Download The role of carbohydrate in sperm

Molecular Human Reproduction vol.2 no.10 pp. 767-774, 1996
The role of carbohydrate in sperm-ZP3 adhesion
Neil R.Chapman1-2 and Christopher L.R.Barratt2-3
Departments of 1Molecular Biology and Biotechnology, Firth Court, University of Sheffield, Sheffield S10 2UH and
Obstetrics and Gynaecology, Jessop Hospital for Women, Leavygreave Road, Sheffield S3 7RE, UK
•'To whom correspondence should be addressed
2
Key words: carbohydrate/cell adhesion spermatozoa/human zona pellucida/ZP3
Introduction
Fertilization is the most important cell adhesion event in the
life time of any organism. In a number of organisms ranging
from echinoderms to mammals, the recognition of carbohydrate
epitopes by complimentary protein receptors has been proposed
to be a critical factor in gamete interaction (Glabe et al, 1982;
Florman and Wassarman, 1985; DeAngelis and Glabe, 1987).
The following article briefly describes both how glycoproteins
arise in vivo and lists some potential biological roles of the
carbohydrate moieties of the given examples. The majority of
the article will be concerned with the involvement of proteincarbohydrate interactions in mammalian sperm—zona binding.
What is protein glycosylation?
Glycoproteins arise in vivo following the covalent linkage of
carbohydrate moieties to certain amino acid residues within
the polypeptide chain of a newly synthesized protein. There
are two main classes of glycosylation, each defined by the
nature of the covalent linkage of the carbohydrate residues to
the polypeptide backbone.
The first type is AMinked glycosylation. The carbohydrate
moieties in these structures are attached to the protein backbone
through covalent linkages to asparagine (Asn) residues. These
Asn residues are located in an amino acid consensus sequence
Asn-X-Ser/Thr (where X is any amino acid except Pro)
within the protein to be glycosylated. Recently, it has been
demonstrated that the amino acid at the X position of this
sequon is an important determinant of core glycosylation
efficiency (Shakin-Eshleman' et al, 1996). Residues such as
Tip, Asp, Glu and Leu were associated with poor N-glycosylation, while Lys, Arg and His had a positive effect on Nglycosylation (Shakin-Eshleman et al, 1996). However, it
must be emphasized that the actual presence of this tripeptide
within the primary amino acid sequence does not necessarily
imply that the protein will be yV-glycosylated at that site
(Komfeld and Komfeld, 1985). It is believed that other factors
including the nature of neighbouring amino acids and local
conformation of the protein are also important determinants
of whether a consensus sequence becomes modified. All Nlinked carbohydrates are derived from a common precursor
© European Society for Human Reproduction and Embryology
which is the sequence of sugar residues as follows (terminal
residue first): Manal-3(Manal-6)Manpl^NAGpi^NAGAsn. (Man = mannose, NAG = A/-acetylglucosamine, Asn =
Asparagine.)
There are three major types of A'-glycosylation: (i) high
mannose, where all subsequent sugars attached to the core
sequence are mannose; (ii) complex, where both the a l - 3 and
a l - 6 terminal mannose residues of the core are linked to Nacetylglucosamine; and (iii) hybrid where the glycosylated
structure contains elements of both (i) and (ii). (For a more
detailed review of A/-linked glycosylation, see Komfeld and
Komfeld, 1985.)
The second type of glycosylation is O-linked. In this form
of modification, the carbohydrate chain is covalently attached
through a glycosidic linkage to a (J-hydroxy amino acid such
as serine or threonine. As with AMinked glycosylation, it is
believed that local protein structure may be an important
determinant as to which Ser or Thr residues are modified
(Elliott et al, 1994). In contrast to A'-glycosylation, there is
no consensus amino acid sequence flanking the modified
amino acid (Wilson et al, 1991). However, comparisons of
glycosylated and non-glycosylated amino acids within an Olinked glycoprotein demonstrate that proline residues are often
found at the - 1 , - 3 , -6 and +3 positions when this form of
glycosylation occurs (Wilson et al, 1991).
