Department of Biochemistry, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
Received on September 21, 2000; revised on December 4, 2000; accepted on December 15, 2000.
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Abstract |
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Key words: boar sperm/carbohydrate binding molecule/oligosaccharide probe
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Introduction |
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In a previous study, we have demonstrated that mouse sperm recognize ß-galactosyl residues of the ZP on the basis of the sperm adherence to beads coupled with fetuin preparations containing enzymatically modified glycans and the effect of glycosidase digestion of fixed egg on sperm binding (Mori et al., 1997). We have also shown in the similar way that the acrosome-intact boar sperm prepared from cauda epididymis, but not acrosome-reacted sperm, have abilities to recognize the sialyl and nonsialyl N-acetyllactosamine units included in the outer chain moieties of N-glycans and the Lewis X (Lex) structure (Mori et al., 2000
). These structural moieties are indeed included in the glycans of porcine ZP glycoproteins. Thus, it is suggested that the distinct carbohydrate-binding molecules are expressed on the acrosome-intact sperm of various species and may function in the gamete interaction. Solving this issue is important for the further molecular approach. Antibodies and lectins are widely used for histochemical or cytochemical detection of membrane components or glycoconjugates. However, detection of carbohydrate-binding molecules requires specific probes containing high-affinity ligands. Recently, we established the method for synthesis of fluorescence-labeled or biotinylated dextran (Dex)-based multivalent oligosaccharide probes (Yoshitani and Takasaki, 2000
). In the present study, we apply various oligosaccharide-dextran probes prepared by this method to cytochemical detection of the putative carbohydrate-binding molecules on the cell surface of boar sperm. In addition, we quantitatively examined binding of the probes to isolated boar sperm plasma membrane by a solid-phase assay and inhibitory effects of various glycoconjugates on their binding.
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Results |
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Discussion |
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Some proteins of the boar spermadhesin family are known to have carbohydrate binding activities (Topfer-Petersen et al., 1998). These proteins are abundant in seminal fluid, but a protein of this family, called AWN-1, also exists in cauda epididymal fluid and has been suggested to coat the plasma membrane overlying the acrosomal cap of sperm head in vivo (Dostalova et al., 1994
). As we used cauda epididymis sperm, one may suspect that one of the carbohydrate-recognition molecules detected in this study correspond to AWN-1. However, this is not the case, because they differ in the following respect. AWN-1 exhibits much higher binding affinity to core 1 O-glycans having the structure of ±Neu5Ac
2
3/6Galß1
3GalNAc than to N-glycans carrying the sialyl and nonsialyl N-acetyllactosamine sequence ±Neu5Ac
2
3/6Galß1
4GlcNAc (Dostalova et al., 1995
). On the other hand, binding of Fet- or AsFet-Dex containing sialo-/asialo-N-acetyllactosamine sequence to sperm was inhibited less efficiently by
-casein (Figure 7D), which contains mainly core 1 O-glycans (van Halbeek et al., 1980
), but almost completely by fetuin and asialofetuin at very low concentrations (Figure 7A,B). Indeed, we have previously demonstrated that boar sperm do not adhere to the beads coupled with
-casein (Mori et al., 2000
).
There are a few reports suggesting the occurrence of fucose-binding proteins on boar sperm. First, the head of freshly ejaculated, uncapacitated boar sperm has been visualized by fluorescence-labeled fucosyl peroxidase (Topfer-Petersen et al., 1985). However, it was later pointed out that proacrosin, the molecule detected by the fucosyl peroxidase, is not localized on the plasma membrane of intact boar sperm (Bozzola et al., 1991
). Another candidate of the fucose binding protein is P-selectin, a member of the selectin family with an affinity to sialyl Lex or sialyl Lea, that has been shown to occur in boar sperm (Geng et al., 1997
). However, P-selectin is localized on the acrosomal membrane, and mediates the acrosome-reacted sperm to oocytes in a Ca2+-dependent manner. These are in contrast with the fact that the Lex-binding protein detected in this study is found only on the acrosome-intact sperm and it binds to the oligosaccharide ligand in a Ca2+-independent manner.
Surprisingly, dextran sulfate (500 kDa) and fucoidin almost completely inhibit Fet- and AsFet-Dex binding to sperm plasma membrane at very low concentrations (Figure 9A,B). However, a less inhibitory potency of low molecular weight dextran sulfate (5 kDa) suggests that the inhibitory activity of sulfated polysaccharides might be due to a steric effect. Possibly the sulfated polysaccharides bind to some sites apart from the carbohydrate-binding sites on the putative recognition molecules, or to other unrelated molecules on the plasma membrane, and interfere with binding of the molecules with their carbohydrate ligands. Anyway, it is a remarkable finding that fucoidin and dextran sulfate can efficiently inhibit the Fet- and AsFet-Dex binding to sperm plasma membrane because they are well known to block boar spermegg binding (Peterson et al., 1984). Quite recently, we have also reported that N-glycans from fetuin and asialofetuin inhibited porcine sperm-egg binding (Mori et al., 2000
). Taking them into consideration, the sialo-/asialo-N-acetyllactosamine recognition molecule(s) detected in this study is strongly suggested to be involved in the initial spermegg binding. On the other hand, the Lex recognition molecule seems to be less important in the initial binding process. This is because LNFP IIIDex binding to sperm plasma membrane is inhibited almost completely by 0.15 mM of LNFP III (Figure 8C) but spermegg binding is slightly inhibited by 8 mM of LNFP III (Mori et al., 1998
). However, the Lex structure may have a cooperative effect on the spermegg binding because the structure is found on porcine ZP glycoproteins (Mori et al., 1997
).
