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Regular Article |
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 Mental Retardation Research Center, University of California Los Angeles, Los Angeles, California 90024
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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acrosome reaction, fertilization, gamete biology, sperm, sperm capacitation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have shown that both SGG and its binding protein (i.e., SLIP1/P68/AS-A) on the sperm head surface are involved in sperm-zona pellucida binding [2, 6, 10–12]. Specifically, SGG liposomes and AS-A can bind to the zona pellucida (ZP). In contrast, galactosylglycerolipid (GG) liposomes do not have this binding ability [2]. Interestingly, SGG and AS-A coexist in the same sperm head area (i.e., the convex ridge and postacrosome) [2, 6], and although SGG has been shown to be the enzyme's substrate in solution in the presence of saposin B or detergent, which solubilizes the sulfoglycolipid to fit into the enzyme's active site pocket [13, 14], SGG molecules on sperm remain intact [15]. This suggests that saposin B is absent in the male reproductive tract, and that sperm SGG cannot access the active site pocket of the neighboring AS-A. However, AS-A contains a number of exposed, positively charged amino acids on the surface of its three-dimensional structure [16], and the enzyme may thus bind to SGG without desulfating it. Indeed, this may be the mechanism by which AS-A in the epididymal fluid deposits onto the sperm surface during epididymal transit [17]. Both AS-A and SGG on the sperm surface may act synergistically in ZP binding. Because AS-A can be extracted by a simple low-salt solution [10] and lacks a transmembrane domain [18, 19], it is likely to be a peripheral plasma membrane protein, and it may interact with the ZP via binding to sulfated sugar residues present on the egg ZP glycans [20, 21]. Based on a previous infrared spectroscopic study [22] revealing interaction between the galactose residues of sulfogalactosylceramide (SGC, a structural analogue of SGG) and galactosylceramide, it is possible that the galactose moiety of SGG molecules that are unbound to AS-A may likewise interact with peripheral galactose residues present in ZP glycans. Although protruding a short distance above the lipid bilayers, SGG's interaction with the ZP glycans may be stabilized by its abundance on the sperm surface.
In the present study, we verified that AS-A could bind to SGG without sequential desulfation of the sulfoglycolipid. To demonstrate that the high molar ratio of SGG to AS-A is essential for sperm-ZP binding, we added purified AS-A exogenously to capacitated sperm. Our results revealed that the treated sperm had reduced ability to bind to the ZP, although the mechanism of this decrease was from premature acrosome reaction.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The AS-A was purified from human liver as described previously [23]. The purified enzyme appeared as a single band on SDS-PAGE with an apparent molecular mass of 63 kDa. Its desulfation activity on an artificial substrate, p-nitrocatecholsulfate (NCS), was 2000 U/mg [23], and on a natural substrate, SGG, in the presence of 0.1% (w/v) taurodeoxycholate, its desulfation activity was 6.15 U/mg (unpublished results; 1 U = 1 µmol of substrate desulfated per hour). AS-A activity was irreversibly impaired by treating the enzyme at 95°C for 1 h, followed by immediate chilling on ice, as assessed by using NCS and SGG as substrates [14, 23]. AS-A was conjugated with Alexa 430 (Molecular Probes, Eugene, OR) following the manufacturer's instructions.
Polyclonal anti-AS-A immunoglobulin (Ig) G antibody and polyclonal anti-SGG IgG antibody were generated in rabbits in our laboratory [2, 6] against purified human liver AS-A and SGG containing liposomes, respectively. ELISA revealed that anti-SGG antiserum specifically recognized SGG and SGC but not their parental glycolipid, GG, or galactosylceramide [2]. Immunoblotting revealed that the anti-AS-A antiserum specifically recognized human liver AS-A (63 kDa) and pig and mouse sperm AS-A (both 
68 kDa) [6]. The IgG fractions were purified from anti-AS-A and anti-SGG antisera as described previously [2, 6]. Mouse monoclonal anti-SGG IgM antibody (O4), produced by Dr. J. Trotter (Heidelberg, Germany) and specifically recognizing SGG and its analogue SGC [24], was provided to us by Dr. B. Gadella (Utrecht University, Utrecht, The Netherlands). Mouse IgM was purchased from Sigma (St. Louis, MO). Secondary IgG antibodies, Immunopure goat anti-rabbit IgG (H + L), and Cappel goat anti-mouse IgM as well as horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H + L) were obtained from Pierce (Rockford, IL), Organon Teknika Corp. (West Chester, PA), and Bio-Rad (Hercules, CA), respectively.
