Mol. Hum. Reprod. Advance Access originally published online on November 12, 2004
Molecular Human Reproduction 2005 11(1):43-51; doi:10.1093/molehr/gah126
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-SNAP and NSF are required in a priming step during the human sperm acrosome reaction
1Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología (IHEM-CONICET), Facultad de Ciencias Médicas, CC 56, Universidad Nacional de Cuyo, 5500 Mendoza, Argentina, 2 Department of Cell Biology, Center for Recombinant Gamete Contraceptive Vaccinogens, University of Virginia, Room 3-101 Jordan Hall, 1300 Jefferson Park Ave, Charlottesville, VA 22903, USA
3 To whom correspondence should be addressed at: Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología. Facultad de Ciencias Médicas. CC 56 Universidad Nacional de Cuyo, 5500 Mendoza, Argentina. Email: ctomes{at}fcm.uncu.edu.ar
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Abstract |
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The acrosome is a membrane-limited granule that overlies the nucleus of the mature spermatozoon. In response to physiological or pharmacological stimuli it undergoes a special type of Ca2+-dependent exocytosis termed the acrosome reaction (AR), which is an absolute prerequisite for fertilization. Aided by a streptolysin-O permeabilization protocol developed in our laboratory, we have previously demonstrated requirements for Rab3A, N-ethylmaleimide-sensitive factor (NSF), several soluble NSF-attachment protein receptor (SNARE) proteins, and synaptotagmin VI in the human sperm AR. Here, we show that
-soluble NSF-attachment protein (-SNAP), a protein essential for most fusion events through its interaction with NSF and the SNARE complex, exhibits a direct role in the AR. First, the presence of -SNAP is demonstrated by the Western blot of human sperm protein extracts. Immunostaining experiments reveal an acrosomal localization for this protein. Second, the Ca2+ and Rab3A-triggered ARs are inhibited by anti--SNAP antibodies. Third, bacterially expressed -SNAP abolishes exocytosis in a fashion that depends on its interaction with NSF. Fourth, we show a requirement for -SNAP/NSF in a prefusion step early in the exocytotic pathway, after the tethering of the acrosome to the plasma membrane and before the efflux of intra-acrosomal Ca2+. These results suggest a key role for -SNAP/NSF in the AR, and strengthen our understanding of the molecular players involved in the vesicle-to-plasma membrane fusion taking place during exocytosis.
Key words:
acrosome/exocytosis/NSF/-SNAP/sperm
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Fertilization is the process where individual gametes from the female (the oocyte) and male (the sperm) unite to produce an offspring (Yanagimachi, 1994
). Interaction with the oocyte 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) is a terminal morphological alteration that must occur prior to penetration of the extracellular coat of the oocyte (zona pellucida). As is the case in somatic cells' regulated exocytosis, Ca2+ is an essential mediator of the AR (Florman et al., 1998
). The AR differs from other known exocytotic events in several ways, mainly in that the sperm contain a single secretory vesicle whose release is a singular occurrence. Nonetheless, it is suspected that sperm use the same conserved fusion machinery and regulatory components as characterized for other secretory events (Tomes et al., 2002
and references therein).
Fusion of biological membranes requires the formation of protein complexes including integral components of the vesicle (R-SNAREs) and of the plasma membrane (Q-SNAREs), in addition to soluble factors such as N-ethylmaleimide-sensitive factor (NSF) and -soluble NSF-attachment protein (-SNAP) (Jahn and Sudhof, 1999
; Chen and Scheller, 2001
). Soluble NSF-attachment protein receptors (SNAREs) form a superfamily of proteins that reversibly assemble into tightly packed helical bundles, the core complexes (Sutton et al., 1998
). Assembly is thought to pull the fusing membranes closely together, driving bilayer fusion. Disassembly requires the concerted action of -SNAP and NSF, whereby NSF uses energy from ATP hydrolysis to dissociate SNARE complexes. Interestingly, -SNAP and NSF are not compartment-specific but participate in both constitutive secretion and regulated exocytosis (Hay and Scheller, 1997
). Exocytosis is a highly regulated, multistage process including the targeting, tethering, priming, docking and fusion of secretory vesicles with the plasma membrane. At some point in the cycle of vesicle traffic, fusion-incompetent cis-SNARE complexes (i.e. on the same membrane) must be disassembled before productive trans-SNARE complexes (i.e. on the opposite membranes) can be assembled. Defining when SNARE complexes dissociate has proven difficult and controversial. For instance, in high output synapses, where vesicles are re-used many times, NSF/SNAP play a role ‘after’ membrane fusion, allowing the individual SNARE proteins to be recycled for subsequent rounds of fusion (Littleton et al., 2001
; Jahn et al., 2003
). In contrast, at the low output crayfish neuromuscular junction, NSF/SNAP mediate SNARE priming before fusion (He et al., 1999
). A similar prefusion site of action has been suggested in dense-core granules in adrenal chromaffin cells, where exocytosis involves a one-shot event (Xu et al., 1999
).