Glycosylation can therefore give rise to three classes of
proteins, those that contain only O-linked glycosylation, those
that contain only A'-linked glycosylation and those that possess
both classes of carbohydrate modifications. A more detailed
review of protein glycosylation can be found in Dwek (1994).
Why glycosylate proteins?
Proteins appear to become glycosylated for a number of
reasons. For example, O-glycosylation appears to be important
for the maintenance of structure in the polypeptide chain of
ovine submaxillary mucin (Gerken et al, 1989). A'-glycosylation appears to have a number of roles in protein function
ranging from the correct folding of polypeptide subunits such
as the a-subunit of the nicotinic acetylcholine receptor from
Torpedo californica (Rickert and Imperiali, 1995) to the
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N.R.Chapman and C.L.R.Barratt
secretion and dimerization of human interferon-v (Sareneva
etal., 1994).
At the molecular level, the survival of an organism can
depend upon the success of interactions between proteins and
carbohydrates; for example cell adhesions between leukocytes
and endothelial cells of blood vessels and capillaries are vital
for leukocyte extravasation into damaged tissue giving rise to
inflammation. Conversely, these protein-carbohydrate interactions may also bring an untimely end to the organism's
existence, examples being the binding of some viral particles
(influenza) and bacterial toxins (cholera toxin from Vibrio
cholerae and pertussis toxin from Bordetella pertussis) to the
plasma membrane of eukaryotic cells which is mediated by
protein-carbohydrate interactions (see Karlsson, 1995 for a
detailed review of microbial recognition of target-cell glycoconjugates).
Glycosylation and fertilization
In the context of mammalian fertilization, it is generally
accepted that a glycoprotein within the zona pellucida termed
ZP3 is responsible for specifically binding to complementary
receptor molecules located on the head of the spermatozoa.
This has been demonstrated to be the case in mice (Bleil and
Wassarman, 1980), hamsters (Moller et al., 1990) and pigs
(Sacco et al, 1989).
Florman and Wassarman (1985) initially demonstrated that
the sperm-binding capacity of mouse ZP3 resided within the
O-glycosylation present on this glycoprotein since removal of
O-linked carbohydrates alone was sufficient to prevent spermzona interaction. Removal of AMinked carbohydrates was
found to have negligible effect on sperm-binding activity.
More recently, Kinloch et al. (1995) have demonstrated that
when five Ser residues located in the C-terminus of mouse
ZP3 were mutated to either Gly, Val or Ala then inactive
recombinant mouse ZP3 was secreted from transfected
embryonal carcinoma cells implying that O-linked carbohydrate located within the C-terminal portion of the ZP3 polypeptide chain was vital for sperm-binding activity. Further work
demonstrated that enzymatic removal of terminal galactose or
fructose residues from carbohydrate attached to mouse ZP3
with either a-galactosidase or cc-fucosidase respectively caused
a reduction in sperm-zona pellucida interaction. The observation that sperm-zona pellucida interactions could be perturbed
by the oxidation of the C6 position alcohol of terminal
galactose residues present in the 0-linked carbohydrate of
mouse ZP3 was also noted in this study (Bleil and Wassarman,
1988). Concomitant with the subsequent reduction of this
sugar aldehyde, sperm-binding activity was restored (Bleil
and Wassarman, 1988). Taken together, these observations
imply that terminal galactose residues of 0-linked oligosaccharides are essential for initiating mouse gamete interaction.
In contrast to the observations of Bleil and Wassarman
(1988), however, other groups have demonstrated that the
amino sugar A'-acetylglucosamine (NAG) is the key terminal
monosaccharide involved in sperm—zona interaction in the
mouse (Lopez etal., 1985). This view is supported by evidence
that implies mouse spermatozoa express a membrane-bound
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p-galactosyltransferase overlying the acrosomal region (Shur
and Neely, 1988). This enzyme utilizes UDP-galactose in
binding to NAG moieties terminally located on ZP3 0-linked
oligosaccharides (Miller et al., 1992).
