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Regular Article |
a Departments of Cellular and Molecular Medicine and
b Obstetrics and Gynecology (Division of Reproductive Medicine), Ottawa Health Research Institute, University of Ottawa, Ottawa, Ontario, Canada K1Y 4E9
c Department of Biology, University of California, Riverside, California 92521
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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acrosome reaction, fertilization, gamete biology, male sexual function, sperm
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In neurons, the release of neurotransmitters and peptide hormones stored in secretory vesicles is also triggered by an increase in [Ca2+]i [7]. The vesicles that participate in such Ca2+-regulated exocytosis are first transported from the Golgi complex to the plasma membrane, dock with the plasma membrane, and subsequently fuse with it, with specific protein complexes mediating each step [8]. The Ca2+-sensitive step in neuronal exocytosis is the fusion of the secretory vesicles with the plasma membrane, which occurs after docking and is controlled through the action of an intracellular Ca2+ sensor. Because the acrosome is closely apposed to the sperm plasma membrane, it resembles a large docked secretory vesicle and may therefore utilize a mechanism to mediate acrosomal exocytosis in response to an increase in [Ca2+]i similar to that controlling fusion of docked vesicles in neurons.
The synaptosome-associated protein (SNAP) receptor (SNARE) hypothesis has been proposed as a model to elucidate the molecular events in vesicle docking and fusion [8–10]. The current model states that specific interaction between a v-SNARE on the vesicle with its cognate t-SNARE on the target membrane forms the core fusion complex. This core SNARE complex is comprised of a v-SNARE, vesicle-associated membrane protein (VAMP), or synaptobrevin [11, 12] and 2 t-SNAREs, syntaxin (Stx) [13] and a 25-kDa SNAP (SNAP-25) [14–16]. Another protein, N-ethylmaleimide-sensitive factor (NSF) [17], and its soluble attachment protein 
-SNAP [18] bind to the core complex and mediate vesicle docking [19, 20]. Thus, the docked complex also contains NSF and -SNAP. In addition to SNARE molecules, the Rab family of small GTPases is necessary for exocytosis. They are responsible for the recruitment of Rab-associated proteins, which are involved in tethering vesicles to the site of fusion [21].
There is growing evidence that SNARE complex proteins may be involved in regulating the AR. Recent work has shown that the core complex components Stx, VAMP, and SNAP-25 are present in sea urchin sperm [22] and are tightly associated in shed acrosomal vesicles following acrosomal exocytosis [23]. Furthermore, Stx 2 and VAMP have been detected in mammalian sperm and lost following the AR [24, 25]. In addition Rab3A has been identified in mammalian sperm [26–28] and has been shown to be essential for Ca2+-mediated acrosomal exocytosis [29].
Established candidates for the Ca2+ sensor that mediates fusion of docked vesicles in neurons are members of a family of 
65-kDa integral membrane glycoproteins known collectively as synaptotagmins (Syts). There are currently 13 reported, distinct mammalian isoforms of Syt expressed in neuronal and nonneuronal cells [30–37]. All Syt isoforms are transmembrane proteins with a glycosylated N-terminal luminal domain. They possess a large C-terminal cytoplasmic domain that projects from the surface of the vesicle and has 2 Ca2+-binding motifs with homology to the C2 domains of a number of other Ca2+-sensitive proteins, designated C2A and C2B [38]. Functional studies have provided direct evidence that Syts are involved in triggering exocytosis in response to increases in [Ca2+]i [39–43]. Furthermore, Syts interact with core complex SNARE proteins such as Stxs [44]. Syts also bind calcium channels [45–47] and are therefore ideally situated to quickly sense increases in [Ca2+]i mediated by influx through activated calcium channels.
