Murine Sperm-Zona Binding, A Fucosyl Residue Is Required for a High Affinity Sperm-binding Ligand
A SECOND SITE ON SPERM BINDS A NONFUCOSYLATED, beta -GALACTOSYL-CAPPED OLIGOSACCHARIDE*

Daniel S. JohnstonDagger §, William W. WrightDagger , Joel H. Shaperpar , Cornelis H. Hokke**, Dirk H. Van den Eijnden**, and David H. Joziasse**

From the Dagger  Division Of Reproductive Biology, Department of Population Dynamics, The Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205-2179, the  Cell Structure and Function Laboratory, Oncology Center, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287-8937, and the ** Department of Medical Chemistry, Vrije Universiteit, 1081 BT Amsterdam, The Netherlands

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

An essential initial step in murine fertilization is the binding of acrosome-intact sperm to specific O-linked oligosaccharides on zona pellucida glycoprotein 3. While there is agreement on the primary role of O-linked glycans in this process, there is a lack of consensus on both the terminal monosaccharide(s) required for a functional sperm binding site and the corresponding protein on the sperm cell surface that recognizes this ligand. Much current debate centers on an essential role for either a terminal N-acetylglucosaminyl or, alternatively, a terminal alpha -galactosyl residue. To gain insight into the terminal saccharides required to form a functional sperm-binding ligand, dose-response curves were generated for a series of related tri- and tetrasaccharides to evaluate their relative effectiveness to competitively inhibit the in vitro binding of murine sperm to zona pellucida-enclosed eggs. A GlcNAc-capped trisaccharide, GlcNAcbeta 1,4GlcNAcbeta 1,4GlcNAc,was inactive (1-72 µM range). In contrast, a beta 4-galactosyl-capped trisaccharide (Galbeta 1,4GlcNAcbeta 1, 4GlcNAc) and an alpha 3-galactosyl-capped trisaccharide (Galalpha 1,3Galbeta 1,4 GlcNAc) inhibited sperm-zona binding with low or moderate affinity (ED50 = 42 µM and 5.3 µM, respectively). The addition of an alpha 3-fucosyl residue to each of these two competitive inhibitors, forming Galbeta 1,4[Fucalpha 1,3] GlcNAcbeta 1,4GlcNAc or Galalpha 1,3Galbeta 1, 4[Fucalpha 1,3]Glc NAc, resulted in ligands with 85- and 12-fold higher affinities for sperm, respectively (ED50 = 500 and 430 nM). Thus, the presence of a fucosyl residue appears to be obligatory for an oligosaccharide to bind sperm with high affinity. Last, mixing experiments with pairs of competitive inhibitors suggest that murine sperm-zona binding is mediated by two independent oligosaccharide-binding sites on sperm. The first (apparently high affinity) site binds both the alpha 3-galactosyl-capped trisaccharide and the two fucosylated tetrasaccharides. The second (apparently low affinity) site binds a nonfucosylated beta -galactosyl-capped trisaccharide.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The initial event in mammalian fertilization is the binding of a sperm to the zona pellucida (ZP),1 the extracellular glycoprotein matrix surrounding the egg. In the mouse, capacitated, acrosome-intact sperm initially bind to zona pellucida glycoprotein 3 (ZP3), one of three glycoproteins comprising the ZP (1, 2). The sperm binding activity of ZP3 is localized to a subset of O-linked oligosaccharides on ZP3 with an estimated molecular mass of about 3.9 kDa (3-5). While there is general agreement that the nonreducing terminal monosaccharides are critical for binding (4, 5), there is a lack of agreement on the identity of the terminal monosaccharide(s) required for a functional sperm-binding ligand and the corresponding binding site(s) on the sperm surface.

Two different models of murine sperm-ZP binding have been proposed that address the basic requirements for both a functional sperm-binding ligand and for the corresponding sperm surface ZP-binding protein. The first model posits that sperm surface beta 1,4-galactosyltransferase (beta 4GT) binds its acceptor sugar substrate, an N-acetylglucosaminyl residue (GlcNAc), located at the nonreducing terminus of an O-linked oligosaccharide on ZP3 (5, 6). In support of this model, beta 4GT has been localized immunocytochemically with a polyclonal antiserum to the sperm plasma membrane that overlies the acrosome (6). Biochemical and immunological probes that can potentially interact with cell surface beta 4GT block the ability of sperm to bind zona-intact eggs in vitro (6). The activity of ZP3 in a competitive sperm-ZP binding assay was reduced either by enzymatic removal or beta 4-galactosylation of terminal GlcNAc residues (5). In conflict with this model are observations from two laboratories that independently report the generation of mice in which the beta 4GT gene is inactivated by homologous recombination (7, 8). The null mice survive to term, a significant percentage survive to maturity, and the males are fertile.

The second model postulates that a sperm surface protein, sp56, binds nonreducing terminal alpha -galactosyl residues on ZP3 (4, 9-12). This model is supported by the observation that enzymatic removal of terminal alpha -galactosyl residues eliminated the inhibitory activity of ZP3 in the competitive sperm-ZP binding assay (4). Tetraantennary, alpha 3-galactosyl-capped oligosaccharides have also been shown to inhibit sperm-ZP binding (12). Additionally, it has been demonstrated that sp56 binds galactose residues and competitively inhibits murine sperm-zona binding in vitro (10, 11). Also consistent with this model is the observation that murine alpha 1,3-galactosyltransferase, the candidate enzyme for the addition of terminal alpha -galactosyl residues on O-linked oligosaccharides on ZP3, is expressed in female but not male germ cells (13). In potential conflict with this model, however, is the observation that inactivation of the alpha 1,3-galactosyltransferase gene by homologous recombination does not affect the fertility of female mice (14). Additionally, recent evidence indicates that sp56 is located primarily in the acrosomal contents and not on the plasma membrane (15). It has been postulated, however, that dynamic pores in the plasma membrane of acrosome-intact murine sperm may allow sp56 to bind ZP3 (15).

