Glycobiology, 2001, Vol. 11, No. 4 313-320
© 2001 Oxford University Press
Detection of carbohydrate recognition molecules on the plasma membrane of boar sperm by dextran-based multivalent oligosaccharide probes
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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Two kinds of molecules, one recognizing the sialo-/asialo-N-acetyllactosamine structures and the other recognizing the Lewis X structure in a divalent cation–independent manner, were detected on the head of boar sperm prepared from cauda epididymis by fluorescence-labeled or biotinylated dextran-based multivalent oligosaccharide probes. The N-acetyllactosamine recognition molecule(s) is weakly detected on uncapacitated sperm and becomes strongly detectable on capacitated sperm. On the other hand, the Lewis X recognition molecule is detected at a moderate level before capacitation and at a high level after capacitation. Both molecules disappear from the sperm head after induction of acrosome reaction and also by mild detergent treatment. Thus, the two kinds of carbohydrate molecules are expressed on the plasma membrane of boar sperm depending on their physiological state. Inhibition study of the oligosaccharide-dextran probe binding to isolated sperm plasma membrane by various glycoproteins, oligosaccharides, and sulfated polysaccharides also supported the occurrence of the two distinct kinds of molecules.
Key words: boar sperm/carbohydrate binding molecule/oligosaccharide probe
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Introduction |
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Sperm–egg binding is an early and essential step in mammalian fertilization, and the importance of glycans on the zona pellucida (ZP) in the binding process is well accepted (Wassarman, 1988
). Structural analysis of mammalian ZP glycans are essential to identify the physiological ligands for sperm, but it difficult to accomplish due to their limited quantity and structural diversity. By taking advantage of being prepared in relatively large quantities, however, structures of porcine ZP glycans have been analyzed in detail by us (Mori et al., 1991, 1998; Hirano et al., 1993) and others (Noguchi et al., 1992; Noguchi and Nakano, 1992; Hokke et al., 1994). Mouse ZP glycans have also been elucidated (Noguchi and Nakano, 1993; Easton et al., 2000). Thus, it becomes possible to investigate the sperm–egg interaction based on the structural information about ZP glycans in limited species (Takasaki et al., 1999). Extensive efforts have also been made to discover carbohydrate recognition molecules on sperm, but accumulating evidence arouses much controversy (Tulsiani et al., 1997; Takasaki et al., 1999, Dell et al., 1999).
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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Detection of carbohydrate-binding molecules on boar sperm head by oligosaccharide-dextran probes
Multivalent oligosaccharide-dextran probes used in this study were listed in Figure 1. To take fine pictures of sperm stained by the oligosaccharide-dextran probes, it is desirable that sperm are immobilized. However, when capacitated sperm were fixed with 1% formaldehyde (w/v) in phosphate-buffer saline (PBS) before addition of the probes, no significant sperm staining was observed. This indicates that it is necessary to immobilize live sperm without inactivation of carbohydrate-binding proteins. Therefore, sperm were immobilized but not fixed by using a very low concentration of formaldehyde (0.01%) as previously described (Dott et al., 1976
) and then stained with the oligosaccharide-dextran probes, resulting in the successful staining as before immobilization. Because washing of sperm by centrifugation even at a low speed after their incubation with the oligosaccharide-dextran probes disrupted the staining pattern, we observed sperm without washing. Under the condition, fluorescence from the probes bound to the sperm head formed a striking contrast to that from the background.
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When Lucifer yellow–labeled Fet-Dex, AsFet-Dex, and lacto-N-fucopentaose III (LNFP III)–Dex probes were used under the condition as above mentioned, strong fluorescence staining of capacitated boar sperm were obtained (Figure 2A). As compared with capacitated sperm, uncapacitated sperm were stained very weakly by Fet-Dex and AsFet-Dex and moderately with LNFP III–Dex, respectively (Figure 2B). On the other hand, almost no fluorescence signal was observed using Ova-Dex from both uncapacitated and capacitated sperm (Figure 2A,B). AgFet-Dex exhibited no fluorescence signal either (data not shown). These results indicate that the molecule(s) recognizing the sialyl and nonsialyl N-acetyllactosamine structures included in fetuin and asialofetuin is expressed on the sperm head more prominently after capacitation. It is also evident that the molecule recognizing LNFP III (Lex structure) is already expressed at a moderate level on the head of uncapacitated sperm, and its expression further increases after capacitation. When sperm were stained by Fet-, AsFet-, or LNFP III–Dex in the presence of 2.5 mM EDTA, the observed fluorescence signal was very similar to that observed in its absence (data not shown), supporting the previous result that calcium ion is not essential for the binding (Mori et al., 2000
).