In a previous study, Ramalho-Santos et al. [24] proposed that the Syt isoform Syt I is present in the acrosomal region of sperm from various species, based on immunologic analysis. This finding conflicts with previous reverse transcription (RT) polymerase chain reaction (PCR) results suggesting that Syt I mRNA is not expressed in murine testis [34]. Here, we report the identification of Syt VIII rather than Syt I as the Syt isoform in spermatogenic cells and the sperm acrosome, where it may participate in the AR.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PCR amplification of Syt I and Syt VIII was performed using 1 µg of mouse brain cDNA and pachytene spermatocyte and round spermatid phage cDNA libraries at 2 x 108 plaque-forming units each (generously provided by Dr. J. McCarrey, Southwest Foundation for Biomedical Research, San Antonio, Texas). Mouse brain cDNA was prepared from 2 µg of freshly isolated total RNA (total RNA isolation kit; Qiagen, Valencia, CA) using a RETROscript first strand synthesis kit (Ambion, Austin, TX). The PCR amplification was performed using isoform-specific primers for Syt I (5'-GCG GAT CCC TAC TGC CGG CAA GCT GAC T-3' and 5'-CTT CTA GAC AGC CAG CAT GGC ATC AAC C-3') and Syt VIII (5'-CTG GAT CCC TGA AGG CTG AGG GCA CA-3' and 5'-CGG AAT TCT AAC GGG AAC CAG GAA GAC G-3'). Samples were resolved on a 1% agarose gel and transferred to nitrocellulose membrane for Southern blot analysis. The blot was prehybridized for 1 h at 60°C in 6x standard sodium citrate (SSC), 5x Denhardt solution, 100 µg/ml of sheared and denatured salmon sperm DNA, and 0.5% SDS. 32P-Labeled probes were prepared from purified DNA constructs of the cytoplasmic regions of Syt I and Syt VIII using a random hexamer labeling kit (Invitrogen, Carlsbad, CA), and unincorporated nucleotides were removed using NuncTrap columns (Stratagene, La Jolla, CA). The blots were hybridized overnight at 60°C with 2 x 106 cpm/ml of prehybridization buffer and washed twice with 2x SSC, 0.1% SDS for 10 min at room temperature and twice with 0.2x SSC, 0.1% SDS for 10 min at 60°C.
Anti-Syt VIII Antiserum
New Zealand White rabbits were immunized at 2-wk intervals with 0.2 mg of the Syt VIII-specific peptide TVDLQHVLESWYQ mixed with Freund adjuvant. The antigen was injected i.m. in the hind limbs, and antiserum was collected biweekly.
Bacterial Expression and Isolation of Syt
Escherichia coli transformed with GST-Syt isoforms (generously provided by Dr. T.C. Sudhof) were induced to express the GST-Syt fusion proteins by a 4-h incubation with 1 mM isopropyl-ß-D-thiogalactoside at 37°C. The bacteria were centrifuged at 5000 x g for 10 min, and the pellets were resuspended in lysis buffer containing 25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 2 mM PMSF, 2 mM dithiothreitol (DTT), and 1 mg/ml lysozyme. The bacteria were lysed with 0.1% Nonidet P40 (NP40), the DNA was sheared by sonication at 35% power for 1 min (Sonic Dismembrator model 300; Fisher, Pittsburgh, PA), and the debris was pelleted at 10 000 x g for 10 min. The fusion proteins were purified from the homogenate by incubation with glutathione agarose beads (Sigma, St. Louis, MO) for 1 h with shaking. The beads were then pelleted and washed extensively with wash buffer containing 25 mM Tris-HCl, pH 7.5, 100 mM KCl, 1 mM EDTA, 2 mM DTT, and 0.1% NP40 prior to elution of the protein with SDS loading buffer. GST-Syt fusion proteins were resolved by SDS-PAGE and blotted to a nitrocellulose membrane. The blot was then probed with anti-Syt VIII or anti-Syt (Transduction Laboratories, Lexington, KY) at a dilution of 1:500 or with anti-GST at a dilution of 1:1000. Horseradish peroxidase-labeled secondary antibodies (Chemicon, Temecula, CA) and enhanced chemiluminescence (Amersham, Piscataway, NJ) were used to visualize the bound antibodies.
AR of Murine Sperm
Sperm were collected from cauda epididymides of 12- to 15-wk-old mice and allowed to swim up for 15 min in Krebbs Ringer bicarbonate buffer (KRB) medium containing 1.7 mM CaCl2, pH 7.4 (37°C with 5% CO2). An aliquot of the sperm was then added to an equal volume of KRB, which contained either 0.4% dimethyl sulfoxide (DMSO) or 20 µM of A23187 in 0.4% DMSO and incubated at 37°C with 5% CO2 for 30 min. Sperm intended for immunohistochemistry were fixed by addition of paraformaldehyde to a final concentration of 4% and incubated at 4°C overnight. Sperm intended for Western blotting were counted, resuspended in SDS loading buffer at a concentration of 1 x 105 sperm/µl, and sonicated for 15 sec at 30% power to reduce viscosity. The proteins were resolved on a 10% SDS polyacrylamide gel and blotted on nitrocellulose. The resulting blots were probed with Anti-Syt VIII at 1:500. Densitometric analysis was performed using a BioRad Molecular Analyst program (Bio-Rad, Hercules, CA).
