoa were incubated for 15 min at 37°C in the presence of 48 ␮g/ml purified SNAP-23 antibody (anti-SNAP23), 50 ␮g/ml of nonspecific rabbit IgG fraction (control Ab), or the indicated quantities of purified recombinant SNAP-23-His 6. Acrosomal exocytosis was evaluated by lectin binding after an additional 15-min incubation at 37°C in the absence (control bar) or presence (all other bars and curve) of 0.5 mM CaCl 2 (10 ␮M free ion). The values were normalized as explained in Materials and Methods. Actual percentages of reacted sperm for negative (control, 0 mM CaCl 2) and positive (calcium, 0.5 mM CaCl 2, no further additions) controls ranged between 11–19% and 25–30.5%, respectively. The data represent the mean ⫾ SEM of at least three independent experiments.

proaches. SNAP-25 is highly susceptible to BoNT/A and BoNT/E attack in monomeric form, whereas it is protected to various degrees in macromolecular complexes (Hayashi et al., 1994). Treatment with these BoNTs inhibits exocytosis in a variety of systems (Link et al., 1994; Xu et al., 1998; Sadoul et al., 1995). In a plasma membrane fraction from PC12 cells (Gerona et al., 2000), BoNT/A inhibition of neurotransmitter release is overcome by strong Ca 2⫹ stimuli. BoNTs/A and -E are particularly interesting amongst clostridial neurotoxins because they cleave SNAP-25 at its very C terminus, removing 9 and 26 amino acids, respectively, while not interfering with membrane attachment or ternary complex assembly. Their inhibitory effect on secretion is caused by hindering the SNARE complex stability (Hayashi et al., 1994). To evaluate the role of SNAP-25, 10 ␮M Ca 2⫹ was added to SLO-treated sperm to trigger the AR, and the effect of neutralizing antibodies, specific toxins, and recombinant proteins was analyzed. SLO-treated sperm were incubated in the presence of BoNT/A or BoNT/E. Both neurotoxins inhibited the Ca 2⫹-triggered AR by 80% (Fig. 3A). Since BoNTs activity is Zn 2⫹-dependent, the lack of effect of BoNT/A in the presence of the Zn 2⫹ chelating compound TPEN confirmed the specificity of the toxin effect (Fig. 3A). Furthermore, since ternary complexes are toxin-resistant, our results suggest that stable SNARE complexes must be assembled upon Ca 2⫹ stimulation for exocytosis to proceed.

In neurons, BoNT/A action on SNAP-25 produces a dual effect: a reduction in functional SNAP-25 and production of a fragment that can support neurotransmitter release, albeit with diminished sensitivity to Ca 2⫹. A rise in intracellular Ca 2⫹ concentrations restores vesicular release (Keller and Neale, 2001; Gerona et al., 2000). We analyzed the effect of BoNT/A at increasing Ca 2⫹ concentrations to further elucidate SNAP-25 function in sperm exocytosis. Sperm SNAP-25 was cleaved by pretreating SLO-permeabilized cells with 30 nM BoNT/A before challenging with different concentrations of Ca 2⫹. As shown in Figs. 3A and 3B, BoNT/A caused a significant inhibition of the AR triggered by 10 ␮M Ca 2⫹. This inhibition was completely reversed by Ca 2⫹ concentrations of 100 ␮M and higher (Fig. 3B). These results confirm the requirement for SNAP-25 in the human sperm AR. They also show that BoNT/A treatment reduces the Ca 2⫹ sensitivity of human sperm’s exocytotic machinery, similarly to observations reported in neuronal exocytosis. The monoclonal antibody Cl 71.1 binds to an epitope encompassing amino acid residues 20-40 (Bruns et al., 1997), a region in the N-terminal portion of SNAP-25 that is part of the SNARE motif and participates in complex formation. Cl 71.1 reduces Ca 2⫹-dependent exocytosis in chromaffin cells, likely through inhibition of ternary complexes formation (Xu et al., 1999). Interestingly, this antibody does not interact with its antigen after assembly of the ternary SNARE complex is complete (Xu et al., 1999). When added to SLO-permeabilized human sperm, Cl 71.1 effectively abrogated Ca 2⫹-triggered AR (Fig. 3C). The effect of the anti-SNAP-25 monoclonal was abolished by preincubation with heat-inactivated recombinant SNAP-25 (Fig. 3C), suggesting that the inhibitory effect of the antibody was specific and due to binding to endogenous SNAP-25. Specificity was further demonstrated by the finding that neither a nonimmune nor an anti-membrin antibody had any effect on acrosomal exocytosis (Fig. 3C). Membrin is a membrane integral Q-SNARE which appears to mediate an early step in endoplasmic reticulum to Golgi transport (Lowe et al., 1997). These results confirm a requirement for SNAP-25 in human sperm acrosomal exocytosis. They also provide further evidence that engagement of SNAP-25 in ternary SNARE complexes upon Ca 2⫹ stimulation is necessary for the AR to take place. Last, addition of bacterially expressed SNAP-25 inhibited the Ca 2⫹-triggered AR in a typical dose-response fashion (Fig. 3C). Recombinant SNAP-25 (20 ␮g/ml) completely abrogated acrosomal exocytosis (Fig. 3C). Since this inhibition is likely due to displacement of the corresponding endogenous SNARE, these data are in complete agreement with all our previous observations, lending further support to a role for SNAP-25 and the SNARE complex in the AR.

