Molecular Human Reproduction, Vol. 6, No. 8, 699-706,
August 2000
© 2000 European Society of Human Reproduction and Embryology
Embryo development |
Evidence for the participation of ß-hexosaminidase in human sperm–zona pellucida interaction in vitro
1 Instituto de Biología y Medicina Experimental (CONICET), Buenos Aires, 2 Fertilab, Buenos Aires, Argentina and 3 Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada
Abstract
Mammalian sperm–zona pellucida (ZP) interaction is mediated by sperm lectin-like proteins and ZP glycoproteins. We have previously reported the participation of binding sites for N-acetylglucosamine (GlcNAc) residues in human sperm function, including sperm interaction with the ZP. Additionally, previous results from our laboratory suggested that some of these events may be mediated by the glycosidase N-acetylglucosaminidase (ß-hexosaminidase, Hex, in mammals). In this study, we report the possible participation of Hex in human sperm–ZP interaction. Human recombinant Hex (hrHex) was obtained by expression in a stable transfected CHO cell line. When the recombinant enzyme was present during hemizona (HZ) assays, the number of sperm bound per HZ was significantly reduced. The same result was obtained when HZ were preincubated with hrHex. Additionally, the presence of a Hex-specific substrate during the HZ assay produced the same inhibitory effect. These results suggest the participation of a sperm Hex in the interaction with human ZP in vitro.
ß-hexosaminidase/fertilization/spermatozoa/sperm-zona binding/zona pellucida
Introduction
Fertilization is a complex process comprising several steps. Once the spermatozoon reaches the oocyte, complementary molecules on both gametes mediate their recognition and `primary' binding. The aggregation of several binding sites on the sperm plasma membrane by a multivalent ligand on the oocyte's extracellular matrix, the zona pellucida (ZP), induces the exocytosis of the sperm acrosome, known as the acrosome reaction (AR) (Leyton and Saling, 1989
). Acrosomal enzymes promote ZP penetration, allowing acrosome-reacted sperm to remain bound (`secondary binding') and/or digest the zona, and continue with the fertilization process.
Sperm–ZP interaction is a critical step during fertilization. An inefficient sperm binding or response to ZP account for some cases of male infertility (Overstreet et al., 1980; Oehninger et al., 1989; Lih et al., 1994; Liu and Baker, 1994). Although the whole mechanism of sperm–ZP interaction is far from being determined, the participation of glycoconjugates has been widely supported (Chapman and Barratt, 1996; Tulsiani et al., 1997; Töpfer-Petersen, 1999). The proposed model sustains that carbohydrate-binding proteins on spermatozoa interact with ZP glycoproteins. Although this mechanism has been extensively studied in animal models (Miller and Ax, 1990; Benoff, 1997), there is a scarcity of similar studies concerning human sperm–ZP interaction.
Previous studies from our laboratory proposed the participation of N-acetylglucosamine (GlcNAc) residues in human sperm function (Brandelli et al., 1994, 1995; Miranda et al., 1997). Treatment of human capacitated spermatozoa with bovine serum albumin (BSA)-GlcNAc induced the AR (Brandelli et al., 1994). This induction exhibited several similarities with the ZP-induced AR, for example, the requirement of capacitated spermatozoa, the kinetics, and the transduction mechanisms involved (Brandelli et al., 1994, 1996). These results lead us to assume that BSA-GlcNAc would simulate the ZP and, under this assumption, GlcNAc residues would be involved in human sperm–ZP interaction. The response of sperm samples to BSA-GlcNAc was found to be correlated with the in-vitro fertilization outcome and seemed to be specifically related to sperm–ZP interaction (Brandelli et al., 1995). Recently, we reported that when human capacitated spermatozoa were preincubated with GlcNAc, their ability to bind to ZP was reduced (Miranda et al., 1997), supporting our basic assumption of GlcNAc involvement in this event.
N-acetylglucosaminidase (NAG, E.C. 3.2.1.30) is the glycosidase responsible for hydrolysing the non-reducing terminal GlcNAc residues from ß-glycosidic linkages in several glycoconjugates. In mammals, it is named ß-hexosaminidase (Hex, E.C. 3.2.1.52) since it is also responsible for the hydrolysis of the other acetylated hexose, N-acetylgalactosamine.