SNAPs stimulate the ATPase activity of NSF to disassemble the SNARE complex (Sollner et al., 1993a
). Three variants of SNAPs exist in mammals, referred to as -, ß-, and 
-SNAP (Whiteheart et al., 1993
). Sequence comparisons show that these proteins are well conserved between yeast (Sec17p is the yeast homologue of -SNAP) and mammals (Whiteheart et al., 1993
). They exhibit differential tissue distribution, with - and -SNAP being expressed in many tissues but ß-SNAP mainly restricted to neurons. It is not yet clear whether they can be substituted for each other or serve different roles. Evidence supporting both the possibilities has been presented in chromaffin cell exocytosis (Sudlow et al., 1996
; Xu et al., 2002
). Interestingly, the mRNA for -SNAP is particularly enriched in the mouse testis, in addition to the central nervous system (Whiteheart et al., 1993
).
To further characterize the molecular mechanisms of sperm secretion, we focused on -SNAP-dependent SNARE activation, based on our previous findings that active NSF (Michaut et al., 2000
) and several SNARE proteins (Tomes et al., 2002
) are required for the human sperm AR. Aided by a streptolysin-O (SLO) permeabilization protocol developed in our laboratory, we demonstrate a requirement for -SNAP in Ca2+ and Rab3A-triggered acrosomal release. -SNAP/NSF function in a prefusion priming step, following tethering by Rab3 prior to the efflux of intra-acrosomal Ca2+.
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Materials and methods |
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Reagents
SLO was obtained from Corgenix (Peterborough, UK). Mouse monoclonal anti-
/ß-SNAP antibodies (whole ascites, clones 77.1, IgG2b and 77.2, IgG1) (Hanson et al., 1995
), and a rabbit polyclonal anti-NSF (whole serum) were from Synaptic Systems (Göttingen, Germany). Horse-radish peroxidase and tetramethyl-rhodamine isothiocyanate (TRITC)-conjugated goat anti-mouse-Immunoglobulin G(IgG) were from Kirkegaard & Perry Laboratories, Inc. (KPL, Gaithersburg, MD). Nickel–nitrilotriacetic acid agarose was from Qiagen (Hilden, Germany) and glutathione–sepharose from Amersham Biosciences Argentina (Buenos Aires, Argentina). Prestained molecular mass standards were from BRL-Life Technologies, Inc. (Gaithersburg, MD). O-nitrophenyl EGTA acetoxymethyl ester (NP-EGTA-AM) was from Molecular Probes (Eugene, OR). Methanol was purchased from Merck Química (Buenos Aires, Argentina). All other chemicals were reagent or analytical grade and were purchased from Sigma Chemical Co. (St. Louis, MO).
Recombinant proteins
Plasmids encoding -SNAP and NSF in pQE9 (Qiagen) were a kind gift from Dr S.W. Whiteheart (University of Kentucky, Lexington, KY, USA). Recombinant His6-ß-SNAP was kindly gifted by Dr M.I. Colombo (Cuyo National University, Mendoza, Argentina). We modified the expression and purification of His6-tagged -SNAP and NSF described in Whiteheart et al. (1993)
to maximize the yield. Briefly, the DNA encoding His6--SNAP was transformed into E. coli XL1-Blue (Stratagene, La Jolla, CA, USA) and induced overnight at 20°C with 0.2 mM isopropyl-thio-ß-D-galactoside (IPTG). The plasmid construct encoding His6-NSF was transformed into E. coli M15pRep4 (Qiagen) and induced for 4 h at 30°C with 1 mM IPTG. Purification of recombinant proteins was accomplished according to The QIA expressionist (http://www.qiagen.com), except that 0.5 mM ATP, 5 mM MgCl2, and 2 mM dithiothreitol (DTT) were added to all the buffers involved in the purification of His6-NSF. The expression plasmid pGEX2T containing the cDNA-encoding human Rab3A was generously provided by Dr M.I. Colombo and Dr P.D. Stahl (Washington University, St. Louis, MO, USA). GST-Rab3A was expressed in E. coli strain XL1-Blue (Stratagene), purified, prenylated and activated as previously described (Yunes et al., 2000
).
