Developmental Biology 247, 182196 (2002) doi:10.1006/dbio.2002.

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Arylsulfatase A Is Present on the Pig Sperm Surface and Is Involved in SpermZona Pellucida Binding
Euridice Carmona,* Wattana Weerachatyanukul,* Tanya Soboloff,* , Arvan L. Fluharty, Dawn White,* , Limthong Promdee,* Marc Ekker,* , Trish Berger, Mary Buhr, and Nongnuj Tanphaichitr* , ,# ,1
*Hormones/Growth/Development Research Group, Ottawa Health Research Institute, Ottawa, Ontario, Canada, K1Y 4E9; Department of Biochemistry, Microbiology and Immunology, Department of Cellular and Molecular Medicine, and #Department of Obstetrics and Gynecology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada, K1H 8M5; Mental Retardation Research Center, University of California, Los Angeles, California 90024; Department of Animal Science, University of California, Davis, California 95616; and Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada, N1G 2W1

We have previously described the afnity of a pig sperm surface protein, P68, to mammalian zonae pellucidae (ZP). In this report, we identied P68 as arylsulfatase A (AS-A) based on the presence of P68 tryptic peptide sequences in the pig testis AS-A cDNA sequence. Our objective was to demonstrate the presence of AS-A on the sperm surface and to elucidate its role in ZP binding. Immunogold electron microscopy revealed the presence of AS-A on the sperm surface. Furthermore, live pig sperm and the extract of peripheral sperm plasma membrane proteins exhibited AS-As desulfation activity. Signicantly, the role of pig sperm surface AS-A in ZP binding was demonstrated by dose-dependent decreases of spermZP binding upon sperm pretreatment with anti-AS-A IgG/Fab, and by the binding of Alexa-430-conjugated sperm surface AS-A to homologous ZP. ZP pretreatment with anti-pig-ZP3 antibody abolished AS-A binding, suggesting that ZP3, recognized as the pig sperm receptor, was AS-As binding ligand. This was further conrmed by the ability of exogenous ZP3 to competitively inhibit AS-AZP binding. Similarly, puried ZP3 , a major sperm receptor component of ZP3, exhibited great inhibitory effect on AS-AZP binding. All of these results designated a new function of AS-A in gamete interaction. 2002 Elsevier Science (USA) Key Words: arylsulfatase A; sperm surface enzyme; sperm egg interaction; desulfation activity; sulfoglycolipids; sulfated glycans; fertilization; zona pellucida.

INTRODUCTION
Binding of sperm to the zonae pellucidae (ZP) is the rst step of mammalian sperm egg interaction that culminates in fertilization (Yanagimachi, 1994). Accumulated evidence indicates that protein carbohydrate interaction is one of the main mechanisms of spermZP binding (TopferPetersen et al., 1997; Wassarman, 1999). Zonae pellucidae from all mammals invariably consist of three to four
To whom correspondence should be addressed at Ottawa Health Research Institute, 725 Parkdale Ave., Ottawa, Ontario, Canada, K1Y 4E9. Fax: (613) 761-5365. E-mail: ntanphaichitr@ohri.ca.
1

polypeptides that are highly glycosylated (Prasad et al., 2000; Nakano and Yonezawa, 2001). In mice, ZP3 (ZPC) is the primary sperm receptor and its O-linked oligosaccharides are important for this binding event (Wassarman and Litscher, 2001). In pigs, ZP3, consisting of hetero-oligomers of ZP3 (ZPB) and ZP3 (ZPC), is the sperm receptor with ZP3 being a major player in sperm binding (Sacco et al., 1989; Yurewicz et al., 1993, 1998). Both N- and O-linked oligosaccharides of pig ZPB/ZPC have been shown to mediate spermZP binding (Yurewicz et al., 1991; Noguchi et al., 1992). In addition, sulfated glycans present in both pig and mouse ZP (Shimizu et al., 1983; Noguchi et al., 1992;
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AS-A Exists on the Pig Sperm Surface and Binds to ZP

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less clear despite the fact that AS-A is transcribed at the highest level in testes (Kreysing et al., 1994b). In this report, the identity of P68 as AS-A was established by the presence of three P68 tryptic peptide sequences in the pig testis AS-A sequence, obtained by molecular cloning. This nding suggested that AS-A existed on the sperm surface (besides the acrosome) and was involved in ZP binding. To validate this postulation, we demonstrated the presence of AS-A on the pig sperm surface by immunogold electron microscopy, as well as its afnity to the ZP. These results herein uncover a new attribute of AS-A in mammalian fertilization.

Nakano et al., 1996) have been suggested to be involved in spermZP binding (Jones, 1991; Chapman and Barratt, 1996; Takasaki et al., 1999). To date, a number of sperm surface proteins have been reported to recognize ZP (Wassarman, 1999; Dell et al., 1999), with several of them having lectin properties, including spermadhesins (Dostalova et al., 1995; Calvete et al., 1996), zonadhesin (Gao and Garbers, 1998), Sp17 (Yamasaki et al., 1995), and sp56 (Cheng et al., 1994). In addition, a number of ZP-binding proteins on the sperm surface are glycoenzymes, such as 1,4-galactosyltransferase (Nixon et al., 2001), -fucosyltransferase (Thaler and Cardullo, 1996), and -Dmannosidase (Cornwall et al., 1991). These enzymes are believed to bind to their substrates, ZP sugar residues, but do not complete their catalytic reaction. This enzymesubstrate binding may be a basis of conjugation between sperm and the egg ZP. A sperm surface protein, called sulfolipidimmobilizing protein 1 (SLIP1), has also been described by our group to have ZP binding ability (Tanphaichitr et al., 1993). SLIP1 was rst isolated from rat testis homogenate by afnity chromatography using sulfogalactosylglycerolipid (SGG, HSO 3-O3Galp 13-sn-alkylacylglycerol, also known as seminolipid) (Lingwood, 1985) as the binding ligand. Like SGG (Murray and Narasimhan, 1990), SLIP1 is present selectively on male germ cell plasma membranes (Law et al., 1988). SLIP1 was further characterized to consist of a few proteins of similar molecular mass (68 kDa), including a ZP binding component, a heat shock protein 70 (HSP70), and albumin (Tanphaichitr et al., 1999; Boulanger et al., 1995). Using chromatofocusing, we have puried P68, the ZP binding component of SLIP1, free from HSP70 and albumin, from the pig sperm peripheral plasma membrane extract. As expected, P68 also has afnity to SGG (Tanphaichitr et al., 1998). Our recent results indicate high identity of three P68 tryptic peptide sequences to the human testis arylsulfatase A (AS-A) sequence, suggesting that P68 is AS-A (Tanphaichitr et al., 1999). AS-A (E.C. 3.1.6.8) is well recognized as a lysosomal and acrosomal enzyme, and has been puried from several tissues and sperm (Allison and Hartree, 1970; Saraan et al., 1982; Dudkiewicz, 1984; Fujii et al., 1992). It has a specic ability to desulfate various arylsulfates at the pH optimum of 4.55, including small articial substrates, such as p-nitrocatecholsulfate (NCS) and p-nitrophenylsulfate (Baum et al., 1959), and natural sulfoglycolipid substrates, i.e., SGG and sulfogalactosylceramide (SGC) (present in brain, epithelial cells, and sperm of lower vertebrates and invertebrates) (Mehl and Jatzkewitz, 1968; Rahi and Srivastava, 1983). However, desulfation of these sulfoglycolipids is only possible following their solubilization by saposin B (a coactivator) or a detergent making the lipids accessible to AS-As active site pocket (Fluharty et al., 2001). The physiological signicance of AS-A in the neurological system has long been recognized. Individuals genetically decient in AS-A suffer from the disorder of metachromatic leukodystrophy due to the accumulation of SGC in the myelin sheath (Kolodny and Fluharty, 1995). However, the enzymes functions in testes and sperm are