Interestingly, binding of Fet- or AsFet-Dex to the acrosome-intact sperm greatly increases in the course of capacitation, but that of LNFP IIIDex moderately increases (Figure 2). As indicated in Figure 3, these two probes show distinct staining profiles of the sperm heads. Thus, the two kinds of carbohydrate-recognition molecules are expressed in a temporally and spatially different manner, suggesting that they have distinct physiological roles in fertilization. Because the molecule recognizing the Lex structure is considerably expressed on uncapacitated sperm, it may have other functions, such as sperm binding to the oviductal epithelium, which is considered to function as sperm reservoir (Suarez, 1998). Further study on the molecular basis will elucidate the possible functions of the sperm carbohydrate binding proteins.
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Materials and methods |
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Preparation of uncapacitated and capacitated sperm
Washing and capacitation of boar sperm were performed as previously described (Lynham and Harrison, 1998) with minor modifications. Sperm were collected from cauda epididymis by retrograde displacement with gentle air pressure, and were washed three times at room temperature (200 x g, 5 min) with 20 mM HEPES-buffered saline adjusted to pH 7.4 with NaOH, containing 137 mM NaCl, 2.5 mM KCl, and 10 mM glucose (medium H). The resulting washed sperm were incubated in the medium H at 25°C for 60 min and were used as uncapacitated sperm. For capacitation, the washed sperm were resuspended to about 1 x 108 cells/ml with capacitation medium (medium BHC) and incubated in a CO2 incubator at 39°C for 60 min. Medium BHC contained 20 mM HEPES, 96 mM NaCl, 15 mM sodium bicarbonate, 3.1 mM KCl, 5 mM glucose, 21.7 mM sodium lactate, 1 mM sodium pyruvate, 0.3 mM sodium phosphate, 0.4 mM MgSO4, 4.5 mM CaCl2, 2 mM caffeine, and BSA (5 mg/ml). Before use, medium BHC was incubated in a CO2 incubator at 39°C and adjusted to pH 7.4 with NaOH.
Staining of uncapacitated and capacitated sperm with fluorescence-labeled or biotinylated oligosaccharide-dextran probes
Capacitated or uncapacitated sperm (about 1 x 108 cells/ml) were washed with PBS (100 x g, 5 min) and immobilized by 0.01% (w/v) formaldehyde in PBS (Dott et al., 1976). The suspension was mixed gently, allowed to stand for 15 min at room temperature, centrifuged, and replaced with PBS. Fifteen microliters of capacitated or uncapacitated, immobilized sperm (about 5 x 107 cells/ml) were added to 15 µl of PBS containing BSA (1 mg/ml) with or without 5 mM EDTA and allowed to stand for short time, or 15 min in case EDTA was included. The sperm suspension (30 µl) was mixed with 3 µl of biotinylated oligosaccharide-dextran probes in PBS, each of which was made at a concentration of approximately 70 nM as dextran molecule. After incubation at room temperature for 10 min, 3 µl of streptavidinAlexa 546 (50 µg/ml in PBS) were added to the suspensions, and the mixture was further incubated for 15 min. Fluorescence microscopic observation was performed without washing. When Lucifer yellowlabeled oligosaccharide-dextran probes were used, fluorescence microscopic observation was performed 20 min after addition of the probes.
Induction of acrosome reaction
Capacitated sperm, which were not immobilized with 0.01% formaldehyde, in medium BHC (50 µl, about 5 x 107 cells/ml) were mixed with 5 µl of biotinylated oligosaccharide-dextran probes in PBS for 10 min, and then with 5 µl of streptavidinAlexa 546 in PBS for 10 min at room temperature. For induction of acrosome reaction, 60 µl of 20 µM of calcium ionophore A23187 in medium BHC, which was freshly prepared from a 10 mM stock in DMSO, were added to the prestained, capacitated sperm suspension. After incubation for 60 min at 39°C in a CO2 incubator, the suspension was pipetted and observed under the microscope.