Human sperm preparation procedure
After at least 2 days of abstinence, semen samples were provided by masturbation from healthy volunteer donors who were free from sexually transmitted diseases. Semen was allowed to liquify for 30–60 min at 37°C. Except for cytosol and membrane fractionation experiments, highly motile sperm were recovered following a swim-up separation for 1 h in gamete preparation medium (GPM) (Serono, Switzerland) at 37°C in an atmosphere of 5% CO2/95% air. For subcellular fractionation sperm were isolated on a discontinuous two-step Percoll® (Pharmacia, Uppsala, Sweden) gradient. We followed the protocol described by Bohring and Krause (1999)
. Briefly, semen was allowed to liquefy, loaded onto Percoll gradients (90–47% in protein-free Hams F-10 medium), and centrifuged for 25 min at 400 g. Sperm pellets were recovered, washed twice with phosphate-buffered saline (PBS), and diluted 1:9 in hypo-osmotic swelling buffer (Jeyendran et al., 1984
). After 2 h at 37°C in a water bath, at least 80% of the cells were swollen. Suspensions were transferred to ice, sperm disrupted by sonication, and centrifuged at 4000 g (4°C, 15 min) in a Beckman OptimaTM ultracentrifuge to remove cell debris. The supernatant was centrifuged at 10 000 g (4°C, 10 min), and the resultant supernatant underwent a further ultracentrifugation at 208 000 g (4°C, 1 h). The final pellets containing the membrane proteins were resuspended and dissolved in sample buffer (see below). The final supernatant containing the soluble fraction was added to protease inhibitors and concentrated in a Centricon 30 (Amicon, Inc., Beverly, MA, USA).
SLO permeabilization and AR assay
After swim up, sperm concentration was adjusted to 5–10 x 106/ml, and incubated for at least 2 h under conditions that support capacitation (GPM, 37°C, 5% CO2/95% air). Permeabilization was accomplished as previously described (Yunes et al., 2000
). Briefly, washed spermatozoa were resuspended in cold PBS containing 0.4 unit/ml SLO for 15 min at 4°C. Cells were washed once with PBS, resuspended in ice-cold sucrose buffer (250 mM sucrose, 0.5 mM EGTA, 20 mM HEPES, pH 7) containing 2 mM DTT, inhibitors were added when indicated, and further incubated for 15 min at 37°C. After addition of stimulants to the sperm suspensions, incubation proceeded at 37°C for 15 min. For the experiments with the photoinhibitable calcium chelator NP-EGTA-AM, SLO-permeabilized sperm were preloaded with 10 µM NP-EGTA-AM before incubation in the presence of inhibitors and stimulants as described, except that all procedures were carried out in the dark. Alternatively, NP-EGTA-AM-preloaded sperm were incubated first with 0.5 mM CaCl2 and second with the inhibitors to test, also in the dark. In all cases photolysis of the chelator was induced, at the end of the second incubation, by 2 min exposure to an UV transilluminator. Incubations proceeded for an additional 5 min at 37°C. Ten microlitres of each reaction mixture was spotted on 8-well slides, air dried and fixed/permeabilized in ice-cold methanol for 30 s. Acrosomal status was evaluated by staining with fluorescein isothiocyanate (FITC)-coupled Pisum sativum agglutinin (PSA) according to Mendoza et al. (1992)
. At least 200 cells were scored using a Nikon microscope equipped with epifluorescence optics. Negative (no stimulation) and positive (10 µM Ca2+) 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.
SDS-PAGE and immunoblot analysis
Sperm were washed in PBS, permeabilized or not, with SLO and lysed in cold 50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 2 mM DTT, 5 µg/ml trypsin inhibitor, 5 µg/ml pepstatin, 0.5 mM phenylmethylsulphonyl fluoride, 0.5 mM benzamidine, and 1% Triton X-100. After sonication for 2 x 15 s and extraction for 30–60 min at 4°C, the sperm extracts were clarified by centrifugation at 12 000 g for 5 min and used immediately or stored at –20°C. For the Western blots depicted in Figure 1A
, extracts were made in sample buffer as described (Tomes et al., 1998
). Proteins were separated on polyacrylamide slab gels according to Laemmli (1970
), and Coomassie blue-stained or transferred to 0.45 µm polyvinylidene difluoride membranes (Millipore, Bedford, MA) (Towbin et al., 1979
). Non-specific reactivity was blocked by incubation for 1 h at room temperature with 5% non-fat dry milk 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, 77.1) for 60 min at room temperature. Horse-radish peroxidase-conjugated goat anti-mouse-IgG was used as secondary antibody (0.25 µg/ml) with 45 min incubations. Excess first and second antibodies were removed by washing 5 x 10 min in washing buffer. Detection was accomplished with an enhanced chemiluminescence system (SuperSignal® West Pico Chemiluminescent Substrate, Pierce, Rockford, IL) and subsequent exposure to Kodak XAR film (Eastman Kodak, Rochester, NY) for 5–30 s.
