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Gamete Biology |
Division of Reproductive Biology,3 Department of Biochemistry and Molecular Biology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland 21205
The Johns Hopkins Kimmel Cancer Center and Department of Pharmacology and Molecular Sciences,4 Johns Hopkins University School of Medicine, Baltimore, Maryland 21231
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3]GlcNAc-R) was a potent, competitive inhibitor of in vitro sperm-ZP binding (Johnston et al. J Biol Chem 1998; 273: 1888–1895). However, those experiments did not define the particular step in the fertilization pathway that was blocked. The experiments described herein test the hypothesis that Lex-containing glycans are specific, competitive inhibitors of the binding of Alexa Fluor 568 fluorochrome (Alexa568)-labeled ZP3 to sperm and, thus, bind the same sperm surface sites as ZP3. Dose-response analyses demonstrated that these glycans are potent inhibitors (IC50 
180 nM), which at saturation, reduced Alexa568-ZP3 binding by 70%. A Lewis A (Lea)-capped glycan (Galß3[Fuc4]GlcNAc) was also a potent inhibitor (IC50 150–200 nM), but at saturation, it reduced Alexa568-ZP3 binding by only 30%. In contrast, nonfucosylated glycans with nonreducing GlcNAcß4 or Galß4 residues did not compete; neither did sialyl-Lex (Neu5Ac 3Galß4[Fuc3]GlcNAc-Lewis X) nor sulfo-Lex (3'-O-SO3-Lewis X). However, at saturation, Gal3Galß4GlcNAcß3Galß4Glc reduced Alexa568-ZP3 binding by 70% but with moderate apparent affinity (IC50 3000 nM). Fluorescence microscopy revealed that Alexa568-labeled Lex-Lac-BSA, Lea-Lac-BSA, and ZP3 bound to the same sperm surface domains. However, Lea-Lac did not inhibit binding of Alexa568-Lex-Lac-BSA, and Lex-Lac did not inhibit binding of Alexa568-Lea-Lac-BSA. Finally, Lex-Lac and Lea-Lac had an additive inhibitory effect on Alexa568-ZP3 binding. Thus, Lex is a ligand for a major class of ZP3 binding sites on mouse sperm, whereas Lea binding defines a different but less-abundant class of sites.
fertilization, gamete biology, sperm
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Current models of the fertilization pathway propose that adhesion of acrosome-intact mouse sperm to the ZP is mediated by a subset of O-linked glycans of ZP3 [8, 9]. Experiments to assess structural features of these functional glycans have relied mainly on a competitive sperm-ZP binding assay (for details, see [10]). In this assay, capacitated sperm are incubated with ZP-enclosed eggs in the presence of potential competitors and bound sperm are enumerated. The first competitors tested were purified ZP3 and partially purified mixtures of glycans released from ZP3 that had been modified by the addition or removal of a specific monosaccharide [11]. Modifications that altered inhibitory activity were interpreted to reveal important structural features of a sperm-binding glycan. Subsequently, glycans with defined structures were analyzed for their potential inhibitory activity [10, 12–14]. The most potent inhibitor was assumed to best mimic the essential structure of a sperm-binding glycan on mouse ZP3.
To gain insight into the essential structures of sperm-binding glycans on ZP3, we previously asked whether a related series of glycans of defined structures could inhibit the in vitro binding of sperm to ZP-enclosed eggs [10]. Results identified a glycan with a terminal Lewis X (Lex) structure, (Galß4[Fuc3]GlcNAc-R), as a specific and potent inhibitor (IC50 500 nM). This result suggested that Lex was a good mimic for the intrinsic sperm-binding glycans on ZP3 implicated in sperm-ZP adhesion. We recognized, however, that the competitive sperm-ZP binding assay did not prove that Lex was a ligand for a ZP3 binding site. Neither did this assay identify the specific step in the first half of the fertilization cascade that was inhibited. It was possible that Lex did not bind a ZP3 binding site, but rather a site mediating other steps in the cascade, ranging from loose attachment of sperm to the ZP, to ZP2-mediated sperm adhesion. Indeed, this potential problem is shared by all other studies employing the competitive sperm-ZP binding assay and may explain why different laboratories have proposed different glycan structures for a sperm-binding ligand on ZP3 [13]. Clearly, a more direct experimental strategy was needed to determine whether a glycan was a ligand for a ZP3 binding site on capacitated, acrosome-intact mouse sperm. The strategy used in the experiments described herein tested whether Lex was a potent and specific inhibitor of the binding of purified ZP3 to complementary sites on acrosome-intact mouse sperm. A positive outcome of this test would directly support the conclusion that Lex is a structural mimic for a subset of sperm-binding glycans on ZP3, which, based on current models, mediates the firm adhesion of sperm to the ZP.
