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Biology of Reproduction 67, 212-219 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Role of Sperm Surface Arylsulfatase A in Mouse Sperm-Zona Pellucida Binding1

Julierut Tantibhedhyangkula, Wattana Weerachatyanukula,d, Euridice Carmonaa, Hongbin Xua, Araya Anupriwana, Dominick Michauda,b, and Nongnuj Tanphaichitr2,a,b,c

a Hormones/Growth/Development Group, Ottawa Health Research Institute, Ottawa, Ontario, Canada K1Y 4E9 b Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada K1H 8M5 c Department of Obstetrics and Gynecology, Division of Reproductive Medicine, University of Ottawa, Ottawa, Ontario, Canada K1Y 4E9 d Department of Anatomy, Faculty of Science, Mahidol University, Bangkok 10400, Thailand


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have previously described the zonae pellucidae (ZP) binding ability of a pig sperm surface protein, P68. Our recent results on peptide sequencing of 3 P68 tryptic peptides and molecular cloning of pig testis arylsulfatase A (AS-A) revealed the identity of P68 as AS-A. In this report, we demonstrate the presence of AS-A on the mouse sperm surface and its role in ZP binding. Using anti-AS-A antibody, we have shown by immunoblotting that AS-A was present in a Triton X-100 extract of mouse sperm. The presence of AS-A on the sperm plasma membrane was conclusively demonstrated by indirect immunofluorescence, immunogold electron microscopy, and AS-A's desulfation activity on live mouse sperm. The AS-A remained on the head surface of in vivo capacitated sperm, as revealed by positive immunofluorescent staining of oviductal/uterine sperm. Significantly, the role of mouse sperm surface AS-A on ZP binding was demonstrated by dose-dependent decreases of sperm-ZP binding on sperm pretreatment with anti-AS-A IgG/Fab. Furthermore, Alexa-430 conjugated AS-A bound to mouse ZP of unfertilized eggs but not to fertilized ones, and this level of binding increased and approached saturation with increasing Alexa-430 AS-A concentrations. Moreover, in vivo fertilization was markedly decreased when mouse sperm pretreated with anti-AS-A IgG were artificially inseminated into females. All of these results designated a new function for AS-A in mouse gamete interaction.

fertilization, gamete biology, sperm


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Binding of sperm to the zona pellucida (ZP) is the first step of gamete interaction that leads to fertilization. In mice, ZP3 and ZP2 sulfoglycoproteins are the primary and secondary receptors for acrosome intact and acrosome reacted sperm, respectively [1]. Both ZP3 and ZP2 are highly glycosylated [2], with evidence of sulfation on their saccharide moieties [3]. Specifically, oligosaccharides of ZP3 are significant for initial sperm binding [2, 4, 5]. Corroborating these findings is the demonstration that a number of mouse sperm surface lectins and glycoenzymes, including sp56 [6], galactose binding protein [7], ß-1,4-galactosyltransferase [8], fucosyltransferase [9], and {alpha}-D-mannosidase [10], have ZP binding ability. The presence of these ZP-binding sperm surface proteins, as well as others with less clear ZP binding mechanisms, may be due to the possibility that these proteins act synergistically or sequentially to one another in ZP binding [11].

Sulfated glycans and/or sulfoglycolipids are also implicated in sperm-ZP binding. This implication is from the finding that polysulfated glycans can inhibit sperm-ZP binding [12–15]. The significance of sulfated ZP glycans in sperm binding especially in the secondary step has been suggested based on the observation that sperm proacrosin, an acrosomal component, possesses ZP binding ability and a polysulfate binding domain [16, 17]. On the sperm side, we have recently shown that sulfogalactosylglycerolipid (SGG), the mammalian male germ cell-specific sulfoglycolipid, plays a significant role in both mouse and human sperm binding to the ZP [18, 19]. P68 (formerly termed SLIP1-sulfolipidimmobilizing protein 1), a protein on the mammalian sperm surface that has specific affinity to SGG, is also involved in mouse and human sperm-ZP binding [20–22]. Since P68 and SGG are localized to the same region on the mouse sperm head, they are likely to function together in ZP binding [23]. Interestingly, 3 tryptic peptides of P68 (FTDFYVPVSLXTP, TLFFYPAYPDEVR, and AQFDAAVTFSPSQIAR) reveal high identity to human testis arylsulfatase A (AS-A) [23, 24]. This finding therefore suggests that AS-A is present on the mouse sperm surface and is involved in ZP binding. Results described in this report confirm this hypothesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Human Liver AS-A and Its Antibody

