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Originally published In Press as doi:10.1074/jbc.M412920200 on March 17, 2005

J. Biol. Chem., Vol. 280, Issue 21, 20197-20203, May 27, 2005
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Synaptotagmin VI and VIII and Syntaxin 2 Are Essential for the Mouse Sperm Acrosome Reaction*

Darren M. Hutt{ddagger}§, Jay M. Baltz{ddagger}, and Johnny K. Ngsee{ddagger}||

From the {ddagger}Ottawa Health Research Institute, Department of Cellular and Molecular Medicine and Department of Obstetrics and Gynecology, Division of Reproductive Medicine, University of Ottawa, Ottawa, Ontario K1Y 4E9, Canada

Received for publication, November 15, 2004 , and in revised form, March 2, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The sperm acrosome is a large secretory granule that undergoes calcium-stimulated exocytosis by a mechanism analogous to neuronal secretion. In neurons the core SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex, composed of syntaxin (Stx), SNAP-25, and VAMP2, mediates vesicle fusion, whereas calcium regulation is thought to be accomplished by the synaptotagmin (Syt) family, some of which exhibit calcium-dependent binding to syntaxin and SNAP-25. Sperm express Syt VI and VIII and Stx2, which are co-localized to the acrosomal compartment where they might mediate exocytosis in response to calcium influx. Therefore, we examined the calcium dependence and isoform-specific interaction of Syt and Stx. We found that Stx2 binds to Syt I, VI, and VIII in a calcium-dependent manner with EC50 values of 175, 233, and 96 µM calcium, respectively. We also determined that the EC50 for calcium of the acrosome reaction in streptolysin O-permeabilized sperm is 87 µM, which closely coincides with the calcium sensitivity of Stx2 and Syt VIII interaction. Consistent with this is the greater potency of recombinant Syt VIII, VI, and Stx2 compared with other isoforms in inhibiting the acrosome reaction in streptolysin O-permeabilized sperm. Similarly, introduction of Syt VIII-specific antibodies was equally effective in inhibiting the acrosome fusion. Taken together, our data suggest a critical role for Syt VIII and Stx2 in membrane fusion and acrosome reaction in the sperm.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Upon stimulation by direct contact with the zona pellucida or progesterone signaling via an unidentified pathway, the sperm undergoes the regulated exocytosis of its single secretory granule, the acrosome. This acrosome reaction (AR)1 must occur before the sperm can penetrate the zona pellucida, since the acrosome contains hydrolytic enzymes necessary for degradation of the surrounding zona. In addition, the newly exposed membrane surface, the inner acrosomal membrane, contains secondary binding sites that mediate continued binding to the zona pellucida during penetration. Therefore, the AR is a prerequisite for sperm-egg plasma membrane binding and fusion (1). It has been established that a rise in intracellular calcium (Ca2+i) subsequent to zona pellucida or progesterone binding triggers the AR (2–10).

In other cells the calcium-regulated release of secretory peptide hormones and neurotransmitters is mediated by a family of proteins commonly referred to as the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) (11–13) complex. The core SNARE complex is composed of two proteins on the plasma membrane, syntaxin (Stx) (14) and SNAP-25 (15), collectively termed target SNARES (t-SNARES), and the SNARE protein on the vesicle (v-SNARE), called vesicle-associated membrane protein (VAMP) or synaptobrevin. Formation of the SNARE complex brings the fusing membranes into close apposition and is a prerequisite for fusion. A number of isoforms of each SNARE protein have been identified, including 18 Stxs (16), 3 members of the SNAP-23/25/29 family (15, 17, 18), and 8 VAMP isoforms in mammals (19–22).

There is growing evidence that isoforms of SNARE proteins are involved in the fusion of the outer acrosomal membrane and the plasma membrane in the sperm AR, consistent with the AR being a form of Ca2+-mediated exocytosis. All three SNARE proteins, Stx, VAMP, and SNAP, were first shown to be present in sperm in the sea urchin, where they remained tightly associated with the shed acrosomal vesicles after the acrosome reaction, indicating co-localization with the fusion machinery (23, 24). In mammalian sperm, VAMP and SNAP-25 proteins were also found to be present, and disruption of VAMP with botulinum neurotoxin inhibited AR (25). The expression and functional importance of Stx isoforms in sperm has been controversial. Stx1A, -1B, -4, and -6 have all been reported in human sperm based upon their detection with antisera (25, 26). However, botulinum neurotoxin C, which selectively cleaves Stx1 and -2, inhibited the AR, suggesting that Stx4 and -6 in human sperm are not able to compensate for the inactivation of these isoforms (25). In addition, the identification of Stx isoforms may also suffer from non-specificity of the antisera used, since the Stx1 antiserum appears to detect a protein in sperm lysate that is smaller than predicted for Stx1 (26). In the mouse, mRNA for Stx2 and -4, but not Stx1 or -3, is expressed in the testis and Stx2 protein, but not Stx4 protein (27) is present in the sperm, suggesting that it is Stx2 that plays an active role in the AR in sperm.