The induction of AR by BSA-GlcNAc could be blocked when ligands for NAG/Hex were present during the assay (Brandelli et al., 1994). Moreover, when NAG was included during hemizona assays, it was able to inhibit sperm binding to ZP (unpublished data). These results lead us to propose that the homologous enzyme on human spermatozoa (Hex) could be mediating the GlcNAc-induced AR, and consequently be involved in human sperm–ZP interaction. To further analyse this hypothesis, we explored the effect of human recombinant Hex (hrHex) and a specific Hex substrate on sperm–ZP interaction in vitro.
Materials and methods
Hex B expression construct
The cDNA from pHexB43 (ATCC 57350, previously named pHex1) (O'Dowd et al., 1985; Korneluk et al., 1986) was digested with BamHI and ligated into pSL301 (Invitrogen, San Diego, CA, USA) to get the pSL301-ß (Hou et al., 1996). The EcoRI/NotI fragment containing the cDNA from pSL301-ß was then subcloned into the expression vector pEFNEO (kindly supplied by Dr Anson) (Anson et al., 1992) which has a neomycin (G418) resistance marker, to give pEFNEO-ß. This construct does not include any tag for later purification.
Stable transfection
Chinese hamster ovary (CHO) cells plated on a 100 mm dish at 60% confluency were transfected with pEFNEO-ß by the Calcium Phosphate Transfection System following the manufacturer's recommendations (Gibco BRL, Life Technologies Inc., Gaithersburg, MD, USA). The cells were maintained in selection media containing 0.4 mg/ml G418 (Sigma Chemical Co., St Louis, MO, USA). Non-transfected CHO cells died after 2 weeks and remaining cells were split 1 in 20 into selecting media. After individual clones appeared, colonies were picked and grown in 24-well dishes and Hex activity assayed in the media. This method allowed for the establishment of a clonal cell line with a high level of production of hrHex B. Although no secretion signal was included within the construct, the high level of expression of the protein overloads the intracellular pathways and a secreted active precursor of the enzyme may be found in the culture media.
Cell culture
CHO cells were initially grown on cell culture dishes (Corning, New York, USA) with 
-MEM medium (Gibco) containing 10% fetal bovine serum (Bioser, Buenos Aires, Argentina) (ß-MEM+S) for cell proliferation. After 2 days, cells were harvested and grown in a suspension culture with homogeneous mixing. The cell suspension, containing 100x106 cells, was added to 0.75 g of Citodex 1 microcarriers (Pharmacia, Upsala, Sweden), previously suspended in 500 ml of -MEM+S medium. The suspension was incubated in a spinner bottle and continuously stirred at low speed. After 2 days, the suspension was washed five times with PBS (pH 7.4), and 500 ml of CHO-S-SFM-II medium (GIBCO) was added. Medium was collected every 2 days, and after the third harvesting, cells were discarded.
Purification of hrHex
Whole medium was centrifuged at 450 g for 20 min at 4°C to pellet free cells. Clear supernatant was loaded onto a 5 ml Concanavalin A Sepharose (Pharmacia) column previously equilibrated with 10 mmol/l Tris, 1 mmol/l CaCl2, 1 mmol/l MnCl2, 1 mol/l NaCl, pH 7.5. All the Hex activity remained bound to the column and was eluted with methylmannoside 0.4 mol/l. After dialysing against 10 mmol/l Na2HPO4, pH 7.1, the enzyme activity was loaded onto a 10 ml AffiGel Blue column. After column washing with 10 volumes of the same buffer, Hex activity was eluted with 0.5 mol/l NaCl. This enzyme preparation was concentrated 23-fold with a CX-10 immersible ultrafiltration unit (Millipore, Bedford, MA, USA) and used for functional assays.