Determination of Contaminating Protease Activity of Human Liver AS-A
Two synthetic substrates conjugated to methylcoumarinyl amide (MCA) susceptible to trypsin and chymotrypsin (i.e., Z-Ala-Lys-Lys-MCA and Ala-Ala-Pro-Phe-MCA, respectively) were chemically synthesized by Dr. Ajoy Basak (Ottawa Health Research Institute, Ottawa, ON, Canada) and used for assaying whether our human liver AS-A contained any trypsin- and chymotrypsin-like activities. Briefly, 63 nM human liver AS-A (molarity calculated based on the molecular mass of an AS-A monomer [i.e., 63 kDa]) was incubated (1 h, 37°C) with 100 µM of each synthetic substrate in 100 µl of Kreb Ringer bicarbonate (KRB) medium supplemented with 0.3% (w/v) BSA (KRB-BSA) [25], the medium used for resuspending sperm during the AS-A treatment (see below). At the end of the incubation, fluorescence of the released product, MCA, was measured using a Spectramax GeminiXS fluorometer (Molecular Devices, Sunnyvale, CA) with excitation and emission wavelengths of 380 and 440 nm, respectively.
Casein zymograms were also used to investigate whether human liver AS-A contained any other proteolytic activity. Casein zymography was conducted according to procedures outlined by Savopoulos et al. [26] and Caballero et al. [27]. Briefly, the samples (20 µl), which were our human liver AS-A (63 nM) and trypsin (6.3 nM and 0.63 nM; Sigma), were electrophoresed in separate lanes in a 12% (w/v) polyacrylamide gel containing 0.1% (w/v) casein under nonreducing conditions. Following electrophoresis, the gel was soaked twice for 30 min in 2.5% (w/v) Triton X-100 in water, washed briefly with incubation buffer (50 mM Tris-HCl and 100 mM NaCl; pH 7.6), and incubated in fresh incubation buffer at 37°C for 18 h. The gel was then stained with 0.5% (w/v) Coomassie blue R-250 in acetic acid:isopropyl alcohol:distilled water (1:3:6) for 20 min and destained with distilled water until clear bands of 24 kDa, the molecular mass of trypsin, that showed casein degradation were visualized.
Binding of AS-A to SGG
Five hundred nanograms of SGG, isolated as described previously [28] and dissolved in 100% ethanol, were immobilized into each well of a black, 96-well polystyrene plate (Corning, Inc., New York, NY) by letting it dry overnight in a dessicator at room temperature. After blocking with 2% (w/v) BSA in Tris-buffered saline (TBS; 20 mM Tris-HCl and 137 mM NaCl, pH 7.6), the lipids were incubated (1 h, room temperature) with different concentrations of AS-A conjugated with Alexa 430 (5–20 nM). The unbound enzyme was removed by washing with TBS containing 0.2% (w/v) BSA, and the fluorescence intensity of AS-A bound to SGG in the wells was measured using a Spectramax GeminiXS fluorometer with excitation and emission wavelengths of 425 and 520 nm, respectively. The amount of AS-A bound to each SGG-coated well was determined from the Alexa-430 AS-A standard curve. The data obtained were analyzed for the Kd value of AS-A-SGG binding by Scatchard plotting using Grafit 4.0 for Windows software (Erithacus Software Ltd., Surrey, U.K.). The regression coefficient (R2) of the Scatchard plot linear regression was calculated using the Microsoft Excel 97 software (Microsoft, Redmond, WA). Alternatively, Alexa-430 ovalbumin was used in place of Alexa-430 AS-A for incubation with SGG under the same conditions as described above.
Binding of Alexa-430 AS-A (20 nM) to GG (SGG's desulfation product; produced in our laboratory as described previously [24]), phosphatidylserine (negatively charged like SGG), and phosphatidylcholine was also evaluated, following the same procedure as described above, except that SGG was replaced with GG or phospholipid. All phospholipids used were from bovine brain and purchased from Doosan Serdary Research Laboratories (Inglewood Cliffs, NJ).