MATERIALS AND METHODS

Production of Rabbit Polyclonal Antibodies against Puried Human Liver AS-A (Anti-AS-A) and P68 Peptide (Anti-P68pep)
Two different antigens were used to immunize the rabbits: human liver AS-A, puried previously (Saraan et al., 1982), and a P68 peptide, AQLDAAVTFGPSQVAR (Tanphaichitr et al., 1999), chemically synthesized by Dr. Ajoy Basak (Protein Chemistry Unit, Ottawa Health Research Institute, Ottawa, ON, Canada). A standard regimen was followed to produce rabbit polyclonal antibodies (Harlow and Lane, 1988). Approximately 0.5 mg of the antigen, human liver AS-A, or P68 peptide, plus Freunds adjuvant was used for each of the three sequential intramuscular injections. The titers of the antibodies produced were assessed by immunoblotting using puried human liver AS-A and pig sperm P68. The IgG fraction of anti-AS-A was prepared by ammonium sulfate precipitation and further puried by using an Afnity Pak Immobilized Protein A Kit (Pierce, Rockford, IL). Anti-AS-A Fab fragments were generated from the puried IgG fraction by using the Immuno Pure Fab Preparation Kit (Pierce). Afnity puried anti-AS-A IgG was prepared, as previously described (Ahnonkitpanit et al., 1999), by adsorbing 1 ml of the IgG antibody (10 g) to 1 g puried human liver AS-A band electroblotted onto the nitrocellulose membrane. The adsorbed antibody was then eluted with 1 ml of 100 mM glycineHCl, pH 2.5, washed, and concentrated to 100 l in CM (capacitation medium, see below) by Microcon 30 (Amicon, Beverly, MA). The nonadsorbed IgG solution (termed AS-A-nonadsorbed IgG) was also collected and used as a negative control of afnity puried anti-AS-A IgG. The anti-AS-A IgG solution of the same concentration and volume, used for afnity purication, was also incubated with a blank blot (no protein) under the same conditions described above. Since this anti-AS-A IgG solution was not expected to adsorb to the blot, it was recovered and concentrated to 100 l in CM (i.e., its concentration became 100 g/ml), and used in comparison with afnity puried anti-AS-A IgG in the in vitro sperm egg binding experiments.

Molecular Cloning of Pig Testis AS-A

A pig testis UniZAP cDNA library was constructed according to the manufacturers instructions (Stratagene, La Jolla, CA). Approximately 800,000 recombinant plaques were screened for their hybridization with a full-length human testis AS-A cDNA (kindly provided by Dr. V. Gieselmann, University of Bonn, Germany) randomly labeled with [ - 32P]dCTP. Positive clones were plaque

2002 Elsevier Science (USA). All rights reserved.

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puried, and the selected phages were in vivo excised. Plasmids were then puried and subjected to long-range nucleotide sequencing (ThermoSequenase; Amersham Pharmacia Biotech, Piscataway, NJ) of the cDNA insert, using an automated Li-Cor DNA Sequencing Analyzer Image 4000 and uorescent M13 forward and reverse primers (Li-Cor Biosciences, Lincoln, NE). The sequences obtained were analyzed for their identity to known sequences available in the GenBank data bank through NCBIs Blastn services. The sequence similarity of the pig testis AS-A to human and mouse testis AS-A was assessed by Clustal W (1.81) Multiple Sequence Alignments software.

Carmona et al.

Purication of Pig Sperm Surface AS-A

The presence of AS-A throughout the purication steps was detected by NCS desulfation activity, following the method described by Fluharty and Edmond (1978). The reaction was performed at 37C for 30 min in 250 l of 250 mM sodium acetate, pH 5.0, containing 5 mM NCS, 0.25 mM Na 4P 2O 7, 0.1% bovine serum albumin (BSA), and 5% NaCl, and was stopped by adding 0.8 ml of 1 M NaOH. The reaction product, p-nitrocatechol, was quantied by its absorbance at 515 nm. One unit of activity was dened as 1 mol of NCS hydrolyzed in 1 h. The AES extract containing AS-A activity was subjected to chromatofocusing (Polybuffer exchanger 94; Sigma, St. Louis, MO) following the same method described for P68 (Tanphaichitr et al., 1998). Proteins were eluted with Polybuffer 74 (Sigma), followed by a NaCl gradient (0 1 M) and 1 M HCl. The salt fractions containing AS-A activity were pooled, dialyzed against 20 mM ammonium bicarbonate, pH 7.4, lyophilized, and resuspended in 0.15 M NaCl, 50 mM TrisHCl (pH 7.0) for subsequent Sephacryl S-200 (Amersham Pharmacia Biotech) size-exclusion chromatography (column dimension: 85 1.5 cm). Proteins were eluted by using the same Trissalt buffer. Calibration of the Sephacryl S-200 column was performed by chromatographing molecular mass standards, including rabbit IgG (150 kDa), BSA (65 kDa), and ovalbumin (45 kDa). The molecular mass of pig sperm AS-A was estimated from the linear plot of the standards molecular masses vs their peak elution volumes. Sperm surface AS-A was further characterized by immunoblotting using anti-AS-A and anti-P68pep antibodies, and then used for studies of its kinetic properties and binding ability to the ZP (see below).

Immunolocalization of AS-A on Live Pig Sperm

Indirect immunouorescence. Live ejaculated pig sperm, washed free of seminal plasma by centrifugation (350g, 10 min, 25C), were incubated with afnity puried anti-AS-A (30 min, room temperature). Following two washes in PBS, the sperm were incubated (30 min, room temperature) with 50 g/ml Alexa-488conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR) and then washed twice with PBS. The sample was mounted onto a glass slide under a coverslip and viewed under a Zeiss epiuorescent IM 35 inverted microscope. Transmission electron microscopic immunoprotein-A gold labeling. Live ejaculated pig sperm were incubated with afnity puried anti-AS-A (30 min, room temperature). Following two washes in PBS, the sperm were incubated (30 min, room temperature) with protein A-gold (8 nm) solution, prepared as described by Slot and Geuze (1985) and provided by Dr. F. Kan (Queens University, Kingston, ON, Canada). The sperm were then washed twice in PBS and xed (1 h, room temperature) with a mixture of 4% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, containing 0.2 M sucrose. The xed samples were then serially dehydrated in ethanol and embedded in LR White (London Resin Co., Berkshire, UK). Thin sections of these sperm were stained with saturated uranyl acetate and Reynolds lead citrate, and were viewed with a Hitachi 7100 transmission electron microscope at 75 kV. A negative control sample was prepared in parallel by using 100 g/ml preimmune rabbit serum (PRS) IgG instead of afnity puried anti-AS-A.

Immunoblotting of Puried Pig Sperm Surface and Human Liver AS-A with Anti-AS-A and AntiP68pep Antiserum
Puried pig sperm surface AS-A and human liver AS-A were subjected to SDSPAGE (10% polyacrylamide) as described by Laemmli (1970), followed by silver staining using Bio-Rad SilverStain Reagent (Bio-Rad Laboratories, Hercules, CA) or by electroblotting to a nitrocellulose membrane (0.45 m; Bio-Rad Laboratories) (Towbin and Gordon, 1984). For immunoblotting, blocking of nonspecic binding was performed by using TBS (20 mM TrisHCl, 137 mM NaCl, pH 7.4) supplemented with 5% milk (TBSmilk). The immobilized proteins were incubated (1 h, room temperature) with anti-AS-A antiserum (1:500 dilution in TBS milk), followed by secondary antibody anti-rabbit IgG conjugated with horseradish peroxidase (Bio-Rad Laboratories) (1:3000 dilution in TBSmilk). Proteinantibody recognition was detected by using an ECL Western Blotting Detection Kit (Amersham Pharmacia Biotech). The bound anti-AS-A was then stripped from the blot by incubation (55C, 30 min) with 2% SDS, 0.8% mercaptoethanol in 50 mM TrisHCl, pH 6.8. The blot was washed with TBS and reprobed with anti-P68pep antiserum (1:500 dilution in TBSmilk) using the same conditions as those for anti-AS-A.