The experiments in this study employ a recently described quantitative assay of the binding of ZP3 to live, acrosome-intact mouse sperm [6]. The ligand for this assay is purified ZP3 labeled with the fluorochrome Alexa Fluor 568 (Alexa568). Fluorescence microscopy in combination with image analysis of bound Alexa568-ZP3 allows simultaneous localization of ZP3 binding sites and measurement of the amount of bound fluorescent ligand. Using this assay, we previously identified ZP3 binding sites on two plasma membrane domains of capacitated mouse sperm. One domain overlies the acrosomal crest and the other overlies the posterior sperm head. Dose-response analysis demonstrated saturable, high-affinity (Km 63 nM) binding of Alexa568-ZP3 to the sites on both membrane domains. Inhibition by ZP3 but not by ZP2 or ZP1 demonstrated that this binding was specific. Additionally, it was shown that capacitation of sperm and extracellular calcium was required for this specific, high-affinity binding to occur. Thus, the binding of Alexa568-ZP3 to sperm is specific, saturable, and, like fertilization, requires calcium and capacitation of sperm. It follows that a glycan that is a specific and potent competitive inhibitor of sperm-ZP3 binding must share essential structural features with one or more intrinsic sperm-binding glycans on ZP3.
Results of the experiments described herein demonstrate that Lex-containing glycans are potent, competitive inhibitors of approximately 70% of ZP3 binding sites on sperm. A glycan that terminates in the structure of Lewis A (Lea; Galß3[Fuc4]GlcNAc) was also a potent, competitive inhibitor of a second class of ZP3 binding sites not recognized by Lex. In contrast, a series of glycans with other nonreducing termini implicated in sperm-ZP binding were not inhibitors [12, 15]. We conclude that Lex is a structural mimic of a glycan on ZP3 that binds to a major class of ZP3 binding sites on capacitated, acrosome-intact mouse sperm. Lea is a structural mimic of a different glycan on ZP3 that binds a second but less-abundant class of sperm surface sites.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The structures of the unconjugated glycans and neoglycoproteins, and their corresponding abbreviations are provided in Figure 1. Both neoglycoproteins and glycans were dissolved in water as 2 mg/ml stock solutions and stored at –20°C.
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Isolation of ZP3
ZP3 was separated from ZP2 and ZP1, and purity was established as previously described [6]. In brief, ZPs were isolated from frozen ovaries of 6- to 7-wk-old ICR mice (Harlan BioProducts for Science, Indianapolis, IN) using Percoll (Amersham-Pharmacia Biotech, Piscataway, NJ) gradient centrifugation. ZP3 was isolated from heat-solubilized ZP by high-performance liquid chromatography using a 250 Bio-Sil Select size exclusion column (Bio-Rad Laboratories, Hercules, CA). Proteins in selected chromatographic fractions were resolved by SDS gel electrophoresis. Purity of ZP3 in these fractions was established by silver staining and by Western blot analysis as previously described [6]. Chromatographic fractions containing only ZP3 were pooled, dialyzed exhaustively against water, concentrated, and stored at –20°C.
Conjugation of the Fluorochrome Alexa568 to ZP3 and to Neoglycoproteins
Alexa568 was conjugated to ZP3 and to neoglycoproteins according to the manufacturer's instructions (Molecular Probes, Eugene, OR). The level of substitution was approximately 1 mole of fluorochrome per mole of protein. Formulas for determining both protein concentrations and levels of substitution with Alexa568 fluorochrome were provided by the manufacturer and used as previously described [6].