Binding of AS-A to Sperm
All studies involving the use of mice were approved by the Animal Care Committee of the Ottawa Health Research Institute. Caudal epididymal and vas deferens sperm were collected from CD-1 mice and subjected to Percoll gradient centrifugation to select motile sperm with normal morphology as described previously [15]. Percoll gradient-centrifuged (PGC) sperm at the concentration of 10 x 106 sperm per milliliter of KRB buffered with Hepes and supplemented with 0.3% (w/v) BSA (KRB-Hepes-BSA) [25] were incubated (30 min, 37°C, 5% CO2) with 63 nM Alexa-430 AS-A. The sperm were then washed 3 times by centrifugation (350 x g, 10 min, 28°C) to remove the unbound enzyme with KRB-Hepes-BSA, resuspended in the same medium, mounted onto a slide in PBS:glycerol (1:1, v/v), and viewed for fluorescent staining under a Zeiss IM35 epifluorescent microscope (Carl Zeiss Canada, Toronto, ON, Canada). Phase-contrast and fluorescent images of sperm were recorded by a Spot Junior CCD camera (Carl Zeiss Canada) and processed through Corel PhotoPaint software (Ottawa, ON, Canada).
Alternatively, the experiment was performed by incubating PGC sperm pretreated (1 h, 37°C, 5% CO2) with 100 µg/ml of anti-SGG IgG to mask SGG on the sperm surface.
A negative-control experiment was performed by incubating PGC sperm with 63 nM Alexa-430 ovalbumin in place of Alexa-430 AS-A using the same conditions as described above.
Treatment of Sperm with AS-A or Anti-SGG and Secondary Antibodies
The PGC sperm at 5–10 x 106 sperm per milliliter of KRB-BSA were capacitated in the same medium for 30 min at 37°C under 5% CO2. For in vitro sperm-egg binding experiments, PGC sperm were incubated (1 h, 37°C, 5% CO2) with various concentrations of purified native AS-A (4, 16, and 63 nM) or with 63 nM heat/chilled-inactivated AS-A (HI-AS-A). Any AS-A unbound to sperm was then removed by centrifugation (see above), and the sperm were used for coincubating with eggs (see In Vitro Sperm-Egg Binding Assay below). For assessment of acrosomal status and sperm motility and viability (see below), incubation was done over various times (30 min, 1 h, and 2 h) but with only one concentration of AS-A or HI-AS-A (63 nM). For lipid characterization (see below), PGC sperm were treated with 63 nM AS-A for 1 h under the same conditions as described above. Sperm treated with 63 nM ovalbumin served as negative controls in all experiments.
Capacitated PGC sperm in KRB-BSA were also treated (1 h, 37°C, 5% CO2) with 25 µg/ml of affinity-purified rabbit anti-SGG IgG or mouse anti-SGG IgM (1:40 [v/v] dilution). After washing with the same medium, sperm were incubated (1 h, 37°C, 5% CO2) with 50 µg/ml of goat anti-rabbit IgG (H + L) or with 50 µg/ml of goat anti-mouse IgM, respectively. Unbound antibody was removed by centrifugation (see above), and sperm were assessed for their acrosomal status and viability. Negative-control samples were prepared by treating sperm with 50 µg/ml of mouse IgM, followed by 50 µg/ml of goat anti-mouse IgM/IgG antibody, under the same conditions as those used for anti-SGG treatment.
In Vitro Sperm-Egg Binding Assay
The in vitro sperm-egg binding assay was performed as described previously [11]. Cumulus masses were collected in KRB-Hepes-BSA from the oviduct of female CD-1 mice superovulated with sequential hCG and eCG injections and treated with hyaluronidase (Sigma) to free zona-intact, unfertilized eggs from each other [25]. Sperm pretreated with AS-A, HI-AS-A, or ovalbumin (see above) were then coincubated with these eggs (30 min, 37°C, 5% CO2) in a 60-µl droplet (60 000 motile sperm with 20–30 eggs). Subsequently, sperm-egg complexes were washed 3 times in KRB-BSA using a drawn Pasteur pipette (bore size, 200 µm) to eliminate the loosely attached sperm. These eggs were placed into a well of a sera culture slide and topped with paraffin oil, and the number of sperm bound per egg was counted under an inverted Nikon microscope (Nikon, Tokyo, Japan) at 200x. Because numerous sperm bound to the ZP, only those in the same focal plane as the ZP diameter were counted. Under our experimental conditions, 20–30 untreated or ovalbumin-treated control sperm bound per egg, and the data from these 2 control sperm samples did not show any significant differences from each other. The background level of sperm binding was assessed by incubating sperm with fertilized eggs and was 1 to 4 sperm per fertilized egg (i.e., <15%–20% of the positive-control level), retrieved as described previously [12]. Data regarding AS-A-treated sperm samples are expressed as percentages of the control values.