Highly purified human liver AS-A [25], a generous gift from Dr. Arvan Fluharty, University of California, Los Angeles, was shown by us to be a single band on SDS-PAGE, with an apparent molecular mass of 63 kDa. Rabbit polyclonal IgG antibody was then generated against this purified human liver AS-A following standard protocols [26]. The animal was bled before and after immunization to obtain preimmune rabbit serum (PRS) and anti-AS-A antiserum, respectively. The anti-AS-A antiserum, as shown by immunoblotting (Fig. 1), recognized human liver AS-A and pig sperm AS-A, as expected, and was therefore used for detection of mouse sperm AS-A. The IgG fraction was isolated from PRS and anti-AS-A antiserum using the Immuno Pure Immobilized Protein A Affinity Pak Column (Pierce, Rockford, IL), and Fab fragments were then generated from the purified IgG fraction using the Immuno Pure Fab Preparation Kit (Pierce). Affinity-purified anti-AS-A IgG was prepared following the previously described method [27]. Approximately 250 ng of purified human liver AS-A was subjected to SDS-PAGE followed by electroblotting onto nitrocellulose (see below). Anti-AS-A IgG (10 µg/ml in 1 ml of Tris-buffered saline (TBS): 137 mM NaCl in 20 mM Tris-HCl, pH 7.6) was adsorbed to the excised human liver AS-A band overnight at 4°C. Affinity-purified anti-AS-A IgG was then removed from the AS-A blot by treatment with 1 ml of 100 mM glycine-HCl, pH 2.5 (25°C, 30 min). The pH of the eluate was adjusted to neutral with Krebs-Ringer bicarbonate (KRB) medium [28], and the eluate was concentrated to 50 µl using a Microcon 30 microconcentrator (Amicon, Beverly, MA). The anti-AS-A IgG solution (10 µg/ml in 1 ml TBS) was also incubated with a blank blot (no protein) under the same conditions described for the AS-A blot. This anti-AS-A IgG solution was not expected to adsorb to the blot and was concentrated to 50 µl in KRB using a Microcon 30 (i.e., its concentration became 200 µg/ml) in parallel with affinity-purified anti-AS-A IgG. This anti-AS-A IgG solution (200 µg/ml) was used in comparison with affinity-purified anti-AS-A IgG in the in vitro sperm-egg binding experiments.



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  		FIG. 1. Immunoblot of mouse sperm with anti-AS-A antiserum. A) Reactivity of anti-AS-A antiserum with human liver AS-A, pig sperm AS-A and recombinant human testis AS-A. Lane 1: 1.4 µg purified pig sperm AS-A; lane 2: 1.0 µg recombinant human testis AS-A; lane 3: 1.0 µg purified human liver AS-A. B) Immunoblot of intact and Triton-X-100-treated mouse sperm and the Triton X-100 extract. Mouse sperm (1.2 x 106) were treated with 0.625% Triton X-100 solution, and the Triton X-100 extract is shown in lane 1, whereas lane 2 displays proteins from the Triton X-100-treated sperm solubilized in SDS-PAGE sample buffer. Lane 3: proteins from 2 x 106 intact mouse sperm solubilized in SDS-PAGE sample buffer. C) Immunoblot of intact mouse sperm and the AES extract. Mouse sperm (4 x 106) were treated with the AES solution, and the anti-AS-A-reactive band in the AES extract is shown in lane 2. Shown in comparison in lane 1 is anti-AS-A-reactive band of mouse sperm (2 x 106). Positions of molecular mass markers (in kDa) on the polyacrylamide gel are marked by arrows on the left of the panel. Results shown here are representative of those obtained from two replicate experiments

AS-A Activity of Intact and Sonicated Mouse Sperm

To assay for sperm surface AS-A desulfation activity, caudal epididymal and vas deferens sperm (10 x 106 sperm/ml), washed free from the bathing fluid, were incubated (30 min, 37°C) at pH 5.0 in an isotonic reaction solution (0.1 M sodium acetate buffer, pH 5.0, 0.5 mM Na4P2O7 and 0.6% NaCl) [29] containing 10 mM p-nitrocatecholsulfate (NCS), an AS-A artificial substrate. Following centrifugation at 350 x g for 10 min at 28°C, 1 M NaOH was added to the supernatant for brown color development of p-nitrocatechol, the desulfated product of NCS, which was then quantified spectrophotometrically [30]. The viability of sperm exposed to the reaction solution at pH 5.0 was assessed by exclusion of propidium iodide (Molecular Probes, Eugene, OR) used at 1 µg/ml following manufacturer's instruction. The AS-A activity was also measured in sonicated mouse sperm, resuspended in 250 mM sodium acetate buffer, pH 5.0. Sonication was performed using a Branson B220 water bath sonicator (Branson Cleanings Equipment Company, Shelton, CT) for three 30-sec cycles, followed by vigorous vortexing for 30 sec. One unit of activity was defined as 1 µmole of NCS hydrolyzed in 1 h.

Extraction of AS-A from Mouse Caudal Epididymal and Vas Deferens Sperm

Sperm, collected from the caudae epididymis and vas deferentia of CD-1 mice [20], were washed free from the bathing fluid in TBS by centrifugation (350 x g, 10 min, 28°C). The plasma membranes and soluble components of the acrosome were then extracted from the sperm pellet following the described method [31]. Briefly, sperm were resuspended at a concentration of 20 x 106/ml in 0.625% Triton X-100 and 0.15 M NaCl, 5% sucrose, protease inhibitor cocktail (concentration used as instructed by the manufacturer; Roche Diagnostics, Laval, PQ, Canada) and 20 mM sodium acetate, pH 5.2 (5 min, 4°C). The sperm suspension was further homogenized by passing through a 26-gauge syringe needle twice. The soluble and insoluble fractions of the Triton X-100-treated sperm were obtained by centrifugation (10;th000 x g, 10 min, 4°C) of the sperm suspension and were then subjected to protein quantification using a BioRad Protein Assay Solution (Bio-Rad Laboratories, Hercules, CA) and to SDS-PAGE/immunoblotting (see below).

