From the 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
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ABSTRACT |
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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
-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, GlcNAc
1,4GlcNAc
1,4GlcNAc,was
inactive (1-72 µM range). In contrast, a
4-galactosyl-capped trisaccharide (Gal
1,4GlcNAc
1, 4GlcNAc) and
an
3-galactosyl-capped trisaccharide (Gal
1,3Gal
1,4 GlcNAc) inhibited sperm-zona binding with low or moderate affinity
(ED50 = 42 µM and 5.3 µM,
respectively). The addition of an
3-fucosyl residue to each of these
two competitive inhibitors, forming Gal
1,4[Fuc
1,3] GlcNAc
1,4GlcNAc or Gal
1,3Gal
1, 4[Fuc
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
3-galactosyl-capped trisaccharide and
the two fucosylated tetrasaccharides. The second (apparently low
affinity) site binds a nonfucosylated
-galactosyl-capped trisaccharide.
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INTRODUCTION |
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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
1,4-galactosyltransferase (
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,
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
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
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
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 -galactosyl residues on ZP3 (4, 9-12). This
model is supported by the observation that enzymatic removal of
terminal
-galactosyl residues eliminated the inhibitory activity of
ZP3 in the competitive sperm-ZP binding assay (4). Tetraantennary,
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
1,3-galactosyltransferase, the
candidate enzyme for the addition of terminal
-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
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 - and
-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
4GT in an in
vitro enzymatic assay, is not a competitive inhibitor of sperm-ZP
binding. We also demonstrate that a terminal
3-galactosyl residue is
not sufficient for an oligosaccharide to be a high affinity competitive
inhibitor of sperm-ZP binding. Rather, an
3-fucosyl residue in the
context of the LewisX trisaccharide,
Gal
1,4[Fuc
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
-galactosyl-capped oligosaccharide.
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MATERIALS AND METHODS |
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Preparation of Oligosaccharides
Oligosaccharide 1--
GlcNAc1,4GlcNAc
1,4GlcNAc
(
GN-
GN-GN) was purchased from Sigma.
Oligosaccharide 2--
Gal1,4GlcNAc
1,4GlcNAc
(
G-
GN-GN) was synthesized by incubation of GlcNAc
1,4GlcNAc
(Sigma) with purified bovine colostrum
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--
Gal1,4[Fuc
1,3]GlcNAc
1,4GlcNAc
(
G-[F]-
GN-GN) was synthesized by incubation of
G-
GN-GN
with recombinant human
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
G-
GN-GN.
Oligosaccharide 4--
Gal1,3Gal
1,4GlcNAc (
G-
G-GN)
was synthesized by incubation of Gal
1,4GlcNAc with soluble
recombinant bovine
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
G-
G-GN were purchased from V-Labs (Covington, LA).
Oligosaccharide
5--
Gal1,3Gal
1,4[Fuc
1,3]GlcNAc (
G-
G-[F]-GN) was
synthesized by incubation of
G-
G-GN with purified human milk
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 G-
G-GN or
G-
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 G-
G-GN or
G-
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 G-
G-[F]-GN or a concentration of
G-
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.
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RESULTS |
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Structural Identification of the Synthetic Oligosaccharides
G-
GN-GN,
G-[F]-
GN-GN,
G-
G-GN, and
G-
G-[F]-GN
G-
G-GN and
G-
G-[F]-GN were synthesized and
characterized as described previously (17). Their chemical shift values
are included in Table I to allow
comparison with
G-
GN-GN and
G-[F]-
GN-GN.
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The presence of the terminal 4-linked Gal residue in
G-
GN-GN
is confirmed in the 1H NMR spectrum by the Gal H-1 and H-4
signals at
4.468 and
3.925, respectively. These values are
comparable with those of the Gal residue in the Gal
1,4GlcNAc element
(17).
In the 1H NMR spectrum of G-[F]-
GN-GN, the
presence of the
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
G-
G-[F]-GN (see
Table I). In addition, the
3-linkage of the Fuc residue to the
internal GlcNAc-2 is deduced from the upfield shifts of the GlcNAc-2
NAc (
0.011) and
Gal H-1 (
0.022) signals compared
with
G-
GN-GN. Similar upfield shifts are observed when comparing
the same signals in
G-
G-GN and
G-
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 G-
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
G-
GN-GN,
G-
G-GN,
G-
G-[F]-GN, and
G-[F]-
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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Dose-response Analysis of Individual Oligosaccharides in the Competitive Sperm-ZP Binding Assay
The Trisaccharide GN-
GN-GN Does Not Inhibit Sperm-ZP
Binding--
If sperm-zona binding is mediated by a sperm surface
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
GN-
GN-GN,
which is an appropriate substrate for
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
GN-
GN-GN (data not shown).