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Localization of carbohydrate-binding molecules
The indirect fluorescence microscopy using biotinylated oligosaccharide-dextran probes with streptavidin–Alexa 546 also gave the results that are similar to those obtained with fluorescence-labeled oligosaccharide-dextran probes as shown above. Typical examples are shown in Figure 3. The half rim-like area of the anterior region of the sperm head was strongly stained by both AsFet-Dex (Figure 3A) and LNFP III–Dex (Figure 3B). In addition, the whole anterior region of the majority of sperm was also stained by AsFet-Dex (Figure 3A). Biotinylated Fet-Dex stained the sperm similarly to the AsFet-Dex (data not shown). Thus, the staining patterns with Fet-, AsFet-, and LNFP III–Dex are distinguishable, suggesting that two kinds of carbohydrate-binding molecules occur differently on the sperm head.
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When capacitated sperm prestained with Fet-, AsFet-, or LNFP III–Dex underwent an induction of acrosome reaction by calcium ionophore A23187, which results in the release of plasma membrane and outer acrosomal membrane, most sperm were found to exhibit slight or no fluorescence signals on their heads in many fields (Figure 4). Instead, strong fluorescence signals were observed from membraneous materials (indicated by arrows in Figure 4) that were beside or apart from sperm heads. Such fluorescent membraneous materials were not observed before addition of calcium ionophore A23187 or when the capacitated sperm prestained by Ova-Dex were used (data not shown). When the capacitated sperm were poststained—that is, stained with the oligosaccharide-dextran probes after induction of acrosome reaction—similar results was also obtained (data not shown). These results indicate that the carbohydrate-binding molecules were released from sperm head through acrosome reaction.
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In addition, capacitated sperm treated with 0.1% Triton X-100 were not stained by Fet-Dex (Figure 5A), AsFet-Dex or LNFP III–Dex (data not shown), whereas the acrosomal shape of the detergent treated sperm was well preserved as evidenced by a strong staining with peanut agglutinin (PNA) (Figure 5A), which specifically binds to the outer acrosomal membrane but not to the plasma membrane (Fazeli et al., 1997
). When the sperm prestained with biotinylated Fet-Dex and streptavidine–Alexa 546 were treated with a lower concentration of Triton X-100 (0.025%), the signals on sperm heads were reduced with release of fluorescent membranous particles (white arrows in Figure 5B). These results suggest the occurrence of the carbohydrate-binding molecules on the plasma membrane. It is not likely from the following observation that the recognition molecules are also localized on the outer acrosomal membrane. Capacitated sperm reacted with a plasma membrane–impermeable biotinylating reagent, sulfosuccinimidyl-6-(biotinamido)hexanoate sodium salt (Hurley et al., 1985) were still motile and remained impermeable as evidenced by a negative staining with PNA–Texas Red, but were not stained with Lucifer yellow–labeled Fet-, AsFet-, or LNFP III–Dex. Thus, biotinylation blocked binding activity of the cell-surface recognition molecules. After freeze-thawing, biotinylated sperm became permeable as evidenced by a positive staining of the outer acrosomal membrane with PNA, but no significant fluorescent signal appeared from the permeable sperm, which were incubated with the Lucifer yellow–labeled oligosaccharide probes. The carbohydrate-binding activity itself sufficiently remained on the freeze-thawed unbiotinylated sperm. Considered together, it is suggested that the carbohydrate binding molecules are mostly localized on the plasma membrane.
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Specificity of the oligosaccharide-dextran probe binding to the isolated boar sperm plasma membrane
To get more quantitative results on sperm recognition of carbohydrates, we isolated plasma membrane from capacitated boar sperm and examined binding of the oligosaccharide-dextran probes to the membrane by a solid-phase assay. As shown in Figure 6, biotinylated Fet-, AsFet-, and LNFP III–Dex bound to the plasma membrane–coated wells in a dose-dependent manner, whereas biotinylated AgFet- and Ova-Dex did not. When the isolated plasma membrane was stained with Lucifer yellow–labeled oligosaccharide-dextran probes, the similar result was also obtained (data not shown). The observed binding specificity well reflects the fluorescence microscopic observation of capacitated boar sperm stained by the probes (Figure 2A). Thus, it is evident that the isolated plasma membrane sufficiently retains the multiple carbohydrate-binding activities.