Artificial Insemination and Assessment of In Vivo Fertilization
Artificial insemination was performed with superovulated CD-1 mice as previously described [29]. Briefly, sperm-containing fluid, extruded from each cauda epididymis and vas deferens of a CD-1 mouse into KRB-BSA, was adjusted for the sperm concentration in the same medium to be of 5 x 107 sperm/ml. AS-A (630 nM) was added to the suspension of sperm retrieved from one side of the organs, whereas 630 nM ovalbumin was added to the suspension of sperm collected from the other side. Fifty microliters of the AS-A- or ovalbumin-containing sperm suspension were used for transcervical injection into each superovulated female approximately 2 h before the expected ovulation time (10 h post-hCG injection [25]). Records were maintained for the paired females that were inseminated with sperm from contralateral epididymis and vasa deferentia, with sperm from one side of the organs treated with AS-A and sperm from the other with ovalbumin. The inseminated females were killed 22–24 h post-hCG injection, and the eggs were retrieved from the oviduct for microscopic assessment of fertilization occurrence (i.e., presence of 2 pronuclei). Fertilization was expressed as the percentage fertilization of eggs (fertilized + unfertilized) retrieved from each female inseminated with the sperm suspension containing either ovalbumin (control) or AS-A. In addition, inhibition of in vivo fertilization in paired females inseminated with the sperm suspension from the same male that was included with either ovalbumin or AS-A was analyzed. The incidence of fertilized eggs retrieved from individual females inseminated with the sperm suspension including ovalbumin was normalized to 100%.
Assessment of Sperm Motility, Viability, and Acrosomal Status
Capacitated PGC caudal epididymal and vas deferens sperm treated with 63 nM AS-A, 63 nM HI-AS-A, or anti-SGG IgG/IgM + secondary IgG antibody, as described above, were assessed for their motility (both percentage motile sperm and vigor of the sperm movement) under a Nikon inverted microscope at 200x magnification. To assess for sperm viability, sperm were incubated (5 min, 37°C, 5% CO2) with 1 µg/ml of propidium iodide in KRB-BSA, washed twice in KRB-Hepes-BSA, and viewed under a Zeiss IM35 epifluorescent microscope. Viable sperm were those that excluded the dye, whereas dead sperm were those that incorporated propidium iodide into their nuclei. The sperm acrosomal status was assessed following the method described by Bleil and Wassarman [30]. Specifically, aldehyde-fixed, acrosome-intact sperm were stained with Coomassie blue at their head convex ridge (i.e., the site of the acrosome). At least 200 sperm from each sample were assessed for their viability and acrosomal status.
Characterization of Sperm Lipids by High-Performance, Thin Layer Chromatography
Lipids were extracted from control sperm (treated with 63 nM ovalbumin) and from sperm treated with 63 nM AS-A using a modification [31] of the method described by Bligh and Dyer [32]. The chloroform extract was dried under a stream of nitrogen and dissolved in chloroform:methanol (1:1, v/v). An aliquot of the lipid solution extracted from approximately 30 x 106 sperm was subjected to high-performance thin layer chromatography (HPTLC) using an HPK silica gel 60 Å plate (thickness, 200 µm; dimensions, 10 x 10 cm; Whatman, Clifton, NJ) and the running-solvent system containing chloroform:methanol:water (65:25:4, v:v) + 0.2% CaCl2 [33]. The chromatographed lipids were then developed with 0.2% orcinol in 75% H2SO4 and dried at 100°C. Under these conditions, the glycolipid (e.g., SGG and GG) bands became violet-purple, whereas phospholipids and cholesterol became light brown and red, respectively [31].