Culture Media
The medium used for all experiments was Medium 199 (M199; Life Technologies, Gaithersburg, MD) supplemented with 4 mg/ml crystalline BSA and 273 µM sodium pyruvate (M199-M; Sigma, St. Louis, MO) [6]. During incubation with either ZP3 or neoglycoproteins, this medium was supplemented with 50 µM 2-aminoethoxydiphenylborate (2-APB; Calbiochem, San Diego, CA), a cell-permeable inhibitor of inositol 1,4,5-triphosphate-induced Ca2+ release [16]. This inhibitor blocks the ability of sperm to undergo an agonist-induced acrosome reaction [17]. Double-strength M199-M was prepared from powdered M199 (Life Technologies) to which was added 8 mg/ml BSA and 546 µM sodium pyruvate. Prior to each experiment, 2x M199-M was mixed 1:1 (vol:vol) with the stock solutions of glycans, neoglycoproteins, or ZP3.
Isolation and Capacitation of Sperm
Sperm were collected from the cauda epididymides of two 8- to 12-wk-old proven-breeder CD-1 mice (Charles River Breeding Labs, Wilmington, MA) and allowed to swim up for 45 min into 2 ml of M-199M. This procedure and all incubations of sperm were conducted at 37°C in an atmosphere of 95% air, 5% CO2. The capacitated sperm in the upper 0.5 ml of medium were collected and used for the experiments described herein [6]. As measured by the ability of ionophone (50 µM A23187) to induce sperm to undergo the acrosome reaction, 87% ± 1% of the sperm were capacitated following this incubation (see [6] for methods used to distinguish acrosome-intact from acrosome-reacted sperm). The use of animals for these experiments was approved by the Animal Care and Use Committee of the Johns Hopkins University Bloomberg School of Public Health.
Incubations of Sperm with Alexa568-Labeled ZP3 and Neoglycoproteins in the Presence or Absence of Unlabeled Glycans
Experiments described herein had three objectives. The first objective was to determine whether Lex-containing glycans or nine other glycans were dose-dependent competitive inhibitors of the binding of Alexa568-ZP3 to capacitated mouse sperm. The other nine unconjugated glycans that were tested were either structurally related to Lex or had nonreducing termini found on other glycans implicated in sperm-ZP binding [12, 15]. Approximately 75 000 live capacitated mouse sperm were incubated for 1.5 h with 36 nM Alexa568-ZP3 in the presence or absence of 2-, 5-, 10-, 50-, 100-, or 1000-fold molar excess of unlabeled glycan. The sperm were then washed, fixed in 4% formaldehyde, and dried onto glass slides as previously described [6]. The fixed sperm were then examined microscopically and fluorescence was quantified (see Fluorescence Microscopy and Quantitative Analysis, below).
The second objective was to determine whether Alexa568-Lex-Lactose (Lac)-BSA and Alexa568-Lea-Lac-BSA bound to one or both of the sperm surface domains bound by Alexa568-ZP3 [6]. In the first of two experiments, we examined neoglycoproteins bound to live sperm surrounded by an intact membrane. This experiment did not discriminate between the possibilities that the acrosomal crest was covered by the plasma membrane or by the inner acrosomal membrane. Live capacitated sperm were incubated for 1 h with 180 nM Alexa568-Lex-Lac-BSA, Alexa568-Lea-Lac-BSA, or Alexa568-ZP3. Next, sperm were washed, pelleted, and resuspended in M199-M supplemented with 50 nM SYTOX Green (Molecular Probes, Eugene, OR), a membrane-impermeant fluorescent nucleic acid dye. Sperm were then allowed to adhere to CellTak-coated glass coverslips (BD Biosciences, Bedford, MA), and the live sperm were immediately examined by fluorescence microscopy [6]. The objective of the second experiment was to confirm that the neoglycoproteins were binding to acrosome-intact sperm. Capacitated sperm were incubated for 1 h with 180 nM Alexa568-Lex-Lac-BSA or Alexa568-Lea-Lac-BSA, washed, fixed in 70% ethanol, and the acrosome was stained with fluorescein isothiocyanate-conjugated peanut (Arachis hypogea) agglutinin (FITC-PNA; Vector Labs, Burlingame, CA) [6]. Sperm were then dried onto glass slides and examined by fluorescence microscopy as described below.