A determination of whether either or both of these models of sperm-ZP binding are correct has been hampered by the limited amounts of oligosaccharides that can be obtained from murine ZP3 for thorough structural and functional analyses and by the potential structural complexity of these oligosaccharides. To circumvent these limitations, we have used an in vitro sperm-ZP binding assay to generate dose-response curves for a series of tri- and tetrasaccharides with different nonreducing ends that these two current models for sperm-ZP binding predict might compete for ZP-binding sites on sperm. Precedent for this approach is provided by the demonstration that tri- and tetraantennary alpha - and beta -galactosyl-capped oligosaccharides inhibit sperm-ZP binding (12) and by the extensive analysis of potential ligands for the family of cell surface receptors, the selectins, which mediate the binding of lymphocytes to the vascular endothelium (16). In this study, we show that a small oligosaccharide with a nonreducing terminal GlcNAc, which is a substrate for beta 4GT in an in vitro enzymatic assay, is not a competitive inhibitor of sperm-ZP binding. We also demonstrate that a terminal alpha 3-galactosyl residue is not sufficient for an oligosaccharide to be a high affinity competitive inhibitor of sperm-ZP binding. Rather, an alpha 3-fucosyl residue in the context of the LewisX trisaccharide, Galbeta 1,4[Fucalpha 1,3]GlcNAc, is required to create a high affinity ligand for a ZP-binding site on murine sperm. Additionally, we present evidence that murine sperm-ZP binding is potentially mediated, in part, by a second, lower affinity site on sperm that preferentially binds a nonfucosylated beta -galactosyl-capped oligosaccharide.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Preparation of Oligosaccharides

Oligosaccharide 1-- GlcNAcbeta 1,4GlcNAcbeta 1,4GlcNAc (beta GN-beta GN-GN) was purchased from Sigma.

Oligosaccharide 2-- Galbeta 1,4GlcNAcbeta 1,4GlcNAc (beta G-beta GN-GN) was synthesized by incubation of GlcNAcbeta 1,4GlcNAc (Sigma) with purified bovine colostrum beta 1,4-galactosyltransferase and UDP-[14C]Gal (0.23 Ci/mol). The product was purified by sequential anion exchange and Bio-Gel P4 gel filtration chromatography and analyzed by 400-MHz 1H NMR spectroscopy. The mass of product was determined by measuring radioactivity.

Oligosaccharide 3-- Galbeta 1,4[Fucalpha 1,3]GlcNAcbeta 1,4GlcNAc (beta G-[F]-beta GN-GN) was synthesized by incubation of beta G-beta GN-GN with recombinant human alpha 1,3-fucosyltransferase VI (produced in insect cells) and GDP-[3H]fucose (0.10 Ci/mol). The product was purified and analyzed as described above for beta G-beta GN-GN.

Oligosaccharide 4-- Galalpha 1,3Galbeta 1,4GlcNAc (alpha G-beta G-GN) was synthesized by incubation of Galbeta 1,4GlcNAc with soluble recombinant bovine alpha 1,3-galactosyltransferase and UDP-[14C]Gal (0.23 Ci/mol), and the product was purified and analyzed as described previously (17-19). The mass of product was determined as described above. Two additional preparations of alpha G-beta G-GN were purchased from V-Labs (Covington, LA).

Oligosaccharide 5-- Galalpha 1,3Galbeta 1,4[Fucalpha 1,3]GlcNAc (alpha G-beta G-[F]-GN) was synthesized by incubation of alpha G-beta G-GN with purified human milk alpha 1,3-fucosyltransferase and GDP-[3H]fucose (0.10 Ci/mol). The product was purified and characterized as described previously (17).

1H NMR Spectroscopy

Products obtained from the enzyme-assisted synthesis of each oligosaccharide were identified using 400-MHz 1H NMR spectra, which were recorded on a Bruker MSL-400 spectrometer (Department of Physics, Vrije Universiteit, Amsterdam) as described (20).

Collection of Mouse Gametes and Embryos

Eggs and sperm were collected from CD-1 mice and prepared as described by Bleil and Wassarman (1). Prior to use in the competitive sperm-ZP binding assay, sperm were capacitated by incubation for about 1 h in medium 199 supplemented with 2 mg/ml BSA and 30 µg/ml sodium pyruvate (M199-M). ZP were isolated from oocytes in Whitten's medium (21) plus 3 mg/ml BSA (WM-BSA) and acid-solubilized as described by Bleil and Wassarman (2). Two-cell embryos were obtained by culturing fertilized eggs from mated females for 18 h in WM-BSA.

Pipettes

Micropipettes were fire-pulled from 4-mm internal diameter borosilicate glass tubing. A bore diameter of 228 µm was obtained by inserting a 0.009-inch diameter piano wire (Small Parts Inc.) down the pipette until it stopped; a flat break was made at that point. Pipettes having a tapered length of 3-4 cm were fire-polished, and the bore diameter was remeasured to ensure that the bore diameters were not reduced.

The Competitive in Vitro Sperm-ZP Binding Assay

This assay was performed essentially as described (1). Briefly, the test oligosaccharide was incubated at 37 °C in 95% air, 5% CO2 for 30 min with 30,000 sperm in a total volume of 25 µl of M199-M under oil, and then at least 10 eggs and three two-cell embryos were added in 5 µl of medium. Sperm, eggs, and embryos were incubated for an additional 15 min. Published results demonstrate that the sperm that bind zona-enclosed eggs during this 15-min incubation do not undergo the acrosome reaction (22). Sperm-egg and sperm-embryo complexes were then serially transferred through 40-µl drops of media until 2-5 sperm remained bound to the embryos. For each experiment, the cultures were pipetted an equal number of times with the same pipette. Cells were immediately fixed with an equal volume of 1.0% formaldehyde, 0.4% polyvinylpyrrolidone-40 in HEPES (pH 7.4) saline, transferred to a glass slide, and examined by phase contrast microscopy. Bound sperm were enumerated using (× 40; N.A. = 0.70) phase contrast objective. Sperm on the ZP overlying the upper 40% of an egg or a two-cell embryo were counted as the focal plane was moved down through the egg or embryo. This region of the ZP was examined because all sperm on this but not on lower regions could be counted rapidly and accurately. The number of bound sperm/egg was defined by subtracting the average number of sperm remaining on the embryos from the average number of sperm on the eggs. The accuracy of this method of counting sperm on eggs or embryos was verified by two methods. First, for a subset of samples, the number of sperm on the total surface of the ZP surrounding eggs and embryos was also determined. These numbers were consistently 2.5 times greater than when sperm on the upper 40% of the zona were enumerated. Second, selected samples were recounted independently by a second investigator, and similar results were obtained. In the experiments described, 69 ± 6 (mean ± S.E.) sperm bound to the entire surface of the eggs in the absence of any added competitor. Data are expressed as percentage of inhibition of sperm-ZP binding, where numbers of bound sperm in the absence of competitor equaled 0% inhibition.