Preparation of an AS-A Crude Extract from Pig Sperm Plasma Membranes

The method previously used for P68 (Tanphaichitr et al., 1998) was applied here. Briey, ejaculated pig sperm, washed free of seminal plasma, were treated with an AES solution (320 mM sucrose containing 1 mM ATP, 1 mM EDTA, and 0.2 mM TLCK (N- -p-tosyl-L-lysine chloromethylketone) for 1 h at 4C. Following centrifugation (350g, 15 min, 25C) to pellet the AES-treated sperm, the supernatant containing AS-A activity, assayed as described below, was ultracentrifuged (100,000g, 1 h, 4C) to remove cellular debris. The nal supernatant obtained was used for further purication of the enzyme (see below). To verify that only the plasma membrane was removed from sperm by AES treatment, the AES-treated sperm were processed for electron microscopy, following the same procedures described above, except that the steps of antibody and gold labeling were omitted.

Enzymatic Properties of Puried Pig Sperm Surface AS-A and Human Liver AS-A
pH prole. Desulfation activity of puried pig sperm surface AS-A and human liver AS-A was measured in 250 mM sodium acetate buffer at various pH values (3.3 6.5) by using NCS (5 mM) as a substrate (see purication section above).

2002 Elsevier Science (USA). All rights reserved.

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afnity puried anti-AS-A IgG. Sperm treated with 100 g/ml PRS IgG/Fab or with AS-A-nonadsorbed IgG (see the section on preparation of afnity puried anti-AS-A IgG) served as controls. After washing with CM-BSA, 1 10 5 sperm were incubated (30 min, 37C, 5% CO 2) in 0.5 ml of CM-BSA with 10 15 cumulus-free ZP-intact oocytes, which were isolated from frozen pig ovaries according to the method of Hedrick and Wardrip (1987). At the end of the incubation, sperm egg complexes were washed free of unbound sperm through four droplets of CM-BSA using a drawn Pasteur pipet having 250- m bore size. The number of sperm bound to the egg ZP was counted under a microscope at 100 magnication. Since a high number of sperm bound to each ZP, only sperm that bound to the periphery of the ZP in the diameter focal plane were counted. Data of anti-ASA-treated sperm samples were expressed as percentages of the control values. Under these experimental conditions, the number of sperm bound per egg in the control samples (untreated or treated with PRS IgG/Fab or AS-Anonadsorbed IgG) was always 30. Differences between the numbers of sperm bound per egg for the control and anti-ASA-treated samples were analyzed by Students t test.

Enzyme kinetics. Experiments were performed at 37C and pH 4.5 using 1.2 g puried sperm surface AS-A or 0.4 g human liver AS-A. Kinetic properties of the enzymes were determined for both articial (NCS) and natural (sulfoglycolipids) substrates. The assay for NCS was as described above, using various substrate concentrations (0.110 mM). For the sulfoglycolipids, the assay was performed with 0.02 0.2 mM of SGG or SGC in 50 l of 250 mM sodium acetate buffer, pH 4.5, containing 0.1% BSA and 0.1% taurodeoxycholate (Fluharty and Edmond, 1978). SGG was puried from ram testes following our previously described method (Tupper et al., 1994), whereas SGC was purchased from Sigma. After incubation, the reaction mixture was subjected to lipid extraction according to the modied (Kates, 1986) Bligh and Dyers method (1959). The extracted sulfoglycolipids (SGG or SGC) and the desulfated glycolipids (GG, galactosylglycerolipid, or GC, galactosylceramide) were concentrated under a stream of N 2 and separated from each other by HPTLC (200 m mesh size, dimension: 10 10 cm; Whatman Inc., Kent, UK) using chloroform/methanol/water (65:25:4, v/v/v) as a solvent system (Levine et al., 1976). The HPTLC plate was stained with 0.2% orcinol in 75% H 2SO 4 at 100C (Kates, 1986) and poststained with 0.03% Coomassie blue G250 in 30% methanol, 100 mM NaCl (Nakamura and Handa, 1984). The amount of the glycolipid, GG or GC, generated from SGG or SGC, respectively, by AS-A was determined by densitometric analysis, using ScionImage software for Windows (Scion Corporation, Frederick, MD). A standard curve of GG (a generous gift from Dr. B. Gadella, Utrecht University, prepared by acid desulfation of SGG) (Alvarez et al., 1990) or GC (Sigma) was constructed from the co-chromatographed glycolipid standards. Kinetic parameters (K m and V max) for desulfation of the three substrates (NCS, SGG, or SGC) by AS-A were calculated from the MichaelisMenten equation using Grat 4.0 software for Windows (Erithacus Software Ltd., Surrey, UK).

SDSPAGE of Isolated ZP, Puried ZP Sulfoglycoproteins, and Deglycosylated ZP

Pig ZP, isolated from homogenized ZP intact oocytes as previously described, were solubilized at 70C for 1 h in 20 mM TrisHCl, pH 7.4 (Hedrick and Wardrip, 1986). Pig ZP3 (55 kDa, consisting of ZP3 and ZP3 ) and partially deglycosylated ZP3 (46 kDa) and ZP3 (42 kDa), both generated from ZP3 treated with endo- -galactosidase, were produced as previously described (Yurewicz et al., 1987a) and were gifts from Dr. E. Yurewicz (Wayne State University, Detroit, MI). Total solubilized ZP were deglycosylated by triuoromethanesulfonic (TFMS) acid treatment (Hedrick and Wardrip, 1987). All of these ZP sulfoglycoproteins and ZP polypeptide cores were subjected to SDSPAGE and silverstaining as described above for AS-A, except that the electrophoresis was performed under a nonreducing condition. Densitometric analysis was performed to estimate the distribution of ZP1 (86 kDa) and ZP3 in total solubilized ZP, as well as that of deglycosylated ZP polypeptides [ZP1 (65 kDa), ZP3 (37 kDa), and ZP3 (32 kDa)] in TFMS-treated solubilized ZP.

AS-A Activity of Intact Pig Sperm

Pig sperm washed free of seminal uid were incubated (30 min, 37C) at a concentration of 3 10 7/ml in 200 l of 0.1 M sodium acetate buffer, pH 5.0, containing 10 mM NCS, 0.5 mM Na 4P 2O 7, and 0.6% NaCl, as described by Chang and Moudgil (1984). Following centrifugation (350g, 10 min, 28C), sperm were resuspended in saline and assayed for their viability by propidium iodide (Molecular Probes) (used at 1 g/ml) exclusion. The supernatant was then measured for p-nitrocatechol, the reaction product of NCS desulfation catalyzed by sperm surface AS-A, following the method described above.