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Antibody preparation
Polyclonal antibodies were raised in mice based on standard immunization protocols (Harlow and Lane, 1988a
). Briefly, each female mouse received nine intra-peritoneal injections of recombinant -SNAP (20 µg each) in complete Freund's adjuvant at weekly intervals. Ascites were collected at the end of the immunization period, cleared by centrifugation and stored at 4°C. Total IgG was purified from the ascitic fluid by sequential caprylic acid and ammonium sulphate precipitations according to standard procedures (Harlow and Lane, 1988b
).
Indirect immunofluorescence
Sperm were washed in PBS and fixed and permeabilized in ice-cold methanol for 10 min at –20°C. After centrifugation (10 000 g for 2 min), sperm were washed twice with PBS containing 0.4% polyvinylpyrrolidone (PVP) (average MW=40 000; ICN, Aurora, Ohio, PBS/PVP). After washing, sperm pellets were suspended in blocking solution [2% bovine serum albumin in PBS/PVP] and incubated for 1 h at room temperature. Two anti-/ß SNAP antibodies were used: a commercial mouse monoclonal (clone 77.2, 1:200), and a mouse polyclonal raised in our laboratory. Whole ascites were diluted 1:50, and purified IgG was used at 100 µg/ml in blocking solution, added to sperm and incubated for 20 h at 4°C, with constant rocking. After washing with PBS/PVP (3x), TRITC-goat anti-mouse-IgG (15 µg/ml in 0.5% horse serum in PBS/PVP) was added and incubated for 1 h at room temperature. After washing as before, samples were mounted on glass slides and air-dried. Slides were examined with an Eclipse TE300 Nikon microscope equipped with a Hamamatsu Orca 100 camera operated with MetaMorph software (offline version 5.Or 1, Universal Imaging Corp., USA).
Protein determination
Protein concentrations were determined by the BioRad Protein assay in 96-well microplates. Bovine serum albumin was used as a standard and the results were quantified on a BioRad 3550 Microplate Reader.
Statistical analysis
Differences between experimental and control conditions were tested by a two-way analysis of variance 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.
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Results |
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Presence and subcellular localization of
-SNAP in human spermThe ATPase NSF and SNAPs are essential for many intra-cellular trafficking events and for the regulated exocytosis of neurotransmitters and hormones. Numerous observations have linked the function of these proteins with SNAREs. Thus, based on our previous findings that active NSF (Michaut et al., 2000
) and various members of all SNARE families (Tomes et al., 2002
) are required for the human sperm AR, we set out to investigate the possible participation of their counterpart -SNAP in this process. We initially investigated the presence of -SNAP in human sperm by Western blot using a monoclonal antibody raised against recombinant -SNAP (clone 77.1). Owing to the high similarity between - and ß-SNAP (83% identity) (Whiteheart et al., 1993
), the antibody recognizes both isoforms (Hanson et al., 1995
). Therefore, this tool alone is inadequate to determine which is the isoform present in sperm. However, given the widespread tissue distribution of -SNAP, including its prominent abundance in testis, and the restricted localization of ß-SNAP to neurons, we will use the term -SNAP throughout this manuscript. This does not rule out the possibility of ß-SNAP being an alternative/additional isoform relevant to sperm physiology. Immunoblot analysis of whole human sperm extracts demonstrated the presence of a single protein band with apparent molecular mass of 33 kDa, corresponding to -SNAP (Figure 1A
). To determine the cellular localization of -SNAP in human sperm, membrane and cytosolic fractions were prepared and probed on Western blot (Figure 1B
). The protein corresponding to 2.4 x 107 cells was loaded per lane. Densitometric analysis revealed that 60–70% of -SNAP was found in the soluble fraction, whereas 30–40% was associated with the particulate fraction. Similar amounts of -SNAP were present in Triton X-100 extracts made from cells treated or untreated with SLO (Figure 1C
). Furthermore, -SNAP did not run-down when permeabilized cells were maintained in buffer at 37°C for progressively longer periods of time (up to 75 min, data not shown).