Caudal epididymal and vas deferens sperm collected and washed in TBS as described above were also subjected to treatment with a sucrose solution (320 mM) containing 1 mM ATP, 1 mM EDTA, and 0.2 mM N-{alpha}-p-tosyl-l-lysine chloromethylketone hydrochloride (AES), which had been used to extract peripheral plasma membrane proteins from intact cells [32], including SLIP1 and P68 from mouse and pig sperm, respectively [20, 21]. Approximately 70 x 106 mouse sperm were incubated in 0.5 ml of AES for 20 min at 4°C. Sperm were then centrifuged (600 x g, 10 min, 4°C), and the collected AES supernatant was concentrated for immunoblotting using a Microcon 30.

SDS-PAGE and Immunoblotting of Purified AS-A and Sperm Proteins with Anti-AS-A and Antiacrosin

Purified human liver AS-A, recombinant human testis AS-A (a gift from Dr. V. Gieselmann, University of Bonn, Germany), pig sperm AS-A (purified in our laboratory as described [33]), and proteins from untreated caudal epididymal and vas deferens mouse sperm as well as those from the soluble and insoluble fractions of Triton X-100-treated sperm were subjected to SDS-PAGE (12% polyacrylamide, 0.75 mm thick) [34], followed by electroblotting onto nitrocellulose membrane [35]. The nitrocellulose blot was blocked for at least 1 h with 5% fat-free milk in TBS to reduce nonspecific binding. Immunoblotting was performed using anti-AS-A antiserum (1:500 dilution in blocking medium) and secondary antibody, goat anti-rabbit immunoglobulin conjugated with horseradish peroxidase (Bio-Rad Laboratories) (1:3000 dilution in blocking medium). Antigen-antibody binding was detected by chemiluminescence using an ECL kit (Amersham Pharmacia Biotech, Buckinghamshire, U.K.). Various concentrations of purified human liver AS-A was coelectrophoresed and immunoblotted. The AS-A signals on the nitrocellulose blot were analyzed by a Typhoon 8600 Variable Mode Imager in the chemiluminescence mode. The images were captured by a Typhoon Scanner Control Version 1.0 and the pixel density was quantified by an Image Quant Version 5.0. The Typhoon Imager and its accessories were from Molecular Dynamics (Sunnyvale, CA). The amount of AS-A in sperm samples was determined from the standard curve constructed with purified human liver AS-A.

To prepare AS-A bands on nitrocellulose for the affinity antibody preparation described above, purified human liver AS-A was loaded onto a few lanes of the polyacrylamide gel. After electroblotting, one lane was used for immunostaining and the position of AS-A was localized on the unstained lanes for proper excision.

To check for the leakage of acrosomal proteins following AES treatment of sperm, immunoblotting of the AES extract was also performed using mouse monoclonal antiacrosin IgG antiserum (a gift from Dr. C. Barros, Pontificial Catholic University of Chile, Santiago, Chile) at 1:1000 dilution in blocking medium and secondary antibody, goat anti-mouse immunoglobulin conjugated with horseradish peroxidase (Bio-Rad Laboratories; 1:3000 dilution in blocking medium).

Immunolocalization of AS-A on Capacitated and Noncapacitated Mouse Sperm

Indirect immunofluorescence Caudal epididymal and vas deferens sperm were subjected to two-step Percoll-gradient centrifugation to select a motile population possessing normal morphology [36]. Live Percoll-gradient centrifuged (PGC) sperm, resuspended in KRB-HEPES-BSA, were incubated (37°C, 30 min) with 10 µg/ml anti-AS-A IgG or affinity-purified anti-AS-A IgG. They were then washed in the same medium and incubated (37°C, 30 min) with 25 µg/ml Alexa-488-conjugated goat anti-rabbit IgG (Molecular Probes). Sperm treated with 10 µg/ml PRS IgG served as controls. In order to confirm that sperm were viable during this treatment, propidium iodide was added to the sperm suspension at a final concentration of 1 µg/ml, 5 min before the incubation time of the secondary antibody ended. Sperm were then washed twice in KRB-HEPES-BSA, resuspended in the same medium, mounted onto a slide in PBS:glycerol (1:1 vol:vol), and topped with a cover slip. The slide was viewed under a Zeiss IM35 epifluorescent microscope (Carl Zeiss Canada, Toronto, ON, Canada). Phase contrast and fluorescent images of sperm were then recorded by a Spot Junior CCD camera (Carl Zeiss Canada) and processed through Corel PhotoPaint software.

Indirect immunofluorescence was also performed with live oviductal/uterine sperm collected as described previously [37]. Briefly, uterine horns and oviducts were dissected from a superovulated female CD-1 mouse mated with a fertile male of the same strain. The organs were cut open and incubated (30 min, 37°C, 5% CO2) in 2 ml KRB-BSA. At the end of the incubation, sperm were flushed from the organs with the same medium and then subjected to PGC using the same procedure applied to epididymal and vas deferens sperm. This procedure removed the surrounding viscous material from the oviductal/uterine sperm, which sedimented as a pellet. Following resuspension in KRB-HEPES-BSA, the isolated oviductal/uterine sperm were subjected to indirect immunofluorescence (IIF) as described above for epididymal and vas deferens PGC sperm.