Binding of Pig Sperm Surface AS-A to Pig ZP

ZP-intact oocytes, washed in TBS 0.1% PVP (polyvinylpyrrolidone), were forced through a Pasteur pipet having 100- m bore size to generate free ZP, which were then cleaned by pipetting through a series of TBS 0.1% PVP droplets. The absence of egg remnants was veried in ZP samplings by the lack of autouorescence under the Zeiss epiuorescent microscope (using a rhodamine lter). The isolated ZP were then incubated (45 min, room temperature) with pig sperm surface AS-A (0.14 M, molarity calculated from the molecular mass of AS-A monomer, i.e., 68 kDa) conjugated with Alexa 430 (Molecular Probes) (conjugation was performed according to the manufacturers instructions). Unbound Alexa-430 AS-A was removed by pipetting the ZP through a series of the TBS droplets, and the ZP were viewed under the Zeiss epiuorescent microscope (using a uorescein lter). ZP incubated with Alexa430-conjugated ovalbumin (0.28 M) served as a negative control. In another set of experiments, isolated pig ZP were pretreated (1 h, room temperature) with rabbit polyclonal anti-pig ZP3 antiserum (1:500 dilution in CM-BSA), produced (Yurewicz et al., 1987b) and

In Vitro SpermZP Binding Assay

Ejaculated pig sperm, washed free of seminal plasma by centrifugation (350g, 10 min, 25C), were resuspended in capacitation medium (CM: 0.1 M NaCl, 0.36 mM NaH 2PO 4, 8.6 mM KCl, 0.5 mM MgCl 2, 11 mM glucose, 10 mM NaHCO 3, 2 mM CaCl 2, 5 mM pyruvate, 23 mM Hepes, pH 7.6) supplemented with 0.3% BSA (CM-BSA) (Melendrez et al., 1994) and subjected to two-step (35%/70%) Percoll-gradient centrifugation to select motile sperm, which sedimented as a pellet. Percoll-gradient centrifuged (PGC) sperm were washed once in CM-BSA, resuspended in the same medium at a concentration of 2 10 7 sperm/ml, and further incubated (2 h, 37C, 5% CO 2) to complete capacitation. Capacitated pig sperm at 4 10 6/ml were then incubated (45 min, 37C, 5% CO 2) with various concentrations of anti-AS-A IgG/Fab or with

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kindly provided by Dr. E. Yurewicz, or with preimmune rabbit serum (1:500 dilution in CM-BSA) before incubation with 0.14 M Alexa-430 AS-A. Kinetics of AS-A binding to both intact and solubilized pig ZP was also determined. For the assay of AS-A binding to intact ZP, 30 pig ZP, individually isolated from zona-intact oocytes as described above, were incubated with various concentrations (0 0.8 M) of Alexa-430 AS-A in TBS droplets by using the same incubation and washing conditions described above. These 30 ZP from a single incubation were then placed in each well of a black 96-well polystyrene plate (Corning Inc., New York, NY) for uorescence intensity reading with a Spectramax GeminiXS spectrouorometer (Molecular Devices, Sunnyvale, CA) at the excitation and emission wavelengths of 425 and 520 nm, respectively. The same number of ZP incubated with 0.8 M Alexa-430 ovalbumin was used as a blank. The amount of AS-A bound to 30 ZP in each well was determined from the Alexa-430 AS-A standard curve. The data obtained were analyzed for the K d value of AS-AZP binding by Scatchard plotting using Graft 4.0 software for Windows. For the assay of AS-A binding to solubilized pig ZP, a black 96-well polystyrene was coated with solubilized ZP (2 g/well) by overnight incubation at 4C in the presence of 100 mM sodium carbonate buffer, pH 9.6. The plate was blocked with 1% BSA in TBS at room temperature for 1 h and washed four times with TBS. Various concentrations (0 0.4 M) of Alexa-430 AS-A in 50 l TBS were then added to different wells of the plate and incubated at room temperature for 1 h. Unbound Alexa-430 AS-A was then removed by washing the plate four times in TBS. Fluorescence intensity of Alexa-430 AS-A bound to solubilized pig ZP was then measured, and the K d value of AS-A-solubilized ZP binding was obtained following the same methods described for intact ZP. Alexa-430 ovalbumin was likewise used as a negative control. To determine whether pig ZP3, ZP3 , and ZP3 , all known to participate in spermZP interaction (Yurewicz et al., 1993, 1998), were involved in AS-AZP binding, these ZP sulfoglycoproteins were assessed for their ability to compete with solubilized ZP in AS-A binding. A competitive binding assay was performed by adding various concentrations (0 4 M) of each of these puried ZP sulfoglycoproteins, solubilized ZP (containing all ZP sulfoglycoproteins), or ovalbumin (negative control) together with 0.15 M of Alexa-430 AS-A in 50 l TBS to each well of the plate precoated with 2 g of solubilized ZP. Following incubation for 1 h at room temperature, the plate was washed and measured for uorescence intensity of Alexa-430 AS-A bound to ZP in the plate wells. Competitive binding curves were plotted as percentage of the control uorescence intensity values (i.e., from incubations of Alexa-430 AS-A with ZP in the wells without any competitors) vs molarity of the competitors. Molarity of total ZP sulfoglycoproteins in solubilized ZP samples was calculated based on the 1:4 molar distribution of ZP1 to ZP3 (determined as described above). In an alternate experiment, the ability of total ZP polypeptide cores (prepared by TFMS treatment of solubilized ZP) to compete with AS-AZP binding was assessed. Total ZP polypeptides were added with Alexa-430 AS-A to the ZP wells. The 1:1.6:2.4 molar distribution of the three ZP polypeptides, ZP1, ZP3 , and ZP3 , was used for calculating molarity. Signicant differences of the ability of a pair of ZP protein samples (total ZP sulfoglycoproteins vs total ZP polypeptide cores, and ZP3 vs ZP3 ) to compete with Alexa430 AS-AZP binding at a specic concentration were analyzed by Students t test. In addition, the levels of AS-A binding to ZP of the control incubations (no competitors added) vs those included with

Carmona et al.

competitive ZP proteins were analyzed for signicant differences by using ANOVA.

RESULTS
Molecular Cloning of Pig Testis AS-A
Previous work from our group described a pig sperm surface protein, P68, with binding ability to ZP (Tanphaichitr et al., 1998). Sequences of three tryptic P68 peptides revealed high identity (81100%) to human AS-A, strongly suggesting that P68 was AS-A (Tanphaichitr et al., 1999). This prompted us to obtain the pig testis AS-A cDNA sequence. Screening 800,000 plaques of our UNIZAP pig testis cDNA library with radiolabeled human AS-A cDNA yielded 9 positive clones after tertiary plaque purication. These clones, having various insert sizes (i.e., 2.0, 1.5, and 1.2 kb), overlapped with each other, and all showed similarity to human (Stein et al., 1989) and mouse (Kreysing et al., 1994a) AS-A cDNA. Two clones were complete, containing the entire coding sequence anked by 5 and 3 untranslated regions, and were used to construct the pig testis AS-A cDNA sequence (GenBank Accession No. AF 316108) that contained 1917 nucleotides, with an open reading frame of 1512 bp (nucleotides 262-1773), a poly(A) tail (19 As) (nucleotides 1899 1917), and an adenylation sequence (nucleotides 1876 1880). The open reading frame encoded 503 amino acids beginning with a putative signal peptide of 17 residues. The deduced amino acid sequence (Fig. 1) contained three potential N-glycosylation sites and 16 cysteines, and was colinear (100% matched) with chemically determined amino acid sequences for the three tryptic P68 peptides (amino acids 58 70, 368 380, and 460 475). The pig testis AS-A sequence showed 86 and 82% identity to human and mouse AS-A, respectively (Fig. 1,*). The positions of the N-glycosylation sites of AS-A were conserved in all three species. The pig sequence contained all 14 cysteine residues present in common in the mouse and human sequences, and had two additional cysteines at positions 134 and 340. Like the mouse sequence, pig testis AS-A did not possess the cysteine residue at position 38 in the human sequence. All of the amino acid residues forming the AS-As active site pocket (i.e., D28, C68, K122, H124, S148, H226, D278, K299), as well as the three amino acid pairs (i.e., H325/P41, S42/S428, and Y435/T404), presumably involved in intermolecular hydrogen bonding important for the enzyme dimerization (Lukatela et al., 1998), were conserved in all three species (Fig. 1). These results identied the sperm surface P68 as AS-A and suggest a new location, the cell surface, for AS-A, previously recognized as a lysosomal/acrosomal enzyme (Allison and Hartree, 1970; Saraan et al., 1982; Dudkiewicz, 1984; Fujii et al., 1992).