Demonstration That the Oligosaccharides Used as Competitors Did Not Trigger the Acrosome Reaction

This study identifies small oligosaccharides of defined structure that inhibit the in vitro binding of acrosome intact sperm to the zona pellucida and correlates the structures of these oligosaccharides with their biological activities in the competitive sperm-zona binding assay. Since the primary receptor of murine spermatozoa for the ZP resides on the plasma membrane overlying the acrosome (1, 2), a prerequisite for this analysis is that the test oligosaccharides themselves do not cause the sperm to undergo the acrosome reaction and, thus, lose their cell surface receptors for the ZP. To verify that the oligosaccharides do not trigger the acrosome reaction, 1 × 105 capacitated sperm were incubated for 45 min in a total volume of 50 µl of medium supplemented with 18 µM oligosaccharide or 10 µM concentration of the calcium ionophore (A23187), the positive control for this procedure. Sperm used as negative controls were incubated in medium to which was added the 2.8 µl of vehicle (water: 2 × M199M; 1:1). Sperm were incubated for 45 min with or without oligosaccharides, because in the competitive sperm-ZP binding assay, sperm were incubated with oligosaccharide competitors for a total duration of 45 min. Following the incubation, sperm were fixed in 4% formaldehyde in phosphate-buffered saline, and acrosomes were stained with 1% Coomassie Brilliant Blue G. A minimum of 200 sperm/sample were analyzed as acrosome-intact or as acrosome-reacted and data were expressed as percentage of acrosome-reacted sperm.

Dose-response Analysis of Individual Oligosaccharides in the Competitive Sperm-ZP Binding Assay: Experimental Design

To compare the abilities of oligosaccharides to inhibit sperm-ZP binding, each oligosaccharide was titrated against sperm and eggs in the competitive sperm-ZP binding assay. Depending on the oligosaccharide, the concentrations tested ranged from 0.25 to 144 µM. The ED50 and maximal percentage of inhibition for each oligosaccharide were calculated from the corresponding dose-response curve (see "Analysis of Data"). The ED50 is defined as the concentration of an oligosaccharide producing half-maximal inhibition of sperm-ZP binding.

Results from the competitive sperm-ZP binding assay are described by the probabilistic model of cell binding of Cozen-Roberts et al. (23). This model predicts that the probability that a sperm will bind a ZP-enclosed egg is a function of the incubation time of sperm with eggs, the number of unoccupied receptors on the sperm, and the rates of association and dissociation of sperm surface binding sites from specific ligands. In addition, this model predicts that saturation of this receptor with the appropriate free oligosaccharide will inhibit sperm-ZP binding by 100% if a single sperm surface receptor mediates this binding. However, if sperm-ZP binding is mediated by multiple zona-binding sites, each binding a different oligosaccharide, saturation of one of these receptors will reduce sperm-ZP binding by less than 100%.

Additive Effects of Paired Oligosaccharides: Experimental Design

Three experiments were conducted to test the hypothesis that different oligosaccharides bind independent ZP-binding sites on sperm. Each experiment consisted of four experimental groups.

Group 1-- Sperm were incubated in a saturating concentration of either alpha G-beta G-GN or alpha G-beta G-[F]-GN. The saturating concentration was defined in the dose-response studies as the concentration above which there was no significant increase in percentage of inhibition.

Group 2-- Sperm were preincubated in twice the concentration of alpha G-beta G-GN or alpha G-beta G-[F]-GN used in group 1. We anticipated that there would be no significant difference in the results obtained between groups 1 and 2, confirming that for a particular experiment, the concentration of oligosaccharide used in group 1 was at apparent saturation.

Group 3-- Sperm were preincubated in a saturating concentration of alpha G-beta G-[F]-GN or a concentration of beta G-beta GN-GN calculated from the competitive sperm-zona binding assay to reduce sperm-ZP binding by 50%.

Group 4-- Sperm were preincubated with the saturating concentration of the oligosaccharide used in group 1 plus the concentration of the second oligosaccharide used in group 3.

Last, to establish the numbers of sperm/egg at 0% inhibition, sperm were preincubated in the absence of any oligosaccharide. Two oligosaccharides were defined as binding independent sites on sperm if the percentage of inhibition with the oligosaccharide pair was greater than the percentage of inhibition with each oligosaccharide alone.

Analysis of Data

Dose response curves for individual oligosaccharides in the competitive sperm-ZP binding assay were fit to a rectangular hyperbola, and regression analysis was performed using the program Sigma Plot; the ED50 and percentage of maximal inhibition were calculated from this regression analysis. The ED50 values provided a measure of the relative inhibitory activities of the oligosaccharides in the competitive sperm-ZP binding assay. Two-way analysis of variance was used to compare the effects of incubating sperm with one or two different oligosaccharides. The latter analysis was conducted using the statistical package, SAS. Statistically significant differences were defined as p <=  0.05.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Structural Identification of the Synthetic Oligosaccharides beta G-beta GN-GN, beta G-[F]-beta GN-GN, alpha G-beta G-GN, and alpha G-beta G-[F]-GN

alpha G-beta G-GN and alpha G-beta G-[F]-GN were synthesized and characterized as described previously (17). Their chemical shift values are included in Table I to allow comparison with beta G-beta GN-GN and beta G-[F]-beta GN-GN.

                              
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Table I
1H chemical shifts of the structural reporter group protons of the constituent monosaccharides of the various enzymatically synthesized oligosaccharides

The presence of the terminal beta 4-linked Gal residue in beta G-beta GN-GN is confirmed in the 1H NMR spectrum by the Gal H-1 and H-4 signals at delta  4.468 and delta 3.925, respectively. These values are comparable with those of the Gal residue in the Galbeta 1,4GlcNAc element (17).