The third objective was to determine whether Lex-Lac or Lea-Lac were ligands for the same or different sites on sperm. The first of two experiments tested whether Lex-Lac or Lea-Lac were competitive inhibitors of the binding of Alexa568-Lex-Lac-BSA or Alexa568-Lea-Lac-BSA to capacitated mouse sperm. Sperm were incubated for 1.5 h with 36 nM of one of the two fluorescent neoglycoproteins and in the presence or absence of 100-fold or 1000-fold molar excess of Lex-Lac or Lea-Lac. The sperm were then washed, fixed in 4% formaldehyde, and dried onto glass slides [6]. The fixed sperm were then examined microscopically and the fluorescence was quantified. The second of two experiments tested whether there was an additive inhibitory effect of Lex-Lac or Lea-Lac on binding to sperm of Alexa568-ZP3. Sperm were incubated for 1.5 h with 36 nM Alexa568-ZP3 with and without a 5-fold molar excess of Lex-Lac, a 50-fold and 100-fold molar excess of Lea-Lac, or a 5-fold and 50-fold molar excess of Lex-Lac and Lea-Lac, respectively. Sperm were then washed, fixed, dried into glass slides, examined by fluorescence microscopy, and the fluorescence was quantified.
Fluorescence Microscopy and Quantitative Analysis
Fluorescence images were captured using a Nikon Eclipse E800 microscope (Nikon, Inc., Melville, NY) equipped with a 40x Plan Apo lens and a Princeton 5-MHz cooled interlined charge-coupled device (CCD) camera (Princeton Instruments, Trenton, NJ). Alexa568 fluorescence was detected using a G2E-RHOD excitation filter, a 565-nm dichroic mirror, and a barrier filter with a bandwidth of 600–660 nm. The binding to sperm of FITC-PNA or of SYTOX-Green was detected using an FITC excitation filter, a 505-nm dichroic mirror, and a barrier filter with a bandwidth of 515–555 nm. Fluorescence filters were manufactured by Chroma, Inc. (Burlington, VT).
The binding of Alexa568-Lex-Lac-BSA, Alexa568-Lea-Lac-BSA, and Alexa568-ZP3 to sperm was quantified using IPLab Spectrum analysis software (Scanalytics, Fairfax, VA) as previously described [6]. Published results document a linear response of the CCD camera over the range of fluorescence measurements made in these experiments (for details see [6]). For each experiment, all treatments were analyzed in duplicate, and every experiment was replicated two to three times. For each treatment, quantitative data were obtained from 200–900 sperm and presented as the percentage of the fluorescence emitted from positive control sperm that were incubated with only the Alexa568-labeled ligand. Dose-response curves for individual oligosaccharides were analyzed as previously described [10]. Briefly, data were fit to a rectangular hyperbola, and regression analysis was performed using the SigmaPlot program (SPSS, Chicago, IL). This program estimated the IC50 value (molar concentration of glycan producing a 50% inhibition in the binding of the Alexa568-ZP3 to sperm), percent maximal inhibition, and fit of the data to the regression line (R2 and P values). In experiments that compared effects of individual treatments on binding of fluorescent ligands to sperm, differences among means were established using analysis of variance, and effects of individual treatments were compared using the Fisher least significant difference test and the Statistical Analysis Systems Statistical Package version 6.1 (SAS Institute, Inc., Cary, NC). For all analyses, statistically significant differences were defined as P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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70% of ZP3 Binding Sites on Capacitated, Acrosome-Intact Mouse Sperm
The hypothesis that Lex-containing glycans are high-affinity ligands for ZP3 binding sites predicted that these glycans are potent, dose-dependent inhibitors of the specific and saturable binding of ZP3 to sperm. To test this prediction, a dose-response curve was generated by incubating capacitated mouse sperm with 36 nM Alexa568-ZP3 alone or also with increasing concentrations of Lex-Lac (72 nM to 36 000 nM). Regression analysis demonstrated that the data fit a rectangular hyperbola (R2 = 0.94, P < 0.05). This statistical analysis estimated an IC50 value of 105 ± 18 nM Lex-Lac with a saturating dose of glycan (1800 nM to 36 000 nM) reducing bound Alexa568-ZP3 to 30% of control (Fig. 2A, closed circles). Identical results were obtained when the Lex trisaccharide was used as a competitive inhibitor (Fig. 2A, open squares). Taken together, these results indicate that oligosaccharides with a nonreducing terminal Lex trisaccharide are high-affinity ligands for about 70%, but not all, ZP3 binding sites on capacitated, acrosome-intact mouse sperm. Additionally, these data indicate that addition of lactose to the one carbon of the GlcNAc residue of Lex, which stabilizes the ring structure of the GlcNAc and locks the number-one carbon of GlcNAc in the ß position, does not affect the affinity of Lex-containing glycans for ZP3 binding sites on sperm.
