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Regular Article |
a Département d'Obstétrique/Gynécologie, Université Laval, Centre de Recherche du CHUQ and Centre de Recherche en Biologie de la Reproduction, Quebec, Canada G1L 3L5
ABSTRACT
This paper presents the partial characterization and the identification of an 80-kDa protein detected in bull spermatozoa using a monoclonal antibody directed against a 16-amino acid long peptide from the N-terminal domain of the protooncogene p60src from the Rous Sarcoma Virus When subjected to two-dimensional electrophoresis, this 80-kDa protein migrated as several isoforms, with an isoelectric point ranging from 7.4 to 8.2. Amino acid sequence analysis of a peptide obtained following trypsin digestion of the bull sperm protein showed homology to the PH-20/hyaluronidase precursor sperm protein. As for PH-20, this bull sperm 80-kDa protein is located at the plasma membrane surface in the postacrosomal region of the head. An increased immunolabeling in the anterior head region of fixed/permeabilized spermatozoa was observed when these cells were incubated under capacitating conditions, whereas most sperm cells challenged with the calcium ionophore A23187 to acrosome react lost their labeling almost completely. As for the PH-20 protein, the 80-kDa bull sperm protein possesses a hyaluronidase activity that is higher at pH 7.0 than at pH 4.0 in an in-gel assay. Unlike what has been observed in the guinea pig, mouse, and human PH-20, this 80-kDa protein was not released from the surface of bull spermatozoa by treatment with phosphatidylinositol-specific phospholipase C or with trypsin. However, this protein was not sedimented by a 100 000 x g centrifugation after nitrogen cavitation, which suggests that the 80-kDa protein is loosely attached to the sperm membrane by a yet-unknown mechanism. These results suggest that the 80-kDa bull sperm protein shares many homologies with the sperm PH-20 protein reported in the literature and, most likely, is the bull sperm homologue of the PH-20.
fertilization, gamete biology, sperm
INTRODUCTION
The success of fertilization depends on the achievement of several events implicating gamete interactions. To reach the egg, the spermatozoa must first go through the cumulus oophorus, which is made of cumulus cells interconnected by a network of polymerized hyaluronic acid. At the egg surface, the spermatozoa encounter the zona pellucida, which is an extracellular matrix produced by the growing oocyte. The spermatozoa bind to the zona pellucida and, on interaction with the matrix's protein ZP3, undergo the acrosome reaction, an exocytotic event during which the sperm cell delivers its acrosomal content. The acrosome-reacted spermatozoa then penetrate through the zona pellucida and reach the egg's plasma membrane, where binding and fusion of the gametes take place.
To interact with the egg and its extracellular components, the sperm cells have to be properly equipped. Capacitation appears to be necessary for the spermatozoa to go through the cumulus and to reach the zona pellucida, whereas acrosome-reacted sperm cells remain outside this matrix [1]. Several sperm proteins have been proposed as being putative mediators of the interaction with the zona pellucida proteins. Some of them, such as primary receptors, have been shown to bind to ZP3, which is the zona pellucida protein responsible for the binding of the acrosome-intact spermatozoa to the egg's matrix and the induction of the acrosomal exocytosis, whereas others, such as secondary receptors, maintain the sperm binding to the zona pellucida through interactions with ZP2 following the sperm acrosome reaction [2–4].
Among this second type of sperm ligand to the zona pellucida is the protein PH-20. The role of sperm PH-20 in gamete interaction was first demonstrated in guinea pig spermatozoa, where monoclonal antibody reacting with different epitopes of the PH-20 antigen inhibited the sperm binding to the egg's zona pellucida [5]. The PH-20 is a glycoprotein associated to the sperm plasma and inner acrosomal membranes by a glycosylphosphatidyl inositol (GPI) anchor [6–13]. More recently, it was shown that PH-20 possesses a hyaluronidase activity [9–11, 13–15], which is located in a domain of the protein separate from its zona pellucida-binding activity [16]. In the bull, the sperm hyaluronidase activity has been characterized for many years [17, 18], but little information is available as to whether PH-20 is "the" sperm hyaluronidase or simply a protein involved in gamete interaction with a hyaluronidase activity.