VAMP Is Required for Calcium-Triggered, Human Sperm AR To investigate the requirement for sperm homologues of all members of the SNARE complex in the Ca 2⫹-dependent

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FIG. 3. (A) Human sperm AR requires SNAP-25. SLOpermeabilized human sperm were treated for 15 min at 37°C in the presence of 357 nM bacterially expressed BoNT/E-LC (BoNT/E), or 30 nM BoNT/A (holotoxin) pretreated (BoNT/A⫹TPEN) or not (BoNT/A) with 5 mM TPEN. Acrosomal exocytosis was evaluated by lectin binding after an additional 15-min incubation at 37°C in the absence (control bar) or presence (all other bars) of 0.5 mM CaCl 2 (10 ␮M free ion). AR was evaluated and data were normalized as indicated in the legend to Fig. 2. Actual percentages of reacted sperm for negative (control, 0 mM CaCl 2) and positive (calcium, 0.5 mM CaCl 2, no further additions) controls ranged between 12-27% and 25-40%, respectively. The data represent the mean ⫾ SEM of at least three independent experiments. (B) BoNT/A inhibits the AR and its effect is reversed by Ca 2⫹. Permeabilized human sperm were incubated in the absence (E) or presence (F) of 30 nM BoNT/A preactivated with DTT for 15 min at 37°C. Sperm suspensions thus treated were aliquoted, challenged with increasing concentrations of CaCl 2, and further incubated for 15 min at 37°C. AR was evaluated and data were normalized as indicated in the legend to Fig. 2. Actual percentages of reacted sperm for 0 and 0.5 mM calcium (no BoNT/A) ranged between 9 –15% and 21–32%, respectively. BoNT/A did not affect the basal % AR (no Ca 2⫹). The data represent the mean ⫾ SEM of at least three independent experiments. (C) SLO-permeabilized human sperm were treated for 15 min at 37°C in the presence of 1:25 (v/v) of an anti-SNAP-25 antibody (Cl 71.1, raw ascites, “anti-SNAP25”), the same antibody preincubated with purified recombinant His 6-SNAP-25 (heat-inactivated for 60 min at 60°C, “Ab⫹SNAP-25”. Final antibody concentration: 1:25 (v/v); final His 6-SNAP-25 concentration: 15 ␮g/m), 20 ␮g/ml anti-membrin antibody (purified IgG, “anti-membrin”), 1:25 (v/v) of nonspecific