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Complementary experiments tested the abilities of two nonfucosylated glycans, [GlcNAc]3 and Gal3-Lac to inhibit the binding of Alexa568-ZP3 to sperm. [GlcNAc]3 at concentrations ranging from 72 nM to 36 000 nM did not inhibit this binding (data not shown). Additionally, Gal3-Lac at concentrations ranging from 72 nM to 1800 nM was an ineffective competitor. However, substantially higher concentrations (3600 nM and 36 000 nM; 100-fold and 1000-fold molar excess, respectively) reduced Alexa568-ZP3 binding to 27% ± 2% of control, producing an estimated IC50 of 3000 nM (data not shown). Results obtained with both glycans are consistent with data previously obtained from the in vitro competitive sperm-ZP binding assay [10].
Removal of the Fucosyl Residue from the Lex Trisaccharide or Addition of Either an 3-Sialyl or a 3'-Sulfo Group to the Galactosyl Residue Abolishes the Glycan's Ability to Act as a Competitive Inhibitor of ZP3 Binding
The results of the experiments described above support the conclusion that Lex-containing glycans are potent, competitive inhibitors of ZP3 binding to sperm. What is the consequence of modifying the Lex structure? To answer this question, we tested the effect of adding a negatively charged sialyl (Neu54Ac3) or sulfo (3'-O-SO3) residue to Lex or of removing the fucosyl residue. Neither sialyl-Lex, sulfo-Lex, nor ßGal, at concentrations as high as 36 000 nM (1000-fold molar excess), inhibited binding of Alexa568-ZP3 to sperm. In contrast, Lex reduced binding to 30% of control (Fig. 2B).
A Lewis A (Galß3[Fuc4]GlcNAc)-Containing Glycan Is a Potent, Competitive Inhibitor of 30% of ZP3 Binding Sites on Capacitated, Acrosome-Intact Mouse Sperm
Lea is a structural isomer of Lex in which the positions of the galactose and fucose residues are interchanged. Because both sialyl-Lex- and sialyl-Lea-containing glycans bind to the human cell surface lectin, L-selectin, Lea-Lac (Fig. 1) might bind the same ZP3 binding sites as Lex-Lac [18, 19]. To test this possibility, a dose-response curve was generated by incubating sperm with 36 nM Alexa-ZP3 alone or with increasing concentrations of Lea-Lac (72 nM to 36 000 nM; 2-fold to 1000-fold molar excess). Results showed that this glycan was a dose-dependent inhibitor of Alexa568-ZP3 binding (Fig. 3). Regression analysis demonstrated that the data fit a rectangular hyperbola (R2 = 0.97, P < 0.05). This statistical analysis estimated an IC50 value of 221 ± 20 nM Lea-Lac with a saturating dose of glycan (1800 nM to 36 000 nM) reducing bound Alexa568-ZP3 to 70% of control. This result suggested that Lea-Lac was recognized by a smaller fraction of sperm-surface ZP3 binding sites than Lex-Lac. To confirm this conclusion statistically, data for both dose-response curves (Lex-Lac and Lea-Lac vs. Alexa568-ZP3) were compared by two-way analysis of variance. This analysis confirmed that Lex-Lac recognized a larger fraction of ZP3 binding sites than Lea-Lac.
