Molecular Human Reproduction, Vol. 7, No. 7, 633-640,
July 2001
© 2001 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
Anti-SLIP1-reactive proteins exist on human spermatozoa and are involved in zona pellucida binding
1 Hormones/Growth/Development Research Group, Loeb Health Research Institute, Ottawa Hospital– Civic Campus, 2 Human In Vitro Fertilization Program, Division of Reproductive Medicine, Department of Obstetrics and Gynecology, Faculty of Medicine, University of Ottawa, 1053 Carling Ave., Ottawa, Ontario K1Y 4E9 and 3 Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Rd., Ottawa, Ontario K1H 8M5, Canada
Abstract
Sulpholipid immobilizing protein 1 (SLIP1) is an evolutionarily conserved 68 kDa plasma membrane protein, present selectively in germ cells. We have previously shown that mouse sperm SLIP1 is involved in sperm–zona pellucida (ZP) binding. In this report, we extended our study to the human system. Immunoblotting demonstrated that anti-SLIP1-reactive proteins (mol. wt 68 and 48 kDa) could be extracted from human spermatozoa by an ATP-containing solution, a result that is consistent with observations in other species. Direct immunofluorescence, using Cy3-conjugated anti-SLIP1 IgG, revealed SLIP1 staining over the acrosomal region, with higher intensity at the posterior area. Using the human sperm–ZP binding assay, we demonstrated that pretreatment of human spermatozoa from three donors with anti-SLIP1 IgG revealed lower numbers of zona-bound spermatozoa, as compared to the corresponding control spermatozoa treated with normal rabbit serum IgG. This decrease in zona pellucida binding was not from an antibody-induced decline in sperm motility or an increase in the premature acrosome reaction. The results strongly suggest that anti-SLIP-reactive proteins on human spermatozoa play an important role in ZP binding.
sperm oocyte interaction/sperm surface protein/sulphoglycolipid binding protein/zona pellucida
Introduction
Mammalian sperm–oocyte interaction that culminates in fertilization occurs in sequential steps, with sperm–zona pellucida (ZP) binding being the initial event. The facts that mammalian ZP consists of only a few glycoproteins and that ovarian ZP can be isolated in quantity have facilitated studies on sperm–ZP binding, especially in the mouse system. Mouse and human ZP3 and ZP2 glycoproteins have been shown to be the primary receptor of acrosome-intact spermatozoa and the secondary receptor of acrosome-reacted spermatozoa respectively (Bleil et al., 1988
; Bleil and Wassarman, 1988; Wassarman, 1988; Whitmarsh et al., 1996; Harris et al., 1999; Tsubamoto et al., 1999). Unlike this simple situation on the ZP side, numerous ZP-associated receptors on spermatozoa have been described. In mice, these include sperm galactosyltransferase (Shur and Hall, 1982a, b; Lopez et al., 1985; Miller et al., 1992b), zona receptor kinase (or p95) (Leyton and Saling, 1989a; Burks et al., 1995), sp56 (Cheng et al., 1994; Bookbinder et al., 1995), sperm trypsin inhibitor sensitive site (Saling, 1981; Benau and Storey, 1987; Boettger et al., 1989), and sulpholipidimmobilizing protein 1 (SLIP1) (Tanphaichitr et al., 1992, 1993).
Whereas sp56 appears to be rodent specific and the presence and significance of galactosyltransferase on human sperm plasma membrane is still inconclusive (Tulsiani et al., 1990; Huszar et al., 1997), the cDNA coding sequence and the derived peptide sequence of human tyrosine kinase (zona receptor kinase) have been reported (Burks et al., 1995). Phosphorylation of a 95 kDa protein on the human sperm surface was also shown to increase after sperm exposure to recombinant human ZP3 (Brewis et al., 1998). FA-1, a human sperm surface protein with apparent mol. wt of 23 and 51 kDa, has been demonstrated to be significant in human sperm–ZP binding (Naz et al., 1984, 1992; Naz and Ahmad, 1994; Kadam et al., 1995), and it is the human ZP3 protein to which FA-1 binds specifically (Naz and Ahmad, 1994). Other human sperm surface proteins that are involved in ZP binding include P34H (Boue et al., 1994, 1996), mannose lectin (Chen et al., 1995), core region peptides of an acrosomal serine protease inhibitor (Moore et al., 1993) and a YLP12-protein (72 kDa) (Naz et al., 2000). As postulated in other species, all of the sperm surface proteins that have been proposed to associate with ZP may act simultaneously and synergistically and/or sequentially (Wassarman, 1999).
