Department of Cell Biology, Graduate Program in Biochemistry, Cell and Developmental Biology, Emory University School of Medicine, 615 Michael Street, Atlanta, GA 30322, USA
* Author for correspondence (e-mail: barry{at}cellbio.emory.edu)
Accepted 20 October 2003
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SUMMARY |
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In this report, we characterize a novel ZP3- and GalT I-independent mechanism for sperm adhesion to the egg coat. Results show that the ovulated zona pellucida contains at least two distinct ligands for sperm binding: a ZP3-independent ligand that is peripherally associated with the egg coat and facilitates gamete adhesion; and a ZP3-dependent ligand that is present in the insoluble zona matrix and is recognized by sperm GalT I to facilitate acrosomal exocytosis. The ZP3-independent ligand is not a result of contamination by egg cortical granules, nor is it the mouse homolog of oviduct-specific glycoprotein. It behaves as a 250 kDa, WGA-reactive glycoprotein with a basic isoelectric point, distinguishing it from the acidic glycoproteins that form the insoluble matrix of the egg coat. When eluted from isoelectric focusing gels, the acidic matrix glycoproteins possess sperm-binding activity for wild-type sperm, but not for GalT I-null sperm, whereas the basic glycoprotein retains sperm-binding activity for both wild-type and GalT I-null sperm. Thus, GalT I-null sperm are able to resolve gamete recognition into at least two distinct binding events, leading to the characterization of a novel, peripherally associated, sperm-binding ligand on the ovulated zona pellucida.
Key words: Fertilization, OGP, Sperm, Zona pellucida, ZP3, Mouse
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Introduction |
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Much of our current understanding of mammalian gamete recognition comes
from studies in mouse. This is due, in large part, to the ability to perform
quantitative in vitro assays of sperm-egg binding along with biochemical
analysis of mouse gametes, as well as the ability to manipulate the mouse
genome. Pioneering studies by Wassarman and colleagues suggest that in mouse,
sperm bind to a specific class of oligosaccharides on ZP3 (also referred to as
ZPC) (Spargo and Hope, 2003),
one of the three glycoproteins that constitute the extracellular coat of the
mouse egg, or zona pellucida. This was demonstrated by the ability of purified
soluble ZP3, as well as its oligosaccharide chains, to competitively inhibit
sperm-egg binding in vitro (Bleil and
Wassarman, 1980a
; Florman and
Wassarman, 1985
). Binding of ZP3 leads to sperm activation of both
pertussis toxin (PTx)-sensitive heterotrimeric G-proteins as well as
voltage-independent and -dependent cation channels that result in elevated
pHi and Ca2+i, thus triggering acrosomal
exocytosis (Arnoult et al.,
1996
; Endo et al.,
1987
; Endo et al.,
1988
; O'Toole et al.,
2000
).
The sperm receptor for ZP3 oligosaccharides has been more difficult to
identify, but most evidence is consistent with sperm surface
ß1,4-galactosyltransferase I (GalT I) performing this function. GalT I
specifically binds to the same class of ZP3 oligosaccharides that possess
sperm-binding activity, and removing or masking the GalT I binding site on
these oligosaccharides removes their sperm-binding activity
(Miller et al., 1992). The
cytoplasmic domain of GalT I binds, directly or indirectly, to heterotrimeric
G proteins that are activated following ZP3-induced aggregation of GalT I
(Gong et al., 1995
). In
support of this, ectopic expression of GalT I on Xenopus oocytes
results in ZP3-specific binding and G-protein activation, and mutagenesis of
the GalT I cytoplasmic domain prevents ZP3-dependent G-protein activation
(Shi et al., 2001
). Transgenic
sperm that overexpress GalT I bind more ZP3 than do normal sperm, have
accelerated G-protein activation and undergo precocious acrosome reactions
(Youakim et al., 1994
). By
contrast, sperm in which surface GalT I has been eliminated by homologous
recombination, but which maintain normal intracellular galactosylation, no
longer bind ZP3 in vitro or undergo zona-induced acrosomal exocytosis
(Lu and Shur, 1997
).