The aim of the present work was to characterize an 80-kDa bull sperm protein that possesses certain homologies to the sperm PH-20 precursor protein using a monoclonal antibody directed against a 16-amino acid long peptide from the N-terminal domain of the tyrosine kinase v-src.
MATERIALS AND METHODS
Sperm Preparation
Freshly ejaculated bull semen was collected at an artificial insemination facility and kindly donated by l'Alliance Semex, Inc. (Ste-Hyacinthe, PQ, Canada). The semen was maintained at 23°C until arrival at the laboratory (within 2.5 h). On arrival, 1 ml of semen was diluted in 5 ml of HBS (10 mM Hepes [pH 7.2] and 150 mM NaCl), spermatozoa were washed from seminal plasma by two centrifugations (250 x g, 10 min), and the sperm concentration was evaluated.
On some occasions, spermatozoa were subjected to capacitation and acrosome reaction as follows: On arrival, the sperm cells were washed by centrifugation (800 x g, 20 min) on a 45%–90% (v/v) Percoll gradient made isotonic with Sp-TALP [19], and the spermatozoa, within the 90% Percoll fraction, were diluted at 50 x 106 in Sp-TALP supplemented with BSA (6 mg/ml) and sodium pyruvate (1 mM). The sperm cells were incubated at 39°C for 5 h in the presence of 10 µg/ml of heparin to induce capacitation. The samples were next washed by two centrifugations in Sp-TALP (250 x g, 10 min) and incubated at 37°C for another 30 min in the presence of 5 µM calcium ionophore A23187 in Sp-TALP to induce the acrosome reaction. The sperm cells were then washed by two centrifugations (250 x g, 10 min) in the same medium. Aliquots were taken before and after incubations with A23187 and processed for immunofluorescence or to evaluate the acrosome reaction.
Protein Electrophoresis and Immunoblotting
Sperm proteins were solubilized in sample buffer (62.5 mM Tris-HCl [pH 6.8], 10% glycerol, 2% [w/v] SDS, 5% ß-mercaptoethanol, and 0.01% [w/v] bromophenol blue) and fractionated on 7.5% SDS-polyacrylamide gels under reducing conditions [20]. In one set of experiments, proteins from bull testis (with spermatids) were first extracted in sample buffer without reducing agent, then diluted in complete sample buffer containing ß-mercaptoethanol before electrophoresis. The proteins were electrophoretically transferred on nitrocellulose membranes (MSI, Inc., Westborough, MA) according to the procedure described by Towbin et al. [21]. After blocking for 1 h in TBSTw (20 mM Tris [pH 7.4], 0.9% (w/v) NaCl, and 0.1% (v/v) Tween 20) containing 5% (w/v) dry skim milk, the membranes were incubated with mouse monoclonal antibody directed against a 16-amino acid long peptide from the N-terminal domain of the protooncogene p60src from the Rous Sarcoma Virus (Quality Biotech, Camden, NJ) for 1 h at room temperature. After extensive washes in TBSTw, the membranes were incubated with horse radish peroxidase-conjugated secondary antibody raised in goat (Jackson Immunoresearch, West Grove, PA) for 1 h at room temperature. Again, the membranes were extensively washed in TBSTw, and positive bands were visualized on x-ray films (Fuji, Tokyo, Japan) by enhanced chemiluminescence according to the manufacturer's instructions (Amersham Life Science, Inc., Oakville, ON, Canada). The protein concentration in the testis extract was determined with the Micro BCA Protein Assay Reagent kit (Pierce, Rockford, IL) according to the manufacturer's instructions.
Sperm proteins were also separated by two-dimensional (2D) electrophoresis to better characterize the 80-kDa bull sperm protein. The first dimension consisted of separating the proteins according to their isoelectric point (pI) by nonequilibrium pH gel electrophoresis, as described by Robertson et al. [22]. Whole-sperm extracts were either concentrated in a Microcon-10 filter apparatus (Millipore, Nepean, ON, Canada) or used as is. The samples were mixed with 2x urea lysis buffer (8 M urea, 2% [v/v] NP-40, 5% [v/v] ß-mercaptoethanol, and 2% [v/v] ampholines [pH 3–10]) and separated according to their isoelectric point (8 M urea, 6% [w/v] acrylamide, 12% [v/v] glycerol, 2% [v/v] ampholines [pH 3–10], and 2% [v/v] NP-40). The proteins were fixed (50% [v/v] methanol and 10% [v/v] acetic acid), then incubated in equilibration buffer (62.5 mM Tris-HCl [pH 6.8], 2.3% [w/v] SDS, 5% ß-mercaptoethanol, and 10% [v/v] glycerol). The second dimension was performed using a 7.5% SDS-polyacrylamide gel. The proteins were stained with either Coomassie blue or silver nitrate or transferred to nitrocellulose for Western blot analysis.