SLIP1 (mol. wt ~68 kDa) was first isolated from a rat testis homogenate by sulphogalactosylglycerolipid (SGG) affinity column chromatography (Lingwood, 1985). Subsequently, it was shown that a crude extract of SLIP1 can be prepared by treating intact testicular cells or gametes (Law et al., 1988; Tanphaichitr et al., 1993; Ahnonkitpanit et al., 1999) with a solution containing sucrose and low concentrations of ATP (1 mmol/l) and EDTA (1 mmol/l) (AES), which has been shown previously to remove peripheral plasma membrane proteins from intact cells (Carter and Hakomori, 1977). Lingwood and his associates have further demonstrated that rat testis SLIP1 can be purified from its AES crude extract to a single 68 kDa band on sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) (Lingwood and Nutikka, 1991). Using this purified SLIP1, we have shown that the protein can bind to the oocyte ZP (Tanphaichitr et al., 1993). Supporting the significance of SLIP1 in ZP binding are the findings that SLIP1 was immunolocalized to the rat and mouse sperm head (Lingwood, 1986; Tanphaichitr et al., 1993), the site of ZP binding (Yanagimachi, 1994). Furthermore, we have shown that pretreatment of mouse spermatozoa with anti-SLIP1 immunoglobulin (Ig) G/Fab results in decreased sperm–ZP binding in vitro and fertilization in vivo (Tanphaichitr et al., 1992, 1993). Immunoblotting revealed that SLIP1 is present primarily in germ cells (both male and female) and to a lesser extent in brain (Law et al., 1988), a result that corroborates SGG tissue specificity (Kornblatt et al., 1972; Kornblatt, 1979; Ishizuka, 1997; Murray and Narasimhan, 1990). In addition, SLIP1 is conserved, existing in the testis of animals across the evolutionary scale, i.e. from fish to birds to mammals (Law et al., 1988).
Further studies in our laboratory revealed that rat testis SLIP1, produced from Lingwood's laboratory, consisted of a number of components including those that have the ability to bind to SGG and ZP (Lingwood, 1985; Tanphaichitr et al., 1993, 1998), as well as albumin and a heat shock protein 70 (HSP70) (Boulanger et al., 1995; Tanphaichitr et al., 1998; our unpublished results). However, the studies have indicated that neither albumin nor HSP70 is involved in sperm–ZP binding (Tanphaichitr et al., 1999). Recently, we have described the purification from pig sperm AES extract (SLIP1 crude extract) of P68, which reacts with anti-SLIP1, but not anti-rat serum albumin (antiRSA) or an antibody against HSP70. Like SLIP1, P68 binds SGG, and when it is included in an excess amount (~10 nmol/l) in mouse gamete co-incubates, it reduces the number of spermatozoa bound to the ZP. And as expected, P68 binds to isolated ZP of various mammalian species, including mice, rats, cats, dogs, pigs and humans (Tanphaichitr et al., 1998). In light of the possibility of using SLIP1/P68 as a non-hormonal contraceptive, we performed studies as reported herein to investigate whether SLIP1 on the human sperm surface is involved in binding to the ZP.
Materials and methods
Antibodies
Anti-SLIP1, a rabbit polyclonal IgG antibody generated against rat testis SLIP1 and purified by SGG affinity chromatography (Lingwood, 1985), and a rabbit polyclonal antiserum (IgG) directed against the recombinant Mycoplasma hyopneumonia heat shock protein 74.5 (HSP74.5) (Boulanger et al., 1995) were provided by Dr C.Lingwood (Hospital for Sick Children, University of Toronto, Toronto, ON, Canada). A Cappel normal rabbit serum (NRS) and rabbit polyclonal antiserum (IgG) generated against rat serum albumin (RSA), anti-RSA, were purchased from Organon Teknika Inc. (Scarborough, ON, Canada). A goat anti-rabbit immunoglobulin conjugated with horseradish peroxidase was purchased from Southern Biotechnology Associated Inc. (Birmingham, AL, USA) and Jackson Immunoresearch Laboratory Inc. (West Grove, PA, USA) respectively. NRS IgG was obtained from Sigma Chemical Co. (St Louis, MO, USA). Previously described procedures were used to prepare IgG fraction from anti-SLIP1 antiserum (Tanphaichitr et al., 1993), to formylate anti-SLIP1 IgG to reduce non-specific binding to spermatozoa (Lingwood, 1985), to generate affinity-purified anti-SLIP1 IgG (Ahnonkitpanit et al., 1999), and to conjugate a fluorochrome, Cy3 (excitation wavelength: 550 nm, emission wavelength: 565 nm), to anti-SLIP1 IgG and NRS IgG (Ahnonkitpanit et al., 1999).