A mouse polyclonal antibody was raised in our laboratory using recombinant -SNAP as immunogen. As was the case with the antibody from commercial sources, bacterially expressed His6-- and ß-SNAP were detected by this antibody in immunoblot experiments (Figure 2A ). Indirect immunofluorescence was used to localize -SNAP on fixed, permeabilized sperm. Both unfractionated ascites (Figure 2B ) and purified IgG (Figure 2C
) reacted with the sperm head in the acrosomal region. This fluorescence pattern was specific since it was not observed when the antibody was previously blocked with recombinant -SNAP (Figure 2D
). In contrast, tail-associated staining was not removed by pretreatment (Figure 2D
). Acrosomal immunostaining was also accomplished with a commercial mouse antibody (clone 77.2, Figure 2E
). Taken together, our data indicate that -SNAP is present in the acrosomal region of human sperm. This localization is similar to that found for its partner NSF (Michaut et al., 2000
). -SNAP partitions between the cytosolic and the membrane-bound compartment. Yet, the majority of the protein does not leak from SLO-permeabilized sperm despite its substantial cytosolic distribution and small size, perhaps reflecting its engagement in large molecular weight protein complexes, unable to exit the cell through the toxin-generated pores. Interestingly, sperm NSF exhibits the same run-down resistant behaviour (Michaut et al., 2000
).
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-SNAP is required for calcium-triggered acrosomal exocytosisTo determine the extent of
-SNAP's involvement in the AR, three antibodies were introduced into SLO-permeabilized human sperm before challenging with Ca2+. Exocytosis was completely inhibited by the addition of two commercially available monoclonal antibodies against -SNAP (Figure 3
, anti--SNAP-77.1
Ca, anti--SNAP-77.2Ca). The antibody raised in our laboratory (unfractionated ascites) inhibited the Ca2+-induced AR at concentrations up to a 1000-fold lower than those from commercial sources (Figure 3
, anti--SNAPCa). To test the specificity of this effect, the active sites of the antibody were blocked by pre-incubation with recombinant -SNAP before addition to permeabilized sperm. Inhibition was not observed when the antibody was pre-incubated with recombinant -SNAP (Figure 3
, anti--SNAP*Ca), suggesting that its inhibitory effect was due to binding to endogenous -SNAP. Given the potency of this antibody, the amount of recombinant -SNAP required to block its active site was quite low. Thus, the final concentration of recombinant -SNAP in the assay (2 µg/ml) was significantly lower than the ones that inhibit exocytosis. As expected, addition of this amount of His6--SNAP to the AR assay had no effect (Figure 3 , -SNAPCa).
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cpatton/maxc.htmlBecause the dissociation of
-SNAP from the SNARE complex is a step in its disassembly, one might imagine that an enhanced binding, accomplished by addition of exogenous -SNAP, would eventually impair disassembly and therefore inhibit fusion. We tested this hypothesis in permeabilized sperm by incubation with recombinant -SNAP prior to Ca2+ stimulation. The results depicted in Figure 4
show that Ca2+-triggered exocytosis was completely abrogated by bacterially expressed -SNAP (Figure 4 , -SNAPCa). This protein had no effect on exocytosis per se (Figure 4
, -SNAP). To characterize -SNAP's inhibition, purified NSF was added to permeabilized cells in the presence or absence of added -SNAP before challenging with the inducer. NSF completely overcame the inhibition due to excess -SNAP (Figure 4 , -SNAP+NSFCa), whereas NSF alone had no effect on the Ca2+-triggered AR (Figure 4
, NSFCa). The ability of NSF to overcome the inhibition by exogenous -SNAP suggests that a selective interaction, not a massive non-directed protein interaction on other aspects of the release machinery, is behind -SNAP's influence on the AR. Furthermore, the absence of effect in the presence of NSF alone suggests that the inhibition elicited by -SNAP is due to the protein and not its His6 tag. Taken together, these data suggest that -SNAP is not only present but also has an essential role in human sperm acrosomal exocytosis.
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-SNAP/NSF mediate Rab3-triggered human sperm ARIn the human sperm, there is a pathway for stimulating acrosomal exocytosis that is activated by Rab3 (Michaut et al., 2000
; Yunes et al., 2000
). Here, we set out to determine whether NSF/SNAP-mediated priming takes place before or after tethering of the acrosome to the plasma membrane elicited by Rab3A. Specific antibodies against -SNAP and NSF were introduced into SLO-permeabilized human sperm before challenging with Ca2+ (grey bars) or GTP-loaded Rab3A (black bars). The IgG fraction isolated from the anti--SNAP ascites raised in our laboratory (see Figure 3
) caused a complete inhibition of the Ca2+-elicited AR (Figure 5
, anti--SNAPCa). Since -SNAP exerts its biological effects in a concerted fashion with NSF, it would be expected that blockage of endogenous NSF would also prevent the AR. Indeed, loading of permeabilized sperm with an anti-NSF prevented the onset of exocytosis triggered by Ca2+ (Figure 5
, anti-NSFCa). The addition of GTP-loaded recombinant Rab3A to SLO-permeabilized human sperm induced an exocytotic response of a magnitude comparable to that of Ca2+ (Figure 5
). As was the case with Ca2+, pretreatment with the anti--SNAP and NSF antibodies inhibited Rab3A-triggered AR by > 90% (Figure 5
). These data indicate that -SNAP and NSF are involved in Rab3A-triggered acrosomal exocytosis in human sperm.