Despite the critical role the core SNARE proteins are proposed to play in triggering vesicle fusion, Ca2+-mediated secretion requires an additional regulatory mechanism that can transduce the increase in cytoplasmic Ca2+ to the SNARE fusion machinery. The established candidates for this calcium sensor are members of the synaptotagmin (Syt) family. There are at least 15 distinct mammalian Syt isoforms expressed in both neuronal and non-neuronal cells (28–37). Functional studies have provided evidence that some Syt isoforms are required for Ca2+-mediated vesicle fusion in neurons and other systems (38–42). Syt isoforms have been shown to interact with the core complex SNARE proteins Stx (32, 43, 44) and SNAP-25 (45) in the presence of calcium. In addition, Syts are capable of binding to various calcium channels (46–50); therefore, they are ideally situated to sense an increase in Ca2+i and to transmit this signal to the core SNARE complex. This has been best studied in neurons where the interactions between Syt I or II and stx1 have been extensively investigated and Ca2+-dependent phospholipid binding has been well established (44). However, the role of other Syt isoforms in Ca2+-mediated exocytosis is less clear.

In sperm, Syt VI and VIII proteins have clearly been shown to be localized to the human and mouse acrosome, respectively, and thus, could potentially function as Ca2+ sensors to trigger the AR (51, 52). A difficulty with this scenario, however, is that the binding of both of these Syt isoforms to the SNARE complex had been reported to be Ca2+-insensitive (32, 53), as had phospholipid binding by Syt VIII (32, 54, 55). In addition, at least in nerve terminals, Syt VI is apparently not localized to exocytotic vesicles (56). On the other hand, no other Syt isoforms had been conclusively identified in sperm. Although Syt I was previously reported to be present in sperm (26), the antiserum used has since been shown to cross-react with almost all other Syt isoforms (52), invalidating this identification. In addition, mRNA for Syt I is not expressed in the testis, unlike Syt VI and VIII, and similarly, Syt I mRNA is absent from spermatogenic cells, whereas Syt VIII is present (52).

Therefore, we have now re-examined whether the Syt and Stx isoforms established to be expressed at the protein level in murine sperm (57–60) might actually be Ca2+-sensitive and mediate the AR in sperm. Indeed, we report here that, despite the interaction of Syt VIII with Stx1 being previously identified as Ca2+-insensitive (32), both actually exhibit isoform-specific, Ca2+-regulated binding. We then used the streptolysin O (SLO)-permeabilized sperm preparation to establish that these Syt and Stx isoforms apparently function in acrosomal exocytosis in the mouse.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—The pGEX-KG Syt constructs were obtained from Dr. T. Sudhof, and the pGEX-2T Stx constructs were obtained from Dr. L. Elferink. SLO was purchased from Sigma, and recombinant SLO was purchased from Dr. S. Bhakdi from the Institute of Medical Microbiology (Mainz University, Germany).

Cloning of Syntaxin to pQE9 —The cytoplasmic region of Stx1 and 2 were PCR-amplified from pGEX-Stx1 or -2 using the following oligonucleotides: Stx1 (forward) 5'-TCCCTGCAGACATGAAGGACCGAACCCAGG-3' and Stx1 (reverse) 5'-GTAAGCTTCTACTTCTTCCTGCGTGCC-3', Stx2 (forward) 5'-ATTAGATCTATGCGGGACCGGCTGCCGGAC-3' and Stx2 (reverse) 5'-AGAAAGCTTTCACCACTTTTTCCGTCTGGCC-3'. The fragments were cut with PstI/HindIII and BglII/HindIII, respectively, and cloned into pQE9 vector (Qiagen), which was modified to contain a hemagglutinin (HA) tag between the His6 tag and the multiple cloning site.

Cloning of Synaptotagmin to pQE9 —The cytoplasmic region of Syt I, VI, and VIII, referred to as C2AB, were PCR-amplified from pGEX-KG Syt I, VI, and VIII using the following oligonucleotides: Syt I (forward) 5'-CGAGATCTGAGAAACTGGGAAAGCTCCAATATTCA-3' and Syt I (reverse) 5'-CAGAAGCTTACTTCTTGACAGCCAGCATGGCATC-3', Syt VI (forward) 5'-CGAGATCTGCCAAGAGCTGTGGGAAGATCA-3' and Syt VI (reverse) 5'-CTGAAGCTTCACAACCGGCGGGTTCCCTCT-3', Syt VIII (forward) 5'-TAGGATCCGTTCAACCAGATGTGGACTGC-3' and Syt VIII (reverse) 5'-TTGAATTCAGGAGCGAGGCCTAAGCAG-3'. The Syt I and VI C2AB were digested with BglII and HindIII and cloned into the modified pQE9 (above). The Syt VIII C2AB was digested with BamHI and EcoRI, which was blunt-ended, and the fragment was cloned into modified pQE9 (above) cut with BglII and SmaI.