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Lex-Lac-BSA and Lea-Lac-BSA Bind to the Same Domains as ZP3 on the Plasma Membrane of Capacitated, Acrosome-Intact Sperm
Alexa568-ZP3 binds to two distinct domains in the plasma membrane of the mouse sperm, the domain over the acrosomal crest and the domain over the posterior sperm head [6]. It was therefore possible that the ZP3 binding sites on a given domain would preferentially bind Lex-Lac or Lea-Lac. To assess this possibility we used as ligands Alexa568-Lex-Lac-BSA and Alexa568-Lea-Lac-BSA. Preliminary experiments demonstrated that the two fluorochrome-labeled neoglycoproteins bound sperm in a saturable manner with half-maximal saturation (Kd) achieved at approximately 150 nM Alexa568-Lex-Lac-BSA and 175 nM Alexa568-Lea-Lac-BSA (data not shown).
The first experiment examined the binding of these fluorochrome-labeled neoglycoproteins to live sperm. Capacitated sperm were incubated with 180 nM of Alexa568-Lex-Lac-BSA, Alexa568-Lea-Lac-BSA, or Alexa568-ZP3, washed, and incubated with SYTOX Green, a membrane-impermeant nucleic acid dye. Living sperm were examined immediately thereafter. Microscopic analysis revealed that Alexa568-Lex-Lac-BSA and Alexa568-Lea-Lac-BSA bound to the same two membrane domains as ZP3, the membrane overlying the acrosomal crest and the posterior sperm head (Fig. 4, A, C, and E). These sperm were not labeled with SYTOX Green and thus, were alive and surrounded by an intact membrane (Fig. 4, B, D, and F). In contrast, dead or dying cells were intensely stained with this fluorescent nucleic acid dye (Fig. 4G).
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To confirm that Alexa568-Lex-Lac-BSA and Alexa568-Lea-Lac-BSA were binding to acrosome-intact sperm, live, capacitated sperm were incubated with these two neoglycoproteins, washed, fixed with 70% ethanol, and then stained with FITC-PNA, which binds to acrosomal glycoproteins [6]. Results confirmed that Alexa568-Lex-Lac-BSA and Alexa568-Lea-Lac-BSA were binding to acrosome-intact sperm (compare Fig. 4, H and J with Fig. 4, I and K). Thus, the data indicate that the entire sperm head, including the acrosomal crest, was covered by the plasma membrane. Taken together, these results indicate that Lex-Lac-BSA, Lea-Lac-BSA, and ZP3 bind to the same plasma membrane domains of capacitated, acrosome-intact mouse sperm. These observations raised the issue of whether Lex and Lea are ligands for different classes of ZP3 binding sites that were present on these two domains.
Lex-Lac and Lea-Lac Recognize Different Classes of ZP3 Binding Sites on Sperm
If Lex-Lac and Lea-Lac are recognized by two distinct classes of ZP3 binding sites, then the two glycans would be predicted not to compete with each other for their respective sites. Additionally, the two glycans would be predicted to have an additive inhibitory effect on the binding of Alexa568-ZP3. To test the first prediction, sperm were incubated with 36 nM Alexa568-Lex-Lac-BSA alone or with either a 100-fold or 1000-fold molar excess of Lex-Lac or Lea-Lac. Results (Fig. 5A) showed that both concentrations of Lex-Lac displaced 60% of the binding of Alexa568-Lex-Lac-BSA, whereas Lea-Lac did not compete. Sperm were also incubated in 36 nM Alexa568-Lea-Lac-BSA alone or with a 100-fold or 1000-fold molar excess of Lex-Lac or Lea-Lac. Similar to the results shown in Figure 5A, significant inhibition of Alexa568-Lea-Lac binding was observed only with Lea-Lac (Fig. 5B).
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To test the second prediction, capacitated sperm were incubated with 36 nM Alexa568-ZP3 and in the presence or absence of 180 nM Lex-Lac (5-fold molar excess), 1800 nM or 3600 nM Lea-Lac (50-fold or 100-fold molar excess), or 180 nM Lex-Lac plus 1800 nM Lea-Lac (Fig. 6). We used saturating concentrations of Lea-Lac but not of Lex-Lac because of the low level of fluorescence from sperm incubated with 36 nM Alexa568-ZP3 plus a saturating concentration of Lex-Lac. Thus, in the presence of a saturating concentration of Lex-Lac, the effect of adding a second competitor would be difficult to detect.