The involvement of terminal galactose residues in an aglycosidic linkage to penultimate sugar moieties in gamete
interaction of the mouse has been questioned recently. Litscher
et al. (1995) utilized oligosaccharides with defined structures
and sequences to inhibit binding of mouse spermatozoa to
unfertilized eggs in vitro. A total of 15 individual oligosaccharides were synthesized; each oligosaccharide differed in
chain length and configuration of the glycosidic linkage at the
anomeric carbon of the terminal sugar residue. This group
demonstrated that a pentasaccharide based on a blood group
type-B oligosaccharide was capable of causing 95% inhibition
of sperm-zona interaction when used at a concentration of
5 raM. Furthermore, at this concentration, this oligosaccharide
structure was seen to promote head-to-head aggregation of
mouse spermatozoa. It is possible these observations may be
artefacts of the experimental conditions employed, i.e. the
spermatozoa were exposed to a very high local concentration
of oligosaccharide resulting in a non-specific inhibition of
mouse gamete interaction. The observed head-to-head aggregates of mouse spermatozoa may arise through the ability of
mouse spermatozoa to recognize and subsequently bind the
reducing (anomeric) carbon of the sugar residue. In vivo, this
anomeric carbon would be conjugated to the polypeptide
backbone of ZP3 via an a-glycosidic linkage: it would therefore
be unavailable for interaction with free swimming spermatozoa
as in the present study.
The study by Litscher et al. (1995) also demonstrated an
apparent lack of specificity concerning the binding of these
oligosaccharides to spermatozoa. Bi- and tetra-antennary oligosaccharides containing at least six monosaccharide units with
the terminal galactose residue in either the a- or pVanomeric
configuration inhibited spermatozoa-zona interaction. Furthermore none of the oligosaccharide molecules were able to elicit
the spermatozoal acrosome reaction; regarding the latter, it has
been suggested by a number of authors that an intact ZP3
polypeptide chain is required for the acrosome reaction to
proceed (Florman et al., 1984; Florman and Wassarman, 1985;
Leyton and Saling, 1989). The absence of the ZP3 polypeptide
backbone in the study by Litscher et al. (1995) is a possible
explanation for the lack of spermatozoal acrosome reaction
observed with these oligosaccharides.
Recently it has been demonstrated that a galactose-al,3galactose (galal,3gal) epitope implicated in sperm-binding to
mouse ZP3 is not required for fertilization. The enzyme UDPgalactose:P-D-galactose-al,3-galactose-galactosyltransferase
(al,3GT) is responsible for gal-al,3-gal synthesis. Female
mice homozygous for the al,3GT transgene developed normally and were able to produce oocytes capable of being
fertilized naturally (Thall et al., 1995). Two implications arise
from this observation: (i) the absence of terminal a-galactose
moieties from O-Iinked carbohydrates in mouse ZP3 are not
detrimental to successful sperm-zona pellucida interaction (Nlinked glycosylation has been shown not to actively participate
in mouse sperm-ZP3 binding (Florman and Wassarman, 1985),
The role of carbohydrate in sperm-ZP3 adhesion
therefore the absence of the galal,3gal epitope from this type
of glycosylation should not effect mouse gamete interaction
to any extent); (ii) if initial gamete interaction in the mouse
is carbohydrate mediated, then other carbohydrate epitopes are
being recognized by ZP3 receptors located on the spermatozoa,
e.g. either NAG (Miller et ai, 1992) and/or fucose (Bleil and
Wassarman, 1988). The latter is based on the observation that
treatment of mouse ZP3 with a-fucosidase reduced spermzona binding (although fucose was not detected by high
performance liquid chromatography analysis). The data concerning al,3GT cannot be extrapolated to human gamete
interactions for the simple reason that, although a human
homologue of the gene encoding this enzyme exists, it is in
fact a pseudogene (an inactive gene) which is not expressed
in humans and other Old World primates (Joziasse et ai, 1991).
The data concerning the involvement of either N- or Olinked glycosylation in other mammals are equivocal: with
regard to gamete interaction in the pig, it is clear that the
glycan chains associated with pig ZP3a (renamed pZPB by
Gupta et ai, 1995) are responsible for mediating sperm-zona
recognition (Sacco et ai, 1989). Whether these glycans are of
the N- or 0-linked type is still open to debate since evidence
for the involvement of both classes has been put forward:
Yurewicz et al. (1991) demonstrated a role for 0-linked
glycosylation in sperm-zona pellucida interaction, while more
recently N-linked glycosylation has been proposed to be
important for successful pig gamete recognition (Yonezawa
etal., 1995).