AR, we searched for the role of the VAMP family. Some isoforms of VAMP are cleaved by a number of neurotoxins, including BoNT/B, -D, -G, and -F and tetanus toxin. None of these toxins are active on preassembled SNARE complexes (Hayashi et al., 1994). BoNT/D and -F-cleaved VAMP can assemble in ternary complexes, but the stability of these complexes is decreased (Hayashi et al., 1994). Treatment with BoNT/F has been shown to inhibit exocytosis of, for instance, synaptic vesicles (Verderio et al., 1999) and growth hormone containing granules (Jacobsson et al., 1997). Because of its broad substrate specificity, at least human VAMP 1, -2, and -3/cellubrevin are susceptible to cleavage (Yamasaki et al., 1994), we chose BoNT/F to investigate whether VAMP is involved in the AR. Treatment of SLO-permeabilized human sperm with BoNT/F significantly inhibited Ca 2⫹-induced acrosome release (Fig. 4). Because cleavage by BoNT/F is Zn 2⫹dependent, we analyzed the consequences of Zn 2⫹ chelation on the activity of this toxin in sperm exocytosis. As expected, preincubation of BoNT/F with 5 mM TPEN prevented AR inhibition, confirming the specificity of the effect of the toxin (Fig. 4). These results indicate that VAMP is involved in human sperm exocytosis. The monoclonal antibody Cl 10.1, raised against gelpurified VAMP, binds to a conserved, middle portion of all VAMPs, including VAMP-1, VAMP-2, and cellubrevin. The epitope recognized by Cl 10.1 is part of the SNARE motif that participates in complex formation (Baumert et al., 1989; Sudhof et al., 1989). The monoclonal antibody Cl 69.1, on the other hand, was raised against the 17 N terminus amino acids of VAMP-2, outside the SNARE motif (Edelmann et al., 1995). Cl 69.1 binds with high affinity to both free VAMP 2 and VAMP 2 assembled in ternary SNARE complexes. Cl 69.1 (200 ␮g/ml) prevents exocytosis in adrenal chromaffin cells (Xu et al., 1999). When added to SLO-permeabilized human sperm, Cl 10.1 attenuated Ca 2⫹-triggered AR (Fig. 4, “anti-VAMP”), whereas a nonimmune antibody did not (Fig. 4, “control Ab”). The anti-VAMP-2-specific monoclonal Cl 69.1 caused a marked inhibition of the Ca 2⫹-dependent AR (Fig. 4, “anti-VAMP2”). These results suggest that VAMP-2 is required for Ca 2⫹-mediated AR. It is worth pointing out that the involvement of other isoform/s cannot be ruled out. Furthermore, our data indicate the need for stable hetero-

mouse ascites (control Ab), or the indicated quantities of purified recombinant His 6-SNAP-25. Acrosomal exocytosis was evaluated by lectin binding after an additional 15-min incubation at 37°C in the absence (control bar) or presence (all other bars and curve) of 0.5 mM CaCl 2 (10 ␮M free ion). AR was evaluated and data normalized as indicated in the legend to Fig. 2. Actual percentages of reacted sperm for negative (control, 0 mM CaCl 2) and positive (calcium, 0.5 mM CaCl 2, no further additions) controls ranged between 14.5– 26% and 30.5–39%, respectively. The data represent the mean ⫾ SEM of at least three independent experiments.

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FIG. 4. Human sperm AR requires VAMP/VAMP2. SLOpermeabilized human sperm were treated for 15 min at 37°C in the presence of 5 nM BoNT/F (holotoxin) pretreated (BoNT/F⫹TPEN) or not (BoNT/F) with 5 mM TPEN, 1:25 (v/v) of an anti-VAMP antibody (Cl 10.1, raw ascites, “anti-VAMP”), 8 ␮g/ml of an anti-VAMP2 antibody (Cl 69.1, “anti-VAMP2”), or 1:25 (v/v) of nonspecific mouse ascites (control Ab). Acrosomal exocytosis was evaluated by lectin binding after an additional 15-min incubation at 37°C in the absence (control bar) or presence (all other bars) of 0.5 mM CaCl 2 (10 ␮M free ion). AR was evaluated and data were normalized as indicated in the legend to Fig. 2. Actual percentages of reacted sperm for negative (control, 0 mM CaCl 2) and positive (calcium, 0.5 mM CaCl 2, no further additions) controls ranged between 12–26.5% and 25–34%, respectively. The data represent the mean ⫾ SEM of at least three independent experiments.

trimeric complexes formation for exocytosis to take place, since an antibody that prevents complex assembly (Cl 10.1) and a toxin that impairs its stability both inhibit the AR.