Assessment of Acrosomal Status
Fixed sperm were harvested by centrifugation at 800 x g for 5 min and washed 3 times with 1 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. Assessment of the status of the acrosome was accomplished by staining sperm with 0.04% Coomassie brilliant blue G-250 in 3.5% perchloric acid for 5 min at room temperature [48]. Slides were washed 4 times with distilled water and mounted with 30% glycerol in PBS. Sperm were observed with bright-field microscopy using a Zeiss Axiophot microscope and scored for acrosomal staining.
Immunocytochemistry
Air-dried slides were prepared as described above and washed with PBS containing 0.1 M glycine prior to staining. Fixed sperm were permeabilized with blocking buffer containing 1% BSA, 2% normal goat serum, and 0.4% saponin in PBS for 30 min at room temperature. Labeling was performed with anti-Syt VIII diluted 1:40 with blocking buffer for 1–2 h. Samples were extensively washed with PBS containing 0.1 M glycine prior to labeling with Alexa 594-conjugated goat anti-rabbit secondary antibody (Molecular Probes, Eugene, OR) for 1 h. The slides were mounted with Slow Fade (Molecular Probes). The sperm were imaged using a BioRad MRC1024 confocal microscope and BioRad Laser Sharp imaging software.
Membrane Fractionation
All buffers contained a protease inhibitor cocktail of leupeptin (20 µg/ml), aprotinin (20 µg/ml), PMSF (1 mM), and benzamidine (200 µg/ml). Sonicated sperm membranes were prepared with a probe sonicator (VirTis, Gardiner, NY) for 15 sec on ice, repeated 3 times at intervals of 1 min. Cell debris was pelleted (500 x g for 15 min), the membrane supernatant was collected and diluted to 5 ml with 1/10 TN (TN: 130 mM NaCl, 20 mM Tris-HCl, pH 7.0), and crude membranes (CMs) were pelleted by ultracentrifugation (SW 50.1 swinging bucket rotor at 108 000 x g for 1 h, 4°C). CMs were fractionated on a sucrose step gradient (0.5 ml membranes, 1.5 ml 30% sucrose, 1.5 ml 40% sucrose, 1.0 ml 45% sucrose) for 2 h at 125 000 x g at 4°C. Membrane fractions were collected from the interfaces: band 1 = 0/30% interface, band 2 = 30/40% interface, band 3 = 40/45% interface, and band 4 = pellet.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We performed PCR amplification of both Syt I and Syt VIII from pachytene spermatocyte and round spermatid 
phage libraries as well as from mouse brain cDNA. Southern blot analysis revealed that the 531-base pair (bp) product of Syt VIII was amplified in all 3 samples, but the 411-bp amplification product of Syt I was detected only in mouse brain (Fig. 1). Having confirmed that Syt VIII mRNA is expressed in germ cells, we generated a Syt VIII-specific antiserum. All Syt isoforms contain 2 C2 domains, and the region between these 2 calcium-binding regions shows the greatest divergence. Hence, we chose a 13-mer peptide in this region (amino acids 218–230) as the antigen to ensure antibody specificity. The specificity of the antiserum was verified using bacterially expressed GST fusion proteins of the cytoplasmic domains of Syt I to VIII. A single band was detected for the Syt VIII fusion protein, but no bands were detected for any other isoforms tested (Fig. 2A). Expression and loading of each Syt isoform was confirmed by detection with anti-GST antibodies. A commercially available Syt antibody (Transduction Laboratories) detected both recombinant Syt I and Syt VIII upon induction with IPTG (Fig. 2B). Hence, these results confirmed that our antiserum is specific to Syt VIII among the isoforms tested in contrast to the commercial antiserum, which cross-reacted with several Syt isoforms.
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Syt VIII Is Present in Sperm and Localizes to the Acrosomal Region
To determine whether Syt VIII protein is present in mature spermatozoa, whole sperm homogenate was probed with Syt VIII antiserum and the commercial anti-Syt and compared with brain homogenate. Both antibodies detected a single clear, prominent band at 65 kDa in sperm homogenates (Fig. 3), which corresponds to the expected size of the Syt family of proteins [30]. Both antibodies also detected a faint band of the same size in the brain homogenate, with a major additional more quickly migrating band detected in brain only by the commercial anti-Syt. This result confirms that Syt VIII is present in mature spermatozoa and indicates that the more quickly migrating isoform prominent in brain is not present in sperm.