Data relating to a role for carbohydrate in human spermzona binding are rare due to the lack of native zonae pellucidae.
Initially it was believed that cloning of the human ZP3 gene
(Chamberlin and Dean, 1990) would circumvent this lack of
material since a 'constant' supply of recombinant material
would be available. However, although biologically-active
human recombinant ZP3 produced by Chinese hamster ovary
(CHO) cells was first reported in 1993 (Barratt et al., 1993),
subsequent purification of biologically active material that
provides consistent results has presented significant problems
in both our own and in other laboratories (Van Duin et ai,
1994; Barratt and Hornby, 1995). We believe characterization
of active glycosylated recombinant human ZP3 is made difficult
due to the fact that a number of different glycoforms have the
potential of being synthesized depending on which cell line is
chosen to express the ZP3 cDNA. There are a number of
examples of this occurring with other glycoproteins that have
been documented in the literature, e.g. tissue plasminogen
activator (t-PA). When t-PA was isolated from a Bowes
melanoma cell line and a human colon fibroblast cell line, it
was shown to contain TV-linked carbohydrate: the nature of
which depended upon the cell line used (Parekh et ai, 1989a;
Wittwer et ai, 1989a). Furthermore, the in-vitro activities of
these t-PA glycoforms were also shown to differ. The reason
for the differences in activity correlates with the nature of the
specific N-glycosylation site on the polypeptide which was
found to differ between the cell lines used (Parekh et ai,
1989b; Wittwer et ai, 1989b).
It is the authors' opinion, that sperm-ZP3 interaction in the
mouse is an event where a protein receptor(s) binds to a
number of different epitopes within the ZP3, i.e. gamete
interaction is a process where recognition of gametes relies
on multivalent ligand interactions. With regard to the molecular
basis of cell adhesion, individual protein-carbohydrate interactions are thought to be very weak and often of broad
specificity (Kiessling and Pohl, 1996). However, it should be
mentioned that bacterial sugar-binding proteins have high
affinities for their substrates, e.g. the arabinose binding protein
has a K<j == 1 uM for L-arabinose (Quiocho and Vyas, 1984).
Structural studies illustrate that the high affinity of the arabinose
binding protein for its substrate arises from the deep binding
cleft within its structure. Conversely, some cell adhesion
proteins (such as E-selectin) contain only very shallow grooves
on their surfaces. These grooves act as the carbohydrate
binding sites resulting in a much lower substrate affinity
(Graves etal., 1994). Kiessling and Pohl (1996) have suggested
that recognition of carbohydrate epitopes by protein receptors
is a process where the receptor protein binds to a great many
sugar residues. Individual monosaccharide-protein interactions
would be very weak but, as the number of interactions
increases, then the strength of the entire protein—oligosaccharide interaction also increases. This results in a strong
and highly specific interaction. The concept of multivalent
ligand binding in high affinity interactions has been suggested
for the binding event between the L- and P-selectin proteins
and a tetrasaccharide structure termed the sialyl Lewis x (sLex)
(Rosen and Bertozzi, 1996).
Based on the observations stated above regarding the number
of different sugar residues reported to be essential for mouse
sperm-ZP3 binding, we suggest that if the sperm-ZP3 binding
interaction relies on recognition of ZP3-based carbohydrate
epitopes by cognate protein receptors on the spermatozoa, then
it is possible the protein receptors involved are recognizing a
range of epitopes, rather than a single sequence, e.g. the protein
receptor has the ability to bind galactose, NAG and fucose
moieties when displayed in a specific geometric pattern on
ZP3 (i.e. a multivalent ligand as opposed to a monovalent
ligand). In this hypothesis, the ligands (the glycans displayed
on ZP3) have a number of different sites with which the
protein receptor on the spermatozoa will interact. In relation
to human sperm-zona recognition, a study conducted by BarShira Maymon et ai (1994) employed lectins to determine the
distribution and identity of carbohydrate moieties on human
zonae pellucidae. A number of sugar residues including mannose, p-galactose and N-acetylglucosamine were shown to be
present within the human zona pellucida. At present one can
only speculate as to whether human gamete interaction also
utilizes a spermatozoa-based receptor protein to recognize
and specifically bind to a multivalent carbohydrate epitope
within ZP3.