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In this experiment, the addition of 180 nM Lex-Lac to sperm reduced the binding of Alexa568-ZP3 binding to 45% of control (Fig. 6). Additionally, both concentrations of Lea-Lac reduced Alexa-ZP3 binding to 75% of control, confirming that 1800 nM Lea-Lac was saturating. However, addition of 180 nM Lex-Lac to a saturating dose of Lea-Lac further reduced the binding of Alexa568-ZP3 to 20% of control. This additive inhibitory effect of Lex-Lac and Lea-Lac on Alexa568-ZP3 binding supports the conclusion that these two glycans recognize distinct classes of ZP3 binding sites on capacitated mouse sperm.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Evidence that Lex Is a Structure-Specific Ligand for a Major Class of ZP3 Binding Sites on Capacitated, Acrosome-Intact Mouse Sperm
Our results demonstrate that at saturating concentrations, Lex and Lex-Lac reduced binding of Alexa568-ZP3 to sperm by 60% to 70%. It was noteworthy that the IC50's of these glycans (180–300 nM) were in the same range as Lex in the competitive sperm-zona binding assay (500 nM) and the affinity of Alexa568-Lex-Lac-BSA (Kd 150 nM) for mouse sperm [10]. Thus, collectively, these results support the conclusion that Lex and Lex-Lac bind the same sites on sperm as ZP3. Furthermore, the apparent high affinity of these glycans supports the conclusion that glycans on ZP3 mediate tight adhesion of sperm to the ZP.
Our experiments also provide some insight into the structural requirements of the ligands for ZP3 binding sites. Consistent with its ability to inhibit the binding of sperm to ZP-enclosed eggs, our current studies identify Gal-Lac as a moderate-affinity (IC50 3 µM) competitive inhibitor of Alexa568-ZP3 binding. However, although Johnston et al. [10] identified a ßGal-capped glycan as a low-affinity competitive inhibitor (IC50 42 µM) of sperm-zona binding, ßGal did not inhibit the binding of Alexa568-ZP3 to sperm. A potential explanation for this apparent discrepancy comes from preliminary results indicating that ßGal-BSA inhibits the binding of Alexa568-ZP2, but not Alexa568-ZP3 to mouse sperm (unpublished results).
The conclusion that ßGal is not a ligand for ZP3 binding sites emphasizes the requirement of the 3-fucosyl residue of Lex for producing a high-affinity ligand. Our results also indicate, however, that the molecular context of the fucose residue determines whether a fucosylated glycan is a ligand for these sites. Neither sialyl-Lex nor sulfo-Lex are ligands for ZP3 binding sites. However, Johnston et al. [10] demonstrated that 3-galactosyl-Lex and Lex are equally effective inhibitors of sperm-zona binding. Thus, we suggest that the reason sialyl-Lex and sulfo-Lex are not ligands for a ZP3 binding site is due to their negative charges.
Although Lex is a ligand for about 60%–70% of all ZP3 binding sites on acrosome-intact mouse sperm, results in Figures 5 and 6 indicate that those sites do not recognize the structural isomer of Lex, Lea. However, our data indicate that Lea is recognized by a second class of ZP3 binding sites that have an apparent low affinity, if any, for Lex. Thus, an interesting conclusion from these studies is that mouse sperm has two distinct ZP3 binding sites that can be distinguished by their capacity to recognize Lex and Lea. On capacitated mouse sperm, the sites that recognize Lex appear to be twice as abundant as the sites that recognize Lea.
What Is the Structural Basis for the Specific Binding of Lex and Lea to Distinct Classes of ZP3 Binding Sites?
The apparent high affinity of Lex, Lex-Lac, and Lea-Lac for ZP3 binding sites on sperm supports the conclusion that these glycans are structural mimics of sperm-binding glycans on ZP3. Our data also indicate that Lex-containing glycans and Lea-containing glycans are ligands for distinct sperm-surface ZP3 binding sites. What are the structural differences between Lex and Lea that would allow them to be differentially recognized by these two sites? As pointed out by Bush and colleagues, the minimal energy confirmations of Lex and Lea, determined by nuclear magnetic resonance, reveal that these two trisaccharides have remarkably overall similar structures [22, 23]. Both glycans are compact, rigid structures with essentially identical stacking between the fucose and galactose rings. The only major structural difference between these two trisaccharides is the orientation of the GlcNAc residue relative to the stacked rings. The GlcNAc residue of Lex is rotated approximately 180° relative to the GlcNAc residue of Lea [23, 24]. Consequently, the C2 acetoamido group and the C6 hydroxymethyl group of GlcNAc are in opposite orientation in the two trisaccharides. Thus, the opposite orientations of the GlcNAc residue of Lex and Lea may explain at the molecular level why these two glycans are ligands for distinct ZP3 binding sites on mouse sperm.
