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Articles |
a Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología (IHEM-CONICET), Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, 5500 Mendoza, Argentina
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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S-stimulated acrosome reaction. Our results indicate that 1) permeabilized spermatozoa can be used to study the role of macromolecules in the acrosome reaction, 2) Rab3A is present in human spermatozoa, and 3) Rab3A or another Rab3 isoform is involved in the exocytosis of the acrosomal granule in human spermatozoa.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It is well known that exocytosis involves several steps: recognition, docking, and, finally, fusion of membranes [9]. Different members of the Rab family of monomeric guanosine triphosphatases (GTPases) are involved in the interaction and fusion of intracellular membrane-bound compartments. Specifically, various isoforms of Rab3 regulate exocytosis in different cell types [10–12]. In fact, a stimulatory role for Rab3 has been proposed for the acrosome reaction of ram spermatozoa by using Rab3A peptides [13]. On the other hand, an inhibitory role has recently been reported when Rab3 effector domain peptides were used concomitantly with the calcium ionophore A23187 [14]. At least two problems arise in evaluating the role of Rab3 in the acrosome reaction of human spermatozoa: 1) there is no evidence for the presence of Rab3 proteins in human spermatozoa and 2) the possibility of studying the role of macromolecules like Rab3 is severely limited because such macromolecules are unable to cross the cell membrane. However, in a previous work [15], we have shown that streptolysin O-permeabilized mouse spermatozoa undergo calcium-dependent acrosome content release. This observation raised the possibility of using a similar approach for human spermatozoa, provided that cells were able to respond differentially to several well-characterized stimuli after permeabilization.
In the present work, we report the presence of Rab3A in human spermatozoa by Western blot analysis and immunofluorescence. We also describe a fully permeable sperm cell preparation capable of undergoing a regulated acrosome reaction. By using this model, we present evidence suggesting that a Rab3 protein is actively involved in the acrosome exocytotic process in human spermatozoa.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Streptolysin O (SLO) was obtained from Wellcome Diagnostics (Dartford, UK). Glutathione-sepharose 4B beads and Sephadex G25 were obtained from Pharmacia (Uppsala, Sweden). LB medium was purchased from Life Technologies (Paisley, UK). Gamete Preparation Medium (GPM, Serono, Madrid, Spain) was used as culture medium. The peptides Rab1 (12mer: GVDFKIRTIELD) and Rab3A (16mer: VSTVGIDFKVKTIYRN) were generously provided by Dr. J. Fernández and Dr. A. Oberhauser [16]. All other reagents were from Sigma Chemical Company (St. Louis, MO). Water for all solutions was purified with a batch-fed system (Barnstead EASYpure; Barnstead/Thermolyne Corp., Dubuque, IA).
SLO Permeabilization of Human Spermatozoa
Human semen samples from healthy donors of proven fertility (motility 
50%, motile spermatozoa 60 x 106/ml) were used. After swim-up separation [17] for 1 h at 37°C in an atmosphere of 95% air:5% CO2 using GPM (based on Earle's balanced salt solution with the addition of phenol red and 20 mg/ml of human serum albumin; osmolality 278–288 mOsm/kg), highly motile sperm cells were recovered. The concentration was adjusted to 5–10 x 106/ml in fresh GPM, and incubation proceeded for additional 2 h at 37°C and 5% CO2, accounting for a total incubation time of 3 h under capacitating conditions. Permeabilization was performed as previously described [15]. In brief, cell suspensions were centrifuged for 1 min in a microfuge (at least two cycles) at 10 000 rpm, and the pellets were resuspended in cold PBS containing 0.4 U/ml of SLO for 15 min at 4°C. After incubation, the cells were washed twice with PBS to eliminate excess SLO, and the pellets were resuspended in ice-cold sucrose buffer (250 mM sucrose, 20 mM Hepes-K, 0.5 mM EGTA, pH 7) with 2 mM dithiothreitol (DTT) to activate membrane-bound SLO. After 15 min of incubation at 37°C, the sperm suspension was divided in 100-µl aliquots to proceed with the different treatments. Permeabilization at this step of the protocol was 100%, as assessed by supravital stain with eosin-Y (5 g/L). Several combinations of reagents were tested. As a rule, inhibitors were added at 37°C 15 min before the addition of the stimulants, and the reaction was stopped after an additional 15-min incubation at 37°C. A sample of nonpermeabilized spermatozoa was used as an additional control group during the first set of experiments. The doses used in this study were selected from the literature and/or from preliminary experiments.