Purification of GST-Synaptotagmin—The purification of GST Syts was performed as described by the manufacturer for GST fusion proteins. Briefly, the bacterial cells were homogenized in 25 mM Tris-HCl, pH 8, 150 mM NaCl, and 2 mM phenylmethylsulfonyl fluoride. After centrifugation at 10,000 x g for 20 min, the supernatant was incubated with glutathione-Sepharose beads (Amersham Biosciences) overnight at 4 °C. The beads were washed with wash buffer 1 (25 mM Tris-HCl, pH 7.5, 500 mM NaCl, and 0.5% Triton X-100) followed by wash buffer 2 (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.05% Triton X-100). The amount of bound GST fusion protein was quantified by Coomassie Blue staining of a polyacrylamide gel using BSA as a standard.

Purification of His6/HA-tagged Proteins—The purification of His6/HA-tagged Stx for in vitro binding assay was performed as described by the manufacturer. Briefly, the cells were homogenized in 50 mM sodium phosphate, pH 8, 300 mM sucrose, and 2 mM phenylmethylsulfonyl fluoride. The homogenate was supplemented with Triton X-100 to a final concentration of 1%. After centrifugation at 10,000 x g for 20 min, the supernatant was added to nickel nitrilotriacetic acid beads preequilibrated in lysis buffer and incubated overnight at 4 °C. The beads were successively washed with wash buffer 1 (50 mM sodium phosphate, pH 7, 500 mM NaCl, and 1% Triton X-100) and wash buffer 2 (50 mM sodium phosphate, pH 7, 150 mM NaCl, and 0.01% Triton X-100). The fusion protein was eluted in wash buffer 2 containing 250 mM imidazole and quantified by Coomassie Brilliant Blue staining of polyacrylamide gel using BSA as a standard. The purification of His6/HA fusion proteins for inhibition of acrosome reaction was performed as described by the manufacturer for His6-tagged fusion proteins. Briefly, the cells were resuspended in 50 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM dithiothreitol, and protease inhibitor mixture (Sigma) and disrupted by a French press. After centrifugation at 35,000 x g for 25 min at 4 °C, the supernatant was added to nickel nitrilotriacetic acid beads and incubated overnight at 4 °C. The column was washed extensively with wash buffer (50 mM Tris-HCl, 500 mM NaCl, and 25 mM imidazole) until the A280 was below 0.05 and was followed by Krebs-Ringer buffer (KRB), pH 7.4, without CaCl2. The His6-tagged fusion proteins were eluted in KRB containing 250 mM imidazole and quantified by Coomassie Brilliant Blue staining of polyacrylamide gel using BSA as a standard.

Calcium Buffers—10x stock calcium buffers were prepared by mixing the appropriate amount of 1 M CaCl2 and 0.5 M EGTA to obtain the predicted 10x final free calcium concentration. The precise free calcium concentration of the 1x buffer for each concentration used was determined by a calcium-selective electrode (Corning) calibrated with calcium standards.

In Vitro Binding Assay—Determinations of binding of Stx1 and -2 to Syt I, VI, and VIII were performed using bacterially expressed proteins in a pull-down assay. The GST-tagged cytoplasmic region of Syt was purified by glutathione-Sepharose as described above and incubated with equal amounts of His6/HA-tagged Stx1 or -2 for 4 h at 4 °C in binding buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.05% Triton X-100) with the indicated free Ca2+ concentration added from 10x stock solutions. After the incubation, the beads were washed three times with the appropriate ice-cold binding buffer, and bound proteins were subjected to Western immunoblot analysis using anti-HA (Roche Applied Science) antibody and Alexa-488 goat anti-mouse secondary antibody (Invitrogen) to detect bound Stx. Densitometric analysis was performed using a Typhoon 8600 imager (Amersham Biosciences). The results are an average of at least three separate experiments.

Ca2+ Titration of the Acrosome Reaction in Streptolysin O-permeabilized Mouse Sperm—Sperm were collected from cauda epididymides of 12–15-week-old CD-1 mice and allowed to "swim-up" for 15 min in KRB, pH 7.4, media (5.6 mM glucose, 0.55 mM sodium pyruvate, 25 mM sodium bicarbonate, 53 mM sodium lactate, 99.6 mM sodium chloride, 4.8 mM potassium chloride, 1.2 mM potassium dihydrogen phosphate, and 1.2 mM magnesium sulfate) containing 3 mg/ml BSA (37 °C, 5% CO2). Sperm were diluted to a concentration of 5.0 x 106 sperm/ml and permeabilized with 0.6 units/ml SLO (Sigma) for 5 min at 37 °C in 5% CO2 and subsequently supplemented with 10x CaCl2 solutions to the indicated final concentration. Sperm were incubated for an additional 20 min at 37 °C in 5% CO2 and fixed with 4% paraformaldehyde. The extent of acrosome reaction was determined by Coomassie Brilliant Blue staining as indicated below.