Triton X-100 treatment of sperm
After washing with PBS, the capacitated sperm were incubated with 0.1% Triton X-100 in PBS containing 2 mM p-aminobenzamidine at room temperature for 5 min and washed with the buffer without Triton X-100. The sperm were stained by adding one-tenth volume of 100 µg/ml of PNATexas red in PBS or subjected to staining with biotinylated oligosaccharide-dextran probes. The capacitated sperm, which were prestained with biotinylated oligosaccharide-dextran probes and streptavidinAlexa 546, were incubated with an equal volume of 0.05% Triton X-100 in PBS containing 2 mM p-amino-benzamidine at room temperature for 5 min and were observed under the microscope.
Cell surface biotinylation and freeze-thawing of sperm
Capacitated sperm (2 x 108 cells/ml) were washed with medium H and centrifuged. After removing the supernatant, sulfosuccinimidyl-6-(biotinamido)hexanoate sodium salt (4 mg/ml), which was dissolved with medium H immediately before addition, was added to the packed cells. The cell suspension was incubated at 25°C for 30 min with occasional gentle mixing. After centrifugation the cells were resuspended with 0.01% formaldehyde in PBS, allowed to stand for 15 min at room temperature, centrifuged, and finally replaced with medium H containing 0.5 mg/ml of BSA. Aliquots of the cell suspension were immediately placed in a deep freezer (80°C) for 30 min and then thawed in a water bath at 25°C. The sperm before and after freeze-thawing were stained with Lucifer yellowlabeled oligosaccharide-dextran probes or PNATexas red as described above.
Preparation of boar sperm plasma membrane
Sperm plasma membrane was prepared according to the previous methods (Kaplan et al., 1984; Bellve, 1993
) with some modifications. Boar epididymal sperm collected with Beltsville thawing solution (Johnson et al., 1988
) were centrifuged at 400 x g (20 min, room temperature) and then resuspended with medium H followed by centrifugation again. Approximately 1020 ml of the packed cells were capacitated in 200 ml of medium BHC as described above. The capacitated sperm were centrifuged at 250 x g (15 min, room temperature), resuspended with PBS, and centrifuged again. To the packed cells were added ninefold volumes of chilled hypotonic medium composed of 5 mM KCl, 3 mM MgCl2, 1 mM EGTA, 2 mM p-aminobenzamidine, 1 mM phenylmethylsulfonyl fluoride (PMSF) in 10 mM HEPES, pH 7.2 adjusted with NaOH, and the suspension was pipetted vigorously and incubated for 5 min on ice. The cell suspension was homogenized with 10 strokes by a Dounce homogenizer. Immediately afterward, one-tenth volume of 1.6 M NaCl, 30 mM MgCl2, and 50 mM KCl in 100 mM TrisHCl, pH 7.4, was added to the suspension to restore isotonicity. The homogenized cell suspension was centrifuged at 3000 x g (10 min, 4°C), and then the supernatant was centrifuged at 5,000 x g (10 min, 4°C). Thus obtained supernatant was ultracentrifuged at 100,000 x g (30 min, 4°C), and the sperm plasma membrane pellet was collected. Afterward, the plasma membrane was resuspended with 0.8 mM PMSF in HBS and reultracentrifuged at 230,000 x g (30 min, 4°C). This washing step was repeated three times, but the final washing was done without PMSF. The isolated sperm plasma membrane was kept at 20°C after complete removal of the supernatant.
Solid-phase binding inhibition assay
Protein concentration of the isolated plasma membrane was quantitated by a BCA assay kit (Pierce) using BSA as standard. Wells of microtiter plates (Nunc-Immunoplate Maxisorp) were incubated overnight at 4°C with 100 µl of the sperm plasma membrane (6 µg/ml as a protein concentration) suspended in 20 mM HEPES and 150 mM NaCl, pH 7.2 (HBS). The membrane suspension could be stably kept at 4°C without addition of protease inhibitors at least for 1 week. All the procedures were thereafter carried out at room temperature. The well contents were discarded, and the wells were washed three times with 0.01% Tween 20 in HBS (HBS-Tween) and blocked with 200 µl of 2% BSA in HBS-Tween by incubation for 30 min. After washing with HBS-Tween once, 50 µl of each glycoconjugate in HBS-Tween at various concentrations (1100 µg/ml for glycoproteins and sulfated polysaccharides, 3300 µM for oligosaccharides) was added to the wells. After incubation for 15 min, 50 µl of various biotinylated oligosaccharide-dextran probes in HBS-Tween (1 nM as a dextran concentration) was added, and the plate was further incubated for 2 h. The well contents were discarded, and the wells were washed three times with HBS-Tween. To the wells were added 50 µl of horseradish peroxidaselabeled avidin (2 µg/ml in HBS-Tween). After incubation for 1 h, the wells were washed four times and then to the wells were added 300 µl of 0.1 M phosphate-citrate buffer (pH 4.8) containing 0.04% o-phenylenediamine and 0.003% hydrogen peroxide. The color was developed for 15 min and the reaction was stopped by addition of 25 µl of 8 N sulfuric acid. Absorbance was read at 492 nm in a microtiter plate reader.
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Abbreviations |
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Footnotes |
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References |
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