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We next determined the subcellular localization of Syt VIII. Western immunoblot analysis of sperm subcellular membrane fractions using our anti-Syt VIII antiserum (Fig. 4) revealed that Syt VIII is found almost entirely within membrane fraction band 2. This fraction contains membranes derived from the sperm head, as indicated by the presence of several proteins known to be localized to the sperm head, including the sperm-specific ß-1,4 galctosyl transferase and PH20/hyaluronidase [49]. This finding suggests that Syt VIII partitions with membranes derived from the sperm head. To further define the cellular localization of the protein, we used immunocytochemistry to examine fixed sperm using our anti-Syt VIII antiserum. Syt VIII was localized to the head of murine sperm in a crescent-shaped pattern that is characteristic of the acrosome (Fig. 5). These results confirm that Syt VIII is indeed present in the acrosomal region of murine sperm.
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Syt VIII Is Shed upon AR
Following the AR, components of the fusion machinery are lost with the shed vesicles and should therefore become undetectable in acrosome-reacted sperm. To determine whether Syt VIII is shed upon AR, we used the calcium ionophore A23187 to induce acrosomal exocytosis and compared the immunoreactivity of Syt VIII in mock-treated and A23187-treated sperm. We assessed an aliquot of each sample to determine the fraction of AR sperm using the Coomassie blue staining method [48] and assessed a parallel sample for Syt VIII immunoreactivity. A representative sample of the triplicate experiments is shown in Figure 6 (A and B). The acrosome, which appears as a darker crescent-shaped structure when stained with Coomassie blue, was lost upon fusion (Fig. 6A). Likewise, Syt VIII immunoreactivity was lost upon acrosomal exocytosis (Fig. 6B). The fluorescent signal observed in the tail of the sperm represents nonspecific immunoreactivity because it was present in sperm stained with preimmune serum (data not shown). A23187 caused approximately 52% of the sperm to undergo AR as assessed by Coomassie blue staining (Fig. 6C). This percentage was correlated with the 47% loss in Syt VIII-immunoreactive sperm upon A23187 treatment. To further confirm this observation, we quantified the loss of Syt VIII immunoreactivity in the A23187-treated sperm by densitometric analysis of the Western immunoblot (Fig. 7). The mock- and A23187-treated samples were adjusted to a concentration of 105 sperm/µl, and 0.5, 1.0, and 2.0 x 106 sperm were solubilized and processed for Western immunoblot. Densitometric analysis of the Syt VIII signal intensity showed a 54% decrease in Syt VIII immunoreactivity in the A23187-treated sperm (Fig. 7A). This decrease in Syt VIII immunoreactivity was correlated with the number of AR sperm as assessed by Coomassie blue staining or by Syt VIII immunocytochemistry. The Coomassie staining and the immunocytochemical and Western analysis data are averages of 3 separate experiments. The immunohistochemical and densitometric analyses indicate that Syt VIII is lost upon AR.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several lines of evidence presented here indicate that Syt VIII is expressed in murine sperm. We have detected Syt VIII cDNA in murine spermatogenic cell cDNA libraries by PCR (Fig. 1). This observation is consistent with the results of a previous study in which RT-PCR revealed that of the synaptotagmin isoforms tested (Syt I through Syt VIII, excluding Syt IV), only Syt VI and Syt VIII mRNA were detectable in testis [34]. To determine whether Syt VIII protein is expressed and present in mature sperm, we raised an antiserum against a peptide corresponding to a region of Syt VIII between the 2 C2 domains; this area shows the highest degree of divergence among the known Syt isoforms. This antiserum specifically detected Syt VIII and did not cross-react with Syts I–VII (Fig. 2). Because of the lack of availability of clones for Syts IX–XIII, we could not conclusively exclude the possibility that our antiserum might cross-react with these Syt isoforms. However, sequence comparisons revealed a lack of significant sequence similarity between any other known Syt isoforms and Syt VIII within the region used to generate the peptide antibody, rendering cross-reactivity highly unlikely. Using this newly raised antiserum for Western blotting, we showed that Syt VIII is present in whole sperm homogenate. To determine the precise localization of the protein, we performed immunocytochemical analysis of fixed and permeabilized sperm using anti-Syt VIII antiserum. Our results confirmed the presence of Syt VIII in mature mouse spermatozoa and localized it to the acrosomal region of the sperm head. This localization was also supported by data from subcellular fractionation of murine sperm membranes, which revealed that Syt VIII localizes to a membrane fraction positive for the sperm membrane proteins ß-1,4 galactosyl transferase and PH20/hyaluronidase [49].