Introduction of Recombinant Proteins and Antisera into Streptolysin O-permeabilized Mouse Sperm—Sperm were collected from cauda epididymides of 12–15-week-old CD-1 mice and allowed to swim-up for 15 min in KRB, pH 7.4, media containing 1.7 mM CaCl2 and 3 mg/ml BSA (37 °C, 5% CO2). Swim-up sperm were pelleted at 300 x g for 5 min and resuspended in Ca2+-free KRB, pH 7.4. Sperm were diluted to a concentration of 5.0 x 106 sperm/ml with KRB (–Ca2+) and permeabilized with 0.6 units/ml (Sigma) or 5 µg/ml recombinant SLO for 5 min at 37 °C in 5% CO2 and subsequently supplemented with CaCl2 to a final concentration of 1 mM. Sperm were incubated for an additional 20 min at 37 °C in 5% CO2 and fixed with 4% paraformaldehyde. Recombinant proteins or antibodies were added at the permeabilization step from a concentrated stock in KRB to the indicated final concentrations and maintained throughout the subsequent incubation.

Assessment of Acrosomal Status—Fixed sperm were harvested by centrifugation at 800 x g for 5 min and washed 2 times with 0.5 ml of 0.1 M ammonium acetate, pH 9.0. The sperm were subsequently resuspended in 0.1 M ammonium acetate, pH 9.0, and air-dried on glass slides. The slides were then washed with water, methanol, and water for 5 min each. The sperm were stained with 0.625% Coomassie Brilliant Blue G-250 in 50% methanol and 10% acetic acid for 10 min at room temperature (61). The slides were subsequently washed 4 times with distilled H2O and mounted with 30% glycerol in phosphate-buffered saline. Sperm were imaged by bright-field microscopy using a Zeiss AxioPhot microscope and scored for acrosomal staining.

Data Processing and Curve Fitting—Data for the SLO-treated sperm were corrected for spontaneous AR by subtracting the percentage of AR in non-permeabilized sperm from that of SLO-permeabilized sperm, and the maximal value was subsequently normalized to 100%. Two models were compared for fit to the data obtained from the Ca2+ titration of the AR in SLO-permeabilized sperm. These included a model that assumed a single binding site for Ca2+ of the form y = max/(1 + 10(logEC50 – {chi}) x Hill slope), where max is the maximum AR at saturating Ca2+, EC50 is the Ca2+ concentration at half-maximal binding, and Hill slope is the Hill coefficient that reflects cooperativity. The data were fit by nonlinear least squares regression (SigmaPlot) with max and EC50 as parameters of the fit. In addition, the fit to the data was compared assuming either a fixed Hill slope of 1 (no cooperativity) or a variable Hill slope fit to the data. The Hill coefficient was also calculated from a Hill plot of the data as log(AR/nAR) versus log[Ca2+], where AR is the fraction of acrosome-reacted sperm, and nAR is the fraction of acrosome intact sperm.

The other model that was tested assumed two binding sites with different Ca2+ affinities (EC1 and EC2 representing the first and second EC50, respectively), which had the form y = max x (F1/(1 + 10(logEC1 – {chi}) x Hill slope 1) + (1 – F1)/(1 + 10(logEC2 – {chi}) x Hill slope 2)), where F1 is the fraction of the population assumed to react with EC1 (i.e. first EC50 component), and the remainder are assumed in this model to react with EC2, the second EC50 component. The comparison between the fits afforded by the one- and two-site models was done by an F-test of the residuals. The in vitro Syt-Stx binding was fitted to a one-site model with a fixed slope using the equation as described above with a Hill slope of 1.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Calcium Dependence of the Acrosome Reaction—To estimate the calcium dependence of the AR in mouse sperm, the free calcium concentrations in the media were varied from 0 to 2 mM, and the extent of AR was assessed in sperm whose plasma membranes had been selectively permeabilized with SLO to allow equilibration of calcium at the site of fusion with external calcium. As predicted, increasing the extracellular calcium concentration resulted in a dose-dependent increase in AR in SLO-permeabilized sperm (Fig. 1), which appeared to reach saturation as calcium concentrations approached the millimolar range. Analysis of the data using a 1-site sigmoidal-dose response model as described under "Materials and Methods" yielded an EC50 of 87 µM Ca2+. The Hill slope thus determined was 0.67, which suggests that the AR does not exhibit cooperativity. A Hill plot of these data was linear, and linear regression yielded a similar slope of 0.73 (Fig. 1, inset). These data are consistent with a Ca2+ dependence of AR with a Hill slope of ~1 as a nonlinear regression of the same curve, but assuming a fixed Hill slope of 1 did not perceptibly alter the curve and yielded essentially the same EC50 (98 µM; not shown).

We then tested whether a model assuming two independent binding sites for Ca2+ could provide a significantly better fit to the data, as there was a slight inflection in the data around log[Ca2+] =–4. This yielded EC50 values of 36 and 1000 µM for the 1st and 2nd sites, respectively (not shown). The first EC50 is of the same order as that obtained with the one-site model; however, the second was well above the likely physiological range. In addition, a comparison of the fits provided by the 1- versus 2-site models by an F-test of the residuals indicated that the more complicated 2-site model did not provide a significantly better fit (F = 0.45, p = 0.72). Therefore, a simple one-site model provided the simplest and best fit and was used for further comparison.