Syntaxin Is Required for Calcium-Triggered, Human Sperm AR Syntaxin is by far the largest SNARE family in mammalian cells. As was the case with other SNARE proteins, the importance of syntaxins became particularly clear from extensive characterization of the neuronal isoforms 1A and 1B. Several syntaxins have been shown to localize to the plasma membrane, including syntaxins 1A and 1B, 2, 3, and 4 (Jahn and Sudhof, 1999) and display a role in regulated exocytosis. Syntaxins 1A and 4 appear to be important for Ca 2⫹-regulated secretion in PC12 cells (Bennett et al., 1993; Scales et al., 2000a). In permeabilized human platelets, anti-syntaxin 4 antibodies inhibit ␣-granule release (Lemons et al., 2000; Flaumenhaft et al., 1999). Syntaxin 6 also displays a role in exocytosis, as demonstrated by the blockage of human neutrophils secretion by specific antibodies (Martin-Martin et al., 2000). An array of specific antibodies was used to investigate the presence of members of the syntaxin family in human

sperm. Immunoblot analysis of human sperm extracts demonstrated the presence of proteins with apparent molecular mass of 35 kDa, corresponding to syntaxin 1B, 32 kDa, corresponding to syntaxin 4, and 31 kDa, corresponding to syntaxin 6 (Fig. 5A). We were unable to detect syntaxin 1A by Western blot, although we have functional evidence of the presence of this protein in human sperm (see below). When added to SLO-permeabilized sperm, a polyclonal antibody raised against a synthetic peptide corresponding to residues 143-160 in the linker region of human syntaxin 1A, significantly inhibited Ca 2⫹-triggered exocytosis (Fig. 5B). Likewise, preincubation of permeabilized human sperm with a polyclonal antibody raised against a sequence contained in the linker portion of syntaxin 1B decreased the exocytotic response to Ca 2⫹ (Fig. 5B). Ca 2⫹ failed to trigger exocytosis in sperm pretreated with anti-syntaxin 4 and anti-syntaxin 6 monoclonal antibodies (Fig. 5B), demonstrating that all these members of the syntaxin family are required for human sperm AR. All inhibitions caused by specific antibodies differed significantly from their corresponding nonimmune counterparts used as negative controls (data not shown). BoNT/C is a Zn 2⫹-endopeptidase that cleaves syntaxin 1A at the Q253-A254 and syntaxin 1B at the Q252-A253 peptide bonds, whereas syntaxin 4 is resistant (Schiavo et al., 1995). Human syntaxin 6 lacks the consensus sequence for cleavage by BoNT/C and is predicted to be toxininsensitive. BoNT/C cleaves syntaxin just outside of the transmembrane region, producing a soluble cytoplasmic fragment which continues to assemble into a ternary complex, albeit of reduced stability (Hayashi et al., 1994). Syntaxin cleavage by BoNT/C blocks neurotransmitter release (Blasi et al., 1993) and chromaffin cells exocytosis (Xu et al., 1998). Human sperm AR is sensitive to BoNT/C, since sperm incubation with this toxin prior to Ca 2⫹ challenging caused a 70% reduction of the exocytotic response compared with the untreated control (Fig. 5B). The effect of BoNT/C on the AR was dependent on Zn 2⫹, because chelation with TPEN completely abolished the inhibition (Fig. 5B). These data indicate that the syntaxin family is present and displays a crucial role in human sperm exocytosis. Antibody inhibition studies show a requirement for syntaxins 1A, 1B, 4, and 6 in human sperm exocytosis. Among these isoforms, syntaxin 1A and 1B are susceptible to cleavage by BoNT/C. Hence, inhibition of AR by BoNT/C can be ascribed to its proteolysis of syntaxin 1. Last, our data suggest the need for membrane-bound, stable heterotrimeric complexes formation for exocytosis to take place.