A number of studies have demonstrated that a polysaccharide
termed fucoidan from Fucus vesiculosus has the ability to
inhibit a number of cell adhesion events including selectinmediated cell-cell interactions (Foxall et ai, 1992) and spermzona pellucida binding (Oehninger et ai, 1990). Fucoidan is
a complex polymer of ocl-3 linked fucose residues, with
sulphate covalently attached to the 4-position of certain fucose
moieties (Patankar et ai, 1993). To our knowledge, the
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N.R.Chapman and C.L.R.Barratt
mechanism by which fucoidan inhibits human gamete interaction has yet to be fully addressed. According to Oehninger
et al. (1991), fucoidan is acting in a competitive manner and
as such is masking the zona pellucida receptor(s) located on
spermatozoa. It is possible that the nature by which fucoidan
inhibits human sperm-zona pellucida interaction depends upon
its ability to present a number of sulphated fucose residues in
relatively fixed, spatial orientations to the receptor(s) located
on the spermatozoa, thereby filling binding sites normally used
by ZP3 associated glycans. This would imply that the zona
receptor located on the spermatozoa has the ability to recognize
a number of sulphated fucose ligands simultaneously, i.e.
fucoidan is acting as a multivalent ligand.
It is possible to immobilize sugar residues on inert matrices
and subsequently examine their effect upon particular binding
events. For example, in an attempt to probe the mechanisms
underlying intercellular recognition and adhesion, Weigel et al.
(1979) demonstrated that rat hepatocytes were able to specifically bind to polyacrylamide gels modified to contain galactose
moieties at fixed intervals along the polymer backbone. This
system provided a synthetic polyvalent ligand with which
the hepatocytes could interact in a specific manner (other
monosaccharides such as NAG were utilized but failed to
promote hepatocyte adhesion to the gel support). More recently,
the use of polyacrylamides to present multiple epitopes to a
protein receptor has been successfully employed in the inhibition of influenza virus haemagglutinin attachment to erythrocytes (Spaltenstein and Whitesides, 1991). This group
demonstrated that polyacrylamides with multiple a-sialic acid
residues attached were more effective inhibitors of haemagglutination than structurally similar monosaccharides. Neuraminidase-treatment of these sialic acid-derived gels completely
abolished the inhibition of haemagglutination.
It would be of great interest to utilize these types of modified
gel to examine the nature of the carbohydrate involved in
mammalian sperm-ZP3 interactions. Monosaccharides of
interest could be conjugated to acrylamide as described by
Kallin (1994). Once poured and set, adhesion of spermatozoa
to the glycosylated gel is examined in a similar fashion to that
employed by Weigel et al. (1979) for adhesion of rat hepatocytes to immobilized sugars. As stated above, this system
would provide a polyvalent carbohydrate ligand to which
spermatozoa could bind, with the immobilized sugar acting as
a 'terminal' monosaccharide (see Figure 1A-C).
However, there are a number of drawbacks associated with
a study of this nature. The first problem is concerned with
how one defines whether a spermatozoon has bound to the
glycosylated gel or not. The study by Weigel et al. (1979)
employed rat hepatocytes, which were unable to undergo
exocytosis upon binding to their ligand. This is in contrast to
spermatozoa from some mammals (e.g. mice) which, in vivo,
undergo the acrosome reaction (a specialized form of cell
exocytosis) shortly after binding to their ligands (Florman and
Storey, 1982). It is possible acrosome-reacted spermatozoa
would be removed from the gel during the experminental
procedure and give rise to a false negative result, i.e. a
proportion of spermatozoa bind the ligand, undergo the acro770
some reaction and are subsequently lost with the removal of
non-specifically bound debris.