Human sperm preparation
With approval from the Human Ethics Committee, Loeb Health Research Institute, Ottawa Hospital, freshly ejaculated human semen samples were obtained from fertile donors with normal semen parameters (volume 2–6 ml, density >20x106 spermatozoa/ml, motility >50%, normal morphology >30%, World Health Organization criteria, 1999). Motile spermatozoa were prepared from these semen samples via discontinuous Percoll gradient centrifugation (Tanphaichitr et al., 1988). The sperm pellet, containing mainly motile spermatozoa, was washed once (500 g, 25°C, 5 min) in HTF (101 mmol/l NaCl, 4.69 mmol/l KCl, 0.2 mmol/l MgSO4, 0.37 mmol/l K2HPO4, 2.04 mmol/l CaCl2, 2.78 mmol/l glucose, 0.33 mmol/l sodium pyruvate, 21.4 mmol/l sodium lactate, 25 mmol/l NaHCO3, penicillin G 100 units/ml, streptomycin sulphate 50 mg/ml, and phenol red 0.001 g/ml) resuspended to 20x106 spermatozoa/ml in HTF supplemented with 0.5% bovine serum albumin (BSA) (HTF–BSA) and incubated under 5% CO2 at 37°C for 4 h until used for the acrosome reaction test and sperm–ZP binding assay. SLIP1 extraction and direct immunofluorescence were also performed on these spermatozoa following their washing in HTF without protein supplementation.
Extraction of SLIP1 from human spermatozoa
SLIP1 was extracted from human spermatozoa with AES solution (1 mmol/l ATP, 1 mmol/l EDTA, 340 mmol/l sucrose, 0.2 mmol/l N-a-p-tosyl-L-lysine chloromethylketone hydrochloride in 20 mmol/l Tris–HCl, pH 7.6), following the method described earlier for rat testis cells and mouse and pig spermatozoa, but omitting the ultracentrifugation step (Law et al., 1988; Tanphaichitr et al., 1993, 1998). Briefly, the sperm pellet from Percoll gradient centrifugation, prewashed three times in phosphate-buffered saline (PBS: 137 mmol/l NaCl, 2.7 mmol/l KCl, 4.3 mmol/l Na2HPO4, 1.4 mmol/l KH2PO4) was extracted twice with 9 volumes of AES solution with continuous agitation (4°C, 1 h). Spermatozoa were still viable and acrosome intact under these conditions, as assessed by the hypo-osmotic test and specific lectin staining (see below). The sperm suspension was centrifuged (500 g, 4°C, 25 min) after each treatment, and the supernatants were pooled and dialysed extensively (4°C, overnight) against Milli-Q water (Millipore Canada, Nepean, ON, Canada) and lyophilized.
SDS–PAGE and immunoblotting
SDS–PAGE (10% polyacrylamide, 0.75 mm thick) (Laemmli, 1970) followed by immunoblotting (Towbin and Gordon, 1984) of sperm AES extracts was performed, as previously described (Tanphaichitr et al., 1998). The nitrocellulose blot was blocked for at least 1 h with 5% fat-free milk in Tris-buffered saline (20 mmol/l Tris–HCl, 137 mmol/l NaCl, pH 7.6) to reduce non-specific binding. Immunoblotting was performed using formylated anti-SLIP1 antiserum (1:500 dilution), anti-RSA (1:1000 dilution), anti-HSP74.5 (1:1000 dilution) and NRS antiserum (1:500 dilution), all of which were diluted in the blocking medium, and the secondary antibody, goat anti-rabbit immunoglobulin conjugated with horseradish peroxidase (1:3000 dilution). Antigen–antibody binding was detected by chemiluminescence using an ECL kit (Amersham Canada Ltd., Oakville, ON, Canada).