SNAP-25 and VAMP Mediate Rab3-Triggered Human Sperm AR Rab3 represents a small subfamily of Rab proteins that are highly concentrated on secretory vesicles (Lledo et al., 1994). Rab3A dissociates from synaptic vesicles and undergoes GTP hydrolysis during or after exocytosis (Jahn and Sudhof, 1999). A body of evidence points to a direct role of

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FIG. 5. Several syntaxins are present in human sperm and required for the AR. (A) Proteins extracted from 3 ⫻ 10 6 human sperm cells (whole sperm lysates in sample buffer; Tomes et al., 1998) were resolved by 10% SDS-PAGE and analyzed by Western blot using anti-syntaxin 1B (lane 1B), anti-syntaxin 4 (lane 4), and anti-syntaxin 6 (lane 6) antibodies as probes, and the immunodecorated proteins were detected by chemiluminescence. Molecular weight standards are indicated on the left. (B) SLO-permeabilized human sperm were treated for 15 min at 37°C in the presence of 11 nM BoNT/C (holotoxin) pretreated (BoNT/C⫹TPEN) or not (BoNT/C) with 5 mM TPEN, 1:25 (v/v) of anti-syntaxin 1A (whole serum, anti-syx1A), 1:17 (v/v) anti-syntaxin 1B (whole serum, anti-syx1B), 10 ␮g/ml anti-syntaxin 4 (purified IgG, anti-syx4), or 10 ␮g/ml anti-syntaxin 6 (purified IgG, anti-syx6) antibodies. Acrosomal exocytosis was evaluated by lectin binding after an additional 15-min incubation at 37°C in the absence (control bar) or presence (all other bars) of 0.5 mM CaCl 2 (10 ␮M free ion). AR was evaluated and data were normalized as indicated in the legend to Fig. 2. Actual percentages of reacted sperm for negative (control, 0 mM CaCl 2) and positive (calcium, 0.5 mM CaCl 2; no further additions) controls ranged between 10 –34% and 25– 49%, respectively. The data represent the mean ⫾ SEM of at least three independent experiments.

active Rab3A in human sperm AR (Yunes et al., 2000; Michaut et al., 2000). Interestingly, in permeabilized sperm, Rab3A-triggered AR does not require addition of Ca 2⫹ (McBride et al., 1999). Addition of GTP-loaded recombinant Rab3A to SLOpermeabilized human sperm, induced an exocytotic response of magnitude comparable with that of Ca 2⫹ (Fig. 6). Pretreatment of the sperm suspension with BoNT/A completely prevented Rab3A from eliciting the AR (Fig. 6). Similarly, addition of recombinant Rab3A following sperm intoxication with BoNT/F and -C, did not result in acrosomal exocytosis (Fig. 6). Our results show a requirement for SNAP-25, syntaxin, and VAMP in Rab3A-triggered sperm exocytosis. Furthermore, they constitute, to the best of our knowledge, the first piece of evidence linking neurotoxin inhibition to a Rab-promoted membrane fusion event.

DISCUSSION Fertilization depends on the regulated fusion of the acrosome with the spermatozoon’s plasma membrane, leading to the exocytotic process termed the AR. In several mammalian species, secretion is triggered during gamete contact

by ZP3, a glycoprotein component of the egg’s extracellular matrix, or zona pellucida (reviewed in McLeskey et al., 1998; Wassarman, 1999; Wassarman et al., 2001). ZP3 produces a sustained increase of the internal Ca 2⫹ concentration, which subsequently leads to the AR (O’Toole et al., 2000) The molecular mechanisms operating in the exocytosis of the acrosome are only now beginning to emerge. Sperm’s biggest drawback is their inability to synthesize proteins, thus ruling out the use of widespread powerful techniques such as transfection and overexpression. To overcome this limitation, we have established a permeabilization protocol which permits entry of toxins, proteins, Ca 2⫹, and other molecules into the cell. This system is readily manipulable and reflective of the in vivo organization of the cell and allows us to measure a functional, Ca 2⫹-regulated fusion event. Despite the fact that exocytosis of the acrosomal vesicle is somewhat unique, it is suspected that sperm use the same conserved machinery and regulatory components as characterized for other secretory events. Thus, we have demonstrated a role for Rab3A (Yunes et al., 2000), NSF (Michaut et al., 2000), and synaptotagmin VI (Michaut et al., 2001) in the Ca 2-dependent AR of human sperm. Inter-

