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Gamete Biology |
Department of Biomedical Sciences and Technology,3 University of L'Aquila, 67100 L'Aquila, Italy
Department of Evolutionary Biology,4 University of Siena, 53100 Siena, Italy
Obstetrics and Gynecology,5 Arcispedale S. Maria Nuova, 42100 Reggio Emilia, Italy
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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acrosome reaction, fertilization, kinases, signal transduction, sperm
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Intracellular signaling systems used by somatic cells in secretion seem to be involved in the zona pellucida–induced acrosome reaction [11]. There is some evidence that protein kinase C (PKC) plays a primary role in activating plasma membrane calcium channels, an event essential for the acrosome reaction [11, 12].
Sperm-egg interaction at fertilization in the mollusc bivalve Unio elongatulus is mediated by fucosylated O-linked oligosaccharide chains of gp273, one of the two glycoproteins constituting most of the vitelline coat in this species [13]. Using affinity-purified immunoglobulins against gp273, we recently demonstrated that they recognize a carbohydrate epitope present on gp273 O-linked oligosaccharide chains and bearing a Lewisa-like structure with fucose as a key determinant in the antibody-antigen interaction [14]. In a preliminary study, we used this immunoglobulin G (IgG) to determine whether Unio epitope was present in the glycoprotein coat of different vertebrate and invertebrate species. Among others, we found it in one of the glycoproteins of the human zona pellucida. On the basis of its electrophoretic pattern, we tentatively identified it as ZP3 [14]. We also demonstrated that human sperm bind to gp273 in different in vitro assays and that binding was specifically inhibited by our purified IgG [14]. Our finding suggests that this epitope is involved in sperm-egg interaction at fertilization in humans and that a strict species-specificity at primary binding therefore does not exist [1].
In the present study, we describe the results of experiments carried out to determine whether human zona pellucida proteins interfere with gp273 binding to human sperm, where gp273 binds to human sperm and whether it induces the acrosome reaction. Because the answer was positive, we then investigated whether a PKC-activated signal transduction pathway was involved.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Digoxigenin-3-O-methylcarbamyl-
-aminocaproic acid-N-hydroxysuccinimide ester and anti-digoxigenin (DIG) antibodies were purchased from Roche Diagnostics (Mannheim, Germany). A protein assay was purchased from Bio-Rad Laboratories (Munich, Germany). Bisindolylmaleimide I (BIM) was obtained from Calbiochem (San Diego, CA). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO).
Purification of gp273 and gp180 Vitelline Coat Glycoproteins
Vitelline coat (VC) glycoproteins of the Unio elongatulus egg were prepared and purified by electroelution as previously described [15].
Sperm Collection
Semen samples, obtained from healthy donors after 3 days of sexual abstinence, were allowed to liquefy at room temperature for 30 min and were then analyzed for volume, pH, sperm concentration, motility, viability, and morphology. Samples were processed as previously described [16]. Briefly, spermatozoa were washed three times by centrifuging at 780 x g for 10 min, and the final pellets were resuspended in Biggers, Whitten, and Whittingham (BWW) medium supplemented with 3% BSA. Motile spermatozoa separated by swim-up technique were counted and diluted to a concentration of 10 x 107/ml. To allow capacitation, spermatozoa were then incubated at 37°C in a 5% CO2 atmosphere for 4 h before use in the following experiments. Sperm viability was assessed by supravital staining with propidium iodide (5 µg/ml) and motility by counting motile sperm at the end of each centrifugation step.
Human Zonae Pellucidae Isolation
Human oocytes were obtained from patients of the in vitro fertilization program of the Department of Obstetrics and Gynecology, Santa Maria Nuova Hospital, Reggio Emilia (Italy). All women involved in the in vitro fertilization program signed informed consent authorizing use of nontransferred, unfertilized oocytes for the isolation of zona pellucida. Cells were disrupted by repeated cycles of freezing and thawing and the cytoplasmic material removed at 6500 rpm for 5 min. The resulting zonae pellucidae were checked for purity under the microscope and then solubilized by heating at 70°C for 70 min in sodium bicarbonate, pH 10. Solubilized samples were centrifuged at 14 000 x g for 30 min to sediment all insoluble particles. The supernatant was stored at -20°C until use.