Syt I Is Not Present in Sperm
In a recent study, Ramalho-Santos et al. [24] used antiserum raised against a conserved region of Syt isoforms and reported that Syt was present in the heads of mammalian sperm of several species. They then identified the isoform in sperm as Syt I based on immunocytochemical and immunoblot analysis performed with a commercial anti-Syt antibody (Transduction Laboratories). In contrast, we determined that the commercial antibody is not specific for Syt I but instead cross-reacts with Syt I and Syt VIII (Fig. 2) and with other isoforms tested (Syts II–VII; data not shown). This finding raises the strong possibility that the sperm immunoreactivity with this commercial antibody might have been due to the presence of a Syt isoform other than Syt I. Closer examination of the immunoblot probed with the commercial antibody also supports the presence of other Syt isoforms in the brain homogenate (Fig. 3). Syts I and II are the major isoforms in the brain and thus probably are represented by the more quickly migrating band, which differs from that detected in the sperm. These data indicate that Syt VIII, and not Syt I, is present in mouse sperm. The identification of the isoform in sperm as Syt VIII rather than Syt I is also supported by our inability to detect Syt I cDNA in spermatogenic cell libraries although Syt VIII cDNA is present and is further supported by a previous report that Syt I mRNA is not expressed in testis but Syt VIII mRNA is expressed [34].
Syt VIII Localizes to the Acrosomal Region and May Participate in the AR
The Syt family of proteins participates in membrane fusion events, where these proteins may serve as calcium sensors [43], and Syt VIII could play a similar role in the AR. Syt VIII is localized to the acrosomal region of the mouse sperm, making it appropriately situated for regulating acrosomal exocytosis. Proteins that are localized to the site of exocytosis should be shed along with the acrosomal vesicles, as has been established for the SNARE molecules VAMP and Stx in sea urchin and hamster sperm [23, 24] and for the small GTPase Rab3A in rat sperm [27]. We have determined by immunofluorescence and Western immunoblot of sperm that Syt VIII decreased following the AR. When sperm were induced to undergo the AR using the Ca2+ ionophore A23187, there was a large increase in the number of sperm showing little or no Syt VIII immunofluorescence on the head. Quantitative analysis of 3 separate experiments of Syt VIII immunoreactivity in fixed sperm and densitometric analysis of Syt VIII from sperm homogenate showed that Syt VIII is lost following AR, and this loss was correlated with the number of sperm undergoing acrosomal exocytosis, as determined by Coomassie blue staining.
Is Syt VIII the Only Syt Isoform in Sperm?
Although it appears highly likely that Syt VIII is present in mouse sperm, our results do not preclude the presence of other Syt isoforms. Messenger RNA encoding the isoforms Syt VI [34] and Syt XIII [37] is expressed in the testis, and Syt IV has been sequenced from a mouse testis cDNA library (GenBank accession BB070672). While the present manuscript was under revision, Syt VI was reported as localized to the outer acrosomal membrane of human sperm and as essential for acrosomal exocytosis [50]. Thus, assuming Syt VI is also expressed in mouse sperm, it appears that there may be at least 2 Syt isoforms present in sperm. Whether both function as Ca2+ sensors during the AR or whether the separate isoforms have distinct functions remains to be determined. Moreover, the Syt isoforms form hetero-oligomers, raising the possibility that such different combinatorial interactions might confer a broader repertoire of Ca2+ sensitivity to the AR. Other Syt isoforms expressed in the testis, such as Syts IV and XIII, may also be expressed in spermatogenic cells or instead may only be expressed in other compartments of the testis. Thus, the total number of Syt isoforms present in sperm and their functions remain to be determined.
Conclusion
The AR takes place at multiple loci at which fusion of the outer acrosomal membrane with the overlying plasma membrane occurs in response to an increase in [Ca2+]i. Recent evidence suggests that the membrane fusion event utilizes protein complexes that are similar to the SNARE complexes operating in neuronal vesicle exocytosis [22–25, 27–29]. The identification of Syt VIII in the acrosomal region of murine sperm supports this hypothesis by localizing this putative calcium sensor to the acrosomal membrane of mammalian sperm. Further work is required to confirm the direct interaction of Syt VIII with members of the core SNARE complex in sperm and to determine whether this protein is required for the AR.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by a grant from the Natural Sciences and Engineering Council of Canada (NSERC). D.M.H. is a recipient of a NSERC postgraduate scholarship. 
2 Correspondence: Johnny K. Ngsee, Department of Cellular and Molecular Medicine, Ottawa Health Research Institute, University of Ottawa, 725 Parkdale Ave., Ottawa, ON, Canada K1Y 4E9. FAX: 613 761 5365; jngsee{at}ohri.ca
Accepted: August 3, 2001.
Received: May 18, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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