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FIG. 6. Rab3A-triggered acrosomal exocytosis requires SNARE proteins Permeabilized human spermatozoa were incubated for 15 min at 37°C in the absence (no BoNT) or presence of 30 nM BoNT/A (BoNT/A), 11 nM BoNT/C (BoNT/C), or 5 nM BoNT/F (BoNT/F). Acrosomal exocytosis was evaluated by lectin binding after an additional 15-min incubation at 37°C in the absence (control bar) or presence of 0.5 mM CaCl 2 (10 ␮M free ion, gray bar) or 300 nM GST-Rab3A loaded with GTP[␥S] (open bars). AR was evaluated and data were normalized as indicated in the legend to Fig. 2. Actual percentages of reacted sperm for negative (control, 0 mM CaCl 2) and positive (calcium, 0.5 mM CaCl 2, no further additions) controls ranged between 14 –20% and 31–39%, respectively. The data represent the mean ⫾ SEM of at least three independent experiments.

estingly, published data on the presence of members of the SNARE complex in sperm are scant, comprising all three protein homologues in sea urchin (Schulz et al., 1997, 1998), and VAMP and syntaxin in mammals (RamalhoSantos et al., 2000; Katafuchi et al., 2000). Here, we report the presence of SNAP-23/25 (Fig. 1) and syntaxin 1B/4/6 (Fig. 5A) in human sperm by Western blot. Following several different approaches, we demonstrate a requirement for all three members of the SNARE complex in the AR. Furthermore, our data suggest that the onset of sperm’s exocytosis relies on the productive assembly of ternary SNARE complexes. The first line of evidence comes from the use of botulinal neurotoxins. Treatment with BoNT/A, -E, -F, and -C resulted in a Zn 2⫹-dependent inhibition of Ca 2⫹-induced acrosomal release (Figs. 3–5), indicating a need for toxinsensitive members of all three SNARE families in the AR. Examples of these proteins described in this paper include SNAP-25, VAMP-2, syntaxin 1A, and syntaxin 1B. Because all the toxins tested compromise SNARE complex stability, we conclude that stable ternary complexes are required for human sperm AR to proceed. BoNT/A cleaves the nine C-terminal amino acids off of its substrate SNAP-25. BoNT/A-treated sperm exhibited an 80% inhibition of acrosomal exocytosis in response to a 10-␮M Ca 2⫹ stimulus compared with controls (Fig. 3A). However, this inhibition

was no longer observed when Ca 2⫹ concentration increased to 100 ␮M and higher (Fig. 3B). Our data fit a model recently proposed by Xu et al. (1998), in which synaptotagmin interdigitates with the tail of a partially “zippered” coiledcoil structure in the ternary SNARE complex, and following a Ca 2⫹-dependent conformational change, put additional torsion on that structure, forcing the two membrane anchors together and initiating fusion. BoNT/A-truncated SNAP-25 is still able to form SNARE complexes (Hayashi et al., 1994), but its binding to synaptotagmin is impaired (Gerona et al., 2000). Thus, cleavage of SNAP-25 would probably reduce the efficacy of the Ca 2⫹ sensor in transmitting the signal to the SNARE complex. High Ca 2⫹ concentrations restore binding of truncated SNAP-25 to synaptotagmin, leading to full SNARE complex “zippering” and, consequently, exocytosis (Gerona et al., 2000). The second line of evidence comes from the use of specific antibodies. As shown in Figs. 2, 3C, 4, and 5B, loading of SLO-permeabilized sperm with antibodies to SNAP-23, SNAP-25, VAMP, VAMP-2, syntaxin 1A, syntaxin 1B, syntaxin 4, and syntaxin 6 reduced sperm’s exocytotic response to Ca 2⫹. These data demonstrate that all those SNARE proteins are implicated in the AR. Epitopes recognized by monoclonal antibodies to SNAP-25 (Cl 71.1) and VAMP (Cl 10.1) map within the SNARE motif of the corresponding proteins. Hence, antibody binding is expected to hinder SNARE complex assembly. Conversely, AR inhibition by these antibodies suggests the need for ternary complexes to assemble for sperm exocytosis to take place. The epitope recognized by Cl 69.1 is part of the proline-rich amino-terminal ⬇24 amino acids of VAMP adjacent to the SNARE-motif. This region is crucial for inhibition of exocytosis by synthetic VAMP peptides (Cornille et al., 1995). The epitopes recognized by polyclonal antibodies to syntaxins 1A and 1B map outside the SNARE motif, in a region known as the linker. Interestingly, a mutation introduced in this region disrupted the closed conformation of syntaxin and abolished munc18-1 binding, thus reducing the effect of syntaxin in secretion (Dulubova et al., 1999). The epitopes recognized by the remaining antibodies used in this study have not been as thoroughly characterized. However, their key role in exocytotic events has been demonstrated in a number of publications, showing that they block secretion in other systems (Chen et al., 2000; Lemons et al., 2000; Flaumenhaft et al., 1999). Third, addition of recombinant SNAP-23 and SNAP-25 caused a marked inhibition of the AR (Figs. 2 and 3C), presumably by displacement of the corresponding endogenous SNARE. A similar inhibitory effect on exocytosis was observed when bacterially expressed SNAREs lacking the transmembrane domain were added to PC12 cells (Bennett et al., 1993; Scales et al., 2000a). Truncated SNARE proteins might have a dominant-negative effect on secretion because they substitute for their endogenous counterparts in protein–protein interactions but are not membranebound, thereby uncoupling the protein–protein interactions from the membrane. In this context, failing to engage in