In the 1H NMR spectrum of beta G-[F]-beta GN-GN, the presence of the alpha 3-linked Fuc residue is reflected in the Fuc H-1, H-5, and CH3 structural reporter group signals, which have similar values as those of the Fuc residue in alpha G-beta G-[F]-GN (see Table I). In addition, the alpha 3-linkage of the Fuc residue to the internal GlcNAc-2 is deduced from the upfield shifts of the GlcNAc-2 NAc (Delta delta -0.011) and beta Gal H-1 (Delta delta -0.022) signals compared with beta G-beta GN-GN. Similar upfield shifts are observed when comparing the same signals in alpha G-beta G-GN and alpha G-beta G-[F]-GN.

Validation of the Competitive Sperm-ZP Binding Assay

Initially, we validated the competitive sperm-ZP binding assay using acid-solubilized total ZP as the competitor. Preincubation of sperm with one, two, or four solubilized ZP/µl for 30 min inhibited sperm-ZP binding in a dose-dependent manner (data not shown). In agreement with Bleil and Wassarman (1), four ZP/µl inhibited sperm-ZP binding by approximately 90% relative to the negative control in which no solubilized ZP was added. By this criterion, results from our assay are comparable with those from other laboratories. Second, we established in blind trials that incubation of sperm under these assay conditions with a 9 or 72 µM concentration of each oligosaccharide did not alter either sperm motility or viability. Third, we determined in 33 independent experiments that three different preparations of alpha G-beta G-GN, at 9 µM, reproducibly inhibited 50-60% of sperm-ZP binding. In three additional experiments where the positive control fell outside this range, the data were excluded from the statistical analysis. The data for the positive control are shown as the bar graph to the right of the dose-response curves in Fig. 1. Finally, it was demonstrated that incubation of capacitated sperm for 45 min with a 18 µM concentration of each oligosaccharide did not cause the sperm to undergo the acrosome reaction (Table II). In contrast, this concentration of beta G-beta GN-GN, alpha G-beta G-GN, alpha G-beta G-[F]-GN, and beta G-[F]-beta GN-GN inhibited sperm-ZP binding (see Fig. 1). Taken together, these data demonstrate that the competitive sperm-zona binding assay measures the abilities of specific oligosaccharides to inhibit the binding of acrosome-intact murine spermatozoa to the zona pellucida.


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Fig. 1.   Dose-response analysis of a series of short oligosaccharides with different nonreducing ends in the competitive sperm-ZP binding assay. A, beta GN-beta GN-GN was tested at 1, 9, 18, and 36 µM; B, beta G-beta GN-GN was tested at 1, 9, 18, 36, and 72 µM; C, alpha G-beta G-GN was tested at 1, 5, 9, 18, and 36 µM; D, alpha G-beta G-[F]-GN was tested at 0.25, 0.5, 0.75, 1, and 9 µM (inset, 1, 5, 9, 18, and 36 µM); E, beta G-[F]-beta GN-GN was tested at 0.25, 0.5, 0.75, 1, and 9 µM. Data (mean ± S.E. for 4-6 experiments) are expressed as percentage of inhibition relative to negative controls (no competitor). These panels were drawn by connecting the mean value of percentage of inhibition for each oligosaccharide concentration. Results for the internal positive control (9 µM alpha G-beta G-GN) are shown by the bar graph in appropriate panels.

                              
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Table II
The oligosaccharides tested do not cause murine spermozoa to undergo the acrosome reaction
105 capacitated sperm were incubated for 45 min in 50 µl of M199M supplemented with 18 µM of an oligosaccharide, with vehicle (1.4 µl of water plus 1.4 µl of 2 × M199M) or with 10 µM A23187, and sperm were then fixed in buffered 4% formaldehyde. Following staining of the acrosomes with Coomassie Blue G, a minimum of 200 sperm/sample were analyzed for the presence or absence of an acrosome. Sperm without an acrosome are defined as acrosome-reacted. Results shown in this table are from four independent experiments.

Dose-response Analysis of Individual Oligosaccharides in the Competitive Sperm-ZP Binding Assay

The Trisaccharide beta GN-beta GN-GN Does Not Inhibit Sperm-ZP Binding-- If sperm-zona binding is mediated by a sperm surface beta 1,4-galactosyltransferase and its acceptor sugar substrate, then an oligosaccharide with a nonreducing terminal GlcNAc residue would be anticipated to inhibit sperm-ZP binding. To test this prediction, sperm were preincubated with 1, 9, 18, or 36 µM beta GN-beta GN-GN, which is an appropriate substrate for beta 1,4-galactosyltransferase. In four replicate experiments, no competition was observed (Fig. 1A). Competition was also not observed when sperm were incubated with 72 µM beta GN-beta GN-GN (data not shown).

Substitution of the Terminal GlcNAc with a beta 4-galactosyl Residue Results in a Trisaccharide That Inhibits Sperm-ZP Binding-- Bleil and Wassarman (4) reported that enzymatic removal of alpha -galactosyl residues reduced ZP3's ability to compete for ZP-binding sites on sperm. Removal of an alpha -galactosyl residue would be anticipated to yield an O-linked oligosaccharide with Galbeta 1,4GlcNAc-R at its terminus. Shur and colleagues (6) have reported that beta 4-galactosylation of ZP3 also reduced the sperm binding activity of ZP3. This galactosylation would also have produced O-linked oligosaccharides which terminated in Galbeta 1,4GlcNAc-R. Therefore, we anticipated that beta G-beta GN-GN would not inhibit sperm-ZP binding. However, as shown in Fig. 1B, this was not the case. Significant inhibition (14%) was observed with 9 µM beta G-beta GN-GN; inhibition increased to 47% at 72 µM, the highest concentration tested. While this concentration was not saturating, regression analysis indicated that beta G-beta GN-GN would produce an estimated 74% maximal inhibition and an ED50 of 42 µM. This ED50 value, coupled with the fact that this site was difficult to saturate, indicates that beta G-beta GN-GN is a low affinity competitive inhibitor for ZP-binding sites on sperm.