Despite these, and many other observations, the apparent role of ZP3 and
GalT I in sperm-egg binding has recently been thrown into question. For
example, GalT I-null sperm still retain the ability to bind to the ovulated
egg coat, despite their inability to bind ZP3 in solution or undergo
zona-induced acrosome reactions (Lu and
Shur, 1997). This binding enables GalT I-null sperm to penetrate
the zona pellucida matrix, presumably via spontaneous acrosome reactions, and
fertilize eggs (although at only 7% the efficiency of wild-type sperm when
assayed in vitro). Similarly, well-defined oligosaccharides that are not
predicted to be substrates for GalT I competitively inhibit sperm-egg binding
(Amari et al., 2001
;
Bendahmane et al., 2001
;
Johnston et al., 1998
;
Loeser and Tulsiani, 1999
).
Even though these inhibitory oligosaccharides do not appear to be present
within the zona pellucida (Aviles et al.,
1999
; Aviles et al.,
2000b
), their ability to inhibit binding speaks against a role for
GalT I in initial gamete adhesion. Finally, evidence that ZP3 is not the only
sperm-binding ligand in the egg coat comes from the observation that mouse
sperm still bind to eggs in which the mouse ZP3 polypeptide has been replaced
by human ZP3 (Rankin et al.,
1998
; Rankin et al.,
2003
). Some of this confusion probably results from the fact that
the biological activity of putative gamete receptors is usually assayed by
competitive inhibition of sperm binding to the coats of ovulated eggs, whereas
the source of competitive ligands for these experiments is frequently the
ovarian zona pellucida. At any rate, all of these observations suggest that
sperm binding to the zona pellucida may involve multiple receptor-ligand
interactions of which ZP3 binding to GalT I may be one component.
In this report, we test the hypothesis that gamete adhesion involves a ZP3-
and GalT I-independent receptor-ligand interaction. We have detected and
characterized a novel ligand for sperm that is present in the ovulated zona
pellucida, but not in the ovarian zona pellucida. This ligand activity does
not result from contamination by egg cortical granules or by ZP3. In addition,
this ligand is shown to be distinct from the mouse ortholog of
oviduct-specific glycoprotein (OGP), which has been implicated in sperm-egg
adhesion in hamster (Boatman and Magnoni,
1995). Lectin depletion studies and lectin-blotting of
two-dimensional polyacrylamide gels indicate that the ovulated zona pellucida
contains a relatively basic, high molecular weight, WGA-reactive glycoprotein
that is not present in the ovarian zona pellucida. This protein, when eluted
from isoelectric focusing gels, possesses sperm-binding activity for both
wild-type and GalT I-null sperm, whereas ZP3 possesses sperm-binding activity
for only wild-type sperm. These results suggest that the WGA-reactive, basic
glycoprotein within the ovulated zona pellucida functions as a ZP3- and
OGP-independent ligand that facilitates gamete adhesion.
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Materials and methods |
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Sperm-egg binding assay
Ovulated eggs were collected from superovulated CD-1 females and cleaned of
cumulus cells as described. Two-cell embryos were collected into dmKRBT from
the oviducts of superovulated females that were mated 15 hours earlier. The
caudae epididymides of strain-matched wild-type and long isoform GalT I-null
sperm (Lu and Shur, 1997) were
dissected into dmKRBT and shredded. The epididymides were incubated for 15
minutes at 37°C to release the sperm, which were collected after
filtration (3-35/27 Nitex, Sefar America, Kansas City, MO). The sperm
suspension was centrifuged at 66 g for 5 minutes at 24°C
and resuspended in fresh medium containing 10 µg/ml pertussis toxin
(CalBiochem, San Diego, CA), to prevent sperm from undergoing zona-induced
acrosome reactions that would artificially reduce the number of sperm bound to
the egg. Sperm were capacitated at 37°C for 1 hour and diluted to a final
concentration of 4x106 motile sperm/ml. Forty-thousand sperm
were incubated in 50 µl drops of dmKRBT containing 30-40 ovulated eggs and
5-10 embryos (as a control for non-specific binding) for 30 minutes at
37°C. The incubation solution contained either the purified zona
glycoproteins or an equal volume of buffer as control. Eggs and embryos were
washed through sequential drops of dmKRBT until one to three sperm remained
bound to the embryos. The gametes were fixed in 4% paraformaldehyde (Electron
Microscopy Sciences, Fort Washington, PA) and the number of sperm bound to
each egg and embryo was counted at 200x magnification using
phase-contrast optics. The average number of sperm bound/embryo was subtracted
from the average number of sperm bound/ovulated egg. The average of triplicate
drops for each time point was determined and normalized so that the number of
sperm bound in the control is equal to 100%. The data presented are the
average of at least three experiments (±s.e.m.).