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-SNAP/NSF are required before intra-acrosomal calcium effluxWe have shown that NSF/SNAP are required in a priming step downstream of the tethering of the acrosome by Rab3. To further elucidate the site of action of these proteins, we resorted to a reversible blocker of exocytosis that prevents the AR by sequestering intra-acrosomal Ca2+. The reversible blocker of choice was the photolabile Ca2+ chelator NP-EGTA-AM. UV photolysis of NP-EGTA rapidly releases the caged Ca2+ with high photochemical yield (Ellis-Davies and Kaplan, 1994
). In our SLO-permeabilized human sperm model, the membrane-permeable compound NP-EGTA-AM crossed the plasma and the outer acrosomal membranes, accumulated inside the acrosome and thus precluded the availability of intra-acrosomal Ca2+. When permeabilized cells were preloaded with 10 µM NP-EGTA-AM, challenging with 10 µM Ca2+ failed to elicit exocytosis as long as the cells were kept in the dark (Figure 6
, NPCa). When NP-EGTA was converted to products with negligible Ca2+ affinity, by an UV flash, exocytosis was restored (Figure 6
, NPCah
). Having shown the requirement for NSF/SNAP (Figures 3 and 4
) and for intra-acrosomal Ca2+ (Figure 6
, and De Blas et al., 2002
) in the Ca2+-induced AR, we investigated the temporal relationship between the two. Thus, we asked whether SNARE complex disassembly takes place before or after intra-acrosomal Ca2+ release by loading permeabilized sperm with the caged Ca2+ buffer NP-EGTA-AM. In one case, the function-blocking anti--SNAP antibody (purified IgG, see Figure 5
) was added and the cells were pre-incubated for 15 min prior to addition of Ca2+. Following this treatment, uncaging of intra-acrosomal Ca2+ was achieved by flash photolysis of the NP-EGTA-AM (Figure 6
, NPanti-SNAPCah). As expected, the AR was blocked by the continuous presence of the inhibitor throughout the experiment. Our protocol allows for an AR inducer to prepare the fusion machinery up to the point when intra-acrosomal Ca2+ release is required. The inhibitors to test are added, and then the caged Ca2+ is released with a flash of UV light to restore the intra-acrosomal pool. If exocytosis is unaffected under these circumstances, it implies that the target of the inhibitor is required upstream of intra-acrosomal Ca2+ release. We added Ca2+ to NP-EGTA-AM-loaded sperm and incubated for 15 min prior to the addition of anti--SNAP antibody in the dark. UV flash photolysis of the Ca2+ chelator was applied at the end of the second incubation. No inhibition was observed under these conditions, indicating that -SNAP is acting upstream of the release of intra-acrosomal Ca2+ (Figure 6
, NPCaanti--SNAPh). We extended our analysis by using a function-blocking anti-NSF antibody (see Figure 5
). As expected, when added from the start, the antibody abolished fusion (Figure 6
, NPanti-NSFCah). After Ca2+, however, the reaction was completely resistant to the antibody, indicating that neither -SNAP (see above) nor NSF (Figure 6
, NPCaanti-NSFh) were still required. Taken together, our data support a post-tethering, prefusion role for SNAP/NSF-mediated priming in sperm exocytosis taking place prior to intra-acrosomal Ca2+ efflux.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Fusion of a transport vesicle with its target membrane is a fundamental process essential for cellular organization and the function of all eukaryotic cells. Several protein families involved in fusion are conserved from yeast to human, and are shared by constitutive and regulated exocytosis as well as by intra-cellular membrane fusion events (Jahn et al., 2003
; Li and Chin, 2003
; Sollner, 2003
). These families, among others, include SNAREs, SNAPs, NSF, Rab GTPases and Munc proteins. Their conservation has been interpreted as indicative that most, if not all, membrane fusion processes use the same common molecular machinery for fusion. The ATPase NSF, and its yeast homologue Sec18p, exerts its effects following recruitment, via SNAPs, to the SNARE complex. ATP hydrolysis by NSF leads to dissociation of itself and SNAPs from the SNAREs, and the disassembly of the SNARE complex. In the original SNARE hypothesis, disassembly of SNARE complexes would lead to membrane fusion (Sollner et al., 1993a
). That model has been extensively revised, and it was later shown that NSF/SNAP dissociate the cis- (i.e. all proteins in the same membrane, thus fusion-incompetent) but not the trans- (i.e. VAMP and syntaxin/SNAP-25 reside in opposing membranes, thus fusion-competent) (Weber et al., 2000
) SNARE complex.