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  		FIG. 1.
Calcium titration of the mouse sperm acrosome reaction. SLO-permeabilized sperm were incubated in KRB containing various concentrations of free calcium from 34 µM to 2 mM for 20 min at 37 °C in 5% CO2. The sperm were fixed and stained with Coomassie Brilliant Blue, and the extent of AR was assessed by counting at least 200 sperm per slide. The data are expressed as a percentage of maximal AR, which is obtained after subtracting the extent of AR in non-permeabilized sperm from that seen in SLO-permeabilized samples at each calcium concentration and subsequently expressing each as a percentage of the maximal AR. The values represent the average of five independent experiments. The EC50 value for the calcium titration of the AR is 87 µM. Inset, a Hill plot for the Ca2+ titration of the AR is shown. The linear regression reveals a Hill coefficient of 0.73. nAR is the fraction of acrosome intact sperm.

 
Calcium Dependence of Binding between Synaptotagmin and Syntaxin Isoforms in Sperm—The Ca2+ dependence of binding between the isoforms of Syt and Stx reported to be present at the protein level in sperm, namely Syt VI and VIII and Stx2 (27, 51, 52), was determined by an in vitro binding assay using the cytoplasmic regions of recombinant proteins. Recombinant His6/HA-tagged Stx1 or -2 was added to GST-Syt I, VI, or VIII bound to glutathione-Sepharose in a pull down assay in the presence of varying Ca2+ concentrations. The amount of Stx that bound to Syt was measured by Western immunoblot using anti-HA. The data were analyzed on a 1-site binding model with a Hill slope of 1 using the equation described under "Materials and Methods." Analysis of the variable slope model yielded Hill coefficients between 0.68 and 0.92 for all the binding permutations with no significant difference in the EC50 values between the fixed and variable slope models. We utilized the binding of Syt I to Stx1 as a positive control since this interaction has been extensively characterized and shown to exhibit an EC50 value in the order of 100–180 µM Ca2+ (44, 62). Our EC50 value of 117 µM Ca2+ for this same pairing (Fig. 2A) is within the previously determined range. In addition, we have shown here that Syt I binds to Stx2 and determined the EC50 for this interaction to be somewhat higher than that for Stx1, at 175 µM (Fig. 2B).

We next found that Syt VIII bound to Stx2 and that this binding was Ca2+-dependent (Fig. 2B) with an EC50 of 96 µM. Syt VIII binding to Stx1 was also Ca2+-dependent (Fig. 2A), with a similar EC50 of 118 µM. Thus, in contrast to a previous report (32), we found here that Syt VIII interacted in a Ca2+-dependent fashion with both Stx isoforms tested, with similar affinity for Ca2+.



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  		FIG. 2.
Calcium dependence of synaptotagmin binding to syntaxin. GST-tagged Syt I, VI, or VIII were incubated with His6/HA-tagged Stx1 or -2 in Tris-buffered saline with 0.05% Triton X-100 containing the indicated concentrations of free calcium for 4 h at 4 °C. The bound Stx were analyzed by Western blot using anti-HA antibody. The values are expressed as a percentage of maximal binding and are the average of at least three independent experiments. A, Stx1 binding to Syt I (•), VI ({blacktriangledown}), and VIII ({blacksquare}) show that Syt I and VIII have a similar affinity for Stx1 with EC50 values of 117 and 118 µM Ca2+, respectively, whereas Syt VI has a lower affinity for Stx1 with an EC50 of 352 µM. B, Stx2 binding to Syt I (•), VI ({blacktriangledown}), and VIII ({blacksquare}) show that Syt VIII has the highest affinity for Stx2 among the isoforms tested with an EC50 value of 96 µM Ca2+. Syt I exhibits a lower affinity for Stx2 than for Stx1 with an EC50 of 175 µM Ca2+. Conversely, Syt VI exhibits a higher affinity for Stx2 than for Stx1 with an EC50 of 233 µM Ca2+.

 
We also found that Syt VI bound to both Stx isoforms and that the binding was similarly Ca2+-dependent. However, binding occurred only at much higher levels of Ca2+, with an EC50 for binding to Stx2 of 233 µM (Fig. 2B) and for Stx1 of 352 µM (Fig. 2A).

Inhibition of the Acrosome Reaction by Anti-synaptotagmin VIII Antisera—The data reported above indicated that, between the isoforms known to be present in mouse sperm, the interaction between Syt VIII and Stx would occur first as Ca2+ increases due to its lower EC50 relative to Syt VI. Thus, to determine whether Syt VIII has a functional role in the AR, we introduced IgG purified from antisera we raised against recombinant GST-Syt VIII and a separate, previously characterized anti-Syt VIII peptide antiserum (52) into SLO-permeabilized sperm and assessed the extent of AR after stimulation with saturating (1 mM) Ca2+. We found that sperm treated with 100 µg/ml purified anti-GST-Syt VIII IgG resulted in a 58% inhibition of AR relative to untreated sperm or sperm treated with 100 µg/ml preimmune IgG (Fig. 3). Similarly, treatment of sperm with 100 µg/ml anti-Syt VIII (peptide) IgG resulted in an 83% inhibition of the AR relative to the untreated or preimmune IgG-treated sperm (Fig. 3). We also attempted a similar inhibition using a commercially available, affinity-purified anti-Stx2 antibody (Stressgen; 10 µg/ml). However, this did not result in inhibition of the acrosome reaction (Fig. 3; see "Discussion").