Triton X-100 Extraction
Washed spermatozoa were incubated in HBS containing 1% (v/v) Triton X-100 for 5 min at room temperature. The samples were then centrifuged at 10 000 x g for 5 min (4°C), and proteins from either the Triton X-100 extract or the nonextracted pellet were investigated for the 80-kDa protein by Western blot analysis as described above.
Isolation of Sperm Membranes
Sperm membranes were isolated according to the procedure described by Noland et al. [23]. The complete procedure was performed at 4°C. Washed spermatozoa were supplemented with protease inhibitors (500 µM PMSF, 1 µg/ml of pepstatin A, 4 µg/ml of leupeptin, and 4 µg/ml of aprotinin) and subjected to nitrogen cavitation at 750 psi for 20 min at 4°C. After nitrogen cavitation, the sperm suspension was centrifuged at 1000 x g to remove cellular debris. Next, the supernatant was further centrifuged at 100 000 x g for 1 h to pellet the membrane fraction. Aliquots were taken from the pellet and supernatant at each step of the procedure. Protein concentration was determined with the Micro BCA Protein Assay kit, and the proteins were solubilized and processed for electrophoresis and Western blot analysis as described earlier.
Indirect Immunofluorescence
The localization of p80 was determined by immunofluorescence on washed spermatozoa or cells incubated under capacitating/acrosome-reaction conditions in two different manners. In the first set of experiments, the spermatozoa were deposited on slides and kept at room temperature for 30 min. The cells were then fixed/permeabilized with 95% ethanol for 30 min at 4°C. After blocking with 10% porcine serum diluted in PBS (137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, and 8.1 mM Na2HPO4 [pH 7.4]), the slides were incubated with either the monoclonal antibody or with mouse IgG (Sigma, St. Louis, MO) for 1.5 h at 37°C. After extensive washes in PBS, the slides were incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibody raised in donkey for 1 h at room temperature. Also after extensive washes in PBS, 25 µl of the antibleaching agent diazabicyclo[2,2,2]octane (DABCO; 1.5% [v/v] made in 90% glycerol) were deposited on the slides, which were then covered with a coverslip. Spermatozoa were examined by epifluorescence microscopy.
In another set of experiments, the presence of p80 was investigated in nonpermeabilized cells to measure the surface expression of the protein. Washed spermatozoa were diluted (25 x 106 spermatozoa/ml) in sp-TALP medium [19] containing 6 mg/ml of BSA and fixed with 3.7% (v/v) formaldehyde for 10 min at room temperature. After two washes, a 100-µl aliquot was incubated with the antibodies mentioned above for 1 h at room temperature. The cells were next washed twice by centrifugation (250 x g, 10 min), then resuspended in Sp-TALP containing BSA. The FITC-conjugated secondary antibody was added to the suspension, and the spermatozoa were incubated for 1 h. At last, the spermatozoa were washed twice in sp-TALP by centrifugation (250 x g, 10 min), resuspended in DABCO (1.5% in 90% glycerol), and mounted on slides for examination.