Intracellular localization of AS-A was performed with aldehyde-fixed sperm on slides. Briefly, caudal epididymal and vas deferens sperm resuspended in KRB-HEPES-BSA were plated onto slides precoated with 1 mg/ml poly-l-lysine (molecular weight ~75 000 to 150 000; Sigma Chemical Co., St. Louis, MO) in a moist chamber. Sperm loosely adhering to the slide were washed away by flushing the slide gently with PBS. The attached sperm were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Following 2 washes in PBS, sperm were treated with cold methanol for 3–5 min, washed with PBS, and then incubated (1 h, room temperature) with PBS-2% BSA to prevent nonspecific binding of antibodies. The sperm were then incubated (30 min, room temperature) with 10 µg/ml anti-AS-A IgG or with 10 µg/ml PRS IgG (controls), followed by washing with PBS and treatment (30 min, room temperature) with 25 µg/ml Alexa 488-conjugated goat anti-rabbit IgG. The slide mounting and viewing and the recording and processing of sperm images were the same as those described for live sperm (see above).

Indirect immunofluorescence of AS-A was also performed with live and aldehyde/methanol-fixed noncapacitated sperm (collected from the caudal epididymal and vas deferens sperm and resuspended in PBS) as well as with aldehyde/methanol-fixed PGC caudal epididymal and vas deferens sperm that were treated with Triton X-100 (see above). The same immunofluorescent procedures described above were used for localizing AS-A in these sperm.

Electron microscopic immunoprotein-A gold labeling Live caudal epididymal and vas deferens sperm were treated with anti-AS-A IgG as described for IIF. At the transmission electron microscopy level, antibody-antigen complexes were detected by incubating anti-AS-A-treated sperm (1 h, room temperature) with protein A-gold (8 nm) solution [38] (provided by Dr. F. Kan, Queen's University, Kingston, ON, Canada). The sperm were then washed twice in PBS, and the sperm pellet was fixed (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 fixed sperm were then serially dehydrated in ethanol and embedded in LR white (London Resin Co., Berkshire, U.K.). Thin sections of these sperm were stained with saturated uranyl acetate and Reynold lead citrate and viewed using a Hitachi 7100 transmission electron microscope (Hitachi, Ltd, Tokyo, Japan) at 75 kV.

In Vitro Sperm-Egg Binding Assay

The assay was performed using caudal epididymal and vas deferens PGC sperm [20]. To test for the role of sperm-surface AS-A, PGC sperm, resuspended in KRB supplemented with 0.3% BSA (KRB-BSA), were preincubated (30 min, 37°C, 5% CO2) with various concentrations of anti-AS-A IgG/Fab or affinity-purified anti-AS-A IgG. Sperm pretreated with PRS IgG or PRS Fab served as corresponding negative controls for sperm preincubated with anti-AS-A IgG or anti-AS-A Fab, respectively. The number of sperm bound to the egg ZP was counted using our method [20]. Data of anti-AS-A-treated sperm samples were expressed as percentages of the control values. Under our experimental conditions, control samples consistently showed >20 sperm bound per egg. Significant differences between the numbers of sperm bound per egg of the control and anti-AS-A-treated samples were analyzed by ANOVA.

Assessment of Sperm Motility and Spontaneous and Zona Pellucida-Induced Acrosome Reaction

Caudal epididymal and vas deferens sperm treated with 100 µg/ml anti-AS-A IgG or PRS IgG were assessed for their motility under a Nikon inverted microscope at 200x magnification. Spontaneous acrosome reaction was determined by evaluating the sperm acrosomal status following the described method [39]. Specifically, aldehyde-fixed acrosome intact sperm were stained with Coomassie Blue at their head convex ridge (the site of the acrosome). The antibody-treated sperm were also evaluated for the acrosome reaction induced by heat solubilized mouse ovarian ZP [18], isolated and processed as described [20], using 10 zonae/µl of the sperm suspension.

Binding of AS-A to Mouse Zonae Pellucidae

Binding of human liver AS-A to intact unfertilized and fertilized mouse eggs (collected as described previously [21]) was performed by incubating (37°C, 30 min, 5% CO2) 0.15 µM AS-A (molarity calculated based on the molecular mass of AS-A monomer, i.e., 63 kDa) conjugated with Alexa 430 (Molecular Probes; conjugation performed as instructed by the manufacturer) with 20–30 eggs in a 60-µl droplet of KRB-BSA. After successive washes of the eggs in the same medium, they were viewed under a Zeiss IM35 epifluorescent microscope. Unfertilized eggs incubated with 0.15 µM Alexa-430 ovalbumin served as negative controls.

The ability of human liver AS-A to bind to solubilized ovarian ZP was also evaluated. Ovarian ZP free from the egg proper and cytoplasm, isolated as previously described [20], were solubilized with acid Tyrode solution [28] and neutralized and concentrated in TBS by a Microcon 30 microconcentrator to 7 ZP/10 µl TBS. Seventy solubilized ZP, applied to each well of a Limbro/Titerteck 96-well polystyrene plate (ICN Biomedical, Aurora, OH), were allowed to attach to the well bottom surface overnight at 37°C. The attached ZP were blocked (2 h, 37°C) with 100 µl TBS-2% BSA. Following 4 washes with TBS-0.05% Tween20, ZP were incubated (1 h, 37°C) with 100 µl of Alexa-430 AS-A (0–0.9 µM). The unbound enzyme was removed by washing the wells with TBS-0.2% BSA, and the fluorescence intensity of AS-A bound to ZP in the wells was measured using a Spectramax GeminiXS fluorometer (Molecular Devices, Sunnyvale, CA) with excitation and emission wavelengths of 425 and 520 nm, respectively. The amount of AS-A bound to each ZP-coated well was determined from the Alexa-430 AS-A standard curve. The data obtained were analyzed for the Kd (dissociation constant) value of AS-A-ZP binding by Scatchard plotting using Grafit 4.0 software for Windows (Erithacus Software Ltd., Surrey, U.K.). Alternatively, Alexa-430 ovalbumin was used instead of Alexa-430 AS-A as a negative control for incubation with ZP under the same conditions.