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  		FIG. 3.
Inhibition of the acrosome reaction with synaptotagmin VIII antibodies. SLO-permeabilized sperm were incubated with 100 µg/ml preimmune, anti-GST Syt VIII or Syt VIII peptide IgG or 10 µg/ml affinity-purified anti-Stx2 antibody and challenged with 1 mM Ca2+. The extent of AR was determined by counting a minimum of 200 sperm per slide, and the results are the average of at least 3 independent experiments expressed as a percentage of maximal AR. The presence of anti-Syt VIII IgG resulted in inhibition of the AR relative to untreated sperm or sperm incubated with equal amount of preimmune IgG. The addition of anti-Stx2 antibody had no-inhibitory effect on the ability of the sperm to undergo AR.

 
Inhibition of the Acrosome Reaction by Recombinant Synaptotagmin—To further confirm a functional role of Syt in the AR, we introduced varying amounts of the bacterially expressed His6/HA-tagged cytoplasmic region of Syt (C2AB) into SLO-permeabilized sperm. The addition of the C2AB of Syt VIII resulted in a dose-dependent inhibition of the AR. Increasing the amount of recombinant C2AB from 5 to 10 and 20 µg/ml resulted in 37, 49, and 74% inhibition of the AR, respectively (Fig. 4). The addition of an amount equal to the highest concentrations tested for SNARE proteins, 20 µg/ml, of an irrelevant protein (bacterially expressed His6/HA-tagged Abstrakt (Abs), a DEAD-box helicase with no known role in membrane fusion) (63) had no measurable effect (Fig. 4). The addition of the C2AB of Syt VI to the sperm also resulted in a dose-dependent inhibition of the AR that appears to saturate at ~10 µg/ml. However, the addition of the C2AB region of Syt I had no significant inhibitory effect on the AR at either 5 or 10 µg/ml but resulted in a 54% inhibition of the AR at the maximal dose tested, 20 µg/ml. The inhibitory effect of Syt I, VI, and VIII seen at 20 µg/ml was eliminated when the protein was denatured by heating for 5 min at 90 °C before its addition to the sperm (see Fig. 6). These observations suggest that Syt is involved in the regulation of the AR and lends further support of a role for Syt VIII in the AR.



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  		FIG. 4.
Inhibitory effect of recombinant synaptotagmin on the acrosome reaction. SLO-permeabilized sperm were incubated with the indicated amount of recombinant His6/HA-tagged Abs (•), Syt I ({circ}), Syt VI ({triangledown}), or Syt VIII ({blacktriangledown}), and the extent of AR was assessed after challenge with 1 mM free Ca2+. The values represent the average of at least three independent experiments and are expressed as a percentage of maximal AR.

 
Inhibition of the Acrosome Reaction with Recombinant Syntaxin—To assess the role of Stx in the regulation of the AR, we introduced varying amounts of the bacterially expressed His6/HA-tagged cytoplasmic region of Stx1 or -2 into SLO-permeabilized sperm and examined their ability to interfere with the AR. Treatment of sperm with Stx2 resulted in >65% inhibition of AR at all concentrations tested relative to sperm treated with equal amount of Abs (Fig. 5). The addition of 5 µg/ml Stx1 resulted in 26% inhibition of AR relative to mock-treated sperm. Increasing the Stx1 concentration to 10 and 20 µg/ml resulted in a 55 and 49% inhibition of AR, respectively, relative to sperm treated with Abs, where no inhibition was observed (Fig. 5). As seen with the Syts, the addition of 20 µg/ml denatured Stx1 or -2 completely abrogated the inhibitory effect seen with equal concentration of the native recombinant protein (Fig. 6). These observations suggest that both Stx1 and -2 may be important components of the fusion machinery involved in mediating the AR.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
There is increasing evidence that the AR in mammalian sperm is mediated by a SNARE complex acting in concert with the Ca2+ sensor, Syt (25–27, 57, 58, 64), similar to stimulated secretion in other secretory cells such as neurons. The AR is a singular, irreversible event, in contrast to the exocytosis and recycling that occurs in other systems. Thus, the secretory machinery may exhibit different constraints from that in other cells to minimize spontaneous exocytosis, and the SNARE and Syt isoforms used could be specific for this function. A number of isoforms of the SNAREs Stx and VAMP as well as isoforms of Syt have been reported to be present at the protein level in sperm (25, 26, 51, 52). However, their roles in sperm are not yet clear. In addition, as discussed above, interactions of Stx with the Syt isoforms shown to be present in sperm were reported to be Ca2+ insensitive (32), whereas the AR is clearly triggered by Ca2+. Thus, it was unclear how acrosomal exocytosis was regulated downstream of Ca2+.