-SNAP has been implicated in Ca2+-dependent exocytosis in neuronal (DeBello et al., 1995
; He et al., 1999
) and non-neuronal (Morgan and Burgoyne, 1995
; Nagamatsu et al., 1999
; Nakamichi and Nagamatsu, 1999
; Xu et al., 2002
; Abonyo et al., 2003
) cell types. In this study, we investigated the involvement of SNAP/NSF during the Ca2+-dependent acrosomal exocytosis in human sperm. We made use of a permeabilization protocol developed in our laboratory, which permits entry of antibodies, recombinant proteins, Ca2+, and other molecules into the cell (Diaz et al., 1996
; Yunes et al., 2000
). This system is easily adapted, reflects the in vivo organization of the cell, and has been used to demonstrate the roles of Rab3A (Yunes et al., 2000
), NSF (Michaut et al., 2000
), synaptotagmin VI (Michaut et al., 2001
), the SNARE complex (Tomes et al., 2002
), calmodulin (Yunes et al., 2002
), and protein tyrosine kinases and phosphatases (Tomes et al., 2004
) in the Ca2+-dependent AR of human sperm.
Sperm might prove to be a useful model to study SNAP/NSF timing given that, in contrast to the nerve terminal, the extent of exocytosis is not affected by vesicle membrane or SNARE protein recycling. In other words, these proteins can only exhibit prefusion roles. -SNAP has been found previously on the acrosome of the mouse round spermatids (Ramalho-Santos et al., 2001
), although a functional role for this protein in acrosomal exocytosis has not been demonstrated to date. Here we report the presence of /ß-SNAP in the acrosomal region of human sperm and its requirement in the Ca2+-triggered AR. A single band corresponding to the molecular mass of -SNAP was detected by Western blot in whole sperm extracts (Figure 1A
). The protein partitions to cytosolic and membrane-bound compartments after subcellular fractionation and does not leak from the cells following permeabilization with SLO (Figure 1B and C
). Although SNAPs are soluble proteins, they associate with membranes through interactions with SNAREs (Sollner et al., 1993b
). An alternative mechanism of membrane association through AMPA glutamate receptors has also been reported (Osten et al., 1998
; Hanley et al., 2002
). In its best characterized role, -SNAP is an adaptor protein that mediates the binding of NSF to SNARE complexes. In turn, -SNAP stimulates the ATPase activity of NSF, leading to SNARE complex disassembly and SNAP/NSF release (May et al., 2001
). Thus, we would have expected that incubation of permeabilized sperm with Mg2+-ATP to allow NSF-mediated complex disassembly would result in the efflux of -SNAP from the cells. Surprisingly, this was not the case, and sperm retained the same amount of -SNAP when treated with Mg2+-ATP, ATP--S or buffer (data not shown). This finding suggests the existence of poorly characterized binding sites for NSF/SNAP in sperm and is in line with previous reports (Morgan and Burgoyne, 1995
; Colombo et al., 1996
; Tagaya et al., 1996
). It is worth pointing out that, in addition to SNAP/SNAREs, NSF binds the yeast Vtc complex (Muller et al., 2002
), the AMPA glutamate receptors (Osten et al., 1998
; Song et al., 1998
), the ß2 adrenergic receptor (Cong et al., 2001
), and ß-arrestin (McDonald et al., 1999
).