A Trisaccharide with a Terminal alpha 3-Galactosyl Residue Has a Higher Inhibitory Activity than beta G-beta GN-GN-- The conclusion by Bleil and Wassarman (4) that a sperm-binding oligosaccharide from ZP3 has a terminal alpha -galactosyl residue and our demonstration that oocytes transcriptionally express alpha 1,3-galactosyltransferase (13) suggested to us that the product of this enzyme, alpha G-beta G-GN, would be an inhibitor of sperm-ZP binding. To determine how effective an inhibitor, sperm were incubated with 1, 5, 9, 18, and 36 µM alpha G-beta G-GN (Fig. 1C). The resulting dose-response curve was nearly linear from 0 to 9 µM where 53% inhibition was observed. Regression analysis indicated that alpha G-beta G-GN produced an estimated 78% maximal inhibition and an ED50 of 5.3 µM. Thus, the inhibitory activity of this trisaccharide was 8-fold greater than the activity of beta G-beta GN-GN.

The Addition of an alpha 3-Fucosyl Residue to alpha G-beta G-GN, Forming alpha G-beta G-[F]-GN, Yields a Tetrasaccharide with High Inhibitory Activity-- There is circumstantial evidence that an alpha -fucosyl residue may also be a component of a sperm-binding oligosaccharide on murine ZP3. Digestion with alpha -fucosidase reduced the inhibitory activities of ZP3- or ZP-derived O-linked oligosaccharides in the competitive sperm-ZP binding assay (4). Additionally, millimolar amounts of two fucose-containing oligosaccharides inhibit sperm-ZP binding in vitro (12). Consequently, we examined the effect of adding an alpha 3-fucosyl residue to the GlcNAc residue of alpha G-beta G-GN. alpha G-beta G-[F]-GN was initially examined at 1, 9, 18, and 36 µM. Maximal inhibition (approximately 40%) was observed at 1 µM (Fig. 1D, inset), and no further inhibition was observed at concentrations as high as 140 µM (data not shown). To accurately define the dose response, this analysis was repeated with 0.25, 0.5, 0.75, 1, and 9 µM alpha G-beta G-[F]-GN (Fig. 1D). Results show a linear dose response between 0 and 0.75 µM; maximal inhibition and ED50 values were calculated to be 46% and 430 nM, respectively. Thus, alpha 3-fucosylation of the reducing terminal GlcNAc residue increased the inhibitory activity of alpha G-beta G-GN by 12-fold.

The Presence of an alpha 3-Galactosyl Residue Is Not Obligatory for an Oligosaccharide with High Inhibitory Activity-- Female mice that lack a functional alpha 1,3-galactosyltransferase gene are still fertile (14), indicating that an alpha 3-galactosyl residue is not obligatory in vivo for a functional sperm-binding oligosaccharide. To determine whether the presence of an alpha 3-fucosyl residue is sufficient to generate a tetrasaccharide with high inhibitory activity, beta G-[F]-beta GN-GN was titrated against sperm at 0.25, 0.5, 0.75, 1, and 9 µM concentrations. The resulting dose-response curve (Fig. 1E) was essentially identical to that for alpha G-beta G-[F]-GN (Fig. 1D). At 1 µM, a maximal inhibition of 46% was obtained. The ED50 for this fucosylated, beta -galactosyl-capped oligosaccharide was 500 nM. Thus, alpha 3-fucosylation of beta G-beta GN-GN increased its inhibitory activity approximately 85-fold. This result suggests that an alpha 3-fucosyl and not an alpha 3-galactosyl residue is required for forming an oligosaccharide with high inhibitory activity in the competitive sperm-ZP binding assay.

Do Inhibitors Bind at the Same Oligosaccharide-binding Site? Additive Effects of Paired Oligosaccharides

While both alpha G-beta G-[F]-GN and beta G-[F]-beta GN-GN had low ED50 values (430 and 500 nM, respectively) in the competitive sperm-ZP binding assay, each only inhibited approximately 45% of sperm-ZP binding. In contrast, acid-solubilized ZP inhibited 90% of sperm-ZP binding. One potential explanation for this difference in maximal inhibition between solubilized ZP and the oligosaccharides examined is that the ZP binds two or more distinct sites with different oligosaccharide binding specificities on the sperm surface. To test this hypothesis, we added a saturating concentration of one oligosaccharide inhibitor to a second oligosaccharide and compared the inhibition achieved with the paired oligosaccharides with inhibition obtained with each oligosaccharide alone. Saturation of the first oligosaccharide was confirmed, since inhibition was not significantly increased when the concentration of the first oligosaccharide was doubled (Fig. 2, A-C, compare lanes 1 and 2).


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Fig. 2.   A second oligosaccharide-binding site on sperm potentially mediates sperm-ZP binding. To determine whether different oligosaccharides bind the same or different sites, sperm were preincubated with the following pairs of oligosaccharides prior to the addition of eggs and embryos. A, alpha G-beta G-GN and alpha G-beta G-[F]-GN; B, alpha G-beta G-GN and beta G-beta GN-GN; C, alpha G-beta G-[F]-GN and beta G-beta GN-GN. Lane 1, effect of an apparent saturating concentration of alpha G-beta G-GN (A and B) or alpha G-beta G-[F]-GN (C). Lane 2, effect of twice the concentration of the oligosaccharide shown in the first bar (A-C). Lane 3, effect of a saturating concentration of alpha G-beta G-[F]-GN (A) or a dose of beta G-beta GN-GN (B and C) calculated to inhibit 50% of sperm-ZP binding. Lane 4, effect of simultaneous incubation of sperm with two different oligosaccharides. The concentrations of the two oligosaccharides were the same used in the first and third bars, respectively. Data (mean ± S.E.; n = 6) are presented as percentage of inhibition of sperm-ZP binding relative to negative controls (no competitor).

Evidence That alpha G-beta G-GN, alpha G-beta G-[F]-GN, and beta G-[F]-beta GN-GN Bind the Same Site on Sperm-- Fig. 2A compares the effect on sperm-ZP binding of incubating sperm with alpha G-beta G-GN (lanes 1 and 2), alpha G-beta G-[F]-GN (lane 3), or both oligosaccharides together (lane 4). No significant differences were observed among these four groups. Identical results were obtained when sperm were incubated with alpha G-beta G-[F]-GN, beta G-[F]-beta GN-GN, or both oligosaccharides together (n = 4; data not shown). These results suggest that alpha G-beta G-GN, alpha G-beta G-[F]-GN, and beta G-[F]-beta GN-GN bind to the same ZP-binding site on sperm.