To address the Ca2+ sensitivity of the AR, we showed that increased Ca2+ triggers the AR in the SLO-permeabilized sperm system with an EC50 of 87 µM, suggesting that the calcium sensor involved in the regulation of the sperm AR is active at calcium concentrations at or near this value. This value is comparable with the EC50 of about 20 µM obtained by measuring the calcium dependence of fusion between sperm plasma membrane and outer acrosomal membrane vesicles (59), especially when taking into account the wide spacing between Ca2+ concentrations employed in that study. However, in vivo measurements of Ca2+i in sperm using the Ca2+-sensitive fluorophore fura-2 yielded much lower values for free Ca2+i of about 0.16 µM before AR, which rose to only about 0.40 µM when the AR was triggered (60). This discrepancy of 2 orders of magnitude may be due to fura-2 measurements lacking the resolution to detect locally high Ca2+ concentrations near the site of fusion. Indeed, it has been proposed that, in general, the local Ca2+ concentrations in secretory cells at the site of fusion is actually several orders of magnitude higher than the global increase measured in the bulk cytoplasm due to the co-localization of the SNAREs, Ca2+ sensor, and the plasma membrane-based Ca2+ channels that mediate Ca2+ influx (65). However, the much higher Ca2+ needed to cause AR in SLO-permeabilized sperm or isolated vesicles might alternatively reflect a loss of sensitivity of the exocytotic apparatus after such manipulations.



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  		FIG. 5.
Inhibitory effect of recombinant syntaxin on the acrosome reaction. SLO-permeabilized sperm were incubated with the indicated amount of recombinant His6/HA-tagged Abs (•), Stx1 ({triangledown}), or Stx2 ({blacksquare}), and the extent of AR was assessed after challenge with 1 mM free Ca2+. The values represent the average of at least three independent experiments and are expressed as a percentage of maximal AR.

 



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  		FIG. 6.
Inhibition of the acrosome reaction in the presence of native and denatured recombinant synaptotagmin and syntaxin. SLO-permeabilized sperm were incubated with 20 µg/ml native or heat-denatured recombinant His6/HA-tagged Syt I, VI, or VIII or Stx1 or -2, and the extent of AR was assessed after challenge with 1 mM free Ca2+. The values represent the average of at least three independent experiments and are expressed as a percentage of maximal AR.

 
We assumed that, like in other secretory cells, one or more Syt isoforms serve as the Ca2+ sensor(s) in the sperm AR and transmit the increase in cytoplasmic Ca2+ to the SNARE core complex via binding to syntaxin. Therefore, we have re-examined whether the best current candidates for mediating Ca2+-induced exocytosis in sperm, Syt VI, Syt VIII, and Stx2, exhibit Ca2+-dependent interaction. In a previous study (32), the C2AB region of Syt VI was shown to bind to Stx1–4, but it was unclear whether these interactions exhibited Ca2+ sensitivity. In addition, the Ca2+ sensitivity of Stx1 binding was only tested on the C2A region of the Syts. Using the entire cytoplasmic regions (C2AB) of each isoform and the well characterized Stx1 and Syt I as controls in an in vitro pull-down assay, we showed here that both Syt VI and Syt VIII exhibited Ca2+-dependent binding with Stx1 and Stx2. The hierarchy for Stx1 binding upon stimulation by Ca2+ was Syt I {approx} Syt VIII < Syt VI, indicating that binding occurred at a lower Ca2+ concentration for Syt I and VIII than for Syt VI. When Syt binding to the Stx isoform, proposed to be present in sperm, Stx2, was assessed, the hierarchy was Syt VIII < Syt I < Syt VI, indicating that, as Ca2+ increases, Syt VIII-Stx2 binding would occur first. If this corresponds to the situation in vivo, the initial trigger of AR could, thus, be Syt VIII, with Syt VI playing a secondary role. The stimulation of AR by Ca2+ in SLO-permeabilized sperm occurred with an EC50 that closely matched that which produced half-maximal binding between Syt VIII and Stx isoforms. This close match may strengthen the case that these isoforms have a role in the sperm AR. However, such data must be interpreted with caution given the difficulties of determining whether data obtained with SLO-permeabilized sperm reflect transduction in intact sperm (see above) and the possibility that binding between cytoplasmic portions of Syt and Stx isoforms occurs with different affinities from intact, transmembrane proteins. Nonetheless, the coincidence of the Ca2+ dependence of AR and Syt VIII-Stx2 binding under the conditions we used is suggestive of a role in the AR. We have attempted to confirm this interaction in vivo by immunoprecipitation of either Syt VIII or Stx2 from sperm lysate, but neither the GST-Syt VIII, the Syt VIII peptide, nor the commercial Stx2 antibodies was effective in precipitating their respective antigens.