Acrosome Reaction Assay
After stopping the incubation by placing the cell suspensions at 4°C, the acrosome reaction was evaluated by the fluorescein isothiocyanate Pisum sativum lectin (FITC-PSA) according to Mendoza et al. [18]. Briefly, the cell suspensions were centrifuged for 1 min at 10 000 rpm in a microfuge (at least two cycles), and the pellets were resuspended in PBS. A 10-µl drop was then placed on an 8-well slide and air-dried at room temperature. The sperm smears were fixed with cold (4°C) methanol for 30 sec and incubated in the dark with 50 µg/ml FITC-PSA in PBS in a humidified chamber for 30 min at room temperature. After intensive washing with distilled water, unmounted slides were examined on the same day as the staining procedure, and at least 200 cells were scored in an epifluorescence Nikon Optiphot II microscope (x400; Nikon, Inc., Melville, NY) according to the following patterns: 1) selective staining of the whole acrosome (unreacted cell) and 2) no staining at all, or staining limited to the equatorial segment (reacted cells).
Expression and Purification of Glutathione-S-Transferase (GST)-Fusion Proteins
The expression plasmid pGEX2T containing the cDNA-encoding segment of human Rab3A (Gen Bank Accession NP 002857), human rab11A (Gen Bank Accession AF000231), and bovine GDP dissociation inhibitor alpha (GDI 
; GenBank Accession P21856) were generously provided by Dr. M.I. Colombo and Dr. P.D. Stahl (Washington University, St. Louis, MO). The plasmids were used to transform XL1-blue or BL21 Escherichia coli. The cells were grown in LB medium containing 50 µg/ml ampicillin. Expression of the GST-fusion proteins was induced by the addition of 0.5 mM isopropyl-ß-D-thiogalactopiranoside (IPTG) after the bacteria reached an absorbance at 600 nm (A600) of 0.5–0.7. After 4–5 h at 37°C, the cells (250 ml) were pelleted at 12 000 x g for 30 min, resuspended in 3 ml of PBS, and disrupted by sonication. The GST-fusion proteins were purified using glutathione-sepharose 4B, according to the manufacturer's instructions.
In Vitro Prenylation of Rab3A
The protocol used was previously described by Li et al. [19] and was slightly modified as follows: recombinant GST-Rab3A (10 µM) was incubated in PBS containing 200 µM GDP, 1 mg/ml J774-E cytosol (as a source of Rab prenyltransferase), 1 mM DTT, 1 mg/ml BSA, 3 mM MgCl2, protease inhibitors (1 µg/ml leupeptin, 50 µg/ml PMSF, 1 µg/ml pepstatin, 50 µg/ml soybean trypsin inhibitor), and 80 mM geranylgeranyl pyrophosphate. The mixture was incubated for 2 h at 37°C and then stored at -80°C. Just before use, aliquots of the prenylated protein were persistently activated or inactivated by incubating with GTPS or GDPßS, respectively. In brief, 10 µM Rab3A was incubated for 60 min at 30°C in a mixture containing 75 µM GTPS (or GDPßS), 50 mM Hepes/KOH pH 7.2, 5 mM MgCl2, 0.5 mM Nonidet-P40, 1 mM DTT, and 25 mM EDTA. After the incubation was completed, the mixture was filtered in a Sephadex G-25 column previously equilibrated with 20 mM Hepes/KOH, 1 mM MgCl2, 0.5 mM Nonidet-P40, and 0.1% BSA. The filtered aliquots were used immediately. The same procedure was used to prenylate and activate Rab11. Aliquots of the prenylation mixture lacking Rab3A received the same treatment to be used as controls.
SDS-PAGE and Western Blots
Sperm cells were isolated on a discontinuous two-step Percoll (Pharmacia) gradient [20]. This procedure yielded pure sperm preparations with motility higher than 90%. Spermatozoa washed with PBS were lysed to extract proteins in cold 50 mM Tris HCl pH 7.4, 130 mM NaCl, 2 mM EDTA, 10 µg/ml leupeptin, 500 KIU (kallikrien inhibitor units)/ml aprotinin, 1 mM PMSF, and 1% Triton-X 100. After sonication for 15 sec twice and extraction for 60 min at 4°C, the sperm extracts were clarified by centrifugation at 12 000 x g for 5 min at 4°C. Proteins were separated on 12.5% polyacrylamide slab gels according to Laemmli [21] and stained with Coomassie blue or transferred to 0.45 µm Immobilon-P membranes (Millipore Corp., Bedford, MA). Nonspecific reactivity was blocked by incubation for 1 h at room temperature with 3% gelatin-0.5% 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 antibody (clone 42.2, 1:1000 tissue culture supernatant) [22] in blocking solution overnight at 4°C. Horseradish peroxidase-conjugated goat anti-mouse-IgG (heavy and light chains; KPL, Gaithersburg, MD) was used as secondary antibody (0.5 µg/ml, 45 min incubation at 20°C). Excess first and second antibodies were removed by washing 5 times for 10 min each in washing buffer. Detection was accomplished with an enhanced chemiluminescence system (ECL; Amersham Life Science, Arlington Heights, IL) and subsequent exposure to Kodak XAR film (Eastman Kodak, Rochester, NY).