While the orientation of the GlcNAc residue potentially confers the specificity of Lex and Lea for their respective sperm-surface sites, it is the fucosyl residue that confers high affinity. This requirement of the fucosyl residue has also been observed in the binding of both sialyl-Lex and sialyl-Lea to P, E, and L selectins [18, 19, 25–27]. Solution structures of sialyl-Lex and sialyl-Lea are similar to their nonsialylated counterparts with the exception of the addition a more flexible sialic acid residue [24]. Crystallographic studies of sialyl-Lex bound to either P or E selectin reveal that the GlcNAc residue does not form hydrogen bonds with the selectin's glycan-binding pocket. Rather, the fucose residue provides a substantial amount of the binding energy by coordinating the required calcium ion and forming hydrogen bonds with amino acids in the calcium-binding pocket [28]. This raises the possibility that the fucose residue in Lex and in Lea may play a similar role in the binding of these glycans to their respective sites on mouse sperm.
What are the Functions of the ZP3 Binding Sites Recognized by Lex and Lea?
While our experiments indicate that Lex and Lea can serve as ligands for distinct ZP3 binding sites, those experiments do not address the biological functions of those sites. Neither do they address whether the more abundant sites, which recognize Lex, play a more important role in the fertilization cascade than the sites recognized by Lea, it is possible that one or both of these sites are adhesion molecules whose primary functions are to tether sperm to the ZP. In that model, these sites are functionally analogous to L-selectin, which mediates the tethering of lymphocytes to high endothelial venules, whereas different molecules, acting as receptors and ligands, generate the signal transduction cascade that regulates the migration of these cells out of the vascular compartment (reviewed in [26, 29]). Alternatively, the sperm surface binding sites that recognize Lex or Lea may be functional receptors, which both tether acrosome-intact sperm to the ZP and mediate the responses of sperm to ZP3, culminating in the acrosome reaction. Because the glycans on ZP3 must be clustered on a polypeptide backbone to trigger the acrosome reaction, and because unconjugated Lex does not trigger the acrosome reaction, testing this proposal requires that Lex or Lea be clustered on a polypeptide backbone, such as BSA [30]. The next paper in this series [17] employs neoglycoproteins with glycans of defined structures to address whether the ZP3 binding sites recognized by Lex-Lac and Lea-Lac are, in fact, functional receptors.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: William W. Wright, Johns Hopkins University Bloomberg School of Public Health, Department of Biochemistry and Molecular Biology, Room 3508, 615 N. Wolfe Street, Baltimore, MD 21205. FAX: 410 614 2356; wwright1{at}jhem.jhmi.edu
Received: 30 September 2003.
First decision: 21 October 2003.
Accepted: 9 April 2004.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-D-mannosidase inhibits sperm-egg binding in vitro. Biol Reprod 1991 44:913-921[Abstract]This article has been cited by other articles:
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  S. A. Williams, L. Xia, R. D. Cummings, R. P. McEver, and P. Stanley Fertilization in mouse does not require terminal galactose or N-acetylglucosamine on the zona pellucida glycans J. Cell Sci., April 15, 2007; 120(8): 1341 - 1349. [Abstract] [Full Text] [PDF]
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 W. F. Hanna, C. L. Kerr, J. H. Shaper, and W. W. Wright Lewis X-Containing Neoglycoproteins Mimic the Intrinsic Ability of Zona Pellucida Glycoprotein ZP3 to Induce the Acrosome Reaction in Capacitated Mouse Sperm Biol Reprod, September 1, 2004; 71(3): 778 - 789. [Abstract] [Full Text] [PDF]
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