Immunofluorescence of spermatozoa
Approximately 200 000 spermatozoa were washed twice in PBS (500 g, 25°C, 5 min). The sperm pellet was treated with 100 µg/ml Cy3-anti-SLIP1 IgG in PBS or in PBS–1% BSA or with affinity-purified Cy3-anti-SLIP1 IgG (5% CO2, 37°C, 30 min), washed twice in PBS (500 g, 25°C, 5 min), mounted onto a glass slide and viewed under a Zeiss IM35 epifluorescence microscope. Spermatozoa exposed to Cy3-NRS IgG in place of Cy3-anti-SLIP1 IgG served as negative controls. The staining pattern of at least 200 spermatozoa was observed for each sample.
Separate aliquots of human spermatozoa prepared as described above were also incubated with anti-RSA antiserum (1:40 dilution in PBS), anti-HSP74.5 (1:30 dilution in PBS) and NRS IgG (100 µg/ml) under the same conditions as used for Cy3-anti-SLIP1 IgG. After successive washes in PBS, the sperm samples were incubated (30 min, 25°C) with Cy3-conjugated goat anti-rabbit IgG (5 µg/ml). The spermatozoa were then washed to remove excess unbound fluorescent secondary antibody and viewed under the Zeiss epifluorescence microscope.
Human sperm–zona pellucida binding
The human sperm–ZP binding was performed following a published method (Liu et al., 1988). The human oocytes used in this assay were those that had failed fertilization in the Human In Vitro Fertilization Program, Ottawa Hospital – Civic Campus, and were donated by the patients for research purposes, with the approval of the Research Ethics Board, Ottawa Hospital. These oocytes were stored in a high-salt buffer containing 2 mol/l ammonium sulphate, 0.5% dextran and 40 mmol/l HEPES, pH 7.4 (Moore et al., 1993) until use. On the experimental day, the salt-stored oocytes were washed five times and incubated (5% CO2, 37°C, 1 h for each wash) in 1 ml HTF–BSA before co-incubation with spermatozoa, prepared as described below.
Spermatozoa resuspended in HTF–BSA at 10x106 motile spermatozoa/ml were divided into two fractions, one being labelled with fluorescein isothiocyanate (FITC; Sigma Chemical Co.) and the other with tetramethylrhodamine isothiocyanate (TRITC) (Sigma Chemical Co.) as described previously (Liu et al., 1988). The FITC-labelled spermatozoa and the TRITC-labelled spermatozoa were treated (5% CO2, 37°C, 30 min) with 100 µg/ml anti-SLIP1 IgG and NRS IgG (negative control) respectively. In order to ensure that the results obtained from this binding study were not due to the fluorochrome effects, the two fluorochromes were switched between the two IgG-treated sperm fractions in alternate experiments (i.e., the anti-SLIP1 IgG-treated fraction was labelled with TRITC and the NRS-IgG-treated fraction with FITC). In another set of experiments, FITC-labelled spermatozoa from donor 1 were also treated with affinity-purified anti-SLIP1 IgG with 200 µg/ml non-affinity-purified anti-SLIP1 IgG (prepared in parallel with the affinity-purified antibody). Following successive washing and resuspension in HTF–BSA, equal volumes of both sperm fractions were mixed and ~200 000 spermatozoa from the mixture were incubated (5% CO2, 37°C, 4 h) with each zona-intact oocyte in 1 ml HTF–BSA. Sperm–oocyte complexes were washed three times in fresh HTF–BSA through a drawn Pasteur pipette of a 200 µm bore size to eliminate loosely bound spermatozoa. Three to five sperm–oocyte complexes were then transferred to a slide, flattened gently by a coverslip (supported by a dot of Nivea cream at each corner), and evaluated for the number of spermatozoa bound to each ZP under a Zeiss IM35 epifluorescence microscope. FITC-labelled spermatozoa and TRITC-labelled spermatozoa bound to the same zona were counted using the specific filters for fluorescein and rhodamine. Since numerous spermatozoa bound to each ZP, only those that were in the same plane as the oocyte diameter were counted.