Indirect Immunofluorescence
Spermatozoa were washed in PBS and fixed/permeabilized in 2% paraformaldehyde-0.1% Triton-X 100 in PBS for 1 h at 37°C. After centrifugation (10 000 x g for 2 min), sperm cells were incubated in PBS containing 50 mM glycine for at least 15 min at room temperature, pelleted, and washed once with PBS. The pellets were suspended in blocking solution (5% BSA, 1% horse serum in PBS containing 0.4% polyvinylpyrrolidone (PVP, average Mr = 40 000; ICN, Aurora, OH) and incubated for 1 h at 37°C. Anti-Rab3A monoclonal antibody (clone 42.2, 1:25 tissue culture supernatant in blocking solution) was added to the sperm pellets, which were incubated for 1 h at 37°C and then overnight at 4°C, and subsequently washed twice with PBS/PVP. Afterwards, tetramethylrhodamine isothiocyanate (TRITC)-goat anti-mouse IgG (10 µg/ml in 1% BSA in PBS/PVP; Kierkegaard and Perry Laboratories, Inc., Gaithersburg, MD) was added, and incubation followed for 1 h at 37°C. After being washed as before, samples were suspended in Gelvatol (Monsanto, St. Louis, MO) and mounted on glass slides. Slides were maintained at 4°C until examination with epifluorescence optics in a Nikon Optiphot II microscope, using a G-2A filter. Controls included incubations without the primary antibody or with a tissue culture supernatant containing an inactive antibody; in no case was any fluorescence observed.
Statistical Analysis
Differences between experimental and control conditions were tested by one-way ANOVA and Fisher's PLSD tests. Percentages were transformed to the arc sine before analysis. Significant differences were those for which P was < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Our first aim was to establish a model of acrosome-content release in permeabilized human spermatozoa in order to study the participation of macromolecules during this exocytotic process. A method that had proved to be successful in mouse spermatozoa [15] was slightly modified for human spermatozoa. The SLO treatment, which rendered spermatozoa 100% eosin-permeable, showed a differential response to increasing concentrations of calcium, with optimal concentration around 0.5 mM (Fig. 1A). Free calcium in the system—which contained 0.5 mM EGTA—was about 5 µM, as measured by calcium green fluorescence [23]. Nonpermeabilized spermatozoa that were subjected to reaction with calcium ionophore A23187 are shown as a control for the percentage of normal acrosome reactions that would be expected in these samples (Fig. 1A). For the remaining experiments, calcium stimulation at a 0.5 mM concentration was routinely used as a positive control.
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Stimulation of Heterotrimeric GTPases Triggered Acrosome Release
In previous reports, we have shown that stimulators of GTPases can trigger the acrosome reaction [8,24]. Permeabilized spermatozoa also reacted with a nonhydrolyzable analogue of GTP (Fig. 1B). Moreover, other reagents that stimulate heterotrimeric GTPases, such as the aluminum fluoride complexes (AlF4) and the peptide mastoparan, were equally effective in inducing the acrosome reaction, suggesting that stimulation of a G protein can trigger acrosomal exocytosis in permeabilized spermatozoa (Fig. 1B). Interestingly, the acrosome reaction initiated by calcium was inhibited by GDPßS, a general inhibitor of GTPases. Since a cytosolic calcium increase is likely to be an event occurring downstream from the activation of G proteins, this observation suggested that another GTPase was necessary for the calcium-dependent exocytotic process.
Rab3A Was Present in Human Spermatozoa
Rab3 is a small GTPase that has been conspicuously involved in calcium-dependent exocytosis [25]. To address the possibility that Rab3 was involved in the acrosome reaction, we first assessed the presence of this protein in human spermatozoa. A low but detectable signal for Rab3 was observed by Western blot analysis using a Rab3A-specific monoclonal antibody (Fig. 2A). Indirect immunostaining was used to localize Rab3A on fixed, permeabilized sperm. The antibody reacted specifically with the sperm head in the acrosomal region (Fig. 2B). Eighty to ninety percent of the cells displayed acrosomal staining, consistent with a potential role of Rab3A in the acrosome reaction.