Cortical reaction assay
The amount of contaminating cortical granule material in the zona pellucida
preparations (50 egg equivalents) was measured by assaying for
N-acetylglucosaminidase (GlcNAc'ase) as described
(Miller et al., 1993). The
reaction product was determined fluorometrically using a Perkin Elmer LS50B
instrument (Beaconsfield, UK) at an excitation wavelength of 380 nm, an
emission wavelength of 460 nm and a slit width of 2.5 nm. Fluorescence
produced by the substrate solution alone was subtracted as background from the
readings of the zona pellucida solutions. To determine the maximum amount of
cortical granule material released, eggs were incubated with 10 µM A23187
for 30 minutes at 37°C prior to preparation of each zona pellucida
fraction. Aliquots of the solutions assayed for GlcNAc'ase activity were
subsequently tested for biological activity in the sperm-egg binding assay at
a concentration of three zona equivalents/µl.
Anti-mouse ZP3 immunoblot
Twenty-five, 50, 100 and 200 egg equivalents of the peripheral and matrix
fractions of ovulated zona glycoproteins were solubilized in reducing sample
buffer and fractionated by gel electrophoresis. The proteins were transferred
to PVDF (Millipore) and blocked with 5% milk, 0.05% Tween 20, 1xPBS. The
blots were incubated with a 1:1000 dilution of IE-10, a rat monoclonal
antibody against mouse ZP3 residues 336-342
(East et al., 1985), and
subsequently in a 1:1000 dilution of sheep anti-rat IgG-HRP (Amersham). The
blots were washed and the signal developed by chemiluminescence (ECL-Plus,
Amersham). The blots shown are representative of two experiments.
Fecundity of GalT-null males crossed with OGP-null females
Twelve-week-old GalT I-null males were caged with at least three 6-week-old
Ogp+/+ or Ogp/ females
(Araki et al., 2003) for a
period of 3 months. The average litter size resulting from these matings was
calculated (±s.d.).
Lectin depletion of biological activity
WGA-agarose or BS-I agarose beads [100 µl (50% slurry)] (Vector
Laboratories, Burlingame, CA) were pelleted at 1000 g for 5
seconds, washed 10 times in 1xPBS containing 0.1 mM CaCl2 and
resuspended in 50 µl wash solution. This slurry (20 µl) was blocked in
100 µl 0.1% PVP in wash solution at 4°C for 2 hours. The beads were
subsequently resuspended in 1000 egg equivalents of the matrix or peripheral
fraction prepared in 0.1% PVP/PBS and incubated at 4°C for 2 hours. As
controls, 1000 egg equivalents of each solution were incubated in parallel in
the absence of the lectin-agarose beads, or alternatively, lectin-agarose
beads were incubated with buffer rather than with zona glycoproteins.
Following incubation, the beads were pelleted at 1000 g for 5
seconds and the supernatant removed. Five-hundred egg equivalents of the
depleted solution or the undepleted control were assayed for biological
activity in the sperm-egg binding assay.