Artificial Insemination and Assessment of In Vivo Fertilization

Artificial insemination was performed with superovulated CD-1 mice [37]. Briefly, sperm-containing fluid, extruded from both caudae epididymis and vasa deferentia of a CD-1 mouse into 400 µl KRB-HEPES-BSA (concentration ~5 x 107 sperm/ml), was aliquoted into 2 fractions, 1 of which was treated (30 min, 37°C, 5% CO2) with anti-AS-A IgG (200 µg/ml) and the other with PRS IgG (200 µg/ml). A 50-µl aliquot of the antibody-treated sperm suspension was used for transcervical injection into each superovulated female approximately 2 h before the expected ovulation time (10 h post-hCG injection [28]). Insemination was done in a paired sequence with sperm collected from the same male. The first female was inseminated with PRS-IgG-treated sperm and the second with anti-AS-A-IgG-treated sperm. Females were killed 22–24 h post-hCG injection, and the eggs were retrieved from the oviduct for microscopic assessment of the fertilization occurrence, i.e., presence of 2 pronuclei. Fertilization was expressed as a percentage of fertilized eggs in the total egg population (fertilized + unfertilized). Significant differences between the percentages of fertilized eggs retrieved from females inseminated with anti-AS-A-IgG-treated sperm and from those injected with PRS-IgG-treated (control) sperm were analyzed by Student t-test. In addition, inhibition of in vivo fertilization in each female inseminated with anti-AS-A-IgG-treated sperm, relative to its paired female inseminated with PRS-IgG-treated sperm, was calculated by assigning the fertilization rate of the latter as 100%.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Biochemical Characterization of Mouse Sperm AS-A

A rabbit polyclonal antibody, generated against human liver AS-A (see Materials and Methods), recognized pig sperm AS-A and recombinant human testis AS-A as shown by immunoblotting (Fig. 1). Significantly, the amino acid sequences derived from the cDNA sequences of human and pig testis AS-A have high identity to the mouse testis AS-A sequence (85% and 82%, respectively) [24, 33, 40]. Therefore, the anti-AS-A reactive protein band of solubilized mouse sperm on the immunoblot (Fig. 1B, lane 3) would represent AS-A in these cells. Figure 1B shows that mouse sperm AS-A had a similar molecular mass, i.e., in the range of 63–68 kDa, as human liver, pig sperm, and recombinant human testis AS-A. The AS-A was quantified to be ~70 fg/mouse sperm by Typhoon imaging analysis. The presence of AS-A in mouse sperm was further supported by NCS desulfation activity in sonicated mouse sperm, i.e., 7.2 mU/mg protein.

Previous studies reveal that membranes and acrosomal soluble components can be extracted from sperm with 0.625% Triton X-100 solution [31]. Immunoblotting results indicated that ~70% of AS-A was present in this detergent extract, whereas the remainder was in the Triton X-100 insoluble fraction (Fig. 1B, lanes 1 and 2). This suggested that AS-A resided mainly in the membrane and/or the acrosomal soluble fraction. Previous studies have shown that AES, a sucrose solution containing a low amount of ATP (1 mM) and EDTA (1 mM), can extract peripheral plasma membranes from cells [32]. Using AES, SLIP1, and P68 (whose identity is AS-A) have been extracted from mouse and pig sperm, respectively [20, 21]. Figure 1C shows that AS-A was indeed present in the AES extract of mouse sperm. Within 20 min of the first AS-A treatment, there was no leakage of acrosin, an acrosomal protein marker [1]. This was deduced by the immunoblotting results revealing no cross-reactivity of the AES extract with anti-acrosin antibody (data not shown). Therefore, AS-A present in this first AES extract would reflect its entity on the plasma membrane. Notably, plasma membrane AS-A had the same molecular mass (63–68 kDa) as AS-A extracted by Triton X-100 from the sperm plasma membrane and the soluble acrosomal components. The amount of plasma membrane AS-A in the first AES extract was quantified to be ~0.5% of total AS-A in mouse sperm. However, this may not represent the total amount of plasma membrane AS-A. Repeating the AES extraction, which also involved sperm centrifugation, led to leakage of acrosin from sperm, and acrosomal AS-A would be expected to be present in the second AES extract. Therefore, the exact amount of AS-A on the sperm plasma membrane could not be quantified because plasma membrane AS-A and acrosomal AS-A appeared to have the same molecular mass.