Partial Purification of the 80-kDa Bull Sperm Protein
Washed spermatozoa were resuspended in HBS and kept frozen at -20°C in the absence of cryoprotectant until use. When needed, the frozen samples were put on ice, protease inhibitors were added (leupeptin, pepstatin A, aprotinin, and benzamidine to a final concentration of 10 µg/ml each; 500 µM PMSF), and the samples were rapidly thawed, vortexed, and centrifuged at 12 000 x g for 10 min at 4°C. The resulting protein extracts were concentrated by centrifugation in a Microcon-10 filter apparatus, washed in Tris-HCl (10 mM, pH 9.0) by an additional centrifugation (12 000 x g, 10 min, 4°C), and resuspended in this buffer to a final volume of 200 µl. The proteins were first separated by HPLC on a Mono-Q HR5/5 anion exchange column (Pharmacia, Baie d'Urfé, PQ, Canada), conditioned in Tris-HCl (10 mM, pH 9.0), and activated with the same buffer (containing 1 M NaCl) at a flow rate of 1.0 ml/min. The proteins were eluted with an ascending, two-step NaCl gradient from 0 to 0.2 M NaCl over 20 min and then from 0.2 to 1 M NaCl over 10 min. Aliquots of 500 µl were collected, and presence of the 80-kDa bull sperm protein was monitored by Western blot analysis as described earlier. The positive fractions were pooled and concentrated by centrifugation in a Microcon-10 filter apparatus, then washed with 10 mM Tris (pH 7.2) and 150 mM NaCl by a supplementary centrifugation (12 000 x g, 10 min, 4°C). This pool was used for amino acid sequencing and protein identification.
Protein Identification
Two methods were used to identify the protein recognized by the monoclonal antibody. In the first, proteins were separated by 2D gel electrophoresis as described above, then transferred onto a polyvinylidene fluoride (PVDF) membrane. The proteins were stained by Coomassie blue. The protein identified by a corresponding Western blot was cut out, and the N-terminal amino acid sequence was performed by Edman degradation at the Service de Séquence de l'Est (CHUQ, Ste-Foy, PQ, Canada). The second approach consisted of the sequence analysis of peptides obtained by tryptic digestion of the protein partially purified on Mono-Q column and separated on 5%–15% gradient SDS-PAGE. The peptides were separated on a Vydac C18 HPLC column (S.P.E. Limited, Concord, ON, Canada), and the peaks of interest analyzed by mass spectrometry for purity and length, before the amino acid sequence was determined as described above. Homology searches for the amino acid sequences were conducted using the BLAST network service of the NIH (http://www.ncbi.nlm.nih.gov/).
In-Gel Hyaluronidase Activity
Hyaluronidase activity was assayed according to the method described by Cherr et al. [15] using commercial hyaluronidase from bovine testes (Sigma) as a positive control. Briefly, proteins were separated on 7.5% (w/v) SDS-polyacrylamide gels copolymerized with 0.17 mg/ml of hyaluronic acid. Protein aliquots were solubilized in SDS-sample buffer under nonreducing conditions. Following electrophoresis, the gel was incubated for 2 h at room temperature in TBS containing 3% (v/v) Triton X-100, then in 100 mM sodium acetate (pH 7.0 or 4.0) for 16–20 h at 37°C. The gels were next incubated with 0.5% (w/v) Alcian Blue (Bio-Rad, Mississauga, ON, Canada) in 3% (v/v) acetic acid for 2 h at room temperature to stain the hyaluronic acid, then destained in 7% (v/v) acetic acid. The presence of activity was revealed by lack of coloration in the gel. The proteins were separated on four identical gels. Two gels were used for hyaluronidase activity at pH 7.0 and 4.0, another was stained with Coomassie blue, and the fourth was transferred to nitrocellulose for Western blot analysis.
Determination of Trypsin Cleavage and GPI Linkage of Sperm p80 Protein
Washed spermatozoa were resuspended in Dulbecco PBS (6.8 mM CaCl2, 2.68 mM KCl, 1.46 mM KH2PO4, 0.492 mM MgCl2, 136.9 mM NaCl, and 8.06 mM Na2HPO4) at a concentration of 25 x 106 spermatozoa/ml and incubated for 30 min at 30°C in the absence or presence of phosphatidylinositol-specific phospholipase C (PI-PLC; 1 IU/ml; Sigma) or trypsin. After the incubation, the cells were centrifuged at 10 000 x g for 3 min, and the presence of p80 was detected by Western blot analysis in both the pellet and supernatant. As a positive control for PI-PLC activity, hamster spermatozoa were treated with this enzyme, and the release of hamster sperm p26h, a surface antigen known to be GPI-anchored [24], was analyzed by Western blot analysis using an antibody raised against the recombinant protein. The p26h antibody was kindly provided by Dr. R. Sullivan (Centre de Recherche en Biologie de la Reproduction, Université Laval, PQ, Canada).