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productive assembly might be due to lack of palmitoylation of bacterially expressed SNAP-23/25, which might in turn lead to impaired membrane targeting/binding. Once again, our data point to an assembled SNARE protein complex as mediator of sperm exocytosis. As well as being involved in driving membrane fusion, SNARE proteins have been implicated in ensuring the accuracy of vesicle trafficking by pairing specifically with their cognate SNAREs (Scales et al., 2000b). According to the original SNARE hypothesis, each fusion step in membrane trafficking would be mediated by a unique set of SNAREs. These would function only in one fusion step and would be excluded from others. This specificity was thought to be caused by the intrinsic affinity of SNAREs for each other, i.e., only cognate SNAREs were thought to bind to each other. Such specificity has been demonstrated in membrane fusion models, such as reconstituted liposomes (McNew et al., 2000) and cracked PC12 cells (Scales et al., 2000a). But this fidelity is somewhat lost in in vitro experiments, where these proteins apparently participate in the formation of several different SNARE complexes, suggesting that they are able to pair with more than one set of partners (Fasshauer et al., 1999; Yang et al., 1999). Moreover, it has recently become clear that at least some SNAREs can function in multiple trafficking steps (Fischer and Stevens, 1999). The best characterized fusion complex is the one involved in neuronal exocytosis where the participating SNARE cognates are SNAP-25, syntaxin 1A, and VAMP2. Other examples of cognate SNARE complexes involved in exocytosis include SNAP-23/syntaxin 4/ VAMP8 in RBL-2H3 mast cells (Paumet et al., 2000), SNAP-23/syntaxin 4/VAMP2 in 3T3-L1 adipocytes (Kawanishi et al., 2000), SNAP-23/syntaxin 4 and an unidentified VAMP isoform in platelets (Flaumenhaft et al., 1999), and SNAP-23/syntaxin 6 in neutrophils (Martin-Martin et al., 2000). Here, we present evidence of the role in the human sperm AR of all three members of the neuronal SNARE complex as well as cognate SNARE proteins involved in nonneuronal exocytosis. A certain level of redundancy, illustrated by the coexistence of more than one isoform of a given protein carrying out seemingly overlapping functions, is not uncommon in biological systems. For instance, SNAP-23 and SNAP-25 coexist in transformed pancreatic ␤ cells, but SNAP-23 can replace SNAP-25 in the process of insulin secretion only when overexpressed (Sadoul et al., 1997). Both, syntaxins 4 and 2 mediate ␣-granule release in platelets (Lemons et al., 2000). Thus, human sperm contain more than one isoform of each Q-SNARE and all of them seem to be required for acrosomal exocytosis. Interestingly, they do not appear to substitute for each other in antibody inhibition experiments. Furthermore, BoNT/A, -E, and -C inhibit acrosomal exocytosis despite the presence in sperm of toxin-resistant isoforms, i.e., SNAP-23 and syntaxins 4 and 6. The AR involves multiple fusions between the outer acrosomal membrane and the overlying plasma membrane that lead to membrane vesiculation. The site where the point fusions