Localization of AS-A in Mouse Sperm

Indirect immunofluorescence of live mouse caudal epididymal and vas deferens sperm indicated positive staining with anti-AS-A IgG over the convex ridge and the postacrosomal region of the sperm head in all sperm that were viable (excluding propidium iodide, i.e., ~95% of total sperm) (Fig. 2, a and b). In contrast, background fluorescence was observed when these sperm were exposed to PRS IgG instead of anti-AS-A IgG (data not shown). The results suggested the presence of AS-A on the mouse sperm surface, and this was confirmed by immunogold transmission electron microscopy labeling, which showed gold particles on the plasma membrane in the dorsal part of the sperm head and the postacrosomal region (Fig. 3, see enlargements of these regions in a2 and a3). Live sperm exposed to PRS IgG showed minimal numbers of gold particles (Fig. 3b). The presence of AS-A on the sperm plasma membrane corroborated the data showing AS-A activity of intact mouse sperm, i.e., 0.35 mU/106 sperm. Although the assay of AS-A activity was performed at pH 5.0, sperm resuspended in buffer at this pH were viable as evidenced by their exclusion of propidium iodide (>95%). Indirect immunofluorescence of live noncapacitated caudal epididymal and vas deferens sperm showed staining of AS-A with the same pattern as capacitated sperm collected from the same parts of the male reproductive tract, although the intensity of the fluorescent staining was lower in the former (data not shown). This suggested that AS-A was on the surface on noncapacitated sperm but may be partially masked. On the other hand, IIF of oviductal/uterine sperm revealed the presence of AS-A staining on the sperm head at the convex ridge with a similar level of fluorescent staining as that observed in caudal epididymal and vas deferens sperm capacitated in vitro (Fig. 2, c and d). This indicates that the enzyme was retained on the surface of these in vivo capacitated sperm.



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  		FIG. 2. Indirect immunofluorescence of mouse sperm with anti-AS-A IgG. Live sperm (a–d) were incubated with anti-AS-A IgG followed by Alexa-488-conjugated goat anti-rabbit IgG. a and b) Epididymal and vas deferens sperm; c and d) Oviductal/uterine sperm. Immunofluorescent localization was also performed with aldehyde/methanol fixed caudal epididymal and vas deferens sperm (e–j). e and f) Untreated sperm; g and h) sperm treated with Triton X-100. i and j) Negative controls (sperm were exposed to PRS IgG instead of anti-AS-A IgG). Panels a, c, e, g, and i are phase contrast images, and b, d, f, h, and j are their corresponding fluorescent micrographs, respectively. Bar = 10 µm. Results shown here are representative of those obtained from 2 replicate experiments



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  		FIG. 3. Immunogold transmission electron microscopy of live sperm. a1) caudal epididymal and vas deferens sperm were exposed to anti-AS-A IgG followed by protein A-gold. Enlargements of the selected areas of the sperm head (boxed) were shown in a2 and a3. b) Negative control (sperm were exposed to PRS IgG instead of anti-AS-A IgG). Bar = 0.2 µm. Results shown here are representative of those obtained from 2 replicate experiments

The existence of AS-A in the sperm acrosome has been reported in several mammalian species [41, 42] but not in mice. Attempts were therefore made to localize the enzyme intracellularly in mouse sperm. Aldehyde fixation followed by treatment with methanol would permeabilize the sperm membranes. All of these aldehyde/methanol fixed mouse sperm showed IIF staining with anti-AS-A IgG in the acrosomal ridge, suggesting the presence of the enzyme in the organelle (Fig. 2, e and f). However, after treatment of sperm with 0.625% Triton X-100, the fluorescent staining of acrosomal AS-A diminished (Fig. 2, g and h), corroborating the immunoblotting results showing a significant decrease of the AS-A signal in the detergent-treated sperm (see above). However, the decrease observed by IIF was more prominent than that revealed by immunoblotting. This may be due to several washing steps involved in IIF, which may have caused further release of AS-A from sperm. In contrast, aldehyde/methanol-fixed sperm exposed to PRS IgG showed no staining (Fig. 2, i and j).

Role of Sperm Surface AS-A in Sperm-ZP Binding

Because AS-A was present on the sperm head convex ridge and the postacrosome, both of which are involved in ZP binding [1, 43, 44], we investigated the enzyme's role in sperm-ZP binding. Sperm pretreated with anti-AS-A IgG had reduced ability to bind to the egg ZP in a dose-dependent manner, with maximal inhibition of ~70% at the antibody concentration of >100 µg/ml (Fig. 4A). Affinity-purified anti-AS-A IgG also showed inhibitory effects on gamete binding to the same extent as its equivalent non-affinity-purified anti-AS-A IgG (i.e., concentration of 200 µg/ml; see Materials and Methods), and this suggested that the anti-AS-A IgG-induced inhibition of gamete binding was specific to masking of sperm surface AS-A. This inhibition was not due to steric hindrance attributed by the bivalent nature of Ig, since anti-AS-A Fab fragments also exerted similar inhibitory effects (Fig. 4B). However, the degree of inhibition, when compared with IgG concentrations of the same antigen binding valency, was less pronounced with the Fab fragments (i.e., 34% inhibition for 35 µg/ml anti-AS-A Fab versus 54% for 50 µg/ml anti-AS-A IgG and 62% inhibition for 70 µg/ml anti-AS-A Fab versus 70% for 100 µg/ml anti-AS-A IgG). The lower degree of inhibition of sperm-ZP binding caused by anti-AS-A Fab may be due to its lower affinity to AS-A compared with anti-AS-A IgG.