RESULTS
Detection and Cellular Localization of the 80-kDa Bull Sperm Protein
An increase in protein tyrosine phosphorylation occurs during sperm capacitation. Suspecting that protein tyrosine kinases of the src family are involved, we first tried to detect, by Western blot analysis, p60src in bull spermatozoa using a monoclonal antibody directed against amino acids 2–17 of the N-terminal domain of the protooncogene src from the Rous Sarcoma Virus. A single protein of 80 kDa was detected by the monoclonal antibody in sperm extract, whereas four major proteins of 80, 60, 52, and 50 kDa were detected in the testicular extract (Fig. 1). This reaction appears to be specific, because no reacting protein band was observed when the antibody was adsorbed with the immunizing peptide (data not shown). Throughout the present manuscript, the 80-kDa sperm protein recognized by the monoclonal antibody will be called p80.
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To determine whether p80 is associated with the cytoskeleton or with the membrane/cytosol, sperm proteins were extracted in the presence of 1% Triton X-100. As shown in Figure 2, the p80 protein was found predominantly in the Triton X-100 extracts, suggesting that the protein is either cytosolic or associated with the membranes. To discriminate between these last two locations, spermatozoa were subjected to nitrogen cavitation in the absence of detergent and to two sequential centrifugations (1000 and 100 000 x g) to separate membranous and cytosolic sperm proteins. Figure 3 shows that p80 is present principally in the soluble fraction, suggesting that it is cytosolic. However, when the localization of p80 was assessed in nonpermeabilized spermatozoa by indirect immunofluorescence, a strong signal was observed in the postacrosomal region of the head (Fig. 4A), suggesting that the p80 is bound to the membrane but facing the extracellular milieu. On the other hand, p80 is detected both in the acrosomal and postacrosomal areas of the head of spermatozoa fixed/permeabilized by ethanol treatment (Fig. 4C). Taken together, these results suggest that p80 is present both intracellularly in the acrosomal area and extracellularly in the postacrosomal portion of the head.
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Effect of Capacitation and Acrosome Reaction on the Distribution of Sperm p80
When spermatozoa were incubated under capacitating conditions, a change in the immunofluorescence pattern was observed. In nonpermeabilized cells, three different patterns were found (Fig. 5A). The majority of the cells (mean ± SEM) (47.3% ± 27.2%) showed a strong labeling in the postacrosomal region, at the edge of the equatorial segment. Other cells were either strongly (29.3% ± 18.8%) or faintly (23% ± 10.5%) labeled in the entire postacrosomal head region. On the other hand, 84.0% ± 5.2% (n = 4) of ethanol-fixed/permeabilized cells expressed intense labeling in the anterior head region, whereas 8.6% ± 5.7% (n = 4) of the cells showed a signal in both the anterior and postacrosomal head regions and 6.9% ± 2.6% (n = 4) expressed p80 only in the postacrosomal region (Fig. 5B). The percentage of spontaneous acrosome reaction in this capacitated sperm population was 11.8% ± 5.1% (n = 4). When capacitated cells were challenged with the calcium ionophore to acrosome react, the pattern of labeling did not change significantly in nonpermeabilized sperm cells when compared with cells incubated under capacitating conditions (Fig. 5C). In ethanol-fixed/permeabilized spermatozoa, a weak signal (Fig. 5D, arrows) was observed in 56.1% ± 2.7% of the cells (n = 4). In addition, 22.5% ± 2.7% of the cells expressed p80 in the postacrosomal region, 14.9% ± 1.3% in the anterior head region only, and 6.5% ± 3.6% showed a positive signal in both regions (Fig. 5D). Of these A23187-treated cells, 61.4% ± 2.7% had undergone the acrosome reaction.
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Partial Purification and Identification of Sperm P80 Protein
Two-dimensional gel electrophoresis protocols were first used to isolate the p80 sperm protein to determine its amino acid sequence. As shown in Figure 6, the sperm p80 has at least three isoforms, with an isoelectric point (pI) ranging from 7.4 to 8.2. After transfer onto a PVDF membrane, the protein was processed for N-terminal amino acid sequencing. As shown in Table 1, the sequence of a seven-amino acid fragment was determined, showing no homology with other protein sequences after a BLAST search.