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FIG. 1. Amino acid sequence of pig testis AS-A shown in comparison with the human and mouse sequences. The deduced amino acid sequence of pig testis AS-A was obtained from the sequences of pig testis cDNA clones, positively screened with 32P-labeled human AS-A cDNA probe. Amino acid sequences of P68 tryptic peptides (100% matched) are in bold font underneath a line. Open boxes enclose the consensus sites for N-linked glycosylation. Gray boxes enclose the amino acids forming the active site pocket. Black boxes enclose extra cysteine residues present on the pig sequence. Comparison of the pig (GenBank Accession No. AF 316108), human (GenBank Accession No. X 52151), and mouse (GenBank Accession No. X 73230) testis AS-A was performed by the Clustal W (1.81) multiple sequence alignment program. Identical residues are indicated by asterisks, conserved substitutions by colons, and semiconserved substitutions by dots.

Localization of AS-A to the Pig Sperm Surface

Immunolocalization of AS-A was performed at both light and electron microscopic levels, using afnity puried anti-AS-A IgG and Alexa-488-conjugated secondary antibody or protein A-gold, respectively. Since live sperm would exclude antibody entry into the cytoplasm, positive signals would represent only the antigen entity on the surface. Figure 2A (a) shows the presence of immunouorescent staining of pig sperm at the head anterior region, the site of ZP binding (Burkin and Miller, 2000). In contrast, control sperm exposed to PRS IgG did not show any staining (Fig. 2A, c). The presence of AS-A on the sperm head surface was ultimately demonstrated by electron mi-

croscopic immunogold labeling. The gold particles were localized to the sperm head surface, on top of the plasma membrane (Fig. 2B, a and b), which appeared as an electron lucent (i.e., white in the image) layer, since the sperm sample was not subjected to osmication (which xes lipids). A negative control sample, which was exposed to PRS IgG, had only a minimal number of gold particles on the surface (Fig. 2B, c), indicating that the positive results obtained with sperm incubated with anti-AS-A IgG were specic to AS-A. Furthermore, sperm treated with the AES solution had their plasma membrane sporadically removed (Fig. 2C, b), as compared with untreated sperm possessing intact plasma membrane (Fig. 2C, a). This AES extract contained

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AS-A desulfation activity on NCS (Table 1). Therefore, the intensity of the immunouorescent staining of AS-A in these AES-treated sperm was markedly diminished, as expected (Fig. 2A, b), compared with untreated sperm (Fig. 2A, a). This staining presumably represented AS-A on the residual sperm plasma membrane and exposed acrosomal membrane of AES-treated sperm. Finally, the presence of surface AS-A was illustrated by the NCS desulfation activity of intact pig sperm, i.e., 0.15 0.06 mU/10 6 sperm (n 5). Although this assay was performed at AS-As pH optimum of 5, all of the sperm were still viable, as shown by the lack of propidium iodide staining in these cells.

Purication and Biochemical Characterization of Pig Sperm Surface AS-A

Possessing all amino acids that form the active site pocket (Fig. 1), pig testis/sperm AS-A was expected to contain arylsulfate desulfation activity in solution. To verify this, we monitored desulfation activity of the puried pig sperm AS-A, at pH 5.0 (the pH optimum of an AS-A), using an articial substrate, NCS (Fluharty and Edmond, 1978). As described for P68 (Tanphaichitr et al., 1998), chromatofocusing was used as the initial purication step of AS-A from the pig sperm AES extract. An additional step, Sephacryl S-200 gel ltration, was included to increase AS-As purity, as well as to determine its native molecular mass. NCS desulfation activity was present in the AES extract and in the eluted fractions (110 130 ml) from the chromatofocusing column (Fig. 3A), with an 11-fold increase in specic activity, relative to the AES extract (Table 1). When the chromatofocused fractions were pooled and subjected to Sephacryl S-200 chromatography, a peak of NCS-desulfation activity was obtained (Fig. 3B), with 122% yield and specic activity of 141.86 U/mg, i.e., a 39-fold increase from the corresponding value of the AES extract (Table 1). The increase in activity yield may reect removal of inhibitors as observed in AS-A purication from human liver (Draper et al., 1976) and rabbit sperm acrosome (Yang and Srivastava, 1974). All of these ndings revealed that pig sperm AS-A was an active peripheral plasma membrane enzyme. The molecular mass of this puried sperm AS-A in the neutral pH solution was estimated to be 135140 kDa (Fig. 3B). The AES extract and the peaks containing NCS desulfation activity, eluted from both chromatofocusing and Sephacryl S-200 columns, reacted with both anti-AS-A and anti-P68pep antibodies. Reactivity with these two antibodies was also observed with puried human liver AS-A, as expected (Figs. 3D and 3E). Signicantly, the nal purication by Sephacryl S-200 yielded pig sperm AS-A with high purity, appearing as the major band on silver-stained SDS-polyacrylamide gel (apparent molecular mass of 68 kDa; Fig. 3C), consistently migrating slightly slower than the major band of human liver AS-A (apparent molecular mass of 63 66 kDa). Using this highly puried pig sperm AS-A for kinetic studies, its pH optimum for NCS desulfation was shown to be in the

FIG. 2. Localization of AS-A to the pig sperm surface. (A) Indirect immunouorescence: Live pig sperm washed with PBS (a) or treated with AES solution (b) were incubated with afnity puried anti-AS-A IgG followed by Alexa-488-conjugated goat anti-rabbit IgG. A negative control was the PBS-washed sperm sample that was incubated with PRS-IgG instead of afnity puried anti-AS-A IgG (c). Bar, 10 m. (B) Transmission electron microscopic immunogold labeling: Live pig sperm washed with PBS were incubated with afnity-puried anti-AS-A IgG (a, b) or PRS-IgG (c) followed by protein A-gold (8 nm). The sperm were postxed in aldehyde, dehydrated, and embedded in LR White. Bars, 0.2 m. (C) Transmission electron microscopy of AEStreated pig sperm: Control sperm (a), washed with PBS, possessed an intact plasma membrane (PM), outer acrosomal membrane (OAM), and the acrosomal matrix (AC). In contrast, sperm exposed to AES (b) showed scattered detachment of the plasma membrane, while the outer acrosomal membrane and the acrosomal matrix remained intact. N, nucleus. Bars, 0.2 m.

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TABLE 1 Purication of Pig Sperm AS-A Total protein (mg) AES extract Chromato-focusing Sephacryl S-200 6.83 0.850 0.215 Total activity ( mol/h) 24.84 36.1 30.5 Yield (% activity) 100 145 122 Specic activity (U/mg) a 3.64 42.47 141.86 b Purication fold 1 11 39

a AS-A enzymatic activity was assayed at pH 5.0, 37C, using the articial substrate NCS (5 mM). Protein was quantied by using the BioRad Protein Assay. One enzymatic unit was dened as 1 mol of substrate hydrolyzed in 1 h. b The nal specic activity obtained after Sephacryl S-200 chromatography was reproducible throughout different rounds of purication (n 5).

range of 4.55.0 (data not shown), similar to that observed for human liver AS-A (Fluharty and Edmond, 1978). Similar K m values were also obtained for both pig sperm AS-A and human liver AS-A, using either a soluble articial substrate, NCS, or detergent-solubilized natural sulfoglycolipids, SGG and SGC (Table 2). On the other hand, V max values of pig sperm AS-A were consistently lower than those of human liver AS-A for all three substrates used (i.e., 23, 3, and 12% for NCS, SGC, and SGG, respectively).