In sperm, -SNAP exhibits acrosomal localization (Figure 2
), consistent with its proposed role in the exocytosis of this granule and with the previously reported localization of its partner NSF (Michaut et al., 2000
; Ramalho-Santos and Schatten, 2004
) and members of the SNARE protein families (Schulz et al., 1997
; Katafuchi et al., 2000
; Ramalho-Santos et al., 2000
). The first line of evidence for the requirement of -SNAP in the AR comes from the use of specific antibodies. As shown in Figure 3
, loading of SLO-permeabilized sperm with three different anti-SNAP antibodies reduced sperm's exocytotic response to Ca2+. The fact that a specific anti-NSF antibody exhibits identical behaviour (Figure 5
) strengthens the conclusion that SNAP/NSF is required in the human sperm AR. The second line of evidence comes from the use of recombinant proteins. The addition of recombinant -SNAP inhibited the AR (Figure 4
). This inhibition was specific since it was completely reversed by preincubation with recombinant NSF (Figure 4
). As we (Figure 4 and Michaut et al., 2000
) and others (Wang et al., 2000
; Abonyo et al., 2003
) have previously reported, exogenously added NSF alone does not have a measurable effect on exocytosis. An inhibitory role for the exogenously added -SNAP homologue Sec17p has been observed in the yeast vacuolar fusion system. As is the case in sperm, this inhibition is reversed by Sec18p (Wang et al., 2000
). Similarly, overexpression of SNAP in transgenic flies disrupts the secretory pathway, inhibiting synaptic transmission (Babcock et al., 2004
). Co-expression of NSF completely suppressed the phenotypes imparted by the SNAP transgene (Babcock et al., 2004
).
We have previously shown that the acrosome behaves as a Ca2+-storing organelle (De Blas et al., 2002
). Release of intra-vesicular Ca2+ takes place after Rab3-elicited tethering of the acrosome to the plasma membrane and is necessary for the human sperm AR. By using a photosensitive Ca2+ chelator in combination with inhibitory antibodies we have been able to show that -SNAP and NSF are required early in sperm exocytosis, before the release of Ca2+ from the acrosome (Figure 6
). An early role for these proteins in vesicle fusion has been described in a variety of systems (see the Discussion in Mayer et al., 1996
for an overview). Our findings are in agreement with the crucial role assigned to SNAP/NSF in a priming step during vesicle exocytosis (Banerjee et al., 1996
; He et al., 1999
; Xu et al., 1999
; Graham and Burgoyne, 2000
). In the studies of secretory granule exocytosis, priming is a term used to describe any functionally detected ATP-dependent process that occurs before fusion (Burgoyne and Morgan, 2003
). There are several, mutually non-exclusive possibilities for the molecular nature of the priming reaction which requires ATP hydrolysis by NSF. In the most comprehensive view, priming consists of distinct molecular events, including cis-SNARE complex disruption and specific activation of individual SNAREs, for their subsequent role in vesicle docking and fusion. The chaperone-like action of NSF would require one or more cycles of concerted ATP hydrolysis to allow correct re-folding of the SNARE protein to occur (Haynes et al., 1998
). Confusion in the literature has arisen from the use of the term priming for a post-docking step during synaptic vesicle maturation and from the application of dissimilar criteria to define membrane fusion steps (Burgoyne and Morgan, 1995
). Thus, apparently opposite conclusions in terms of whether priming takes place before or after docking have arisen from the fact that morphological, functional or molecular criteria have been used to claim granules are or not docked (see the Discussion in Xu et al., 1999
and Burgoyne and Morgan, 2003
). Our view coincides with that put forth in the yeast system, among others, where priming occurs on separate membranes and prepares them for docking. After priming, attachment occurs in two ordered subreactions comprising reversible tethering and irreversible, trans-SNARE pairing-mediated docking (Ungermann et al., 1998
). In our working model, Ca2+ stimulation would result in the activation of Rab3A, which in turn would mediate tethering of the acrosome to the plasma membrane. Priming, defined as a general conformational activation of SNARE proteins including dissociation of pre-existing cis-SNARE complexes, by NSF/-SNAP would then take place on either or both the plasma and the outer acrosomal membrane. SNARE proteins would assemble in partially zippered trans-SNARE complexes and, in doing so, would elicit SNARE-dependent docking of the acrosome. This docking machinery would contain or interact with the Ca2+ sensor synaptotagmin. Upon binding Ca2+ mobilized from the acrosome, synaptotagmin would undergo a conformational change that would induce full zippering of the SNARE complexes, ultimately promoting fusion (AR). Experiments are underway in our laboratory to test these hypotheses.
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Acknowledgements |
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The authors thank Tirso Sartor and Marcelo Furlán for their excellent technical assistance, Dr Sean Patterson for critical reading of the manuscript, Drs Philip Stahl, María I. Colombo and Sidney Whiteheart for plasmids. MAM and GADB are thankful to the Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina for fellowships. This work was supported by an International Research Scholar Award from the Howard Hughes Medical Institute, a Carrillo-Oñativia Scholar Award (Argentina), and grants from Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina to LSM and from the National Institutes of Health (HD 38082) to PEV.
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Notes |
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4Present address: Biology Department, 3749 Hamilton Walk, 224 Leidy Labs, University of Pennsylvania, Philadelphia, PA 191046018, USA
5Present address: 208 Paige Labs, Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA 01003, USA
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