Determination of Dimer Formation of AS-A
Purified human liver AS-A at 63 nM in 50 µl of TBS was loaded onto a 1-ml Sephadex G-100 column (Amersham Pharmacia Biotech, Piscataway, NJ) pre-equilibrated with TBS. Aliquots (200 µl) of the same buffer were manually applied to the column to elute the enzyme, which was detected by ELISA using 10 µg/ml of anti-AS-A IgG as the primary antibody, HRP-conjugated goat anti-rabbit IgG (1:3000 [v/v] dilution; Bio-Rad) as the secondary antibody, and o-phenylene diamine dihydrochloride as an HRP substrate. The color product was quantified spectrophotometrically by its absorbance at 490 nm. Incubation for the primary and secondary antibodies was for 1 h and 30 min, respectively, and was performed at room temperature. Blocking of nonspecific binding and plate washings were performed using TBS containing 2% BSA and TBS + 0.05% Tween 20, respectively. Purified rabbit IgG (molecular mass, 150 kDa) was also chromatographed separately to the column to determine its eluted position. In addition, IgG was determined by ELISA using only the HRP-conjugated goat anti-rabbit IgG.
Statistical Analysis
Two-way ANOVA was used to compare the differences of all sets of data among AS-A-treated sperm, HI-AS-A-treated sperm, and control (ovalbumin-treated) sperm. Significant differences in the data between each AS-A- or HI-AS-A-treated sperm sample and the control were statistically assessed by the Student t-test. A P value of less than 0.05 was considered to be statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this report, we demonstrate the binding of Alexa-430 AS-A to isolated SGG immobilized to the microtiter plate wells. The level of AS-A-SGG binding increased with AS-A concentrations and approached saturation (Fig. 1). Scatchard plot analysis indicated a Kd for this association of 8.9 ± 1.7 nM (R2 = 0.79). In contrast, Alexa-430 ovalbumin up to 20 nM did not bind to SGG. In addition, 20 nM Alexa-430 AS-A did not show affinity to GG or phospholipids, including phosphatidylserine (negatively charged like SGG) and phosphatidylcholine (the major sperm phospholipid [4]) (data not shown). All these results implicate specific binding of AS-A to SGG.
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The SGG exists in mammalian sperm at a considerable level (i.e., 12% of total sperm lipids [3, 4]), amounting to 0.4 nmol in 106 mouse sperm [5]. In contrast, the amount of AS-A per sperm is markedly lower than that of SGG (i.e., 1 pmol per 106 sperm [6, 34]). In the present study, we wanted to investigate whether the abundant amount of SGG unbound to endogenous AS-A on the caudal epididymal and vas deferens sperm surface could be a binding ligand of exogenous, soluble AS-A and whether this binding may be followed by SGG desulfation. Because sperm SGG is involved in ZP binding [2], we expected that masking or desulfation of SGG by exogenous AS-A would result in a decrease in sperm-ZP binding. Figure 2, a and b, shows that 63 nM Alexa-430 AS-A did, indeed, bind to the head surface at both the convex ridge and the postacrosome (85%–90% of capacitated PGC sperm) or at the postacrosome alone (10%–15% of the same sperm sample). The fluorescent staining was observed in more than 95% of sperm viewed. Notably, SGG has been localized to both these regions on the sperm head [2]. In contrast, 63 nM Alexa-430 ovalbumin did not bind to sperm of the same sample (Fig. 2, c and d). Alexa-430 AS-A binding to sperm was abrogated by pretreatment of sperm with anti-SGG IgG (Fig. 2, e and f), suggesting that AS-A bound to sperm via its affinity with SGG.
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As expected, pretreatment of sperm with exogenous AS-A resulted in inhibition of sperm-ZP binding in a dose-dependent manner (Fig. 3). With 63 nM AS-A, the percentage of sperm bound to the ZP decreased to approximately 24%, which is similar to the background value (defined as nonspecific binding of sperm to the ZP of fertilized eggs or 2-cell embryos, which was 15%–20% of the number of the same sperm sample that bound to the ZP of unfertilized eggs). The native conformation of AS-A appeared to be important for maximum inhibition, because HI-AS-A (with no activity toward NCS substrate; see Materials and Methods) at 63 nM showed only 40% inhibition of sperm-ZP binding (Fig. 3). However, motility of both AS-A- and HI-AS-A-treated sperm was the same as that of ovalbumin-treated sperm (70%–80% motile sperm). In addition, the AS-A-induced inhibition of gamete binding was not from the enzyme's desulfation action on sperm SGG. The level of SGG in AS-A-treated sperm, as shown by HPTLC, was the same as that in the ovalbumin-treated sperm (negative control) with no trace of GG formation (Fig. 4). The purified enzyme also showed an absence of contaminated proteolytic activities in KRB-BSA. Human liver AS-A at 63 nM did not hydrolyze either of the synthetic substrates of trypsin and chymotrypsin (i.e., Z-Ala-Lys-Lys-MCA and Ala-Ala-Pro-Phe-MCA, respectively) as revealed by the background level of the fluorescent product, which was similar to that observed in KRB-BSA without the enzyme (i.e., <20 fluorescence arbitrary units). In addition, no proteolytic activity of human liver AS-A at 63 nM was detected on the casein zymogram, whereas trypsin at 6.3 or 0.63 nM definitely digested casein as revealed by a blank band at 24 kDa, the molecular mass of trypsin. Therefore, the inhibition of sperm-ZP binding following sperm pretreatment with human liver AS-A is unlikely to be from modification of sperm plasma membrane proteins.