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  		FIG. 4. Inhibitory effects of sperm pretreatment with anti-AS-A on sperm-ZP binding. A) Sperm were pretreated with anti-AS-A IgG at various concentrations or with affinity-purified anti-AS-A IgG before coincubation with eggs. Control sperm were pretreated with 100 µg/ml PRS IgG and coincubated with eggs likewise. B) Anti-AS-A Fab (35 and 70 µg/ml) and PRS Fab (70 µg/ml) were used in place of anti-AS-A IgG and PRS IgG, respectively. The number of sperm bound to egg ZP was counted and expressed as percent control, as compared with the corresponding number of PRS IgG/Fab-treated sperm. n, Number of total eggs assessed in each sample. Data are expressed as mean ± SD of percent control of means from three experiments or more, except for the experiments using affinity-purified anti-AS-A IgG or anti-AS-A Fab, which were done once. * Significant difference (P < 0.05) from the control samples as determined by ANOVA of raw data (i.e., number of sperm bound/egg)

The decrease of sperm-ZP binding following sperm pretreatment with anti-AS-A antibody was not from an increase in premature acrosome reaction. The majority of sperm (~90%) treated with either 100 µg/ml anti-AS-A IgG or 100 µg/ml PRS IgG remained acrosome intact, similar to those untreated sperm (Fig. 5, empty boxes). In addition, these antibody-treated sperm, like the untreated sperm, contained the functional machinery necessary for the ZP-induced acrosome reaction; ~90% of them underwent the acrosome reaction when exposed to 10 solubilized ZP/ml (Fig. 5, dot-filled boxes).



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  		FIG. 5. Lack of effects of sperm treatment with anti-AS-A IgG on the acrosome reaction. Empty boxes: spontaneous acrosome reaction (sperm were treated with 100 µg/ml PRS IgG or 100 µg/ml anti-AS-A IgG or untreated [control]) (n = 6); dot-filled boxes: ZP-induced acrosome reaction (sperm were treated as those appearing as empty boxes, followed by solubilized ZP) (n = 6). Data were expressed as box plots of medians with the 25–75th percentile range

The results shown in Figures 4 and 5 implicated a direct function of sperm surface AS-A in ZP binding. To further validate this hypothesis, experiments were conducted to monitor direct binding of purified AS-A to both intact and solubilized ZP. Figure 6A reveals that Alexa-430-AS-A bound to the ZP of unfertilized eggs (panel a), while Alexa-430 ovalbumin (panel b) did not. All unfertilized eggs incubated with Alexa-430 AS-A showed uniformity of fluorescent staining. When nonfluorescently labeled AS-A (100x) was included in the coincubation of Alexa-430 AS-A and unfertilized mouse eggs, the fluorescent signal on the egg ZP was also minimal (data not shown). Furthermore, Alexa-430 AS-A bound to solubilized ovarian ZP adhered to the microtiter well, and the binding level increased and approached saturation with increasing amounts of Alexa-430-AS-A (Fig. 6B). Scatchard plotting (Fig. 6B) revealed a Kd of this binding to be 0.21 µM. In contrast, AS-A did not bind to the ZP of fertilized eggs (Fig. 6A, panel c). These results, therefore, revealed specificity of AS-A binding to the ZP of unfertilized mouse eggs.



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  		FIG. 6. Binding of AS-A to the mouse ZP. A) ZP-intact eggs. Unfertilized eggs (20–30) were incubated with 0.15 µM Alexa-430 AS-A (a) or with 0.15 µM Alexa-430 ovalbumin (b) in a 60-µl droplet of medium. Fertilized eggs (20–30) were also incubated with 10 µg/ml Alexa-430 AS-A (c) under the same conditions as those used for unfertilized eggs. Bar = 0.2 µm. Note that only the unfertilized eggs showed the intense fluorescent staining, indicating AS-A binding. Results shown here are representative of those obtained from two replicate experiments. B) Solubilized ZP. Seventy solubilized ovarian mouse ZPs were attached to each microtiter plate well and incubated with 0–0.9 µM Alexa-430 AS-A, and the fluorescence intensity of Alexa-430 AS-A bound to the ZP was measured spectrofluorometrically. The background fluorescence of ZP incubated with 0.9 µM Alexa-430 ovalbumin was used as a blank. The amount of Alexa-430 AS-A bound to ZP was determined from the standard curve of Alexa-430 AS-A. Open circles represent individual sample points. Linear Scatchard plot analysis (inset) revealed a Kd of 0.21 µM

The physiological significance of AS-A in fertilization was investigated by comparing the number of eggs fertilized in vivo following insemination of the superovulated females with anti-AS-A-IgG- or PRS-IgG-treated sperm. Eighty percent of eggs retrieved from females inseminated with PRS-IgG-treated sperm were fertilized. In contrast, the average percentage of fertilized eggs collected from females inseminated with anti-AS-A-IgG-treated sperm was only 42%, a value significantly lower than that of females inseminated with control (PRS-IgG-treated) sperm (P < 0.001; Fig. 7A). When the numbers of fertilized eggs were compared between the paired females inseminated with sperm treated with anti-AS-A IgG or PRS IgG (control; Fig. 7B), the range of in vivo fertilization inhibition was from 20% to 70% of the control values, with an average of 46%. The lower inhibitory effects on in vivo fertilization by sperm treatment with anti-AS-A, compared with the results obtained from the in vitro binding assay (Fig. 4), may be due to the fact that the treated sperm suspension used for artificial insemination still contained epididymal fluid, which may have impeded the binding of the antibody to the sperm surface.