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Active Rab3A Triggered the Acrosome Reaction in Permeabilized Spermatozoa
The only evidence that Rab3A might be involved in the acrosome exocytosis is the reported effects of peptides corresponding to the effector domain of Rab3A on intact sperm cells [13, 14]. As a first approach to assess a role of this GTPase in permeabilized spermatozoa, the effector domain peptides of Rab3A and Rab1 (as a negative control) were added to the assay. Only the Rab3A peptide was able to induce the acrosome reaction in a dose-dependent manner (Fig. 3).
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Since peptides may have nonspecific effects, recombinant Rab3A (whole protein) was tested in the assay. Rab3A was produced in bacteria as a GST fusion protein, prenylated in vitro, and loaded with either GDP or GTP. Unbound nucleotides were eliminated by gel filtration. Activated Rab3A (GTP form) triggered the reaction, whereas the GDP-bound form had no significant effect (Fig. 4A). Preparations in which Rab3A was omitted but which received the same treatment with nucleotides had no effect, indicating that residual GTPS was not responsible for the stimulation. Moreover, the GTP-bound form of Rab11 was not effective, indicating specificity for the Rab protein and ruling out an effect of residual free nucleotide present in the Rab preparation (Fig. 4A).
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If Rab3 were necessary for the reaction, then depletion of Rab proteins from the membranes should inhibit release of GTPS-stimulated acrosome content. GDI is a protein that forms a complex with several Rab proteins (including Rab3A) in their GDP-bound form and extracts them from the membranes. Preincubation with recombinant GDI was able to block the acrosome reaction triggered by GTPS (Fig. 4B). Heat-inactivated GDI had no effect. Taken together, these results strongly indicate a role for Rab3 in the acrosome reaction of human spermatozoa.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Exocytosis involves recognition, docking and fusion of membranes. It is well known that different members of the Rab family of small GTPases are actively involved in the mechanism of intracellular membrane fusion [27], and, more specifically, different isoforms of Rab3 are known to regulate exocytosis in several cell types. A stimulatory role for Rab3 in the ram spermatozoon acrosome reaction has been suggested by Garde and Roldan [13] on the basis of the effect of Rab3A peptides. Since Rab3 has not been described in human spermatozoa, we first assessed its presence by Western blot. A 28-kDa band was detected by using a monoclonal antibody that recognizes Rab3A. Moreover, immunostaining of fixed, permeabilized cells showed that Rab3A localizes to the acrosomal region of the sperm head. A similar localization has been recently reported in rat and mouse spermatozoa [14, 28] and is consistent with a role of Rab3A in the acrosome reaction.
Since the protein was present, several Rab3 specific reagents were tested. First, the peptide of the effector domain of Rab3A was added to the assay. In the permeabilized spermatozoa preparation, the peptide stimulated the acrosome reaction in a concentration-dependent manner. This peptide and some analogues have been shown to trigger secretion in ram spermatozoa and in several other cell types [16, 19, 29]. The exact mechanisms by which Rab3A peptides activate secretion are not well defined, but it is believed that they act on Rab3A effectors. However, some authors have proposed that they act as a mastoparan-like peptide, i.e., by activating heterotrimeric GTPases [30]. As shown in Figure 1B, mastoparan also triggered acrosome content release in the permeabilized sperm model.
Recently, an inhibitory role for Rab3 effector domain peptides on rat sperm when used concomitantly with A23187 has been reported [14]. We used Rab3A—the whole molecule instead of just the effector domain peptide—as a useful way of assessing more directly the role of Rab3A in the acrosome reaction. Our results strongly indicate that recombinant Rab3A in its active (GTP-bound) form promotes the acrosome reaction. Recombinant Rab proteins have been extensively tested in cell-free assays, and it has been shown that the proteins maintain all their specific properties [31]. Calcium and GTPS can trigger the acrosome reaction in the absence of added cytosol, suggesting that all the required factors are bound to the membranes. To assess whether extraction of Rab proteins would abrogate the acrosome reaction, recombinant GDI was included in the assay. An excess of GDI, a protein that binds several Rab proteins in their GDP-bound form, can efficiently extract these proteins from membranes [32]. Consistent with a requirement of membrane-bound Rab3, this treatment inhibited the GTPS-promoted acrosome reaction in a concentration-dependent manner.