Hex activity
Enzyme activity was assayed by measuring the fluorescent signal produced by the cleavage of N-acetylglucosamine residues from 4-methylumbelliferyl-N-acetyl-ß-D-glucosaminide (Leaback and Walker, 1961). In order to determine the optimum pH for hrHex activity, the enzyme was incubated in 0.25 mol/l citrate or phosphate buffers with different pH values among 2.5–6 and 6–8 respectively. Hex activity was measured by adding 100 µl of buffer and 100 µl of substrate solution (4 mg/ml) to the enzyme preparation. The reaction mixture was incubated at 37°C for 5–60 min depending on enzyme concentration in the extract being measured. The analysis of Hex activity at optimum and HZ assay conditions was done using variable substrate concentrations from 0.08 to 10.5 mmol/l and with citrate buffer and Earle's balanced salt solution (EBSS; Gibco) respectively. The amount of fluorescent product, methylumbelliferone (MU), was measured in a Hoeffer TKO 100 fluorimeter (emission at 380 nm and detection at 460 nm). After preliminary studies, 0.25 mol/l sodium citrate buffer, pH 4.5 was used for subsequent measurements of enzyme activity. A calibration curve was plotted using different concentrations of MU, and the enzyme activity determined by interpolation. Activity was expressed in units/mg protein, with one unit being the amount of enzyme necessary to catalyze the production of 1 nmol of MU/min at 37°C. Protein concentration was determined using the method described by Bradford (1976).
Electrophoresis and Western immunoblot
Samples were diluted in lysis buffer [50 mmol/l Tris, 1% sodium dodecyl sulphate (SDS), 5% mercaptoethanol, pH 6.9] and processed by SDS–polyacrylamide gel electrophoresis (PAGE) in 10% acrylamide denaturing slab gels according to Laemmli (1970). After electrophoresis, gels were electroblotted onto nitrocellulose membranes (Sigma). Membranes were blocked with PBS containing 0.1% Tween 20 and 2% low-fat powdered milk (blocking solution). Hexosaminidase was detected by incubation with a polyclonal antibody developed in chicken against placental Hex B, purified as previously described (Mahuran and Lowden, 1979), and diluted 1/1000 in blocking solution. Anti-chicken-peroxidase conjugated antibody (Sigma) diluted 1/1000 in blocking solution was used as secondary antibody. All incubations were carried out at room temperature for 1 h, and all washes were done with PBS containing 0.1% Tween 20. The ECL detection system (Amersham Pharmacia Biotech Inc., NJ, USA) was used to locate immune complexes. Molecular weights were calculated by interpolation using Low Range Prestained SDS–PAGE Standards (Bio-Rad, California, USA).
Semen samples
Semen samples used throughout this study were obtained from men undergoing assisted fertilization due to female factors. All the samples were normal according to World Health Organization standards (average seminal parameters: 138 ± 23 x106 spermatozoa/ml, 49 ± 2% motile cells, 20 ± 1% normal forms and 96 ± 2% IVF). Motile spermatozoa were selected by the swim-up method. Briefly, fractions of 1 ml of liquefied semen were covered with 1.2 ml of Ham's F-10 or EBSS medium (Gibco). After 1 h incubation, 1 ml of the upper phase was taken and centrifuged at 300 g for 5 min. The sperm pellet was resuspended in EBSS medium with 35 mg/ml human serum albumin (HSA) (4x106 spermatozoa/ml). For sperm preincubation with the enzyme, two sperm aliquots were taken: one was incubated with 0.3 units of hrHex (0.17 µg/100 µl) and the other with medium. After a 2 h incubation, the sperm suspension was centrifuged and resuspended in fresh medium and used for hemizona assay. Sperm motility was monitored prior to and after incubations and no difference was observed (80–90% grade 3 motile sperm in all cases). All the assays were done three to eight times on different days and using different samples.