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Inhibition of oligosaccharide-dextran probe binding to the isolated boar sperm plasma membrane by various glycoproteins and oligosaccharides
To find the background of multiple carbohydrate recognition by boar sperm, we first examined the inhibitory effect of distinct glycoproteins on binding between the oligosaccharide-dextran probes and sperm plasma membrane. As shown in Figure 7A and B, Fet- and AsFet-Dex bindings were almost completely inhibited by fetuin and asialofetuin at very low concentrations (approx. 1 µg/ml by asialofetuin and 10 µg/ml by fetuin), respectively. On the other hand, LNFP III–Dex binding was weakly inhibited by fetuin and moderately by asialofetuin (Figure 7A,B), but their inhibition curves seem to be somewhat peculiar. Similarly, ovalbumin and
-casein tend to weakly inhibit all the probe binding (Figure 7C,D). However, their efficiency is considerably lower than that of fetuin or asialofetuin, and the inhibition curves do not seem to fit a usual mode of inhibition.
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Then, we used N-glycans for the inhibition assay to exclude effects of the peptide moieties of glycoproteins. As shown in Figure 8A and B, N-glycans prepared from fetuin and asialofetuin remarkably inhibited Fet- and AsFet-Dex binding at approximately 10 µM but not significantly LNFP III–Dex binding. By contrast, LNFP III almost completely inhibited LNFP III–Dex binding at 150 µM, but not Fet- and AsFet-binding (Figure 8C). Ovalbumin oligosaccharides weakly inhibited Fet- and AsFet-Dex binding only at higher concentrations but did not significantly inhibit LNFP III–Dex binding (Figure 8D). Considered together, it is strongly suggested that the Lex recognition molecule is different from the sialo-/asialo-N-acetyllactosamine recognition molecule(s).
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Inhibition of oligosaccharide-dextran probe binding to the isolated boar sperm plasma membrane by various sulfated polysaccharides
Fucoidin and dextran sulfate are known to inhibit porcine sperm–egg binding (Peterson et al., 1984
). We examined their effect on the probe binding to the sperm plasma membrane. As shown in Figure 9A and B, fucoidin and dextran sulfate (500 kDa) inhibited Fet- and AsFet-Dex binding more efficiently than LNFP III–Dex binding. The less inhibitory activity of fucoidin to LNFP III–Dex binding is rather surprising. On the other hand, low molecular weight dextran sulfate (5 kDa) weakly inhibited only Fet- and AsFet-Dex binding at high concentrations (Figure 9C). In accordance with its effect on sperm–egg binding (Peterson et al., 1984), heparin showed slight or little inhibition (Figure 9D).
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Discussion |
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In the present study, we could detect at least two kinds of carbohydrate-recognition molecules on boar sperm head by using the various oligosaccharide-dextran probes that were synthesized according to our recently established method (Yoshitani and Takasaki, 2000
). One is the molecule(s) that binds to the oligosaccharide-dextran probes (Fet-/AsFet-Dex) containing the sialo-/asialo-N-acetyllactosamine structures, and the other is the molecule that binds to the probe (LNFP III–Dex) containing the Lex structure. These results accord well with the previous observation that boar sperm adhere to beads coupled with fetuin, asialofetuin, and Lex trisaccharide polymer (Mori et al., 2000). To be noted is that the two groups of molecules disappear from the acrosome-reacted sperm, suggesting that both are expressed on the sperm plasma membrane.
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
3/6Galß13GalNAc than to N-glycans carrying the sialyl and nonsialyl N-acetyllactosamine sequence ±Neu5Ac23/6Galß14GlcNAc (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 sperm–egg 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 sperm–egg binding. On the other hand, the Lex recognition molecule seems to be less important in the initial binding process. This is because LNFP III–Dex binding to sperm plasma membrane is inhibited almost completely by 0.15 mM of LNFP III (Figure 8C) but sperm–egg 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 sperm–egg 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 III–Dex 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
Fetuin, asialofetuin, ovalbumin,
-casein, fucoidin, dextran sulfate (approximately 5 and 500 kDa), calcium ionophore A23187, and p-aminobenzamidine dihydrochloride were purchased from Sigma Chemical Co. (St. Louis, MO). LNFP III, Galß14(Fuc13)GlcNAcß13Galß14Glc was purchased from Seikagaku Corp. (Tokyo). Bovine serum albumin (BSA), heparin sodium salt, o-phenylenediamine dihydrochloride, and dimethyl sulfoxide (DMSO) were purchased from Nacalai Tesque Inc. (Kyoto). Alexa 546–conjugated streptavidin (streptavidin–Alexa 546) was from Molecular Probes Inc. (Eugene, OR) and Texas red–labeled PNA (PNA–Texas red) was from EY Laboratories Inc. (San Mateo, CA). Sulfosuccinimidyl-6-(biotinamido)hexanoate sodium salt was purchased from Vector Laboratories Inc. (Burlingame, CA). Preparation of N-glycans and synthesis of oligosaccharide-dextran probes were carried out as previously described (Yoshitani and Takasaki, 2000).