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Premature Acrosome Reaction Occurred as a Consequence of Sperm Treatment with Exogenous AS-A or Anti-SGG IgG/IgM + Secondary IgG Antibody
An increase in premature acrosome reaction may be the cause of a decrease in sperm-ZP binding following sperm pretreatment with AS-A. Assessment of the acrosomal status of sperm treated with 63 nM AS-A as a function of time supported this hypothesis. Sperm treated with 63 nM ovalbumin showed a minimal increase in spontaneous acrosome reaction (i.e., from 13% to 15% and 18% at 0.5, 1, and 2 h of incubation, respectively, in KRB-BSA) (Fig. 5A). In contrast, AS-A-treated sperm had a more rapid increase in the acrosome-reacted sperm population, reaching 18%, 32%, and 53% within 0.5, 1, and 2 h, respectively. These values were significantly higher than those of the untreated sperm (P < 0.05) (Fig. 5A). Notably, sperm treated with HI-AS-A also showed increases in spontaneous acrosome reaction, although these increases were not as high as those observed with the native form of AS-A and were not statistically different from those observed with the control sperm. Nonetheless, based on the findings that they all excluded propidium iodide present in the incubation medium, all these AS-A- and HI-AS-A-treated sperm were as viable as the control sperm treated with 63 nM ovalbumin or untreated sperm (Fig. 5C).
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Attempts were made to explain this increase of spontaneous acrosome reaction caused by AS-A. The enzyme is known to exist as a dimer at the neutral pH and as an octomer at an acidic pH [16, 35, 36]. To test whether AS-A at 63 nM manifested as a multimer in our treatment condition, the enzyme was subjected to gel filtration at neutral pH using Sephadex G-100 resin, to which the proteins of molecular masses lower than 100 kDa are restrained. On the other hand, proteins of higher molecular masses would elute in the void volume. This was, in fact, the case for AS-A (molecular mass of a monomer, 63 kDa [23]), indicating that AS-A presumably existed as a dimer, with a molecular mass exceeding 100 kDa at the neutral pH (data not shown). With this multimeric nature, AS-A may cause aggregation of SGG on its binding to the sulfoglycolipid, which, in turn, may lead to the spontaneous acrosome reaction.
To further demonstrate that SGG aggregation could initiate signal transduction with sperm exocytosis as the end point, capacitated PGC sperm were treated with bivalent anti-SGG IgG or multivalent anti-SGG IgM followed by an appropriate secondary IgG antibody. Figure 5B shows that the acrosome reaction was, indeed, induced by this treatment. On treatment with anti-SGG IgG for 1 h followed by 30-min exposure to secondary IgG antibody, 28% ± 4% of sperm became acrosome-reacted. This percentage was significantly (P < 0.05) higher than that of untreated sperm incubated in medium and processed in parallel with the antibody-treated sperm sample (i.e., 16% ± 4%). When anti-SGG IgM was used in place of anti-SGG IgG, an increase in the acrosome-reacted sperm population was even more pronounced (i.e., 34% ± 7%; P < 0.02) as compared to the untreated control. More than 95% of sperm treated with anti-SGG IgG/IgM + secondary IgG antibody were viable as revealed by their ability to exclude propidium iodide (data not shown). Treatment of capacitated sperm with anti-SGG IgG or anti-SGG IgM alone, without secondary IgG antibody, did not lead to a significant increase in the acrosome reaction.