Lectin blot of zona glycoproteins
One-thousand egg equivalents of the peripheral fraction were solubilized in
reducing sample buffer and fractionated by gel electrophoresis. The proteins
were transferred to PVDF and subsequently blocked in 1% BSA, 0.1% Tween-20,
0.9%NaCl, 50 mM TrisCl, pH 7.4. To detect glycoproteins, the membranes
were incubated with 1 µg/ml of biotinylated-WGA or BS-I (Sigma). The
membranes were washed and subsequently probed with a 1:50,000 dilution of
streptavidin-HRP (Zymed, S. San Francisco, CA). After washing, the signal was
developed by chemiluminescence.
Two-dimensional polyacrylamide gel electrophoresis of zona glycoproteins and characterization by lectin blot
Five-hundred egg equivalents of ovarian (2.5 µg) and ovulated zona
glycoproteins were precipitated by mixing with eight volumes of ice-cold
acetone and incubating overnight at 20°C. The proteins were
pelleted by centrifugation at 3000 g for 15 minutes at
4°C. After draining the acetone, the proteins were dried briefly at room
temperature and then solubilized in IEF sample buffer [9.5 M recrystallized
urea, 2% deionized NP-40, 5% ß-mercaptoethanol (BioRad), 1.6% Servalyt
5-7 and 0.4% Servalyt 3-10 isodalt (Crescent Chemicals)] for 30 minutes at
24°C. Prior to loading the protein solution, urea-acrylamide tube gels
(dimensions=5.5x0.1 cm) were cast according to the manufacturer's
directions (BioRad, Hercules, CA). The pH gradient was established by
electrophoresing gels in 10 mM NaOH and 10 mM H3PO4 for
10 minutes at 200 V, 15 minutes at 300 V and 15 minutes at 500 V. The buffers
were replaced and the sample was loaded directly onto the surface of the gel
and overlaid with 9 M recrystallized urea, 0.8% Servalyt 5-7, 0.2% Servalt
3-10, isodalt and 0.05% Bromophenol Blue. The proteins were electrophoresed at
500 V for 10 minutes and 750 V for 3.5 hours (until equilibrium). The gels
were stored in SDS-equilibration buffer (62.5 mM TrisCl, pH 6.8, 2.3%
SDS, 8% glycerol, 0.05% Bromophenol Blue) at 80°C. The IEF gels
were warmed to 24°C and equilibrated for 30 minutes with gentle agitation.
The gels were transferred into the well of a 4-12% gradient polyacrylamide gel
(Jule, Milford, CT) and covered with agarose solution (1% low
Mr agarose, 0.1% SDS, 125 mM TrisCl, pH 6.8). The
proteins were fractionated by electrophoresis, transferred to PVDF, and then
probed with biotinylated WGA as described.
Purification of the ZP3-independent ligand from IEF gels
Ovulated zona glycoproteins (5000 egg equivalents) were fractionated by IEF
as described above. After electrophoresis, the tube gel was sliced into 2 mm
pieces and transferred into siliconized 1.5 ml tubes. The gel pieces were
incubated in 200 µl of 50 mM NH4HCO3, pH 7.6
containing 1 µg/ml fatty acid-free BSA for 12 hours at 4°C. This was
repeated two additional times, after which the three wash solutions were
combined and incubated with 0.12 g BioBeads (BioRad, Hercules, CA) for 15
minutes at 24°C. The solutions were concentrated to 100 µl and dialyzed
against 50 mM NH4HCO3 for 1 hour at 24°C. The
solutions were dried and the proteins washed twice in double-distilled
H2O before being solubilized in dmKRBT and tested in the sperm-egg
binding assay.