Evidence for a Second Site on Sperm That Binds beta G-beta GN-GN-- Fig. 2B shows the effects on sperm-ZP binding of incubating sperm with alpha G-beta G-GN, beta G-beta GN-GN or both trisaccharides together. The 75% inhibition of sperm-ZP binding achieved with 9 µM alpha G-beta G-GN plus 72 µM beta G-beta GN-GN (lane 4) was significantly greater than the 45% inhibition achieved with either 18 µM alpha G-beta G-GN (lane 2) or 72 µM beta G-beta GN-GN (lane 3) alone.

Fig. 2C shows the effects on sperm-ZP binding of incubating sperm with alpha G-beta G-[F]-GN, beta G-beta GN-GN, or both oligosaccharides together. The 60% inhibition achieved with 36 µM alpha G-beta G-[F]-GN plus 72 µM beta G-beta GN-GN (lane 4) was significantly greater than the inhibition achieved with either 72 µM alpha G-beta G-[F]-GN (40%) (lane 2) or 72 µM beta G-beta GN-GN (36%) (lane 3) alone. In summary, these results suggest that beta G-beta GN-GN binds to a second, independent oligosaccharide-binding site that is distinct from the binding site occupied by alpha G-beta G-GN, alpha G-beta G-[F]-GN, or beta G-[F]-beta GN-GN.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The impetus for this study is the current lack of a consensus on the molecular basis for mouse sperm-ZP binding. This lack of a consensus is reflected by the fact that different models have been proposed to account for this fundamental biological process. As outlined in the Introduction, each model under consideration in this study is based on the putative identification of a distinct sperm cell surface protein (beta 1,4-galactosyltransferase versus sp56) that has the intrinsic ability to bind to a specific and dissimilar terminal carbohydrate sequence found on a subset of O-linked glycans on ZP3. Because the common denominator for both models is the binding of sperm to a glycan with specified sequence requirements, we have used the experimental strategy of examining a series of short oligosaccharides with differing nonreducing ends to competitively inhibit sperm-ZP binding in vitro. Dose-response curves (as opposed to single concentration assays) were carried out to establish a rank order for the effectiveness of the oligosaccharides as competitive inhibitors. Inherent in our approach is the assumption that the more effective the competitive inhibitor, the more closely the structure of the inhibitor mimics the essential intrinsic structure of the functional sperm-binding oligosaccharide ligand(s). By extrapolation, information on the essential monosaccharides in a functional sperm-binding ligand can aid in the identification of the cognate sperm surface protein.

This analysis required that the competitive sperm-ZP binding assay measures the relative affinities of specific oligosaccharides for ZP-binding sites on sperm. Based on the data presented in Fig. 1, this requirement was met. The relative effectiveness of a given inhibitor was specified by its structure, and the binding of each inhibitor was saturable. Both characteristics are hallmarks of the specific binding of a ligand to its receptor.

A Fucosyl Residue Is Required for an Oligosaccharide to Bind Acrosome-intact Sperm with High Affinity-- The lack of inhibition of sperm-ZP binding with beta GN-beta GN-GN is in agreement with published reports that in vitro sperm-ZP binding is not inhibited by free N-acetylglucosamine or by a tetraantennary oligosaccharide with four terminal N-acetylglucosaminyl residues (12, 24). Additionally, our results are consistent with two independent reports that male mice that lack a functional beta 4GT gene are fertile (7, 8). Collectively, in our view, these observations do not support the requirement for nonreducing terminal N-acetylglucosaminyl residues on ZP3 to form a high affinity ligand for acrosome-intact sperm, nor do these observations support a role for sperm surface beta 1,4-galactosyltransferase in mediating high affinity murine sperm-ZP binding.

In contrast, our data indicate that alpha G-beta G-GN binds, albeit with moderate affinity, a high affinity ZP-binding site on murine sperm. This observation is consistent with a role for terminal alpha -galactosyl residues on ZP3 and sperm surface protein, sp56 (4, 9-12). However, the major new insight from this study is that high affinity sperm binding oligosaccharides require a fucosyl residue and that it is the fucosyl and not alpha -galactosyl residue that is essential for the assembly of this high affinity sperm-binding ligand. Interestingly, the apparent affinities of the two fucosylated tetrasaccharides (ED50 values in the 430 and 500 nM range) are comparable with the affinity reported for gp55 (ED50 = 200 nM), a functional sperm binding glycopeptide isolated from murine ZP3 (25).

The apparent high affinity of the two fucosylated oligosaccharides for ZP-binding sites on sperm raises the question of whether a fucosyl residue in the absence of either an alpha - or beta -galactosyl residue is sufficient to create a sperm-binding ligand. To address this question, chemically synthesized Fucalpha 1,3GlcNAcbeta 1,2Man was incubated with sperm and eggs. Preliminary results indicate that 9 µM of this oligosaccharide inhibited sperm-ZP binding by approximately 40%. This observation reinforces the conclusion that fucosyl residues may be important to forming high affinity sperm binding oligosaccharides on the ZP. Last, the potential importance of the fucosyl residue in sperm-ZP binding is relevant to considering why female mice, which lack a functional, alpha 1,3-galactosyltransferase gene and consequently cannot synthesize alpha 3-galactosyl-capped oligosaccharides, are nevertheless still fertile (14). The observation that a fucosyl-containing tetrasaccharide with or without a terminal alpha -galactosyl residue (alpha G-beta G-[F]-GN and beta G-[F]-beta GN-GN) competes with comparable affinity for the same oligosaccharide-binding site on sperm provides a potential explanation for the observed fertility in these animals.

The alpha 3-Fucosyl Residue in High Affinity Oligosaccharides Is Present in the Context of the LewisX Trisaccharide, Galbeta 1,4[Fucalpha 1,3]GlcNAc-- It is noteworthy that both high affinity tetrasaccharides we have identified contain the LewisX trisaccharide. This trisaccharide is of interest because it has been implicated in other examples of cell-cell interactions, including embryonic compaction, nerve cell adhesion, and breast cancer invasiveness (26-29). Additionally, the sialylated LewisX oligosaccharide mediates binding of lymphocytes to the vascular endothelium (30). Thus, murine sperm-ZP binding may be another example of cell-cell interactions regulated by oligosaccharides containing the LewisX trisaccharide.