Inhibition of gp273-Human Sperm Binding by Solubilized Human Zonae Pellucidae in a Solid-Phase Assay
Capacitated sperm were resuspended in coating buffer (0.05 M sodium carbonate, sodium hydrogen carbonate, pH 9.6) to a final concentration of 2 x 107 sperm/ml and the sperm contained in 100 µl of suspension allowed to adhere to wells of polystyrene microtiter plates (Maxisorp, Life Technologies, Milan, Italy) overnight at 4°C, taking care that wells did not dry up. After a rinse with buffer A (10 mM Tris-HCl, pH 7.4, 0.85% NaCl, 0.05% Tween 20), the immobilized cells were blocked for 1 h using buffer A plus 2% BSA (100 µl/well) and then incubated for 1 h at room temperature in buffer B (buffer A plus 1 mM CaCl2 and 0.5% BSA) with gp273-conjugated with DIG (see below) at a concentration of 250 ng/ml, alone or with increasing concentration of solubilized human zonae pellucidae. After washing with buffer A, the wells were incubated for 1 h with an anti-DIG antibody conjugated with peroxidase at a dilution of 1:1000. The enzyme reaction was developed by 0.3% tetra-methyl-benzamidine plus 0.02 hydrogen peroxide in phosphocitrate buffer (0.1 M sodium dihydrogen phosphate, 0.1 M citric acid, pH 5.0). The reaction was stopped with 0.1 M HCl and absorbance was determined at 450 nm. All experiments were performed three times using three wells per sample per assay.
Localization of gp273-Binding Sites on Human Sperm by Confocal Microscopy
Purified gp273 was labeled with DIG according to the manufacturer's instructions. Conjugation was checked on electrophoresed and nitrocellulose-blotted gp273-DIG using an anti-DIG antibody conjugated with peroxidase at a dilution of 1:1000. Capacitated sperm were smeared onto a glass slide, fixed with 4% formalin and, after blocking the unspecific sites with PBS containing 2% BSA, incubated with 10 µg/ml gp273-DIG in the same buffer for up to 30 min at room temperature. After washing, sperm were incubated with an anti-DIG antibody conjugated with rhodamine diluted 1:5. Slides were then washed, mounted in PBS-glycerol, and observed with a laser scanning confocal apparatus (TCS 4D, Leica, Heidelberg, Germany). In other experiments, sperm were treated with the calcium ionophore A23187 and stained with PSA-fluorescein isothiocyanate (FITC) as described below before labeling with gp273-DIG.
Acrosome Reaction Induced by A23187
In all experiments capacitated human sperm were resuspended in BWW-BSA at a concentration of 10 x 107 cells/ml in a total volume of 100 µl. Test and control tubes were incubated for 1 h at 37°C in 5% CO2 in air in the presence of 5 µM A23187 freshly prepared from a 10 mM stock in dimethylsulfoxide (DMSO) or DMSO alone. After incubation, sperm were washed by centrifugation at 780 x g for 10 min with PBS (50 mM, KH2PO4, 150 mM, NaCl, pH 7.4) and then smeared for the acrosome assessment as described below.
Acrosome Reaction Induced by gp273
For the gp273-induced acrosome reaction, sperm were prepared as described above and then incubated for 1 h at 37°C in 5% CO2 in air in the presence of 1, 5, and 10 µg/ml gp273. As control, sperm were incubated with the same concentrations of gp180, another glycoprotein of the Unio VC, devoid of biological activity [13]. To determine the degree of spontaneous acrosome reaction, a sperm aliquot was always incubated under the same conditions with BWW-BSA alone. Sperm were washed and then smeared for acrosome assessment. In other experiments, sperm were treated with 10 µg/ml gp273 for 10 min, washed, incubated in BWW-BSA to reach a final incubation time of 1 h, and scored for acrosome reaction. All the experiments were performed at least four times.