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Results |
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The lack of ligand activity for GalT I-null sperm in ovarian zona glycoproteins predicts that GalT I-null sperm should be incapable of binding directly to the intact zona pellucida of ovarian oocytes. Zona-intact oocytes were collected from antral follicles, and their follicular cells removed following prolonged hyaluronidase digestion. GalT I-null sperm bound to zona-intact antral oocytes at 50% the level of binding to ovulated oocytes (11±1 sperm/ovarian oocyte versus 21±2 sperm/ovulated oocyte). However, we suspect that the 50% binding seen towards antral oocytes is an artifact of preparation, as the prolonged digestion with hyaluronidase required to remove follicular cells increased the binding of GalT I-null sperm to both ovarian and ovulated eggs (data not shown). To test more directly for the presence of ligand activity towards GalT I-null sperm, the zonae pellucidae of antral oocytes were solubilized and assayed in competitive sperm-egg binding assays; no inhibition of GalT I-null sperm binding occurred, whereas the solubilized zona glycoproteins readily inhibited wild-type sperm-egg binding (similar to that in Fig. 1). Collectively, these results suggest that GalT I-null sperm recognize a ligand present primarily, if not exclusively, on ovulated oocytes, whereas wild-type sperm recognize ligands in both the ovarian and ovulated egg coat.
Although GalT I-null sperm are unable to bind ZP3 or undergo ZP3-induced
acrosome reactions, a small number of sperm (i.e. 7% of wild-type) are able to
penetrate the zona pellucida in vitro (Lu
and Shur, 1997). This low level of zona penetration is thought to
account for the fertility of GalT I-null males in vivo, which may result from
spontaneous acrosome reactions or other `non-physiological' inducers of
acrosomal exocytosis. In any event, we tested whether the sperm-binding ligand
present in the ovulated zona could induce acrosomal exocytosis in GalT I-null
sperm. This was shown not to be the case; ovulated zona glycoproteins failed
to induce acrosomal exocytosis in GalT I-null sperm but, as expected, did
induce acrosome reactions in wild-type sperm, presumably owing to the presence
of ZP3 (data not shown).
The ovulated zona pellucida contains a peripherally associated ligand for sperm binding
At the time of ovulation, epithelial cells lining the oviduct are actively
secreting glycoproteins into the lumen where fertilization occurs. Some of
these components have been postulated to play a role in maintenance of the
oocyte, gamete interaction and development of the embryo
(Buhi et al., 2000). We
therefore tested whether the sperm-binding ligand in the ovulated zona
pellucida resulted from addition to the zona pellucida during transit into the
oviduct. Cumulus cell-free ovulated eggs were stringently washed to remove any
material loosely associated with the zona pellucida, as described in the
Materials and methods, and the wash solution was collected as the `peripheral
fraction'. The remaining intact washed zona pellucida was solubilized as the
`matrix fraction'. Both the peripheral and matrix fractions were tested for
ligand activity in the competitive sperm-egg binding assay.
As expected, the matrix fraction inhibited wild-type sperm binding to eggs in a concentration-dependent manner (Fig. 2A), but had no activity against GalT I-null sperm (Fig. 2B). Thus, the matrix fraction recapitulates the activity of the ovarian zona glycoproteins, indicating that it contains ZP3 but not the ligand to which GalT I-null sperm bind. By contrast, the peripheral fraction inhibited the binding of both wild-type and GalT I-null sperm to eggs in a concentration-dependent manner. This demonstrates that the ovulated zona pellucida contains two distinct ligand activities: ZP3 associated with the insoluble matrix and a ZP3-independent component that is peripherally associated with the zona pellucida. The fact that the ZP3-independent ligand can be removed from the zona pellucida by stringent washing suggests it results from addition to the egg coat upon entry into the oviduct, and not from covalent modification of the zona matrix.
|
No GlcNAc'ase activity above background was detected in the intact ovulated zona pellucida or in the solubilized matrix fractions (five different preparations assayed) (data not shown). This result argues against the possibility that the ZP3-independent ligand results from egg-derived material, as the intact ovulated zona pellucida contains both ZP3 and ZP3-independent ligands (Fig. 1).
However, preparing the peripheral fraction resulted in variable levels of GlcNAc'ase activity, ranging from 5-16% of the total enzyme activity detectable in eggs (1.7±0.6 to 5.2±0.5 fluorescence units relative to 32.2±5.4 fluorescent units in ovulated eggs). Thus, it remained a formal possibility that this contamination was responsible for the sperm-binding activity in the peripheral fraction. To test this possibility, we maximized the amount of potential egg-released material by treating eggs with the calcium ionophore A23187 to induce the cortical reaction, and the peripheral fraction was collected and assayed for GlcNAc'ase activity and sperm-binding activity.