Evidence That the Second, Low Affinity Site on Sperm Participates in Murine Sperm-ZP Binding-- Our data suggest that there are two independent oligosaccharide-binding sites on sperm with different binding specificities. One site binds alpha G-beta G-GN and the two fucose-containing tetrasaccharides tested, with moderate and high affinity, respectively. The second site preferentially binds the linear beta -galactosyl-capped oligosaccharide (beta G-beta GN-GN), with low affinity. The apparent low affinity of this second site raises the question of whether it, in fact, mediates sperm-ZP binding. The following considerations are relevant to this question. First, we have tested only a single beta -galactosyl-capped oligosaccharide, and this compound may be a poor mimic for the naturally occurring ligand on ZP3. Second, for the sake of argument, let us assume that the relatively low binding affinity estimated for this test oligosaccharide reflects the affinity of the "unknown" intrinsic ligand. In model systems, it has been experimentally estimated that the adhesive strength of a noncovalent bond varies as a function of the logarithm of the affinity of the ligand for its binding site (31). Consequently, the predicted strength of the low affinity bond would be only 4-fold less than that achieved with the alpha 3-fucosyl-containing, high affinity tetrasaccharides. However, independent of whether this second site mediates a low or high affinity interaction between a spermatozoan and an oligosaccharide on ZP3, our empirical data indicate that this site does, in fact, participate in sperm-ZP binding. When the high affinity site on sperm was saturated with either of the alpha 3-fucosyl-containing tetrasaccharides, sperm-ZP binding was reduced by only 45%; sperm remaining on the ZP were bound with sufficient strength to resist the shear force produced by multiple pipettings. However, as shown in the mixing experiments in Fig. 2, in specific cases, two inhibitors are better than one. The addition of beta G-beta GN-GN to alpha G-beta G-[F]-GN further inhibited sperm-ZP binding by an additional 20%. Thus, the lower affinity site must create a sufficiently strong bond to participate in the tight binding of sperm to the ZP. The availability of both high and low affinity sites on mouse sperm, as demonstrated by binding studies with 125I-labeled solubilized ZP proteins, has been reported (32).

Are There Candidate Sperm Surface Molecules That Mediate the High and Low Affinity Binding Reactions between ZP3 and Sperm?-- Our conclusion that there are two distinct oligosaccharide ZP-binding sites on the surface of murine sperm raises the question of the identity of these sites. As already noted, our results are consistent with a role for sp56 as the high affinity ZP-binding site on sperm. If sp56 is responsible for the high affinity oligosaccharide-binding site, our results predict that this sperm surface protein would bind the two fucose-containing tetrasaccharides with high affinity.

There are at least two different candidates for the second binding site. A cell surface, calcium-dependent lectin with affinity for galactose has been described on sperm surface from mice, rats, and rabbits; in rabbits, this lectin has been shown to bind ZP3 (33-35). A sperm surface fucosyltransferase enzymatic activity has been identified and suggested to mediate sperm-ZP binding (24, 36). However, we cannot exclude the possibility that these two oligosaccharide-binding sites on sperm represent unidentified receptors. While the identity of the authentic ZP-binding molecule(s) on the sperm surface has yet to be completely resolved, our findings demonstrate that in mice, sperm-ZP binding is a redundant process that involves at least two distinct oligosaccharide-binding sites on the plasma membrane of sperm.

    ACKNOWLEDGEMENTS

We thank Dr. Jeffrey Bleil for instruction in the competitive sperm-ZP binding assay, Pascale Schoenmakers for assistance with synthesis of the oligosaccharides, Dr. Philip Castle for helpful discussions, and Dr. Karen Bandeen-Roche and Dr. Ron Schnaar for insightful discussion on data analysis.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grants HD17989 (to W. W. W.), P30-HD06308 (to the Hopkins Population Center), and CA45799 (to J. H. S.); The Council for Tobacco Research-U.S.A. Grant 4368R1 (to J. H. S.); and The Human Frontiers Science Program Grant RG-414/94M (to D. H. J.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by an Institutional Training grant, HD-07276. The results in this study represent partial fulfillment of this author's requirements for the degree of Doctor of Philosophy at The Johns Hopkins University, under the supervision of W. W. W.

par To whom correspondence should be addressed: The Johns Hopkins University School of Medicine, Oncology Center, Room 1-127, 600 N. Wolfe St., Baltimore, MD 21287-8937. Tel.: 410-955-8879; Fax: 410-502-5499; E-mail: jshaper{at}welchlink.welch.jhu.edu.

1 The abbreviations used are: ZP, zona pellucida; ZP3, zona pellucida glycoprotein 3; beta GN-beta GN-GN, GlcNAcbeta 1,4GlcNAcbeta 1,4GlcNAc; beta G-beta GN-GN, Galbeta 1,4GlcNAcbeta 1,4GlcNAc; alpha G-beta G-GN, Galalpha 1,3Galbeta 1,4GlcNAc; alpha G-beta G-[F]-GN, Galalpha 1,3Galbeta 1,4[Fucalpha 1,3]GlcNAc; beta G-[F]-beta GN-GN, Galbeta 1,4[Fucalpha 1,3]GlcNAcbeta 1,4GlcNAc; beta 4GT, beta 1,4-galactosyltransferase (EC 2.4.1.38); murine beta 4GT refers to the alpha -lactalbumin responsive, UDP-galactose:N-acetylglucosamine beta 4-galactosyltransferase that has been mapped to the centromeric region of mouse chromosome 4; M199M, medium 199 supplemented with 2 mg/ml BSA and 30 µg/ml sodium pyruvate; WM-BSA, Whitten's medium plus 3 mg/ml BSA.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