PKC Activity
Because PKC activity in human spermatozoa is so low [17], collected pooled samples from 15 to 20 subjects were needed for each experiment. The donors were the same in all experiments. PKC activity was determined essentially as previously described [16] by measuring transfer of the phosphate group from [-32P]-ATP to the histone III-S in isolated sperm membrane [18, 19]. Swim-up selected, capacitated spermatozoa (10 x 107/sample) were stimulated with phorbol myristate acetate (PMA, 1,6 mM) [16] for 5 min at 37°C in BWW-BSA or exposed for different times at various concentrations of gp273. As control, sperm were incubated with gp180. The cells were centrifuged and washed twice in a buffer (buffer 1) containing 20 mM Tris, 2 mM EDTA, 1 mM EGTA, 2 mM PMSF, 330 mM sucrose, 50 mM 2ß-mercaptoethanol, 5% glycerol, 5 mg/ml leupeptin, 0.5 mg/ml bacitracin, and 50 mg/ml soybean trypsin inhibitor. Spermatozoa were homogenized and sonicated to obtain a homogenous suspension that was centrifuged for 60 min at 100 000 x g. The final pellet was resuspended in buffer 1 containing 1% NP-40, homogenized, sonicated, and incubated for 30 min at 4°C. The particulate fraction was centrifuged at 500 x g for 15 min and the obtained supernatant applied to previously activated DEAE-cellulose DE-52 columns equilibrated with buffer 1.
Protein concentration was determined by a commercial protein assay kit (Bio-Rad). PKC assay was initiated by adding protein suspension (40 µg/tube) to a final volume of 100 µl buffer 2 containing 1 mM CaCl2, 96 mg/ml phosphatidylserine, 10 mM MgCl2, 100 mM ATP, 3 mg/ml diolein, 1 mg/ml histone IIIS, and 0.5 mCi 32P-ATP. Basal activity was measured in buffer 2 devoid of CaCl2, phosphatidylserine, and diolein and containing 1 mM EGTA. The reaction was allowed to run for 30 min at 37°C and was then stopped in ice. The mixture was collected on membrane filters (GF/C, Whatman, Maidstone, England) that were subsequently washed four times with 1% phosphoric acid. The samples were counted for radioactivity by the Cerenkov method. Blank values were subtracted from all experiments and phosphate transferred per minute per milligram of protein was determined in picomoles. Ratios of experimental to control values were calculated in each experiment, and PKC activity was expressed as mean ± SEM of ratios obtained from three to four replicates.
Effect of a PKC Inhibitor on gp273-Induced PKC Activation
Swim-up selected capacitated human spermatozoa were preincubated for 5 min with the PKC inhibitor, BIM, at a concentration of 1 µM in BWW-BSA. After washing in BWW-BSA, spermatozoa were exposed to gp273 for 10 min and then processed as previously described for the determination of PKC activity.
Effect of a PKC Inhibitor on gp273-Induced Acrosome Reaction
In some experiments, swim-up selected capacitated human spermatozoa were preincubated for 5 min with 1 µM BIM in BWW-BSA. After washing in BWW-BSA, the cells were incubated for 1 h at 37°C in 5% CO2 in air in the presence of 10 µg/ml of purified gp273 as described above, before determination of acrosome status.
Assessment of Acrosome Status
Acrosome status of spermatozoa after the different incubation conditions was determined by the fluorescein isothiocyanate-Pisum sativum agglutinin method (PSA-FITC) [20]. Briefly, an aliquot (50 µl) of spermatozoa (5 x 106 cells/ml) was smeared onto a glass slide and allowed to attach at room temperature, taking care to keep it in a liquid phase to avoid damaging the plasma membranes. Sperm were then permeabilized with cold methanol for 10 min, washed with PBS, and incubated for 30 min with PSA-FITC (25 µg/ml) in PBS containing 2% BSA. Slides were then washed, mounted in PBS-glycerol, and observed with a TCS 4D laser scanning confocal apparatus (Leica Lasertechnik). As many as 300 cells were counted in each sample in a blind fashion. Sperm with 60% or more of the acrosome fluorescent were scored as nonreacted (Class 1). Sperm with fluorescence localized only in the equatorial region (Class 3) or with no fluorescence at all (Class 2) were scored as reacted. Each experiment was preformed at least three times.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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When swim-up selected, capacitated sperm were allowed to interact in a solid-phase binding assay with gp273-DIG in the presence of solubilized human zonae pellucidae [14], hZP protein competed with gp273-human sperm binding in a dose-dependent way and inhibited this binding up to 60% (Fig. 1). These results confirm that the epitopes of gp273 recognized by human sperm are the same or very similar to those recognized in the natural system.