As expected, treatment with A23187 increased the GlcNAc'ase activity of both the matrix and peripheral fractions (Fig. 3A) and of the intact ovulated zona pellucida (data not shown). However, A23187 treatment reduced the ability of the peripheral and matrix fractions to inhibit wild-type sperm binding, relative to solutions prepared from DMSO-treated eggs (Fig. 3B). This result indicates that egg-released material is not the source of the ligand in the peripheral fraction and, in fact, increasing the amount of contaminating egg material decreases the biological activity of the ligand, probably as a result of proteases and glycosidases released from cortical granules.
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Multiple attempts to purify the biological activity from the peripheral
fraction proved unsuccessful because of limiting amounts of protein (estimated
at 1 ng per egg equivalent) and variable amounts of biological activity,
which is probably the result of cortical granule damage during preparation.
Therefore, we returned to the original source of the peripherally associated
ligand, the ovulated zona pellucida, and compared the migration of
WGA-reactive proteins in ovarian and ovulated zonae by two-dimensional
SDS-PAGE (Fig. 7). As expected,
both the ovarian and ovulated zonae pellucidae consist of heavily
glycosylated, relatively acidic proteins that migrate with a pattern
consistent with ZP1, ZP2 and ZP3 (Bleil
and Wassarman, 1980b
). Furthermore, the ovulated zona pellucida
contains a WGA-reactive, relatively basic protein of
250 kDa that is
absent from the ovarian zona pellucida. We determined that this glycoprotein
originated from the zona pellucida and was not a contaminant of the
hyaluronidase or buffers used to prepare the zona glycoproteins (data not
shown). The molecular weight of this protein is comparable with the
WGA-reactive protein detected in the peripheral fraction by one-dimensional
SDS-PAGE, although the conditions of the electrophoresis and the molecular
weight standards differed between the two experiments.
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Discussion |
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The two distinct sperm-binding activities can be attributed to two distinct sperm-binding ligands present in the ovulated egg coat: a ligand in the insoluble zona matrix and a peripherally associated ligand that can be removed by extensive washing. The matrix fraction can account for the behavior of ovarian zona glycoproteins in that it inhibits wild-type sperm-egg binding, but has no effect on GalT I-null sperm. By contrast, the peripheral fraction inhibits both wild-type and GalT I-null sperm binding. These results strongly suggest that the matrix fraction contains ZP3 and the peripheral fraction contains a ZP3-independent component. Furthermore, as wild-type sperm are sensitive to both the peripheral and matrix fractions, this indicates that the ZP3-independent ligand is physiologically relevant to wild-type sperm-egg binding, and not a peculiarity of the GalT I-null sperm phenotype.
The ligand activity in the peripheral fraction is not a byproduct of
cortical granule secretions released during preparation or a result of to
residual ZP3, nor is it a function of mouse oviduct-specific glycoprotein
(OGP). This is consistent with recent data indicating that OGP-null females
are fertile (Araki et al.,
2003). Initial characterization suggests that the ligand is a
relatively basic, WGA-reactive, high molecular weight (
250 kDa)
glycoprotein that can be readily distinguished from the insoluble matrix
glycoproteins by two-dimensional SDS-PAGE. Preliminary estimates indicate that
the peripheral fraction contains
1.7 ng protein/egg, whereas the
insoluble matrix fraction contains
5.1 ng protein/egg. Although the
peripheral fraction probably contains proteins in addition to the
ZP3-independent ligand, this suggests that the ligand is present at levels not
grossly different from the individual zona matrix glycoproteins, which have
both structural as well as sperm-binding capacities.