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Because the quantity of protein obtained with the 2D gel electrophoresis protocol was not sufficient to determine the sequence of a longer fragment of p80, the protein was purified by anion-exchange chromatographic methods. The procedure was based on the results obtained with the 2D gel electrophoresis, indicating the pI of the protein. A typical chromatographic elution profile is shown in Figure 7A. Western blot analysis of the fractions collected revealed that p80 was eluted in fractions 29–34 (Fig. 7B). Consistently (n = 11), p80 was eluted with 75 mM NaCl. In the pool of fractions 29 and 30, the monoclonal antibody reacted with a second band at 60 kDa. It is not known, however, whether this protein results from proteolytic cleavage of the p80 protein. The positive fractions were then pooled and separated on a 5%–15% linear gradient SDS-polyacrylamide gel. The band at 80 kDa was cut out and digested with trypsin, the fragments were separated on a C18 column, and the amino acid sequence of two internal peptides was determined (single-letter code: VDFETAGK and AKNDIAYYIPNDSVGA, 8 and 16 amino acids long, respectively) (Table 1). The 16-amino acid peptide showed significant homologies with the PH-20 sperm surface protein/hyaluronidase precursor, suggesting that p80 may be the bull homologue of PH-20, which was first identified in guinea pig spermatozoa. Furthermore, we found one report in the literature showing the bull sperm PH-20 amino acid sequence as deduced from a cDNA clone [25] and, as shown in Table 1, were able to match our three peptide sequences.
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In-Gel Hyaluronidase Activity in Sperm Protein Extract
Because PH-20 possesses a hyaluronidase activity [9, 11, 13–15], an in-gel hyaluronidase assay was performed to verify whether p80 shares similar enzymatic activity with the sperm surface protein PH-20. Furthermore, because PH-20 hyaluronidase activity is optimal at different pH, whether it is membrane bound (pH 7.0) or soluble (pH 4.0) [15], the hyaluronidase activity of bull sperm p80 was assayed at both pH 4.0 and 7.0 using commercial bovine testis hyaluronidase as a positive control. As shown in Figure 8, p80 possesses a hyaluronidase activity that is stronger at pH 7.0 than at pH 4.0. Although commercial bull testis hyaluronidase displayed enzymatic activity, no reacting protein was detected by Western blot analysis, whereas a strong signal was observed in the sperm extract and the partially purified fraction (Fig. 8A). It is interesting to note that although several bands (i.e., 161, 157, 121, and 60 kDa) are detected in the sperm extract and anion-exchange partially purified p80 when the samples are processed under nonreducing conditions (Fig. 8A), a single 80-kDa reacting band is found under reducing conditions (Figs. 1–3).
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PI-PLC and Trypsin Treatment of Bull Spermatozoa
The PH-20 is linked to the sperm membrane by a GPI anchor [8], and it has been reported that PH-20 released from the membranes through a protease action during the acrosome reaction is associated with a reduction in size [15, 26]. Freshly ejaculated bull spermatozoa were treated with PI-PLC or trypsin to determine whether these enzymes could release p80 in the supernatant and whether trypsin treatment induced a diminution in the molecular weight of the protein as detected by Western blot analysis. Neither the treatment with PI-PLC nor trypsin released the protein in the supernatant (Fig. 9). Trypsin also had no effect on the molecular weight of p80. This inability of PI-PLC to release p80 into the supernatant was not caused by a poor enzymatic preparation, because, as a positive control [24], hamster sperm P26h was successfully released by PI-PLC when the assay was run in parallel (data not shown). The experiments with PI-PLC were also done with higher concentrations of enzyme (5 IU/ml) and/or treated for a longer period of time (1 h) at a higher temperature (37°C), or after a sperm treatment with 1 M NaCl, and always with similar results (data not shown).