Confirming the role of specific gene products requires inhibiting the function of the endogenous proteins. However, sperm are transcriptionally and translationally silent, and therefore, conventional molecular biological approaches such as transfection of dominant negative mutants and gene silencing cannot be easily performed. To accomplish such inhibition, we and others have employed the bacterial pore toxin, SLO, to permeabilize the plasma membrane of the sperm and gain access to the cytoplasm (25, 66, 67). The protocol we used results in the creation of pores in the plasma membrane that are large enough to allow for the introduction of macromolecules that could block the endogenous activity of proteins of interest but which do not breach the acrosome. We introduced IgG purified from two different antisera; one raised against a non-conserved Syt VIII-derived peptide that we have previously shown does not cross-react with other Syt isoforms (52) and a second raised against the C2AB cytoplasmic region of Syt VIII. The second antiserum likely does cross-react with other Syt isoforms, although we have not fully assessed its specificity. The addition of either anti-Syt VIII peptide or anti-GST-C2AB Syt VIII IgG to SLO-permeabilized sperm resulted in an 83 and 58% inhibition of the Ca2+-stimulated AR, respectively, relative to untreated sperm or sperm treated with the equal amount of preimmune IgG. This inhibition, especially by the anti-peptide antiserum that is Syt VIII-specific, indicates that Syt VIII is required for the AR in mouse sperm. We could not similarly assess a role for Syt VI, as no specific antisera were available to us.

We observed a similar dose-dependent inhibition of the Ca2+-stimulated AR in SLO-permeabilized sperm by the addition of recombinant His6/HA-tagged Syt VI or VIII cytoplasmic regions. Both recombinant Syt proteins neared maximal inhibition at a concentration of 10 µg/ml, whereas the addition of His6/HA-tagged Syt I had little effect until the dosage was elevated to 20 µg/ml, suggesting that Syt VI and Syt VIII may be the preferred isoforms to bind to the sperm SNARE complex. The simplest interpretation of these data is that the inhibition of AR by recombinant C2AB of Syt I, VI, or VIII is due to their acting as dominant negative inhibitors, disrupting endogenous Syt binding to target sperm SNAREs. The inhibition was specific, as the addition of equal amounts of an irrelevant protein, His6/HA-tagged Abs, had no effect on AR, and the inhibitory effect seen with recombinant C2AB of Syt I, VI, and VIII was eliminated when the proteins were first denatured by heating.

In contrast to our results for Syt VIII above, the addition of a commercial anti-Stx2 antibody to SLO-permeabilized sperm did not result in inhibition of the AR. One interpretation is that Stx2 is not critical for the AR. However, an alternative explanation may be that Stx2 is already stably assembled into the SNARE core complex with SNAP-25 and VAMP and, thus, may not be accessible to the antibody. Alternatively, antibody binding to its epitope, which spans amino acids (1–19 of rat Stx2) that are not involved in binding to other core SNARE proteins or Syt, may occur but not interfere with Stx2 function. In contrast, the addition of recombinant cytoplasmic domain of Stx2 resulted in 78% inhibition of AR relative to sperm treated with a nonspecific protein, with its inhibitory effect saturated at the lowest dose of 5 µg/ml that we used. The addition of recombinant Stx1 also resulted in inhibition of AR but required 10 µg/ml to saturate and only caused a maximal inhibition of 55% relative to sperm treated with a control protein. Thus, under these conditions, the Stx2 cytoplasmic domain was a more potent inhibitor of the AR than Stx1. As was seen with the addition of recombinant Syt, the inhibition was specific, as denatured Stx1 or -2 had no effect. The simplest interpretation of these data is that the interaction of the endogenous Stx2 with the endogenous Syt or other SNARE proteins in sperm is disrupted by the presence of added cytoplasmic region of Stx isoforms, thus blocking transduction of the Ca2+ signal to the SNARE complex. The higher potency of Stx2 and its presence in sperm suggests that it is a likely candidate for the endogenous Stx in sperm.

In conclusion, we have established here that two Syt isoforms, Syt VI and Syt VIII, can exhibit Ca2+-dependent binding to Stx isoforms and, thus, can potentially mediate Ca2+-stimulated secretion and exocytosis. Furthermore, our data suggest that Syt VIII has a required role in the AR and that Syt VI may also participate but cannot substitute in the role of Syt VIII. The Stx isoform that binds to one or both of these Syts is most likely to be Stx2 among the candidates thus far proposed. We propose that this particular combination of SNARE proteins and putative Ca2+-sensors may help account for the unique character of the sperm exocytotic event.


   FOOTNOTES
 
* This work was supported by a grant from the Natural Sciences and Engineering Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Recipient of an Ontario graduate scholarship. Back

|| To whom correspondence should be addressed: Ottawa Health Research Institute, Dept. of Cellular and Molecular Medicine, University of Ottawa, 725 Parkdale Ave., Ottawa, ON K1Y 4E9, Canada. Tel.: 613-798-5555 (ext. 17079); Fax: 613-761-5365; E-mail: jngsee{at}ohri.ca.

1 The abbreviations used are: AR, acrosome reaction; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; Stx, syntaxin; SNAP, soluble N-ethylmaleimide; VAMP, vesicle-associated membrane protein; Syt, synaptotagmin; SLO, streptolysin O; HA, hemagglutinin; BSA, bovine serum albumin; KRB, Krebs-Ringer buffer; GST, glutathione S-transferase; Abs, Abstrakt. Back



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