We expected that the matrix and peripheral fractions would be less
efficacious in inhibiting wild-type sperm binding than the `unfractionated'
ovulated zona pellucida. Similarly, one would predict that wild-type sperm
would continue to bind to the zona pellucida even in the presence of soluble
ZP3, owing to the presence of the ZP3-independent ligand. However, we, and
others, have observed that sperm-egg binding is effectively blocked when only
one competitive ligand is present, e.g. ZP3 or the ZP3-independent ligand. The
mechanism underlying this is unclear; blocking the binding site of one
receptor may sterically interfere with the ability of the other receptor to
recognize its ligand or may alter its affinity for ligand. Evidence for this
comes from the observation that GalT I-null sperm bind to ovulated eggs in
higher numbers than do wild-type sperm (Lu
and Shur, 1997), suggesting that the affinity of the GalT
I-independent receptor may be modulated by the presence of GalT I. This model
cannot be definitively tested until the ligand and the receptor have been
purified in sufficient quantities to perform binding analyses. It is
noteworthy, however, that cellular interactions known to be mediated by
multiple receptor-ligand pairs, such as the concerted action of selectins and
integrins in mediating lymphocyte interactions with vascular endothelium, are
readily inhibited by low molecular weight competitors of only one of the
relevant receptor-ligand pairs
(Bevilacqua, 1993
;
Lasky, 1992
;
Stoolman and Rosen, 1983
).
There are several reasons that may explain why the ZP3-independent ligand has not been identified previously. First, the removal of GalT I from sperm by homologous recombination allowed us to eliminate, for the first time, the contribution of ZP3, and thereby reveal a novel, previously undetected binding activity. Second, the traditional procedure of extensively washing ovulated eggs before solubilizing their zona pellucida removes most of the peripherally associated sperm-binding ligand, as shown here by the ability to remove ligand activity by extensive washing. Finally, the majority of experiments characterizing the function of zona glycoproteins have used homogenized ovaries as a source of zona glycoproteins, which lacks the ZP3-independent ligand.
The sperm receptor for the ZP3-independent ligand is of great interest. We
tested the possibility that other members of the GalT family may function in
this capacity, as GalT I is now known to be one of six enzymes in the
ß1,4-galactosyltransferase family
(Almeida et al., 1999;
Lo et al., 1998
), some of
which have been reported to be expressed in testis
(Almeida et al., 1997
;
Sato et al., 1998
). This
possibility was shown to be unlikely, as the addition of UDP-galactose readily
inhibited wild-type sperm-egg binding, as previously demonstrated
(Lopez et al., 1985
), whereas
it had no effect on GalT I-null sperm-egg binding
(Fig. 9). As UDP-galactose is
able to force the catalytic dissociation of any putative galactosyltransferase
from its galactosylated product, this result suggests that no other members of
the GalT family function during the binding of GalT I-null sperm to the zona
pellucida. However, any of the other sperm components recently implicated in
ZP3-independent sperm-egg binding, such as SED1 or arylsulfatase A, could
function as the receptor for this novel, ZP3-independent ligand
(Ensslin and Shur, 2003
;
Tantibhedhyangkul et al.,
2002
; White et al.,
2000
).
|
Finally, and perhaps most significantly, is that during fertilization sperm
bind to the zona pellucida of ovulated oocytes, and not to the ovarian egg
coat. In fact, oviductal glycoprotein secretions are known to permeate the
zona pellucida, and, at least in hamster, there is evidence to suggest that
the ovulated zona pellucida has biological activities that are distinctly
different from those in the ovarian zona pellucida
(Boatman and Magnoni, 1995;
Kan et al., 1990
;
Robitaille et al., 1988
;
St-Jacques et al., 1992
). All
of these observations necessitate a re-examination of the simple premise that
sperm-egg binding involves a single receptor-ligand interaction. In this
regard, the fact that GalT I-null sperm fail to undergo acrosomal exocytosis
even though they bind to the ovulated zona pellucida, clearly resolves gamete
interaction into at least two distinct components, a ZP3-independent adhesive
event and a ZP3-GalT I-dependent induction of acrosomal exocytosis.
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ACKNOWLEDGMENTS |
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