Role of AS-A in Pig SpermZP Interaction

Our identication of P68 as AS-A and localization of AS-A to the sperm head surface strongly suggested the involvement of AS-A in spermZP binding. Figure 4 (left panel) shows that pig sperm pretreated with anti-AS-A IgG had reduced ability to bind to the egg ZP in a dosedependent manner, with a marked inhibition of 70% at the antibody concentration of 100 g/ml. The afnity puried IgG antibody showed a similar inhibitory effect (65% inhibition) as the original anti-ASA IgG (100 g/ml, see Materials and Methods). In contrast, IgG in the original anti-AS-A IgG solution that was not adsorbed to AS-A (AS-A-nonadsorbed IgG) did not show any inhibition of spermZP binding. The results suggested that the inhibition observed with sperm pretreatment with anti-AS-A IgG was specically attributed to the masking of sperm surface AS-A. This inhibition was not from the steric hindrance due to the bivalent nature of IgG, since anti-AS-A-Fab fragments also exerted similar inhibitory effects (Fig. 4, right panel). However, when compared with IgG concentrations of the same antigen-binding valency, the degree of inhibition was less pronounced with Fab fragments (i.e., 41% inhibition for 35 g/ml anti-AS-A Fab vs 48% for 50 g/ml anti-AS-A IgG; and 58% inhibition for 70 g/ml anti-AS-A Fab vs 70% for 100 g/ml anti-AS-A IgG). The lower degree of inhibition of spermZP binding caused by anti-AS-A Fab may be due to its lower afnity for AS-A, relative to anti-AS-A IgG. The participation of AS-A in ZP binding was further demonstrated by its direct binding to pig ZP. Figure 5A (a) shows that intact pig ZP, incubated with 0.14 M Alexa430 AS-A, possessed uniform uorescent staining. In con-

trast, pig ZP exposed to 0.28 M Alexa-430 ovalbumin showed background uorescent staining (Fig. 5A, b). An excess amount (100 ) of the unlabeled enzyme, present in the coincubate of pig ZP with Alexa-430 AS-A, also abrogated the uorescent staining of the ZP (data not shown). When increasing amounts of Alexa-430 AS-A were incubated with intact ZP, the uorescence intensity of the ZP-bound Alexa-430 AS-A increased and approached a plateau at 0.8 M of free Alexa-430 AS-A (Fig. 5B). Notably, the amount of Alexa-430 AS-A bound to the intact ZP was only 1/500 1/120 of the free Alexa-430 AS-A in the ZP incubation mixture. Scatchard plot analysis revealed one K d of 0.57 M. A similar K d value (0.50 M) was obtained when solubilized ZP (coated onto the plate well) were used in place of intact ZP (Fig. 5C). All of these results strongly suggested specic binding of Alexa-430 AS-A to ZP. Pretreatment of pig ZP with anti-pig ZP3 antibody, specic to pig ZP3 sulfoglycoprotein (Yurewicz et al., 1987b), abolished the binding of Alexa-430 AS-A to the ZP (Fig. 5A, c). In contrast, control ZP pretreated with PRS showed the same uorescent staining of Alexa-430 AS-A, as seen with untreated ZP (see Fig. 5A, a). The results suggested that ZP3, recognized as the pig sperm receptor (Sacco et al., 1989; Yurewicz et al., 1998), was the binding ligand of AS-A. To further investigate specicity of pig ZP components that bound to AS-A, competitive binding assays were performed in a microtiter plate. As expected, solubilized pig ZP containing total ZP sulfoglycoproteins, added together with Alexa-430 AS-A, effectively inhibited Alexa-430 AS-A binding to the ZP immobilized in the plate well, showing an IC 50 of 0.35 M (Fig. 6A, ). However, the maximum inhibition was 74%. The inability of the added solubilized ZP to further inhibit Alexa-430 AS-AZP binding might be due to incomplete availability of the AS-A binding ligand(s) of solubilized ZP in solution. On the other hand, these ligands may be well exposed when the solubilized ZP were attached to the plate well. When 4 M deglycosylated ZP was present in the Alexa-430-ZP incubation, Alexa-430 AS-AZP binding was also signicantly reduced as compared with control incubations without any competitor (P 0.05). However, the degree of competitive inhibition by deglycosylated ZP (Fig. 6A, ) was much lower than

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binding. The ZP polypeptide cores also participated in AS-AZP binding, but to a lesser extent than the ZP carbohydrate moieties. Puried pig ZP3 also competed effectively with Alexa430 AS-AZP binding, revealing an IC 50 of 0.18 M and maximum inhibition of 90% (Fig. 6B, ). This result is in agreement with the observation that anti-ZP3 antibody blocked AS-A binding to the ZP (Fig. 5A, c). ZP3 , one of the two ZP3 components, also exhibited strong competitive inhibition on Alexa-430-AS-A binding, similar to that observed with ZP3, with IC 50 of the same range, i.e., 0.50 M (Fig. 6B, ). The maximum inhibition induced by ZP3 was also as high as that observed with ZP3 (i.e., 84%). In contrast, exogenous ZP3 (Fig. 6B, ff) possessed a much lower inhibitory effect than ZP3 . At 0.5, 1, 2, and 4 M of ZP3 , competitive inhibition of Alexa-430 AS-AZP binding was at 12, 21, 29, and 45% (vs 50, 63, 82, and 87%, respectively, observed with ZP3 ). The results indicated greater signicance of ZP3 in AS-AZP binding. ZP3 has also been recognized to be a major player in pig spermZP binding, as compared with ZP3 (Yurewicz et al., 1993, 1998). Therefore, our results suggested that AS-A is part of the sperm machinery involved in ZP binding.

DISCUSSION
We have described a pig sperm surface protein, P68, which possesses afnity to both ZP and SGG, and also participates in spermZP interaction (Tanphaichitr et al., 1998). In this report, molecular cloning revealed the presence of three P68 tryptic peptides in the pig testis AS-A sequence (Fig. 1), indicating that P68 was AS-A, an enzyme previously shown to be present in the somatic cell lysosome and sperm acrosome (Allison and Hartree, 1970; Saraan et al., 1982; Dudkiewicz, 1984; Fujii et al., 1992). This result, therefore, uncovered two new physiological attributes of AS-A, i.e., its existence on the sperm surface and its adhesive property to the egg extracellular matrices, the ZP. Immunolocalization was the rst approach used to demonstrate the presence of AS-A on the pig sperm surface. Indirect immunouorescence of live pig sperm revealed AS-A staining in the anterior head region (Fig. 2A), the site of ZP binding (Burkin and Miller, 2000), and this localization was further conrmed by electron microscopic immunogold labeling (Fig. 2B). Furthermore, live sperm possessed NCS desulfation activity at pH 5.0, typical of an AS-A. Finally, AES treatment of pig sperm yielded a soluble extract with NCS desulfation activity, whereas the treated sperm pellet showed only sporadic plasma membrane detachment (Fig. 2C). AS-A puried from this sperm AES extract, through a series of column chromatography, in fact possessed a number of enzymatic properties (i.e., pH optimum of 4.5, substrate specicity, and K m values for sulfoglycolipids and NCS) very similar to those of human liver AS-A (Table 2). In addition, the puried sperm surface AS-A