Oocytes
Human oocytes were obtained from women undergoing ovulation induction in order to achieve assisted fertilization. Non-inseminated, surplus oocytes were used for this purpose with the patient's consent. Multiple follicular development was induced by sequential FSH (Metrodine; Serono, Buenos Aires, Argentina) and human menopausal gonadotrophin (HMG, Pergonal; Serono) injections in patients receiving gonadotrophin-releasing hormone (GnRH) analogues as described (Meldrum et al., 1988). Oocytes were incubated in Ham's F-10 for 6–8 h for maturation. Cumulus cells were removed by treatment with 80 IU hyaluronidase for 20 s, followed by washing with PBS. Oocytes in metaphase II were maintained in 0.1 mol/l Tris, 1.5 mol/l(NH4)2SO4, 0.5% Dextran, pH 7 at 5°C until use.
Hemizona assay
The day before the hemizona (HZ) assay, oocytes were washed four times by pipetting into drops of PBS and placed in a Petri dish (Falcon 1006) at 4°C for 12 h. Oocytes were immobilized with holding pipettes (Cook, Queensland, Australia) attached to a micromanipulator (Narishigue, Tokyo, Japan) coupled to an inverted microscope (Nikon, Diaphot) and bisected into two equal halves with a microscalpel. Hemizonae were placed in a 100 µl drop of EBSS with 35 mg/ml HSA. In those experiments where the factor to be tested was present during the assay, buffer was added to the control HZ while the respective factor was added to the other half. The agents tested were: hrHex, 0.017–0.5 µg/100 µl; proteins from control CHO cells, 0.17 µg/100 µl and Hex substrate, 1 mmol/l. As an additional control, the hrHex preparation was boiled for 15 min and then added to one HZ, while buffer was used for the respective half. Active hrHex and buffer were run in parallel. The hemizona index was calculated as the ratio between the number of sperm-bound/HZ in the presence of hrHex to that bound in buffer for each case (native and heat-denatured enzyme) in order to compare the effect of both preparations. To study the effect of ZP treatment before the assay, one HZ was incubated for 4 h with hrHex (0.3 units, 0.17 µg/100 µl) and the control half in medium. Finally, 4x104 spermatozoa were added per drop. After a 4 h incubation, HZ were washed by repeated pipetting in four drops of medium and the number of spermatozoa tightly bound to the outer surface was counted under x400 magnification using Nomarsky optics.
Expression of results and statistical analysis
Enzyme or substrate preparation and HZ assay were done by different researchers in order to obtain a completely blind experiment. Results were plotted as the number of spermatozoa bound to each hemizona. When average values are displayed, they are expressed as the mean ± SE calculated from the individual values for each HZ. Data processing was done on an IBM-compatible computer using the GraphPad InStat program (GraphPad Software, San Diego, CA, USA). For single comparisons, the number of spermatozoa bound to each HZ was statistically analysed using the Wilcoxon signed rank test for paired samples. The data contained in the dose–response curve were analysed using the Bonferroni multiple comparison test. The hemizona indexes obtained for native and heat-denatured Hex were analysed using the Mann–Whitney test for non-paired samples.
Results
Human recombinant Hex B was obtained from a stable transfected CHO cell line and partially purified by sequential affinity chromatography. The final enzyme preparation used in this study was analysed by SDS–PAGE followed by protein staining or immunoblot with a specific polyclonal anti-Hex B antibody (Figure 1). The enzyme preparation used for HZA showed a major component of 70 kD (lane hrHex, Figure 1A), coincident with the single anti-Hex reactive band found in the immunoblot (see lane hrHex in Figure 1B). The same band was also found in Hex-expressing (lane 5C) but not in control CHO cells medium (lane C) and corresponds to the ß-subunit precursor found in extracellular forms of Hex (Hasilik and Neufeld, 1980). As positive control, a protein extract from human placenta was run and a 59 kDa reactive band representing the proß subunit was found (lane P) (Mahuran, 1995). When preimmune chicken IgY was used as primary antibody, no signal was detected (data not shown).
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Effect of hrHex on sperm–ZP binding
In order to analyse the possible participation of Hex in human sperm–ZP interaction, the existence of a Hex effect on the sperm ability to bind to ZP was tested. For that purpose, HZ binding assays were performed in the presence of different amounts of hrHex. In Figure 2
the average number of spermatozoa bound per HZ at different hrHex concentrations is displayed. When 0.3 and 0.9 units of hrHex were included in the medium, the number of sperm bound/HZ was significantly reduced (P < 0.05), while no effect was observed when using less than 0.1 enzyme units.