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 streptavidin–Alexa 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 yellow–labeled 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 streptavidin–Alexa 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 PNA–Texas red in PBS or subjected to staining with biotinylated oligosaccharide-dextran probes. The capacitated sperm, which were prestained with biotinylated oligosaccharide-dextran probes and streptavidin–Alexa 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 yellow–labeled oligosaccharide-dextran probes or PNA–Texas 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 10–20 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 Tris–HCl, 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 (1–100 µg/ml for glycoproteins and sulfated polysaccharides, 3–300 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 peroxidase–labeled 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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BSA, bovine serum albumin; Dex, dextran; DMSO, dimethyl sulfoxide; Lex, Lewis X; LNFP III, lacto-N-fucopentaose III; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; PNA, peanut agglutinin; ZP, zona pellucida.
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Footnotes |
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1 To whom correspondence should be addressed
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References |
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Bellve, A.R. (1993) Purification, culture and fractionation of spermatogenic cells. Methods Enzymol., 225, 84–113.[Web of Science][Medline]
Bozzola, J.J., Polakoski, K., Haas, N., Russell L.D., Campbell, P., and Peterson, R.N. (1991) Localization of boar sperm proacrosin during spermatogenesis and during sperm maturation in the epididymis. Am. J. Anat., 192, 129–141.[Medline]
Dell, A., Morris H.R., Easton, R.L., Patankar, M., and Clark, G.F. (1999) The glycobiology of gametes and fertilization. Biochim. Biophys. Acta, 1473, 196–205.[Medline]
Dostalova, Z., Calvete, J.J., Sanz, L., and Topfer-Petersen, E. (1994) Quantitation of boar spermadhesins in accessory sex gland fluids and on the surface of epididymal, ejaculated and capacitated spermatozoa. Biochim. Biophys. Acta, 1200, 48–54.[Medline]
Dostalova, Z., Calvete, J.J., Sanz, L., and Topfer-Petersen, E. (1995) Boar spermadhesin AWN-1. Oligosaccharide and zona pellucida binding characteristics. Eur. J. Biochem., 230, 329–336.[Medline]
Dott, H.M., Moor, R.M., and Polge, C. (1976) Artificial insemination with spermatozoa in formaldehyde. J. Reprod. Fertil., 46, 277.
Easton, R.L., Patankar M.S., Lattanzio, F.A., Leaven, T.H., Morris, H.R., Clark, G.F., and Dell, A. (2000) Structural analysis of murine zona pellucida glycans. Evidence for the expression of core 2-type O-glycans and the Sd(a) antigen. J. Biol. Chem., 275, 7731–7742.
Fazeli, A., Hage, W.J., Cheng, F.-P., Voorhout, W.F., Marks, A., Bevers, M.M., and Colenbrander, B. (1997) Acrosome-intact boar spermatozoa initiate binding to the homologous zona pellucida in vitro. Biol. Reprod., 56, 430–438.[Abstract]
Geng, J.G., Raub, T.J., Baker, C.A., Sawada, G.A., Ma, L., and Elhammer, A.P. (1997) Expression of a P-selectin ligand in zona pellucida of porcine oocytes and P-selectin on acrosomal membrane of porcine sperm cells. Potential implications for their involvement in sperm-egg interactions. J. Cell Biol., 137, 743–754.
Hirano, T., Takasaki, S., Hedrick, J.L., Wardrip, N.J., Amano, J., and Kobata, A. (1993) O-linked neutral sugar chains of porcine zona pellucida glycoproteins. Eur. J. Biochem., 214, 763–769.[Medline]
Hokke, C.H., Damm, J.B.L., Penninkhof, B., Aitken, R.J., Kamerling, J.P., and Vliegenthart, J.F.G. (1994) Structure of the O-linked carbohydrate chains of porcine zona pellucida glycoproteins. Eur. J. Biochem., 221, 491–512.[Web of Science][Medline]
Hurley, W.L., Finkelstein, E., and Holst, B.D. (1985) Identification of surface proteins on bovine leukocytes by a biotin-avidin protein blotting technique. J. Immunol. Methods, 85, 195–202.[Web of Science][Medline]
Johnson, L.A., Aalbers, J.G., and Grooten, H.J.G. (1988) Artificial insemination of swin: fecundity of boar semen stored in Beltsville TS (BTS), Modified Modena (MM), or MR-A and inseminated on one, three and four days after collection. Zuchthygiene, 23, 49–55.