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DISCUSSION
The aim of this work was to characterize an 80-kDa bull sperm protein (p80) recognized by an anti-v-src monoclonal antibody. Although this immunoreactive protein possesses a higher mass than the src tyrosine kinase (80 vs. 60 kDa), higher-molecular-weight forms of src-related tyrosine kinases have been reported in the past. A variant of src containing 17 additional amino acids has been described in the brain [27], and a variant of fyn, showing an increase of 10 kDa over the "classical" kinase, has also been demonstrated [28]. In addition, as for src-related tyrosine kinases, experiments with the nitrogen cavitation of spermatozoa and extraction with Triton X-114 (data not shown) suggested that p80 is cytosolic. However, the amino acid sequence of an internal peptide revealed a significant homology to the mammalian protein PH-20, a sperm surface membrane protein involved in sperm/oocyte interactions [5, 6, 10, 13]. The PH-20 protein possesses a hyaluronidase activity [9–11, 13, 15] and a domain involved in the sperm binding to the egg's zona pellucida [16]. The complete amino acid sequence of a 65-kDa bull sperm PH-20 has been deduced from a testis cDNA clone [25], and the sequences of the three peptides we analyzed were present within that deduced sequence (Table 1). Although these authors did not isolate the sperm protein, they showed a high degree of similarity between the amino acid sequence deduced from the bull testis cDNA clone and the amino acid sequence of commercial bovine testis hyaluronidase. The present study is, to our knowledge, the first to characterize the putative bovine homologue of the sperm PH-20, and it shows that this protein, p80, possesses a hyaluronidase activity.
To this point, why the monoclonal antibody that we used recognized the bull sperm p80 protein remains unclear. The antibody used in all the experiments was raised against a synthetic peptide of 16 amino acids (GSSKSKPKDPSQRRHS), corresponding to the amino acids 2–17 of the N-terminal domain of p60v-src, and a search for similarity with proteins in public databases resulted in homology only to p60src. We found one short sequence of four amino acids in the bull PH-20 sequence (KPKD) and two sequences of three amino acids (GSS; RRH) that were conserved in the amino acid sequence of the synthetic peptide. Whether these short sequences allow a specific interaction with the anti-v-src monoclonal antibody remains to be assessed. In addition to the 65-kDa bovine PH-20, a 75-kDa protein is present in commercial bovine testis hyaluronidase, but the amino acid sequence has not been determined [25]. We cannot exclude that the p80 protein we identified could correspond to this isoform. However, that the monoclonal antibody used in the present study was unable to recognize commercial bull testis hyaluronidase in Western blot experiments (Fig. 8A) makes this possibility less likely.
Similarities and differences exist between the bull sperm protein, p80, and the reported sperm PH-20. First, the bull protein migrates at 80 kDa on SDS-PAGE, whereas PH-20, or 2B1 in rats [29], migrates at 58–68 kDa in most species studied [6, 11, 13, 26, 30]. Also, bull sperm p80 possesses more basic isoforms, with a pI ranging between pH 7.4 and 8.2, whereas the 2B1 antigen, the PH-20 rat orthologue [14], focuses with a broad range between pH 5.3 and 6.3 and the human PH-20 protein between pH 6.5 and 7.5 [31].
On the other hand, indirect immunofluorescence on nonpermeabilized spermatozoa revealed the localization of p80 at the postacrosomal region of the head (Fig. 4A). Moreover, this result suggests that p80 is expressed at the sperm surface, facing the extracellular milieu. Similar localization of PH-20 has been reported in the guinea pig [6] and stallion [13], whereas it is present over the entire head in other species [10, 12]. A strong signal, however, was detected on the anterior region of the head of spermatozoa fixed/permeabilized with ethanol, which might represent a population of PH-20 expressed intracellularly. Unlike what is reported for PH-20 in other species [5, 12, 13], no modification in the surface expression pattern of p80 was found during sperm capacitation or acrosome reaction (Figs. 4A and 5, A and B). This suggests that p80 is not present on the inner acrosome membrane. On the other hand, in cells fixed/permeabilized in ethanol, a change is observed during capacitation and acrosome reaction. During sperm capacitation, a decrease in the signal intensity is observed in the postacrosomal region and is associated with an increase in the anterior region of the head. When spermatozoa are challenged to acrosome react, the signal is decreased in most cells (56%). Taken together, these results suggest that p80 is either soluble, associated with the outer acrosomal membrane, or associated with the inner leaflet of the plasma membrane, but not associated with the inner acrosomal membrane.