FIG. 3. Purication of pig sperm AS-A. Pig sperm surface proteins were extracted by using an AES solution (1 h, 4C). Extracts were subjected to chromatofocusing (A), and fractions with AS-A activity were pooled and chromatographed on a Sephacryl S-200 column (B). Column fractions were assayed for the amount of proteins at A 230 ( ) and for AS-A desulfation activity of NCS at A 515 (FF). pH of each chromatofocusing fraction was also measured (). The vertical arrow in (A) indicates where the salt gradient started, whereas the arrowheads in (B) denote the elution positions of protein standards, i.e., rabbit IgG (150 kDa), bovine serum albumin (65 kDa), and ovalbumin (45 kDa). (C) Silver-stained SDSpolyacrylamide gel of the AES extract (lane 1), pooled fractions from chromatofocusing (lane 2) and Sephacryl S-200 chromatography (lane 3), which possessed AS-A activity, and puried human liver AS-A (lane 4). Std, molecular mass markers. Immunoblotting using anti-AS-A (D) or anti-P68pep (E) antibody was performed with the same blot. The horizontal arrows indicate the position of AS-A band on SDSpolyacrylamide gel.

that observed with total ZP sulfoglycoproteins (Fig. 6A, ), i.e., 15, 17, and 43% vs 67, 71, and 75% at 1, 2, and 4 M, respectively. In contrast, ovalbumin up to 8 M, added together with Alexa-430 AS-A to the ZP well, did not show any inhibition on Alexa-430 AS-AZP binding (data not shown). These results suggested that the carbohydrate moieties of ZP sulfoglycoproteins were important for AS-A

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TABLE 2 K m and V max for the Hydrolysis of an Articial Substrate (NCS) and Natural Sulfoglycolipids (SGC and SGG) by Pig Sperm and Human Liver AS-A NCS AS-A Pig sperm Human liver K m (mM) 7.02 6.90 2.84 1.53 V max (U/mg) 387.5 1712 85.97 205 K m (mM) 0.121 0.124 0.061 0.056 SGC V max (U/mg) 0.525 23.04 0.109 5.51 K m (mM) 0.091 0.092 0.052 0.040 SGG V max (U/mg) 0.72 6.15 0.19 1.15

Note. Kinetic experiments (n liver AS-A.

3) were carried out at pH 4.5, 37C using 1.2 g of puried pig sperm AS-A or 0.4 g of puried human

appeared as a dimer in solution at neutral pH (Fig. 3B) like human liver AS-A (Saraan et al., 1982), and as a single 68-kDa band on SDSPAGE, which reacted with the antibody produced against human liver AS-A (Figs. 3C and 3D). Our work using various approaches conclusively revealed the existence of AS-A on the pig sperm surface. Although AS-A was previously implied to be on the rabbit sperm surface, indirect immunouorescence was the only approach used in this study (Nikolajczyk and ORand, 1992). Previous reports indicate transcriptional expression of AS-A in spermatogenic cells (Kreysing et al., 1994b) and the

FIG. 4. Inhibitory effects of sperm pretreatment with anti-AS-A on spermZP binding. Sperm were pretreated with anti-AS-A IgG (left panel) or its Fab fragments (right panel) at various concentrations or with afnity puried anti-AS-A IgG before coincubation with eggs. Control sperm were pretreated with 100 g/ml PRS IgG for the anti-AS-A IgG experiments or with 70 g/ml PRS Fab for the anti-AS-A Fab experiments, and similarly coincubated with eggs. The number of sperm bound to egg ZP was counted and expressed as percent control. The number of PRS IgG/Fab-treated sperm (control) bound to eggs was assigned as 100%. Data were expressed as mean SD of percent control of means from three experiments or more, except for the experiments using afnity puried antiAS-A IgG-treated sperm. These experiments were done twice and the plot represents an averaged percent control from these two experiments. *, denotes signicant difference (P 0.05) from the control samples, as determined by ANOVA of the raw data (i.e., number of sperm bound/egg).

presence of AS-A in the sperm acrosome (Allison and Hartree, 1970; Dudkiewicz, 1984; Nikolajczyk and ORand, 1992). However, since AS-A does not contain any transmembrane domain (Fig. 1), its trafcking from the cytoplasm to the surface of male germ cells needs an explanation. While a postulation has been made that acrosomal proteins may be transported to the sperm plasma membrane through dynamic fusion pores across the acrosomal membranes (Monck and Fernandez, 1996; Foster et al., 1997), our recent results suggest an alternative mechanism for the presence of AS-A on the mature sperm surface (Weerachatyanukul et al., 2001b). Immunolocalization at both light and transmission electron microscopic levels reveal the absence of AS-A on the surface of mouse spermatogenic cells and testicular sperm. However, surface AS-A increasingly appears in sperm transiting through the epididymis, due to the adsorption of the enzyme, present in the epididymal uid, onto the sperm surface. This adsorption is via AS-As afnity to SGG already present in testicular sperm (Weerachatyanukul et al., 2001b) and would likely be part of the events necessary for sperm maturation (Tulsiani and Abou-Haila, 2001). The involvement of sperm surface AS-A in spermZP binding was demonstrated in the present work by two approaches. In the rst, capacitated pig sperm pretreated with anti-AS-A IgG/Fab antibody were shown to have reduced ability to bind to the ZP, in a dose-dependent manner. The fact that inhibition was observed with the monovalent Fab fraction of anti-AS-A to a similar extent as anti-AS-A IgG (Fig. 4) indicated that the inhibition of spermZP binding was due to specic masking of AS-A, and not from steric hindrance induced by the bivalent nature of the anti-AS-A IgG antibody. Nonspecic inhibition caused by other immunoglobulins that do not recognize AS-A was also excluded since afnity puried AS-A inhibited spermZP binding to the same extent as the original antiAS-A IgG solution from which it was puried (Fig. 4), whereas AS-A-nonadsorbed IgG did not show any inhibition on gamete binding (data not shown). In the second approach, Alexa-430 AS-A was shown to have direct and specic binding to both solubilized ZP and intact ZP, with similar K d values (0.50 and 0.57 M, respectively) (Fig. 5).

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FIG. 5. Binding of pig sperm AS-A to the ZP. (A) Pig ZP were incubated with 0.14 M Alexa-430-conjugated puried pig sperm AS-A (a) or 0.28 M Alexa-430-conjugated ovalbumin (b). After washing the excess unbound uorescent protein, the ZP were viewed under an epiuorescent microscope. In (c), pig ZP were pretreated with anti-pig ZP3 antibody before incubation with Alexa-430 AS-A. Control pig ZP, pretreated with PRS IgG and similarly incubated with Alexa-430 AS-A, showed the same binding as in (a). Bar, 10 m. (B) Binding of Alexa-430-conjugated AS-A to intact ZP. Thirty pig ZP were incubated with 0 0.8 M Alexa-430 AS-A, and the uorescent intensity of the ZP was measured spectrouorometrically by using ZP preincubated with 0.8 M Alexa 430-ovalbumin as a blank. The amount of Alexa-430 AS-A bound to the ZP was determined from the Alexa-430 AS-A standard curve. Open circles represent individual sample points. Linear Scatchard plot analysis (inset) revealed a K d of 0.57 M. (C) Binding of Alexa-430 to solubilized ZP. Alexa-430 AS-A (0 0.4 M) was incubated with solubilized pig ZP (2 g), attached to each microtiter plate well, and the binding of Alexa-430 AS-A to ZP was measured as described in (B). K d was likewise analyzed to be 0.50 M.