Kaplan, M., Russell, L.D., Peterson, R.N., and Martan, J. (1984) Boar sperm cytoplasmic droplets: their ultrastructure, their numbers in the epididymis and at ejaculation and their removal during isolation of sperm plasma membranes. Tissue Cell, 16, 455–468.[Web of Science][Medline]
Lynham, J.A., and Harrison, R.A.P. (1998) Use of stored pig eggs to assess boar sperm fertilizing functions in vitro. Biol. Reprod., 58, 539–550.
Mori, E., Hedrick, J.L., Wardrip, N.J., Mori, T., and Takasaki, S. (1998) Occurrence of reducing terminal N-acetylglucosamine 3-sulfate and fucosylated outer chains in acidic N-glycans of porcine zona pellucida glycoproteins. Glycoconj. J., 15, 447–456.[Web of Science][Medline]
Mori, E., Mori, T., and Takasaki, S. (1997) Binding of mouse sperm to ß-galactose residues on egg zona pellucida and asialofetuin-coupled beads. Biochem. Biophys. Res. Commun., 238, 95–99.[Web of Science][Medline]
Mori, E., Takasaki, S., Hedrick, J.L., Wadrip, N.J., Mori, T., and Kobata, A. (1991) Neutral oligosaccharide structures linked to asparagines of porcine zona pellucida glycoproteins. Biochemistry, 30, 2078–2087.[Medline]
Mori, E., Yoshitani, N., Mori, T., and Takasaki, S. (2000) Calcium ion-independent recognition of sialyl and nonsialyl N-acetyllactosamine and Lex structures by boar sperm. Arch. Biochem. Biophys., 374, 86–92.[Medline]
Noguchi, S., and Nakano, M. (1992) Structure of the acidic N-linked carbohydrate chains of the 55-kDa glycoprotein family (PZP3) from porcine zona pellucida. Eur. J. Biochem., 209, 883–894.[Web of Science][Medline]
Noguchi, S., and Nakano, M. (1993) Structural characterization of the N-linked carbohydrate chains from mouse zona pellucida glycoproteins ZP2 and ZP3. Biochim. Biophys. Acta, 1158, 216–217.
Noguchi, S., Hatanaka, Y., Tobita, T., and Nakano, M. (1992) Structural analysis of the N-linked carbohydrate chains of the 55-kDa glycoprotein family (PZP3) from porcine zona pellucida. Eur. J. Biochem., 204, 1089–1100.[Web of Science][Medline]
Peterson, R.N., Russell, L.D., and Hunt, W.P. (1984) Evidence for specific binding of uncapacitated boar spermatozoa to porcine zonae pellucidae in vitro. J. Exp. Zool., 231, 137–147.[Medline]
Suarez, S.S. (1998) The oviductal sperm reservoir in mammals: mechanisms of formation. Biol. Reprod., 58, 1105–1107.
Takasaki, S., Mori, E., and Mori, T. (1999) Structures of sugar chains included in mammalian zona pellucida glycoproteins and their potential roles in sperm-egg interaction. Biochim. Biophys. Acta, 1473, 206–215.[Medline]
Topfer-Petersen, E., Friess, A.E., Nguyen, H., and Schill, W.-B. (1985) Evidence for a fucose-binding protein in boar spermatozoa. Histochemistry, 83, 139–145.[Medline]
Topfer-Petersen, E., Romero, A., Varela, P.F., Ekhlasi-Hundrieser, M., Dostalova, Z., Sanz, L., and Calvete J.J. (1998) Spermadhesins: a new protein family. Facts, hypotheses and perspectives. Andrologia, 30, 217–224.[Web of Science][Medline]
Tulsiani, D.R.P., Yoshida-Komiya, H., and Araki, Y. (1997) Mammalian fertilization: a carbohydrate-mediated event. Biol. Reprod., 57, 487–494.[Web of Science][Medline]
van Halbeek, H., Dorland, L., Vliegenthart, J.F.G., Fiat, A.M., and Jolles, P.A. (1980) A 360-MHz 1H-NMR study of three oligosaccharides isolated from cow kappa-casein. Biochim. Biophys. Acta, 623, 295–300.[Medline]
Wassarman, P.M. (1988) Zona pellucida glycoproteins. Annu. Rev. Biochem., 57, 415–442.[Web of Science][Medline]
Yoshitani, N., and Takasaki, S. (2000) Microscale synthesis of dextran-based multivalent N-linked oligosaccharide probes. Anal. Biochem., 277, 127–134.[Medline]
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