Assessment of the sperm acrosome status after anti-SLIP1 IgG treatment
Capacitated human spermatozoa treated with anti-SLIP1 IgG or NRS IgG, as described in the gamete binding section, were assessed for their viability and acrosome integrity following a published method (Aitken et al., 1993). Spermatozoa were defined as viable by their swollen tails following exposure to a hypo-osmotic solution (Jeyendran et al., 1984). The acrosome intactness was demonstrated by complete staining of the acrosome after the spermatozoa were fixed in ice-cold methanol and treated with FITC-conjugated Pisum sativum agglutinin (PSA; Sigma Chemical Co.) (Cross et al., 1986). In contrast, acrosome-reacted spermatozoa showed FITC-PSA staining in the equatorial segment only, whereas acrosome-reacting spermatozoa displayed partial or patchy staining of the apical segment of the acrosome along with complete staining of the equatorial segment.
To test the acrosome reaction inducibility, spermatozoa pretreated or untreated with anti-SLIP1 IgG were incubated in HTF–BSA, and then exposed to 2.5 µmol/l A23187 [Calbiochem-Novabiochem Co., La Jolla, CA, dissolved in 0.025% dimethylsulphoxide (DMSO)], or to 0.025% DMSO alone. After the treatment, the sperm samples were subjected to the hypo-osmotic test and FITC–PSA staining (Aitken et al., 1993).
Approximately 200 viable spermatozoa were assessed for acrosomal integrity in each treatment.
Assessment of sperm motility after anti-SLIP1 IgG treatment
Motility of Percoll gradient centrifuged spermatozoa, treated with anti-SLIP1 IgG or NRS IgG (see the gamete binding section), as well as that of untreated spermatozoa, was assessed using an automated HTM-S Motility Analyzer, version 7 (Hamilton-Thorn Research Inc., Danvers, MA, USA). The analyser was set to analyse motility parameters in 16 frames at 30 frames/s for each field. The motility parameters analysed included average path velocity (VAP, µm/s), mean tract speed (VCL, µm/s), progressive velocity (VSL, µm/s), mean linearity (LIN, %), mean straightness (STR, %), amplitude of lateral head displacement (ALH, µm) and mean beat frequency (BCF, Hz).
Statistical analysis
Freidman 
2r-test was used to compare the percentages of the acrosome reaction among the anti-SLIP1 IgG-treated sample and the control samples (NRS-IgG, dimethylsulphoxice and A23187 treated spermatozoa). The same test was also employed to compare the motility parameters of spermatozoa after anti-SLIP1 IgG, NRS IgG treatment and no treatment. A paired t-test was performed to compare the effect of sperm pretreatment with anti-SLIP1 IgG or NRS IgG on human sperm–ZP binding within the same experiment. Two-way analysis of variance (ANOVA) was used for the same comparison among several experiments. P < 0.05 was considered to be statistically significant.
Results
AES is able to extract SLIP1 from mouse and boar spermatozoa and testis cells from various mammals and vertebrates (Lingwood, 1985; Law et al., 1988; Tanphaichitr et al., 1993, 1998). Using anti-SLIP1, immunoblotting of the sperm AES extract from three donors shows that SLIP1 (mol. wt ~68 kDa, reactive with anti-SLIP1) was present in human spermatozoa (Figure 1). Donor 3 appeared to possess the highest amount of intact SLIP1, as compared to donors 1 and 2. In contrast, an additional anti-SLIP1-reactive protein band (mol. wt ~48 kDa), possibly a processed product or a precursor of SLIP1, was present in both donors 1 and 2. It was noted that two anti-SLIP1-reactive bands with faster electrophoretic mobility (mol. wt ~55 and 48 kDa) were also present in the boar sperm AES extract (Figure 1). Since formylated anti-SLIP1 still cross-reacts with albumin, also of 68 kDa mol. wt, when present at >1 µg on nitrocellulose, and since albumin could be coating the ejaculated spermatozoa (Tanphaichitr et al., 1998), the nitrocellulose blot of these human AES extracts was reprobed with anti-RSA. The results indicated the absence of albumin in these AES extracts obtained from the human sperm samples, which had been washed extensively in PBS (data not shown). Reprobing was also performed with anti-HSP74.5 for the similar reason that cross-reactivity of anti-SLIP1 to rat testis HSP70 has previously been described (Boulanger et al., 1995). Again, no anti-HSP74.5-reactive bands were observed on the blot of human sperm AES extracts (data not shown).