Furthermore, the intensity of ZP-bound Alexa-430 AS-A increased and approached saturation with increasing amounts of free Alexa-430 AS-A, and the ZP-bound Alexa430 AS-A was only 1/120 of free Alexa-430 AS-A. In addition, Alexa-430 ovalbumin did not show binding to the ZP. Scatchard plot analysis yielded only one K d of 0.50 M for AS-A binding to solubilized pig ZP, which was seven times higher than a K d described for the binding of solubilized mouse ZP to homologous sperm (Thaler and Cardullo, 1996; Kerr et al., 2001). This discrepancy could be due to species differences. In addition, the synergistic action of sperm surface molecules in ZP binding may account for the lower K d of the binding of solubilized mouse ZP to intact sperm. In the case of sperm surface AS-A, this could be SGG, which also has ZP-binding ability (White et al., 2000; Weerachatyanukul et al., 2001a). AS-A and SGG have high afnity to each other (Carmona et al., 2002) and they are localized to the same region on the surface of mature sperm (White et al., 2000; Weerachatyanukul et al., 2001a; Tantibhedhyangkul et al., 2002). In addition, our results indicated that AS-A bound to ZP3, the pig sperm receptor (Sacco et al., 1989), since pretreatment of eggs with anti-ZP3 inhibited the AS-AZP binding (Fig. 5A) and since ZP3 could competitively inhibit AS-AZP binding (Fig. 6B). The competitive binding assay also revealed that ZP carbohydrate moieties were important for AS-A binding (Fig. 6A). In the same assay, ZP3 inhibited AS-AZP binding at a low IC 50 (0.50 M) in contrast to ZP3 showing a much lesser extent in inhibiting AS-AZP binding (Fig. 6B). Both ZP carbohydrate moieties and ZP3 have been shown to be signicant for interaction between ZP and pig sperm plasma membranes (Yurewicz et al., 1991, 1993; Noguchi et al., 1992). Therefore, all of our results strongly suggest that AS-A is an integral component of the sperm plasma membrane machinery for ZP binding. Like the situation with -galactosyltransferase (Nixon et al., 2001) and -mannosidase (Tulsiani and Abou-Haila, 2001), the contribution of AS-A in spermZP binding is not species specic, since AS-A also exists on the mouse sperm surface and is involved in ZP binding (Tantibhedhyangkul et al., 2002). AS-A is known to desulfate glycosulfates, including detergent-solubilized sulfoglycolipids (Fluharty and Edmond, 1978; Fluharty et al., 2001) and sulfated monosaccharides (Mehl and Jatzkewitz, 1968; Rahi and Srivastava, 1983). Recently, we have shown that AS-A, in the absence of a detergent, also binds tightly to SGG and SGC without exerting its desulfation activity (Carmona et al., 2002). Nonetheless, the glycosulfate moiety of SGG/SGC is still essential for this binding (Carmona et al., 2002). Based on these results, we speculate that AS-A might bind to sulfated sugar residues of the acidic ZP glycans, including those present in pig ZP3 (i.e., in both the glycan antennae and the reducing end; Mori et al., 1998). Notably, sperm AS-A had low V max values for desulfation of SGG, SGC, and NCS,

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FIG. 6. Competitive inhibition of Alexa-430 AS-A binding to pig ZP by ZP proteins. Solubilized ZP, coated in microtiter plate wells (2 g/well), were incubated with 50 l of 0.15 M of Alexa-430 AS-A in the presence of various concentrations (0 4 M) of each puried ZP protein, total ZP sulfoglycoproteins, total ZP polypeptides, or ovalbumin (negative control). Binding of Alexa-430 AS-A to ZP in the wells was then measured spectrouorometrically. Competitive binding curves were plotted as percentage of control values (i.e., uorescence intensity of Alexa-430 AS-A bound to ZP in the plate when incubations were performed without any competitors) vs molarity of the pig ZP sulfoglycoprotein competitors. (A) Inhibition by total pig ZP sulfoglycoproteins ( ) and TFMS-deglycosylated ZP proteins ( ). SDSPAGE patterns of total ZP sulfoglycoproteins (lane 1) and TFMS-deglycosylated ZP proteins (lane 2) are shown on the right of the panel. *, denotes signicant difference (P 0.05) of the AS-AZP binding levels compared between incubations with total ZP sulfoglycoproteins and with deglycosylated ZP proteins, at the same molar concentration, as determined by Students t test. The AS-AZP binding levels observed in incubations with total ZP sulfoglycoprotein incubations at all concentrations were signicantly lower from the levels of control incubations (without any competitors), as determined by ANOVA. In contrast, a signicant decrease in AS-AZP binding was observed at only 4 M of deglycosylated ZP. (B) Inhibition by ZP3 (), ZP3 (), and ZP3 (ff). SDSPAGE on the right displays electrophoretic patterns of ZP3 (lane 1), ZP3 (lane 2), and ZP3 (lane 3). *, denotes signicant differences (P 0.05) of the AS-AZP binding levels compared between ZP3 - and ZP3 -containing incubations, at the same molar concentration, as determined by Students t test. The AS-AZP binding levels at all concentrations of ZP3 and ZP3 and at 2 and 4 M of ZP3 were signicantly lower from the control values, as determined by ANOVA.

and this may also be the case for ZP sulfated glycans, should they bind to AS-As active site. The initial binding between sulfated sugars of the acidic ZP glycans may then be retained due to the low capacity of sperm AS-A to hydrolyze their substrates. Subsequently, desulfation of the ZP glycans sulfated sugars may occur, and this may weaken the binding between sperm surface AS-A and ZP glycans. Together with other mechanisms involved in disrupting the interaction between sperm and the ZP (e.g., hydrolysis of peripheral sugars of the ZP glycans involved in sperm binding; Dell et al., 1999), sperm could migrate further through the ZP layer. Interestingly, the concept that sperm surface hydrolases repeatedly bind to their substrates on the egg vestment (e.g., ZP) and surface, and then exert their enzymatic activity, as part of sperm passage process through these egg entities, has been previously described (Primakoff and Myles, 2000). Our results demonstrating the signicance of AS-A on the sperm surface in ZP binding are consistent with the recent report revealing the role of surface N-acetyl glucosamine sulfatase in cell adhesion and signaling during quail embryo patterning (Dhoot et al.,

2001). The postulation that sulfated sugars of the ZP glycans participate in binding to sperm surface molecules (in our case, AS-A) (Huang et al., 1984; ORand et al., 1988; Jones, 1991; Oehninger et al., 1992) is also in accordance with recent observations implicating (poly)sulfated glycans in cell adhesion (Vilela-Silva et al., 2002; Talevi and Gualtieri, 2001; Su et al., 2001; Pinzon-Ortiz et al., 2001; Shikano et al., 2001). Attempts to elucidate the molecular mechanisms of AS-A interaction with the ZP sulfoglycoproteins are ongoing in our laboratory.

ACKNOWLEDGMENTS
We thank Mr. Ali Shoushtarian, Ms Jennifer Miles, and Ms. Barbara J. Nitta for their valuable technical assistance; and Ms. Terri Van Gulik for help in manuscript preparation. We also appreciate the permission from the United Leukodystrophy Foundation to use the human liver arylsulfatase A in this study. This work was supported by the Canadian Institutes of Health Research (MT-10366) and the Rockefeller Foundation (both to N.T.), National Science and Technology Developmental Agency of Thailand

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(graduate study scholarship to W.W.), OMAFRA, and Ontario Pork (to M.B.) and USDA (NRICGP 99-02240) (to T.B.).

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