In Vivo Contraceptive Effect of Sperm Treatment with AS-A
All of the 5 mice inseminated with AS-A-treated sperm consistently had significant decreases (P < 0.001) in their fertilized eggs (i.e., <10% of total eggs retrieved) as compared to the 5 control females inseminated with ovalbumin-treated sperm (i.e., 84% of total eggs) (Fig. 6A). When percentage inhibition was expressed for each pair of females inseminated with sperm from the same male pretreated with either AS-A or ovalbumin (control, assigned as 100%), the average inhibition attributed to AS-A treatment from these 5 females was 75% (Fig. 6B). The results, therefore, indicate the inhibition of in vivo fertilization as a consequence of sperm pretreatment with AS-A. Sperm pretreated with 630 nM AS-A still remained viable and motile, like ovalbumin-treated sperm. Notably, the epididymal/vas deferens sperm used for this artificial insemination were not washed free of the bathing fluid. Thus, the results indicate that AS-A-induced contraceptive effects were not prevented by the epididymal/vas deferens fluid.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We expected that the inhibition of gamete interaction induced by sperm treatment with AS-A would be a result of the enzyme's desulfation activity of SGG, yielding GG. Being structurally related to monogalactosyldiacylglycerol, which induces a hexagonal II phase that results in destabilization of the lipid bilayers [39, 40], GG may likewise enhance the fusion property of the sperm plasma membrane and, hence, increase the acrosome reaction rate as observed (Fig. 5A). To our surprise, SGG remained intact in sperm treated with the highest concentration (63 nM) of AS-A, with no GG formation (Fig. 4). The nonenzymatic binding of AS-A to SGG indicated that SGG on the sperm surface did not access AS-A's active site, presumably due to the anchorage of SGG molecules in the sperm membrane lipid bilayers. Nonetheless, the binding was dependent on the sulfated sugar moiety of SGG. Therefore, SGG may interact with positively charged amino acids (R, K, and H) that are abundant on the surface of AS-A's three-dimensional structure [16, 41]. Yet, twice as many of these AS-A-treated sperm underwent the acrosome reaction prematurely within an hour of treatment as compared to ovalbumin-treated sperm (i.e., 32% vs. 15%) (Fig. 5A). The level of the acrosome reaction rose to 53% at 2 h after the enzyme treatment, whereas only 18% of ovalbumin-treated sperm were acrosome-reacted. These AS-A-treated sperm still remained viable as well (Fig. 5C). These results differed from those observed in sperm treated with another lipid hydrolase, sphingomyelinase, in which ceramide is generated from sphingomyelin to induce the acrosome reaction and sperm apoptosis [42].
Previous observations indicate that immunoaggregation of a number of ZP-binding sperm surface proteins by multivalent antibodies results in sperm exocytosis [43–45]. This aggregation may mimic the binding of multivalent ZP oligosaccharides to their binding ligands on the sperm surface, and this may be a basis of triggering the sperm signal transduction pathway, which ultimately leads to the acrosome reaction [46]. Interestingly, AS-A has been shown to exist in a multimeric form (i.e., a dimer at physiological pH and an octomer at an acidic pH [16, 35, 36]). At 63 nM, AS-A was, in fact, a dimer. With its affinity to SGG, AS-A added exogenously to sperm would likely aggregate the sulfoglycolipid on the sperm surface, and in turn, this aggregation may activate the sperm signaling pathways, culminating in the acrosome reaction. This postulation was supported by our experiments demonstrating that premature acrosome reaction could also be induced by sequential treatment of sperm with multivalent anti-SGG and secondary antibodies (Fig. 5B).