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Direct immunofluorescence of live human spermatozoa with Cy3-anti-SLIP1 IgG diluted in either PBS or PBS–BSA (to block any cross-reactivity of anti-SLIP1 with albumin on the sperm surface) revealed that >95% of spermatozoa exhibited fluorescent staining over the acrosome with the highest intensity at the posterior region of the acrosome (Figure 2
). In contrast, another aliquot of these live human spermatozoa, which were incubated with Cy3-NRS IgG under the same conditions as those used for Cy3-anti-SLIP1 IgG, did not show any fluorescent staining (Figure 2). The results therefore indicated that the fluorescent staining observed specifically represented anti-SLIP1-reactive protein(s). Indirect immunoflourescence studies using anti-RSA or anti-HSP74.5 and Cy3-secondary antibody revealed no fluorescent staining on live human spermatozoa (data not shown), indicating the absence of an albumin-like protein and a HSP70 on the sperm surface.
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Using the sperm–ZP binding assay (Liu et al., 1988
), we demonstrated the role of human sperm SLIP1 in this binding event. Since the number of human oocytes and the amount of anti-SLIP1 available to us were limited, spermatozoa from the same three donors used for SDS–PAGE/immunoblotting analysis were pretreated with only one concentration of anti-SLIP1 IgG, i.e. 100 µg/ml (experiments I, III and IV, Table I), which exhibited maximum inhibition of mouse sperm–ZP binding (Tanphaichitr et al., 1993). Although the number of control spermatozoa (treated with 100 µg/ml NRS IgG in place of anti-SLIP1 IgG) bound to each ZP varied significantly from one experiment to another (ranging from 15–30 spermatozoa per ZP, P < 0.0001, two-way ANOVA), the anti-SLIP1 IgG-treated spermatozoa consistently bound to each ZP at a lower number than their corresponding controls in all three experiments (P < 0.0001, paired t-test; Table I). The degree of sperm–ZP inhibition caused by sperm pretreatment with 100 µg/ml anti-SLIP1 IgG ranged from 49–87%. In order to further substantiate that the blocking of sperm binding to the ZP by anti-SLIP1 was due to the specific masking of the ZP binding component of SLIP1, anti-SLIP1 IgG affinity-purified against pig sperm P68 (which has been shown to have direct ZP binding ability and to be devoid of albumin and HSP7) (Tanphaichitr et al., 1998) was also used for treatment of spermatozoa from donor 1. Results listed in Table I indicated that affinity-purified anti-SLIP1 IgG (experiment IIb) inhibited human sperm–ZP binding to the same degree as the non-affinity-purified anti-SLIP1 IgG (experiment IIa) that was used for affinity purification. All of these results suggested that the specificity of anti-SLIP1-induced inhibition of gamete binding was due to the reaction of the antibody proteins on the sperm surface that participated in human ZP binding.
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The decrease in human sperm–ZP binding following sperm pretreatment with anti-SLIP1 IgG was not due to the inhibitory effects of the antibody on sperm motility nor to an increase in the acrosome reaction induced by immuno-aggregation of SLIP1. Results presented in Table II
indicated that all motility parameters (i.e. % motility, VAP, VCL, VSL, % LIN, % STR, ALH and BCF) of spermatozoa treated with anti-SLIP1 IgG and NRS IgG were not significantly different from each other or from untreated spermatozoa incubated in parallel in medium (Table II). Although there were decreasing trends of some parameters (i.e. VCL and ALH) and increasing trends of % LIN and BCF after the antibody treatment, these changes were not significant among the three groups. The results indicated that the decrease in human sperm–ZP binding following pre-exposure of spermatozoa to 100 µg/ml anti-SLIP1 IgG (Table I
) was not attributed to a decline in sperm motility. Spermatozoa from donor 1 treated with 200 µg/ml anti-SLIP1 IgG also showed the same motility parameters as the control spermatozoa exposed to 200 µg/ml NRS IgG (data not shown). In addition, pretreatment of human spermatozoa with 100 µg/ml anti-SLIP1 IgG did not induce the spontaneous acrosome reaction above the values observed in the negative control (i.e. spermatozoa treated with 100 µg/ml NRS IgG). More than 90% of anti-SLIP1-treated human spermatozoa remained acrosome intact and were expected to bind to the human ZP to the same degree as the control (untreated or NRS IgG-treated sperm). On the other hand, spermatozoa from these donors retained the ability to undergo the acrosome reaction following treatment with 2.5 µmol/l A23187 at ~50% of the total population, as described previously (Aitken et al., 1993), whether they were untreated (data not shown) or pretreated with anti-SLIP1 IgG (Figure 3). All of these results (Tables I and II and Figure 3) taken together strongly suggested that human sperm SLIP1 was involved in ZP binding.