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) is shown (*P < 0.05).To check the specificity of this effect, and given that the hrHex preparation used was not completely pure, proteins from non-transfected CHO cells were used as control. Culture medium was processed following an identical procedure to that used for the purification of hrHex. The effect of the same amount of total protein to that producing an inhibitory effect in the dose–response curve (1.7 µg/ml equivalent to 0.3 hrHex units) was tested. The presence of proteins secreted by control CHO cells during the HZA did not change the number of spermatozoa bound/HZ (45 ± 10 versus 51 ± 9). As an additional control, hrHex was inactivated by heating before HZA. When the enzyme preparation was heat-denatured, its inhibitory capacity was reverted (hemizona index: 0.4 ± 0.1 versus 0.72 ± 0.04, for native and heated hrHex respectively, P < 0.02).
Preincubation with hrHex
The inhibitory effect produced by hrHex when present during the sperm–HZ binding assay did not reveal which component of the co-incubation mixture (spermatozoa or HZ) was being affected by the enzyme. In order to check the hypothesis that Hex was acting on the ZP, HZ were pretreated with hrHex, washed and incubated with a sperm suspension without treatment under control conditions. As shown in Figure 3, HZ pretreatment with hrHex reduced sperm binding (40 ± 12 versus 21 ± 7 spermatoza/HZ, P < 0.004). On the other hand, when spermatozoa were preincubated with hrHex and then added to non-treated-HZ, no inhibitory effect was found (16 ± 3 versus 21 ± 4, P = 0.1, Figure 4). On the contrary, a slight stimulatory effect was noticed.
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Enzyme activity and pH
The effect of hrHex on human HZ suggests the participation of a GlcNAc residue of the ZP in sperm binding. However, hrHex could be affecting human HZ using two possible mechanisms. The enzyme could bind to terminal GlcNAc on the HZ blocking them or it could also be catalysing the hydrolysis of these residues, eliminating the binding sites for spermatozoa. In order to distinguish between the two possibilities mentioned above, the activity of hrHex at different pH values was first determined. The hydrolysis of a specific substrate, 4-methylumbelliferyl-N-acetyl-ß-D-glucosaminide (SMU), was optimal around pH 4–5 and only 14% of the activity remained at pH 7.5 (Figure 5
).
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Subsequently, we measured the activity of hrHex at optimal pH and under the HZ binding assay conditions. As shown in Figure 6
, the catalytic activity of hrHex in HZ assay buffer was negligible even at saturable substrate concentrations.
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) was measured.Effect of Hex substrate on human sperm–ZP binding
The foregoing results suggest the involvement of Hex in human sperm–ZP interaction in vitro. This leads us to propose that a Hex present in human spermatozoa would participate in their binding to ZP. However, our data involve the addition of exogenous enzyme. In order to analyse if a sperm Hex is involved in ZP binding, we tested the effect of the Hex-specific substrate in the HZA. As shown in Figure 7
, when the assays were carried out in the presence of 1 mmol/l SMU, the spermatozoa's ability to bind to the HZ was significantly reduced (31 ± 7 versus 20 ± 3 spermatozoa/HZ, P < 0.03).
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GlcNAc/Hex and successful sperm–ZP binding
The original description of the HZ assay (Franken et al., 1991
) reported a cut off value for fertile men of 20 spermatozoa/HZ. Previously, we have not detect significant differences between samples displaying values above (high binding) or below (low binding) 20 spermatozoa/HZ when testing different agents in HZA. Therefore, they were used indistinctly. However, during the development of this study some interesting features were found.
When analysing the effect of SMU upon sperm–ZP binding, a larger number of oocytes was required to obtain statistical significance when compared to most of our experiments. When data were segregated according to the number of sperm bound to control HZ, the presence of SMU reduced the amount of spermatozoa/HZ in most of the cases with high binding, leading to a significant inhibition (47 ± 7 versus 27 ± 4, see Figure 8A, P < 0.008). In contrast, in the low binding population, the effect of SMU was erratic, not reaching statistical significance (7 ± 1 versus 10 ± 3, Figure 8B).