first take place varies according to species. In human sperm, fusion between outer acrosomal and plasma membranes begins at the most anterior tip of the head and progresses toward the equatorial segment (Yudin et al., 1988). This might reflect an intrinsic heterogeneity in the boundaries of the sites destined to fuse that determines the topology of this peculiar exocytotic process. Thus, complexes in the tip might have a different protein composition than those closer to the equatorial segment, and only the concerted opening of all fusion pores would lead to a successful acrosomal release. The distribution of SNARE proteins restricted to specialized fusion sites has been very recently reported (Lang et al., 2001; Chamberlain et al., 2001) Calcium, initially considered as the universal link between receptor stimulation and the onset of exocytosis in secretory cells, is now recognized as only one of a number of intracellular activators. For instance GTP-␥-S is capable of stimulating exocytosis, either in conjunction with, or independent of, Ca 2⫹ (Pinxteren et al., 2000). In human sperm, there is an alternative pathway for stimulating AR that is activated by the small GTPase Rab3 (Yunes et al., 2000). Rab3- and Ca 2⫹-activated pathways appear to converge at a point prior to or at SNAREs since Rab3-stimulated secretion was effectively inhibited by BoNT/A, -C, and -F (Fig. 6). Ours constitutes the first piece of evidence that a Rabpromoted fusion event can be effectively blocked by specific neurotoxins attacking the SNARE proteins. In the only other study published to date, Rab3 exhibits an inhibitory role in secretion, and a GTPase-negative mutant of Rab3 delays further inhibition of neurotransmitter release by BoNT/A and tetanus toxins (Johannes et al., 1996). Nevertheless, both studies agree on the existence of a functional link between Rab3 and the SNARE complex during vesicle exocytosis. Tools that interfere with assembly of SNARE complexes (Cl 10.1 and 71.1) or allow assembly only of labile or partially membrane-anchored complexes (BoNTs), prevent exocytosis, indicating the need for stable ternary complex formation in the Ca 2⫹-dependent AR. A recent model explaining the widespread observation that SNARE complexes are essential for membrane fusion postulates that trans-SNARE complexes assemble between membraneanchored SNARE partners in the two membranes destined to fuse after they become attached. As a consequence, the membranes are pulled very close together, a prerequisite for initiating fusion. Assembly causes a hemifusion intermediate, and thereafter, the complex is expendable and can be dissociated without affecting the progress of the fusion reaction (Jahn and Sudhof, 1999). Our data are consistent with this model and allow us to propose the following sequence of events operating during human sperm AR. In our working model, Ca 2⫹ stimulation would result in the activation of Rab3A, which in turn would mediate tethering of the acrosome to the plasma membrane. Priming and/or dissociation of preexisting cis SNARE complexes by NSF/␣-SNAP would then take place (at this step the system would be toxin-, antibody-, and recombinant protein-

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sensitive). SNARE proteins would then assemble in productive, fusion-competent trans SNARE complexes, and in doing so, would elicit docking of the acrosome. After that, Ca 2⫹ binding to synaptotagmin would cause a conformational change in this protein, ultimately promoting fusion (see above). Experiments are underway in our laboratory to test these hypotheses.

ACKNOWLEDGMENTS We thank A. Challa and Olga Chertihin for excellent technical assistance; Dr. S. Patterson for the rat brain preparation; Drs. M.I. Colombo, S. Patterson, and R. Yunes for critical reading of the manuscript; Drs. T. Galli and T. Binz for plasmids; Dr. Whiteheart for the anti-SNAP-23 antibody; and Ms. L. de Jong and Dr. R. Ferna´ ndez for BoNT/A, -F, and -C. This work was supported partly by an International Research Scholar Award from the HHMI and by grants from Consejo Nacional de Investigaciones Cientifı´cas y Te´ cnicas de Argentina (to L.M.), and from the National Institutes of Health, HD38082 (to P.E.V.).

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