The increased rate of premature acrosome reaction (32%) induced by sperm exposure to 63 nM of purified AS-A for 1 h would partially explain a pronounced decrease (76%) in ZP-binding ability of these sperm. The markedly low level of sperm binding to the ZP following exposure to AS-A may also reflect the AS-A-induced modification of plasma membrane fluidity/rigidity and/or masking of free SGG molecules, which otherwise would have participated synergistically with AS-A in ZP binding. The same molecular mechanism as postulated for induction of the acrosome reaction may also be involved in membrane modifications of AS-A-treated sperm that were still acrosome-intact. These AS-A-bound sperm may have been activated prematurely in their signal transduction pathways, even though they had not yet reached the final destiny of sperm exocytosis. This preactivation may also have led to plasma membrane modifications that have affected sperm binding to the ZP. The concept that not all sperm are equally mature has been well presented in the literature [47]. This postulation was supported by the fact that the percentage of acrosome-reacted sperm increased from 32% after 1 h treatment of sperm with 63 nM AS-A to 53% after 2 h. Nonetheless, the postulated AS-A-induced sperm membrane modification is unlikely to be through hydrolysis of sperm plasma membrane proteins, because the purified AS-A used in this study was devoid of contaminating proteolytic activity. Like AS-A, HI-AS-A may also have modified the sperm plasma membrane (e.g., by masking sperm surface SGG), although its effect may not have been as efficient as that of native AS-A due to its denatured conformation. Thus, only 40% inhibition of sperm-ZP binding was observed following sperm pretreatment with 63 nM HI-AS-A as compared to 76% inhibition with native AS-A pretreatment (Fig. 3).
Our recent results reveal that testicular sperm do not contain AS-A on their surface. However, AS-A is present in the epididymal fluid, and it deposits onto the sperm head surface during sperm transit through the epididymis. Based on the observation that AS-A in the epididymal fluid could no longer adsorb onto the surface of sperm pretreated with anti-SGG antibody [17], this deposition is presumably through AS-A binding to SGG on the sperm plasma membrane. This adsorption in vivo, however, is highly controlled; the final molar ratio of AS-A to SGG in caudal epididymal mouse sperm is 1:400 [6]. This very low AS-A:SGG molar ratio is also observed in ejaculated pig sperm (1:300; unpublished results), despite the presence of AS-A in the seminal plasma [48]. AS-A and SGG, both localized to the same regions in mature capacitated sperm head, are involved in ZP binding [2, 6, 10], and their action in this binding event may be synergistic, with the possibility that free SGG, which is abundant on the sperm plasma membrane, may stabilize the interaction between sperm and ZP (see Introduction). The masking of SGG on the sperm surface as a result of in vitro pretreatment of sperm with exogenous AS-A would have disturbed the equilibrium of free SGG needed for continued ZP binding. In addition, the dimeric conformation of AS-A would induce SGG aggregation, leading to premature acrosome reaction (Fig. 5). Both these mechanisms thus result in marked inhibition of sperm binding to eggs (Fig. 3). Our observations described in the present study would also explain why AS-A deposition onto the surface of sperm during their epididymal transit is stringently controlled. However, the mechanisms of this regulation still need to be elucidated.
Regardless of the mechanisms by which AS-A induced premature acrosome reaction and inhibition of gamete interaction, the present results indicate the potential use of this enzyme as a nonhormonal contraceptive via specific interference in fertilization. The inhibition of gamete binding by sperm pretreatment with AS-A as observed in vitro was, in fact, confirmed in vivo by the experiments utilizing the artificial insemination technique. In these experiments, sperm were still bathed in the epididymal fluid, yet inhibition of in vivo fertilization did occur (Fig. 6), suggesting a lack of preventive effects of the sperm-surrounding epididymal fluid on the AS-A-induced inhibition. Similarly, inhibition of sperm-ZP binding was observed in ejaculated human sperm containing diluted seminal plasma (unpublished results). All these results suggest the feasibility of including AS-A in a vaginal ring as a nonhormonal contraceptive.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Funded by CIHR (grant no. 10366 to N.T.). W.W. and A.A. are awardees of a scholarship from National Science and Technology Development Agency of Thailand and Thailand Research Funds, respectively. 
2 Correspondence: Nongnuj Tanphaichitr, Ottawa Health Research Institute, 725 Parkdale Ave., Ottawa, ON, Canada K1Y 4E9. FAX: 613 761 5365; ntanphaichitr{at}ohri.ca
Accepted: January 9, 2002.
Received: September 6, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-HII transition. Biophys J 1999; 76:751-767This article has been cited by other articles:
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  W. Weerachatyanukul, H. Xu, A. Anupriwan, E. Carmona, M. Wade, L. Hermo, S. M. da Silva, P. Rippstein, P. Sobhon, P. Sretarugsa, et al. Acquisition of Arylsulfatase A onto the Mouse Sperm Surface During Epididymal Transit Biol Reprod, October 1, 2003; 69(4): 1183 - 1192. [Abstract] [Full Text] [PDF]
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