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A similar differential effect was found when spermatozoa were preincubated with hrHex. As stated above, the overall result showed a non-significant difference between control and treated samples. However, when the individual results were split, those samples with high binding showed a variable result causing a non-significant effect (28 ± 3 versus 28 ± 7; see Figure 9A
). On the other hand, the low binding samples displayed a greater number of sperm-bound/HZ after hrHex incubation, giving a significant stimulatory effect (7 ± 2 versus 17 ± 5, for control and hrHex-treated spermatozoa respectively, P < 0.02; see Figure 9B).
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Discussion
Hexosaminidase is able to hydrolyse not only terminal GlcNAc but also N-acetylgalactosamine (GalNAc). However, we only referred to GlcNAc residues when proposing the involvement of Hex in sperm interaction with the ZP. This assumption was initially based on our evidence that showed no effect of the GalNAc-neoglycoprotein in the induction of human sperm AR (Brandelli et al., 1994) nor of GalNAc in sperm binding to ZP in vitro (unpublished data). Moreover, the results obtained with NAG, the enzyme with the ability to hydrolyse only GlcNAc residues, further sustain the participation of GlcNAc but not GalNAc in sperm–ZP interaction. This possibility is in accordance with the glycosidic composition reported for human ZP (Lucas et al., 1994; Maymon et al., 1994).
Some glycosidic enzymes have been reported to be actively involved in adhesion events (Rauvala et al., 1981; Shur, 1993). One of these is galactosyltransferase (GTAse), which seems to be involved in different adhesive interactions including fertilization (Cooke and Shur, 1994). Mouse sperm GTAse represents the most actively studied example of an enzyme involved in sperm binding to ZP (Shur and Hall, 1982; Lopez et al., 1985; Macek and Shur, 1988; Miller et al., 1992).
In this report, the presence of hrHex during HZA diminished the number of sperm bound to ZP in vitro. Although the hrHex preparation used was not completely pure, the lack of effect of proteins from control CHO cells supports the specificity of this result. The inhibition could be reproduced when the HZ were preincubated with the enzyme, indicating that the ZP is the target for hrHex. This result supports the involvement of GlcNAc residues of the ZP in sperm binding. The inhibitory effect of hrHex on sperm–ZP binding was reproduced when, instead of the exogenous enzyme, a specific Hex substrate was added to the assay mixture. This result suggests that a sperm-derived Hex would be mediating binding to ZP. Although the presence of a sperm GlcNAc binding lectin cannot be ruled out, they usually require a more complex glycosidic environment to achieve recognition and binding. Unfortunately, the anti-Hex antibody only recognizes the denatured enzyme and was not useful to analyse its possible effect in the HZA.
Glycosidases usually display their catalytic activity at acidic pH and the substrate hydrolysis at neutral pH is considerably lower than their optimum. Human Hex from different tissues has only 10–25% of its maximal activity at neutral pH (Srivastava et al., 1974; Marinkovic and Marinkovic, 1977; Miranda et al., 1995). Accordingly, we found that the hydrolysis of the Hex substrate is negligible under the HZ assay conditions. Some explanations for this apparent contradiction (i.e. the effect obtained in the absence of catalytic activity) can be outlined. First, Hex ligand binding activity can be displayed over a broader pH range (Geiger et al., 1974), suggesting that Hex could be acting as a binding molecule rather than an enzyme. This was also reported for other enzymes proposed to be involved in sperm–ZP interaction (Benau and Storey, 1988; Macek and Shur, 1988; Cornwall et al., 1991). However, the actual substrate binding ability of Hex at physiological pH could not be directly measured in this study. As a second explanation, the existence of specific neutral glycosidases in mammalian spermatozoa have been described. Galactosidase changes its characteristic optimum acidic pH against artificial substrates to near neutrality when `natural' substrates are used (Skudlarek et al., 1993). Additionally, a neutral sperm surface mannosidase has been detected in human and rat spermatozoa besides the acrosomal acidic enzyme (Tulsiani et al., 1989, 1990).