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  		FIG. 7. Effects of anti-AS-A treatment of sperm on in vivo fertilization. A) Percent fertilization of total eggs (fertilized + unfertilized) retrieved from each female inseminated with the sperm suspension containing either PRS IgG (control) or anti-AS-A IgG (200 µg/ml). Each female pair (see Materials and Methods) was represented by different symbols. B) Percent inhibition of in vivo fertilization in each female pair. The number of fertilized eggs retrieved from individual females inseminated with the PRS IgG containing sperm suspension was assigned as 100%. Horizontal bars in both A and B represent means of 7 data points


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our laboratory has demonstrated the significance of a pig sperm surface protein, P68 (formerly named SLIP1), in ZP binding [20–22, 37]. Three tryptic peptides of P68 have high similarity to human and mouse testis AS-A, strongly suggesting that P68 is AS-A [23]. Furthermore, our molecular cloning work shows that pig testis AS-A contains the three sequences of P68 tryptic peptides [33]. AS-A and P68/SLIP1 also possess 2 other common features. Both proteins are conserved [24, 40, 45] and have a similar molecular weight, i.e., 63–68 kDa (P68: [21]; human and pig testis AS-A and human liver AS-A: [24, 25, 33]). These results suggest that AS-A would have similar localization and physiological roles as P68, i.e., existing on the sperm surface and participating in sperm-ZP binding. While this hypothesis was verified in pig sperm [33], it needed to be verified in the mouse system, widely used for gamete interaction studies, including our earlier work on the physiological significance of P68 [21].

Immunoblotting and immunofluorescence of both live and aldehyde/methanol fixed sperm, using anti-AS-A antibody, suggested that AS-A was present on the sperm surface and in the acrosome. Our semiquantitative analysis showed that AS-A was present at a low level (i.e., 70 fg/sperm), a result similar to that observed for sp56, another ZP-binding sperm protein in rodents [46]. Similar to the previous result described in live rabbit sperm by indirect immunofluorescence [47], AS-A was shown to exist on the surface of live mouse sperm by the same experimental approach. However, we conclusively documented the existence of AS-A on the sperm surface by electron microscopic immunoprotein-A gold labeling (Fig. 3) and possession of NCS desulfation activity of live sperm. Since AS-A is known as a lysosomal enzyme in somatic cells [48–50] and as an acrosomal enzyme in sperm [41, 42], the presence of AS-A on the sperm surface, as reported here and previously [47], addresses the questions of its origin and its significance. Postulation has been made that acrosomal proteins can be transported through dynamic fusion pores across the acrosomal membranes to the sperm plasma membrane [51, 52]. However, since AS-A is an acidic protein (pI ~4) [53, 54], its shuttling through the acrosomal membranes may not be feasible, and our unpublished results reveal that AS-A is in fact present only in the acrosome but absent on the testicular sperm surface. Our recent work indicates that AS-A is present in mouse epididymal fluid and deposits onto the sperm surface via its affinity to surface SGG during epididymal sperm transit [55]. In this report, AS-A was shown to remain on the surface of sperm capacitated in vitro and in vivo (Fig. 2).

The involvement of AS-A in sperm-ZP binding was also demonstrated in this report. First, purified AS-A bound to both intact and solubilized ZP, and the binding was specific to the ZP of unfertilized eggs (Fig. 6), suggesting AS-A's significance in fertilization. The Kd of Alexa-430 AS-A binding to solubilized mouse ZP (0.21 µM) was also in the same range as the Kd of solubilized mouse ZP binding to sperm (~0.07 µM) [9, 22]. The lower value of the latter could be attributed to the synergistic binding of several sperm surface components to ZP [2]. Furthermore, we showed that AS-A on the mouse sperm surface was involved in ZP binding. When sperm surface AS-A was masked by anti-AS-A antibody, sperm had decreased ability to bind to the egg ZP in a dose-dependent manner (Fig. 3). Because the sperm treatment with anti-AS-A IgG did not result in induction of premature acrosome reaction (Fig. 5), which would lead to a marked decrease in sperm binding to the ZP [56], sperm surface AS-A was likely to be directly involved in ZP adhesion and was relevant to the fertilization process. The inhibition of sperm-ZP binding following treatment of sperm with anti-AS-A IgG occurred within 10 min of gamete coincubation (data not shown), indicating that sperm surface AS-A participated in primary binding [57], although its involvement in the secondary binding could not be excluded, based on this result. Because sulfated monosaccharides have been shown to be substrates of arylsulfatases [53, 58, 59], it is tempting to speculate that sperm AS-A has binding ability to mouse ZP sulfated glycans. This postulation would conform to previous results showing inhibition of sperm-ZP binding by polysulfated glycans [12–14, 60]. Regardless of the mechanisms, our demonstration that sperm AS-A is involved in both in vitro (Fig. 4) and in vivo (Fig. 7) fertilization invites future application of the purified enzyme and/or its antibody as a nonhormonal vaginal contraceptive.


   ACKNOWLEDGMENTS
 
We thank Mr. Maroun Bou Khalil for valuable discussions and Ms. Terri van Gulik and Dr. Krittalak Chakrabandhu for assistance in manuscript preparation.


   FOOTNOTES
 
First decision: 8 October 2001.

1 This work was supported by CIHR (grant 10366) and the Rockefeller Foundation, both awarded to N.T. W.W. and A.A. are awardees of a scholarship from the National Science and Technology Department Agency of Thailand and Thailand Research Funds, respectively. D.M. was awarded an NSERC summer student scholarship (2000, 2001). Back

2 Correspondence: Nongnuj Tanphaichitr, Ottawa Health Research Institute, 725 Parkdale Avenue, Ottawa, ON, Canada K1Y 4E9. FAX: 613 761 5365; ntanphaichitr{at}ohri.ca Back

Accepted: February 4, 2002.

Received: September 20, 2001.


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