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gp273-Binding Sites Were Located in the Acrosomal Region of Human Sperm
When swim-up selected, capacitated sperm were incubated with purified DIG-labeled gp273 and an anti-DIG antibody conjugated with rhodamine, laser confocal microscopy revealed a bright spotted fluorescence restricted to the acrosomal region in most sperm (Fig. 2). When A23187-treated sperm were incubated with DIG-labeled gp273, no fluorescence was observed in most sperm (data not shown). The percentage of acrosome reactions was less than 15% in control capacitated and more than 40% in ionophore-treated sperm.
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gp273 Induced the Acrosome Reaction in Human Sperm
When swim-up selected, capacitated sperm were incubated with purified gp273 at concentrations of 1 and 5 µg/ml and assessed by PSA-FITC method, about 22% showed acrosome reaction (Fig. 3). This percentage was similar to that of spontaneous acrosome reaction in control sperm. About 61% reacted at 10 µg/ml gp273. This percentage was very similar to that induced by calcium ionophore treatment (56%). At all concentrations tested, gp180, the other glycoprotein of Unio VC gave rise to a percentage of acrosome-reacted sperm similar to that shown by control sperm (basal level; Fig. 3).
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When sperm were treated with 10 µg/ml gp273 for 10 min, washed, and scored for acrosome reaction after 1 h, the percentage of acrosome reacted sperm was 51% to 60%, similar to that observed after 1 h of incubation with gp273 (data not shown).
gp273 Activated PKC on Human Spermatozoa
Because PKC was suggested to be involved in the signaling cascade leading to acrosome reaction during homologous fertilization in human [3], we examined whether gp273 stimulated PKC activity in human sperm. When the membrane fraction of untreated and treated sperm was assayed for PKC, we found that sperm treatment with gp273 for 5 min stimulated PKC activity 3- to 4-fold (Fig. 4). Stimulation after sperm treatment at a concentration of 10 µg/ml was the same extent as that observed when sperm were exposed for 5 min to 1 µM PMA. Figure 4 shows that PKC activity decreased to a basal level after 15 min of exposure and remained at this level even when sperm were maintained in the presence of gp273 for 30 min. In contrast, sperm PKC activity was not affected by 10 µg/ml gp180 at any time point investigated (Fig. 4).
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As shown in Figure 5, experimental groups incubated at concentrations of gp273 insufficient to induce acrosome reaction (1 and 5 µg/ml) did not exhibit any significant increase in PKC activity.
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BIM Reversed gp273-Induced PKC Activity and Acrosome Reaction
Because histone III-S might be phosphorylated by kinases other than PKC (Kemp et al., 1994), we tested the effect of BIM, a specific PKC inhibitor, on gp273-induced PKC stimulation. As shown in Figure 6, when BIM was added to the reaction mixture, PKC activity in membranes from sperm stimulated with 10 µg/ml gp273 for 5 min decreased to a level not significantly different from control. Likewise, a good inhibition of PKC activity was observed when sperm were exposed to BIM for 5 min prior to treatment with gp273.