Other similarities are found between p80 and sperm PH-20. The bull sperm p80 protein is extracted by Triton X-100, suggesting that the protein is either membranous or cytosolic. When in-gel hyaluronidase activity is assayed in fractions resulting from Triton X-100 extraction experiments, most of the activity is found in the Triton X-100 extract (data not shown), which is in agreement with previously reported results [14]. In spermatozoa subjected to nitrogen cavitation, most of the p80 was detected in the soluble fraction, which agrees with the hyaluronidase activity reported in the soluble fraction of sonicated bull spermatozoa [14]. These results suggest that p80 is loosely attached to the membrane and is removed by Triton X-100 treatment, nitrogen cavitation/sonication, or even freezing in the absence of cryoprotectant. This statement is reinforced by p80 being detected in the aqueous fraction when bull spermatozoa are extracted with Triton X-114 (data not shown). In most species, PH-20 is attached to the plasma membrane by GPI and is released by PI-PLC treatment [8–11, 13]. However, we were unable to release the bull p80 protein by PI-PLC treatment (Fig. 9), even after a sperm treatment with NaCl (1 M; data not shown). Similar results have been obtained with the rat 2B1 protein [14]. In addition, the amino acid sequence deduced from the red fox PH-20 cDNA suggests this protein, in that species, is neither attached to the membrane via a GPI anchor nor possesses a transmembrane domain [30]. Our results, as well as those mentioned above [14, 30], suggest that additional anchoring mechanisms are involved for the presence of PH-20 at the membrane surface, and that treatments affecting the stability or solubility of the plasma membrane will effectively release the p80 protein.
We have demonstrated (Fig. 8) that the bull sperm 80-kDa protein possesses a hyaluronidase activity that, in an in-gel hyaluronidase assay, is higher at pH 7.0 than at pH 4.0. This result is also in agreement with the association of the protein with the membrane, because membrane-bound protein PH-20 shows an optimal hyaluronidase activity at neutral pH [15]. Harrison and Gaunt [32] have detected a single 80-kDa bull sperm protein by Western blot analysis using an anti-ram sperm hyaluronidase monoclonal antibody in protein samples solubilized under reducing conditions. As for the p80 described in the present study (Fig. 8), we detected several immunoreactive proteins when the samples were processed under nonreducing conditions. This finding might suggest that, in spermatozoa, p80/hyaluronidase forms homopolymers, which are reduced to monomers under reducing conditions, or heteropolymers with other proteins.
The physiological importance of a PH-20 homologue on bovine spermatozoa remains elusive, because the need for a hyaluronidase activity to disperse the egg's cumulus on ovulation is controversial. However, hyaluronic acid is present in the zona pellucida and perivitelline space, and it induces an increase in intracellular sperm calcium through its interaction with PH-20 [33, 34]. Such an action resulted in an increased percentage of acrosome reactions on sperm binding to the egg zona pellucida. The interaction of p80 with hyaluronic acid could result in the priming of capacitated spermatozoa for the subsequent acrosome reaction. This hypothesis will be investigated in the near future.
In the present study, we have demonstrated that an 80-kDa bull sperm protein shares amino acid sequence and functional homologies with PH-20, a sperm protein with a hyaluronidase activity involved in the interaction with the egg. Whether p80 is involved in sperm interaction with the eggs during the process of fertilization, however, needs further study.
ACKNOWLEDGMENTS
The authors are thankful to l'Alliance Semex for providing the bull semen used throughout this study. Our special thanks also go to Dr. R. Sullivan for his careful revision of the manuscript and gift of P26h antibody. In addition, we are grateful to Mr. P. De Grandpré for his help in purification of the p80 by HPLC.
FOOTNOTES
First decision: 1 December 2000.
1 Supported by a grant from the Natural Science and Engineering Research Council of Canada to P.L. 
2 Correspondence: Pierre Leclerc, Endocrinologie de la Reproduction, Pav.
St-François d'Assise, 10, de l'Espinay, Québec, PQ, Canada G1L 3L5.
FAX: 418 525 4195; pierre.leclerc{at}crsfa.ulaval.ca
Accepted: April 5, 2001.
Received: October 27, 2000.
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