Substitution of the Terminal GlcNAc with a 4-galactosyl Residue
Results in a Trisaccharide That Inhibits Sperm-ZP Binding--
Bleil
and Wassarman (4) reported that enzymatic removal of
-galactosyl
residues reduced ZP3's ability to compete for ZP-binding sites on
sperm. Removal of an
-galactosyl residue would be anticipated to
yield an O-linked oligosaccharide with Gal
1,4GlcNAc-R at
its terminus. Shur and colleagues (6) have reported that
4-galactosylation of ZP3 also reduced the sperm binding activity of
ZP3. This galactosylation would also have produced O-linked
oligosaccharides which terminated in Gal
1,4GlcNAc-R. Therefore, we
anticipated that
G-
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
G-
GN-GN; inhibition increased to 47% at 72 µM, the
highest concentration tested. While this concentration was not
saturating, regression analysis indicated that
G-
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
G-
GN-GN
is a low affinity competitive inhibitor for ZP-binding sites on
sperm.
A Trisaccharide with a Terminal 3-Galactosyl Residue Has a
Higher Inhibitory Activity than
G-
GN-GN--
The conclusion by
Bleil and Wassarman (4) that a sperm-binding oligosaccharide from ZP3
has a terminal
-galactosyl residue and our demonstration that
oocytes transcriptionally express
1,3-galactosyltransferase (13)
suggested to us that the product of this enzyme,
G-
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
G-
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
G-
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
G-
GN-GN.
The Addition of an 3-Fucosyl Residue to
G-
G-GN, Forming
G-
G-[F]-GN, Yields a Tetrasaccharide with High Inhibitory
Activity--
There is circumstantial evidence that an
-fucosyl
residue may also be a component of a sperm-binding oligosaccharide on
murine ZP3. Digestion with
-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
3-fucosyl residue to the GlcNAc residue of
G-
G-GN.
G-
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
G-
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,
3-fucosylation of the
reducing terminal GlcNAc residue increased the inhibitory activity of
G-
G-GN by 12-fold.
The Presence of an 3-Galactosyl Residue Is Not Obligatory for an
Oligosaccharide with High Inhibitory Activity--
Female mice that
lack a functional
1,3-galactosyltransferase gene are still fertile
(14), indicating that an
3-galactosyl residue is not obligatory
in vivo for a functional sperm-binding oligosaccharide. To
determine whether the presence of an
3-fucosyl residue is sufficient
to generate a tetrasaccharide with high inhibitory activity,
G-[F]-
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
G-
G-[F]-GN (Fig. 1D). At 1 µM, a
maximal inhibition of 46% was obtained. The ED50 for this
fucosylated,
-galactosyl-capped oligosaccharide was 500 nM. Thus,
3-fucosylation of
G-
GN-GN increased its
inhibitory activity approximately 85-fold. This result suggests that an
3-fucosyl and not an
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 G-
G-[F]-GN and
G-[F]-
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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Evidence That G-
G-GN,
G-
G-[F]-GN, and
G-[F]-
GN-GN Bind the Same Site on Sperm--
Fig.
2A compares the effect on sperm-ZP binding of incubating
sperm with
G-
G-GN (lanes 1 and 2),
G-
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
G-
G-[F]-GN,
G-[F]-
GN-GN, or both
oligosaccharides together (n = 4; data not shown).
These results suggest that
G-
G-GN,
G-
G-[F]-GN, and
G-[F]-
GN-GN bind to the same ZP-binding site on sperm.
Evidence for a Second Site on Sperm That Binds
G-
GN-GN--
Fig. 2B shows the effects on sperm-ZP
binding of incubating sperm with
G-
G-GN,
G-
GN-GN or both
trisaccharides together. The 75% inhibition of sperm-ZP binding
achieved with 9 µM
G-
G-GN plus 72 µM
G-
GN-GN (lane 4) was significantly greater than the 45% inhibition achieved with either 18 µM
G-
G-GN
(lane 2) or 72 µM
G-
GN-GN (lane
3) alone.
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DISCUSSION |
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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 (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 GN-
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
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
1,4-galactosyltransferase in mediating high
affinity murine sperm-ZP binding.
The 3-Fucosyl Residue in High Affinity Oligosaccharides Is
Present in the Context of the LewisX Trisaccharide,
Gal
1,4[Fuc
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 G-
G-GN and the two
fucose-containing tetrasaccharides tested, with moderate and high
affinity, respectively. The second site preferentially binds the linear
-galactosyl-capped oligosaccharide (
G-
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
-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
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
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
G-
GN-GN to
G-
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.
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FOOTNOTES |
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* 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.
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; GN-
GN-GN,
GlcNAc
1,4GlcNAc
1,4GlcNAc;
G-
GN-GN,
Gal
1,4GlcNAc
1,4GlcNAc;
G-
G-GN, Gal
1,3Gal
1,4GlcNAc;
G-
G-[F]-GN, Gal
1,3Gal
1,4[Fuc
1,3]GlcNAc;
G-[F]-
GN-GN, Gal
1,4[Fuc
1,3]GlcNAc
1,4GlcNAc;
4GT,
1,4-galactosyltransferase (EC 2.4.1.38); murine
4GT refers to the
-lactalbumin responsive,
UDP-galactose:N-acetylglucosamine
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.
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REFERENCES |
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