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When capacitated human spermatozoa were preincubated for 5 min with BIM and then challenged with 10 µg/ml of gp273, a significant inhibition of the acrosome reaction was observed. Indeed, gp273 induced acrosome reaction in 54% of sperm cells, and preincubation with BIM reduced this value to the basal level of 26% (Fig. 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, we confirmed this suggestion by demonstrating that: 1) heat-solubilized zonae pellucidae reverse gp273-human sperm binding; 2) gp273-binding sites are restricted to the acrosomal region; 3) gp273 induces the acrosome reaction in human sperm; and 4) gp273 binding results in significant stimulation of PKC activity. In humans, as in other mammals, binding of spermatozoa to the zona pellucida is mediated by glycans and is the natural inducer of the acrosome reaction [7]. Human ZP3 probably plays a key role in this process because recombinant glycosylated human ZP3 induces the acrosome reaction in sperm [21]. Opening of Ca2+ channels is the key step of the exocytotic acrosomal event [22]. The site of calcium entry is detected by fluorescent dye-mediated image analysis in the acrosomal region in which vesiculation and fusion of the plasma membrane and outer acrosomal membrane take place [23]. Here voltage-dependent calcium channels are also found [24]. Restriction of gp273-binding sites to this sperm area and the capacity of solubilized human zonae pellucidae to reverse binding strongly support the idea that human sperm membrane receptors, recognizing the glycans of the heterologous Unio glycoprotein, are the same that interact with homologous glycans. Gp273 bears glycans that not only bind specifically to human sperm but also induce in them the acrosome reaction in a concentration-dependent way as do solubilized human zonae pellucidae [7]. Because glycans generally need to be bound to their peptide to modulate the acrosome reaction [25, 26], we suggest that the gp273-polypeptide backbone bears O-glycans in the correct way to be functional in eliciting the acrosome reaction in human sperm. Indeed, acrosome reaction activity seems more related to the spatial configuration of the structure bearing the glycans than to an exact peptide sequence. In fact in mice, this activity is lost by treating ZP3 with pronase, but it is restored when the fragments are bound to Fab fragments of anti-ZP3 and then cross-linked with goat anti-rabbit IgG [7]. Gp273-binding sites on human sperm function as signaling receptors because exposure of spermatozoa to this glycoprotein results in significant stimulation of PKC activity. Besides, the observation that BIM reverses both PKC activation and the acrosome reaction indicates that this kinase is a key component of the signal transduction mechanism activated by gp273 and leading to the exocytotic event.
Much evidence exists in support of the presence of PKC on the human sperm surface [27] and its role in the ZP-induced acrosome reaction [11, 12]. Different PKC antagonists inhibit the human acrosome reaction induced by oocytes or isolated zonae pellucidae [12, 28]; PMA is known to induce both an increase in PKC activity and the acrosome reaction [16, 29]. Interestingly, the topographical location of PKC [17] matches that of gp273-binding sites. PKC activation is hypothesized [30] to take part in the signaling pathway involving a tyrosine kinase receptor coupled to phospholipase C (PLC) and a Gi-coupled receptor that activates a PLCß1 [11]. The products of PLC-mediated phosphatidylinositol-2 hydrolysis, diacylglycerol and inositol phosphate-3, would translocate PKC to the plasma membrane and activate it [28, 31, 32]. This in turn would open a voltage-dependent Ca2+ channel in the plasma membrane, leading to the increase in Ca2+ that finally triggers the acrosome reaction [33]. In mice, two waves of calcium influx have been described in spermatozoa as a consequence of ZP3 binding: a first relatively small transient wave occurring in the first seconds of ZP3 stimulation [34] and a second one leading to a sustained calcium influx that requires up to several minutes of ZP stimulation to reach maximum levels [22, 35]. However, no involvement of PKC has been suggested in mice. In humans, two waves of calcium influx have been also described, but the relationship with PKC activation during the acrosome reaction is unknown [36]. It is therefore difficult to establish whether stimulation of PKC by gp273, detectable 5 min after sperm incubation in the protein, and of a transient nature can be related to the first or second calcium wave.
Taken together, our results show that gp273 can be regarded as an alter ego of the glycoprotein of the human zona pellucida, functioning as ligand and inducer of the acrosome reaction in humans. Indeed, the amount of gp273 inducing the acrosome reaction in human sperm is in the same molar range as those of solubilized ZP [37] and recombinant hZP3 [21]. Because it is difficult to obtain sufficient hZP proteins from native sources, the Unio protein could have great theoretical and practical utility.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Dr. Riccardo Focarelli, Department of Evolutionary Biology, Via A. Moro 2, 53100 Siena, Italy. FAX: 39 0577 234476; focarelli{at}unisi.it
Received: 4 June 2003.
First decision: 23 June 2003.
Accepted: 9 July 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This article has been cited by other articles:
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  S. Canovas, P. Coy, and E. Gomez First Steps in the Development of a Functional Assay for Human Sperm Using Pig Oocytes J Androl, March 1, 2007; 28(2): 273 - 281. [Abstract] [Full Text] [PDF]
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