  1. Bleil, J. D., and Wassarman, P. M. (1980) Cell 20, 873-882[Medline] [Order article via Infotrieve]
  2. Bleil, J. D., and Wassarman, P. M. (1980) Dev. Biol. 76, 185-203[Medline] [Order article via Infotrieve]
  3. Florman, H. M., and Wassarman, P. M. (1985) Cell 41, 313-324[Medline] [Order article via Infotrieve]
  4. Bleil, J. D., and Wassarman, P. M. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6778-6782[Abstract]
  5. Miller, D. J., Macek, M. B., and Shur, B. D. (1992) Nature 357, 589-593[CrossRef][Medline] [Order article via Infotrieve]
  6. Lopez, L. C., Bayna, E. M., Litoff, D., Shaper, N. L., Shaper, J. H., Shur, B. D. (1985) J. Cell Biol. 101, 1501-1510[Abstract]
  7. Lu, Q, Hasty, P., and Shur, B. D. (1997) Dev. Biol. 181, 257-267[CrossRef][Medline] [Order article via Infotrieve]
  8. Asano, M., Furukawa, F., Kido, M., Matsumoto, S., Umesaki, Y., Kochibe, N., and Iwakura, Y. (1997) EMBO J. 16, 1850-1857[Abstract/Free Full Text]
  9. Bleil, J. D., and Wassarman, P. M. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5563-5567[Abstract]
  10. Bookbinder, L. H., Cheng, A., and Bleil, J. D. (1995) Science 269, 86-89[Medline] [Order article via Infotrieve]
  11. Cheng, A., Le, T., Palacios, M., Bookbinder, L. H., Wassarman, P. M., Suzuki, F., Bleil, J. D. (1994) J. Cell Biol. 125, 867-878[Abstract]
  12. Litscher, E. S., Juntunen, K., Seppo, A, Pentila, L., Niemela, R., Renkonen, O., and Wassarman, P. M. (1995) Biochemistry 34, 4662-4669[Medline] [Order article via Infotrieve]
  13. Johnston, D. S., Shaper, J. H., Shaper, N. L., Joziasse, D. H., Wright, W. W. (1995) Dev. Biol. 171, 224-232[CrossRef][Medline] [Order article via Infotrieve]
  14. Thall, A. D., Maly, P., and Lowe, J. B. (1995) J. Biol. Chem. 270, 21437-21440[Abstract/Free Full Text]
  15. Foster, J. A., Friday, B. B., Maulit, M. T., Blobel, C., Winfrey, V. P., Olson, G. E., Kim, K-S., Gerton, G. L. (1997) J. Biol. Chem. 272, 12714-12722[Abstract/Free Full Text]
  16. Sears, P., and Wong, C-H. (1997) Proc. Natl. Acad. Sci. U. S. A. 93, 12086-12093[Abstract/Free Full Text]
  17. Joziasse, D. H., Schiphorst, W. E. C. M., Koeleman, C. A. M., Van den Eijnden, D. H. (1993) Biochem. Biophys. Res. Commun. 194, 358-367[CrossRef][Medline] [Order article via Infotrieve]
  18. Joziasse, D. H., Shaper, N. L., Salyer, L. S., Van den Eijnden, D. H., Van der Spoel, A. C., Shaper, J. H. (1990) Eur. J. Biochem. 191, 75-83[Abstract]
  19. Lie, J. T., Van den Nieuwenhof, I., Van den Eijnden, D. H., Koeleman, C. A. M., Joziasse, D. H. (1993) Glycoconjugate J. 10, 257-258
  20. Hokke, C. H., Zervosen, A., Elling, L., Joziasse, D. H., Van den Eijnden, D. H. (1996) Glycoconjugate J. 13, 687-692[Medline] [Order article via Infotrieve]
  21. Whitten, W. K. (1971) Adv. Biosci. 6, 129-139
  22. Florman, H. M., and Storey, B. T. (1982) Dev. Biol. 91, 121-130[Medline] [Order article via Infotrieve]
  23. Cozen-Roberts, C., Lauffenburger, O. A., Quinn, J. A. (1990) Biophys. J. 58, 841-856[Abstract]
  24. Thaler, C. D., and Cardullo, R. A. (1996) Mol. Reprod. Dev. 45, 535-546[CrossRef][Medline] [Order article via Infotrieve]
  25. Litscher, E. S., and Wassarman, P. M. (1996) Biochemistry 35, 3980-3985[CrossRef][Medline] [Order article via Infotrieve]
  26. Eggens, I., Fenderson, B., Toyokuni, T., Dean, B., Straud, M., and Hakamori, S. (1989) J. Biol. Chem. 264, 9476-9484[Abstract/Free Full Text]
  27. Hakamori, S. (1992) Histochem J. 24, 771-776[Medline] [Order article via Infotrieve]
  28. Streit, A., Yuen, C.-T., Loveless, R. W., Lawson, A. M., Finne, J., Schmitz, B., Feizi, T., Stern, C. D. (1996) J. Neurochem. 66, 834-844[Medline] [Order article via Infotrieve]
  29. Brooks, S. A., and Leathem, A. J. (1995) Histochem J. 27, 689-693[Medline] [Order article via Infotrieve]
  30. Phillips, M. L., Nudelan, E., Gaeta, F. C. A., Perez, M., Singhal, A. K., Hakamori, S., Paulson, J. C. (1990) Science 250, 1132-1135[Medline] [Order article via Infotrieve]
  31. Kuo, S. C., and Lauffenburger, D. A. (1993) Biophys. J. 65, 2191-2200[Abstract]
  32. Thaler, C. D., and Cardullo, R. A. (1996) J. Biol. Chem. 271, 23289-23297[Abstract/Free Full Text]
  33. Abdullah, M., and Kierszenbaum, A. L. (1989) J. Cell Biol. 108, 367-375[Abstract]
  34. Abdullah, M., Widgren, E. E., and O'Rand, M. G. (1991) Mol. Cell. Biochem. 103, 155-161[Medline] [Order article via Infotrieve]
  35. Goluboff, E. T., Mertz, J. R., Tres, L. L., Kierszenbaum, A. L. (1995) Mol. Reprod. Dev. 40, 460-466[Medline] [Order article via Infotrieve]
  36. Cardullo, R. A., Armant, D. R., and Millette, C. F. (1989) Biochemistry 28, 1611-1617[Medline] [Order article via Infotrieve]


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