Our results indicate that Rab3A is present in human sperm cells and that addition of active Rab3A triggers the acrosome reaction. However, we cannot rule out that other Rab3 isoforms may also be present in the spermatozoa. Addition of recombinant Rab3A may trigger the reaction by mimicking these other isoforms. More work is required to better define the Rab3 isoform participating in the acrosome reaction.
The exact mechanism by which Rab3 participates in exocytosis is not known. In most fusion-dependent transport steps, the Rabs seem to promote fusion by inducing the formation of a multiprotein complex on the membranes that would be necessary for specific docking and SNARE activation [33]. However Rab3A, the Rab participating in synaptic vesicle fusion, one of the best characterized fusion processes, has been shown to be inhibitory in several exocytotic events [24, 34]. Rab3 has been also described as positive regulator in other secretion models [11, 35, 36]. A model whereby Rab3A promotes a fusion-dependent inhibition of nearby vesicle exocytosis has been proposed [37]. Our results suggest that Rab3 acts as a positive regulator of acrosome content secretion. Interestingly, since acrosome exocytosis involves multiple fusion events in the same secretory granule, the fusion-dependent fusion inhibition mechanism is unlikely to participate in the reaction. Probably Rab3 would have similar effects on the different exocytotic processes, and the differences observed would be explained by 1) the step at which regulated exocytosis is halted waiting for the correct stimulus to be triggered, 2) the effectors and other regulating proteins involved in each process, and 3) the isoform of Rab3 involved in the process.
In brief, in this report we have shown, by using a permeabilized human spermatozoa model, that Rab3 is required for acrosome exocytosis. More work will be necessary to better characterize the Rab3 isoform active in the process, the Rab3-interacting factors involved, and the function of each of them.
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ACKNOWLEDGMENTS |
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; and Dr. J. Fernández and Dr. A. Oberhauser (Mayo Clinic, Rochester, MN) for generously providing the peptides we used in this paper. We also express thanks to Dr. R. Jahn (Yale University, New Haven, CT) for the monoclonal antibody used in the Western blots and to Dr. S. Patterson (School of Medicine, National University of Cuyo, Argentina) for the positive controls in the Western blots and critical reading of this manuscript. Finally, we would like to thank M.I. Colombo for useful suggestions on the manuscript and Mr. Daniel Bari for his excellent microscopy assistance. |
FOOTNOTES |
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1 This work was partially supported by an International Research Scholar Award from the Howard Hughes Medical Institute, CONICET PIP (4276/96) and PEI (0423/97), and CIUNC. 
2 Correspondence: Roberto Yunes, Laboratorio de Biología Celular y Molecular, Instituto de Histología y Embriología (IHEM-CONICET), CC 56, Facultad de Ciencias Médicas, Universidad Nacional de Cuyo, 5500 Mendoza, Argentina. FAX: 54 261 4494117; ryunes{at}fmed2.uncu.edu.ar
Accepted: November 22, 1999.
Received: August 9, 1999.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ß subunits in tail membranes of bovine spermatozoa. Biol Reprod 1992; 47:337–346.[Abstract]This article has been cited by other articles:
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M. T. Branham, L. S. Mayorga, and C. N. Tomes Calcium-induced Acrosomal Exocytosis Requires cAMP Acting through a Protein Kinase A-independent, Epac-mediated Pathway J. Biol. Chem., March 31, 2006; 281(13): 8656 - 8666. [Abstract] [Full Text] [PDF]
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D. M. Hutt, J. M. Baltz, and J. K. Ngsee Synaptotagmin VI and VIII and Syntaxin 2 Are Essential for the Mouse Sperm Acrosome Reaction J. Biol. Chem., May 27, 2005; 280(21): 20197 - 20203. [Abstract] [Full Text] [PDF]
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G. De Blas, M. Michaut, C. L. Trevino, C. N. Tomes, R. Yunes, A. Darszon, and L. S. Mayorga The Intraacrosomal Calcium Pool Plays a Direct Role in Acrosomal Exocytosis J. Biol. Chem., December 13, 2002; 277(51): 49326 - 49331. [Abstract] [Full Text] [PDF]
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M. Michaut, C. N. Tomes, G. De Blas, R. Yunes, and L. S. Mayorga Calcium-triggered acrosomal exocytosis in human spermatozoa requires the coordinated activation of Rab3A and N-ethylmaleimide-sensitive factor PNAS, August 29, 2000; 97(18): 9996 - 10001. [Abstract] [Full Text] [PDF]
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