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Discussion
The presence of SLIP1 (68 kDa) as shown by its reactivity with anti-SLIP1 in human spermatozoa confirms previous results describing the evolutionary conservation of SLIP1 in male germ cells, as it exists in various vertebrate classes ranging from mammals to birds and fish (Law et al., 1988). The slight possibility that this 68 kDa anti-SLIP1-reactive band was albumin or HSP70 (due to the cross-reactivity of anti-SLIP1 with these two proteins when present at a microgram amount on nitrocellulose) was completely ruled out by the absence of an anti-RSA or anti-HSP74.5-reactive band on the Western blot of these human AES extracts. The presence of additional anti-SLIP1 bands (48 kDa) may plausibly be due to these bands being proteolytic products or precursors of SLIP1 (68 kDa). This 48 kDa band, along with additional bands, was also present in the boar sperm AES extract, indicating a common property of anti-SLIP1-reactive proteins in the two species. Nonetheless, it cannot be ruled out that these additional anti-SLIP1-reactive bands (including the 48 kDa one) may be proteins, which possess different peptide sequences but yet contain some common epitopes with SLIP1.
Like SLIP1 from male germ cells of other species (Law et al., 1988), human sperm anti-SLIP1-reactive proteins were extractable with AES (Figure 1). AES has been used previously to extract peripheral plasma membrane proteins (Carter and Hakomori, 1977). Therefore, our results indicate that anti-SLIP1-reactive proteins originated from the sperm surface, and this was confirmed by our immunofluorescence studies, which revealed the presence of fluorescent staining of anti-SLIP1-reactive protein(s) on the surface of the live human sperm heads. Since Cy3-anti-SLIP1 affinity-purified against P68 (which has ZP binding ability and is devoid of an albumin-like protein and a HSP70) was also used in this fluorescent study, the fluorescent pattern obtained was representative of the antigen that was expected to have ZP affinity. The absence of an albumin-like protein and a HSP70 on the surface of human spermatozoa was also confirmed by the lack of fluorescent staining on the surface of human spermatozoa that were exposed to anti-RSA and anti-HSP74.5 with subsequent incubation with a fluorescent secondary antibody. Although a HSP70 or albumin or an albumin-like protein (e.g., testibumin) present in the seminal plasma and epididymal fluid (Cheng and Bardin, 1986; Miller et al., 1992a) is expected to be absorbed onto the sperm surface, it would have been removed by the Percoll gradient centrifugation employed in our human sperm preparation.
Localization of anti-SLIP1-reactive antigens to only the acrosomal region of the human sperm head surface (Figure 2) supported our argument that human sperm SLIP1, like mouse sperm SLIP1 (Tanphaichitr et al., 1993, 1992, 1998), is involved in ZP binding. The higher intensity of immunofluorescent SLIP1 staining in the posterior area of the human sperm acrosome (around the equatorial segment) agreed with the concept that binding of the ZP via its multi-oligosaccharides intitiates at the equatorial segment or the postacrosome of the mammalian sperm head (Chen and Cardullo, 1994; Kerr et al., 2000). Presumably, following this initial ZP binding, aggregation and relocalization of SLIP1 and other ZP receptors (Leyton and Saling, 1989b) to the sperm head anterior region take place (Moore et al., 1987; Aarons et al., 1991; Macek et al., 1991; McKinnon et al., 1991; Wolf et al., 1992; Moase et al., 1997). For a number of these ZP binding proteins, this aggregation and relocalization, as mimically induced by their bi/multivalent antibodies, leads to sperm exocytosis (the acrosome reaction), implying that these proteins may regulate sperm signalling events (Aarons et al., 1991; Macek et al., 1991; McKinnon et al., 1991). However, this was not the case for either human sperm SLIP1 or mouse sperm SLIP1 (Tanphaichitr et al., 1993; Moase et al., 1997) since capacitated spermatozoa of these two species remained acrosome intact following treatment with anti-SLIP1 IgG, suggesting that SLIP1 may be involved only in ZP affinity and not the downstream sperm signal transduction.