Concerning human sperm Hex, it has been reported to be present mainly as an acrosomal enzyme, but a certain portion of activity was found to be associated with a crude membrane fraction (Tulsiani et al., 1990). It will be interesting to study the expression of human sperm Hex activity against an oligosaccharide substrate.
It is worthy to note that, although Hex substrate could not be hydrolysed under the HZA conditions, it was able to inhibit sperm binding to ZP, suggesting that at least this substrate is able to compete with ZP for some binding site within the enzyme.
Other enzymes have been proposed to be involved in mammalian sperm–ZP-interaction given their localization and/or fate within the male reproductive tract or spermatozoa (Durr et al., 1977; Saling, 1981; Jones et al., 1988; Ram et al., 1989; Tulsiani et al., 1989, 1990; Cornwall et al., 1991; Hunnicutt et al., 1996; Barksdale et al., 1997). Concerning Hex or N-acetylglucosaminidase, it has been reported to be necessary for the penetration of the oocyte's investments during rabbit and mouse fertilization (Farooqui and Srivastava, 1980; Miller et al., 1993). It has also been proposed as the sperm receptor for the oocyte extracellular matrix in ascidians (Godknecht and Honegger, 1991, 1995).
A complete blockade of sperm–ZP binding could not be achieved even using high hrHex concentration, suggesting the existence of supplementary binding sites. A multiple ligand–receptor system has been previously suggested for sperm–ZP binding (Benau and Storey, 1988; Chapman and Barratt, 1996).
Concerning the differential effect produced by the Hex substrate on samples with control values above or below 20 spermatozoa/HZ, although we cannot make definitive conclusions given the limited number of samples used in this study, some interpretations can be outlined. The result obtained in the binding assay depends not only on the sperm ability to bind to the ZP but also on the quality of ZP to bind spermatozoa. This would mean, concerning this work, the presence of Hex on spermatozoa and GlcNAc residues on the ZP. When the complex `sperm–HZ' had a high binding performance (at least 20 spermatozoa/HZ), the Hex substrate reduced sperm binding, suggesting that this successful event is related to Hex/GlcNAc. On the other hand, for low binding samples (less than 20 spermatozoa/HZ), the effect of the Hex substrate was variable. In these cases, sperm–ZP binding may be independent of Hex/GlcNAc, meaning a failure or absence of these putative binding sites in either gamete. In this regard, when spermatozoa were preincubated with hrHex and those samples with low binding were analysed separately, a significant increase in binding to HZ was found. This result leads us to speculate that during preincubation hrHex could associate with the spermatozoa and improve their binding to ZP. A similar result was reported when a protein mixture obtained from spermatozoa from fertile donors increased binding to ZP when added to other samples, although in that case no specific protein was identified (Jean et al., 1995).
In summary, in this work we reported that human sperm–ZP binding in vitro is significantly reduced when a specific Hex substrate or hrHex was added to the binding mixture. Our present results do not allow us to differentiate an effect on primary binding, secondary binding or penetration of ZP. This point is currently being analysed in our laboratory. However, together with our previous studies (Brandelli et al., 1994, 1995; Miranda et al., 1997), these results further support the relevance of GlcNAc residues in human sperm–ZP interaction in vitro and suggest the participation of Hex in this event.
Acknowledgments
The authors would like to thank Dr George Vavougios for his technical help in isolating the hrHex B high-producing clone. We also thank Patricia Delcourt and Amy Leung for their technical assistance. This work was supported by the World Health Organization (Career Development Fellowship to P.V.M.), Research Institute of The Hospital for Sick Children (Visitor Scientific Award to P.V.M.), The Rockefeller Foundation and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) of Argentina.
Notes
4 To whom correspondence should be addressed at: Instituto de Biología y Medicina Experimental, Vuelta de Obligado 2490, (1428) Buenos Aires, Argentina. E-mail: pmiranda{at}dna.uba.ar 
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Submitted on January 18, 2000; accepted on May 24, 2000.
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