Participation of anti-SLIP1-reactive proteins on the human sperm surface in ZP binding was demonstrated by the in-vitro human sperm–human ZP binding assay, using sperm pretreated with anti-SLIP1 IgG. Consistently, decreased numbers of spermatozoa bound per ZP were observed with spermatozoa from the three donors that were exposed to 100 µg/ml anti-SLIP1 IgG, although the degree of the decreases appeared to be varied among the three donors. The reasons for this variation are still unclear. Besides the possibility that the quality of the failed fertilization oocytes used for each donor may be varied, the amount of anti-SLIP1-reactive proteins may also differ from one donor to another. Based on the proposal that the redundant ZP receptors on the sperm surface may serve as back-ups/surrogates for one another (Wassarman, 1999), those sperm samples having lower amounts of SLIP1 may be compensated by other ZP binding proteins. These spermatozoa would show less inhibition in ZP binding when treated with anti-SLIP1 IgG. In donor 1, we also demonstrated that inhibition of human sperm–ZP binding induced by anti-SLIP1 IgG occurred in a concentration-dependent manner. And since the inhibition of human sperm–ZP binding occurred at the same level when either anti-SLIP1 IgG affinity-purified against P68 (a highly purified form of pig sperm SLIP1 that possesses ZP/SGG binding ability and is devoid of contaminants including albumin and HSP70) or non-affinity-purified anti-SLIP1 IgG was used, the results indicated that the inhibition observed was due to specific masking of the P68 immunologically related protein(s) on the human sperm surface. This should include mainly the ~68 kDa protein (SLIP1) present in the sperm AES extracts of all three donors, although the ~48 kDa protein in donors 1 and 2 should not be excluded. Since there was no increase in spontaneous acrosome reactions (Figure 3) and no changes in sperm motility parameters (Table I), the inhibition of human sperm binding to the ZP was interpreted to be due to the participation of the anti-SLIP1-reactive antigens directly in ZP binding. All of these results described here were in agreement with our previous finding that P68 has the ability to bind to ZP of various mammals including humans (Tanphaichitr et al., 1998). Results from our laboratory also indicate that arylsulphatase A, which also exists selectively in human male germ cells (Stein et al., 1989), is the SLIP1 component, or P68, involved in ZP binding (Tanphaichitr et al., 1999). Notably, the molecular weights of various forms of arylsulphatase A are very similar to those of anti-SLIP1-reactive human sperm proteins (~68 and ~48 kDa) described here, i.e. 63–65 kDa (glycosylated form) (Sommerlade et al., 1994), and ~50 kDa (degraded product of the glycosylated form) (Fujii et al., 1992). Experiments are underway to identify the SLIP1/arylsulphatase A binding ligand on the ZP. We anticipate that SLIP1/arylsulphatase A and/or their antibodies can be used as non-hormonal vaginal/uterine contraceptives by interfering specifically with human gamete interaction.
Acknowledgements
The authors would like to thank Dr Euridice Carmona for her valuable comments on this manuscript, and Ms T.van Gulik for assistance in the manuscript preparation. This work was supported by CIHR and the Rockefeller Foundation (operating grants to N.T.) and the Ministry of University Affairs of Thailand (research training fellowship to M.R.).
Notes
4 To whom correspondence should be addressed at Loeb Health Research Institute, 725 Parkdale Ave., Ottawa, Ontario, Canada, K1Y 4E9. E-mail: ntanphaichitr{at}ohri.ca 
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Submitted on November 23, 2000; accepted on April 27, 2001.
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