Secondly, it is debatable whether the polyacrylamide used
in studies of this nature would be an ideal substitute for the
polypeptide chain, i.e. would a polyacrylamide backbone be
of sufficient flexibility to facilitate the clustering of receptors
on each spermatozoon.
Thirdly, the spatial separation of monosaccharide ligands
covalently linked to polyacrylamide may not reflect the geometric orientation utilized in the natural ligand. This could
potentially lead to a sub-optimal number of receptors on
the spermatozoon being occupied by immobilized ligand,
preventing tight binding of spermatozoa to the glycosylated
gel and causing failure of acrosomal exocytosis (Figure 1D-E).
The final major drawback of this system is that only
monosaccharides are used. Thus, the effects that penultimate
and other deeper saccharides have on both the terminal
sugar and spermatozoa binding are not observed. These other
carbohydrate residues (including the terminal saccharide) may
form part of a sequence with which the spermatozoa interacts;
if they are not present the spermatozoa will either bind with
a much lower affinity or not bind at all to the immobilized
sugar giving the impression that this sugar is not vital for
gamete interaction. An appropriate analogy for this hypothetical
scenario is obtained from studies into the mechanism of
lysozyme. This enzyme is found in a number of body secretions
where its function is to hydrolyse the polysaccharide component of bacterial cell walls. The enzyme hydrolyses the
glycosidic bond between Af-acetylmuramic acid and A'-acetylD-glucosamine within an active site cleft that is able to
accommodate six sugar residues. Binding of carbohydrate to
all six subsites is important for successful catalysis since the
enzyme will hydrolyse a hexasaccharide 107 times more rapidly
than it does a disaccharide (Creighton, 1993). However,
despite these caveats, glycosylated gels have been successfully
employed in a number of studies investigating the functions
of carbohydrates in cell adhesion (see above and references
therein). Furthermore, we believe experminents utilizing monosaccharides immobilized on polyacrylamide backbones could
prove useful tools to investigate the identity of critical carbohydrate residues involved in mammalian gamete interactions.
Is sulphate important
actions?
in sperm-ZP3
inter-
Sulphated proteins arise in vivo through the covalent attachment
of sulphate groups to tyrosine residues within the polypeptide
chain forming tyrosine O-sulphate and also by modifying
carbohydrate residues within certain glycoproteins (Huttner,
1984). Sulphated tyrosine residues and sulphated carbohydrate
moieties can exist within the same protein (Huttner, 1984).
Shimizu et al. (1983) demonstrated that it was possible to
maintain mouse oocytes when grown in follicle culture. This
study investigated the biosynthesis of the three mouse zona
pellucida glycoproteins. Zonae pellucidae grown in this system
were shown to incorporate [35S]-sulphate. Subsequent analysis
of individual zona glycoproteins illustrated that [35S] had been
incorporated only into ZP1 and ZP2, not ZP3. From a study
The role of carbohydrate in sperm-ZP3 adhesion
Spermatozoon
plasms 11KJIIUJ altt
Acrosome
Fig. 1A. Poh/acrylamide backbone
with covaJently attached
monosaccharide units binds
spermatozoa
•"" = Carbohydrate nx)iety
—( = Spermatozoa receptor
forZP3hgand
Fig. IB
Fig. ID
Figure 1. Potential use of polyaerylamide gels to investigate the involvement of selected immobilized monosaccharide moieties in spermzona pellucida interaction. This figure illustrates two hypothetical experimental situations. The polyaerylamide backbone presents a number
of monosaccharides in arelativelyfixedposition to the spermatozoa. (A) The immobilized sugars bind to receptors located on the
spermatozoa, the polyaerylamide backbone facilitates the subsequent clustering of thesereceptors.(B) Acrosomal exocytosis ensues. (C) In
contrast, the synthetic backbone could preclude tight binding to the immobilized monosaccharides. The distance between each sugar bound
to the polyaerylamide backbone may not reflect the spatial separation occurring between individual monosaccharides in vivo (D-E).
of this nature, it is tempting to extrapolate in-vitro observations
to an in-vivo situation and suggest that sulphation of ZP3
in vivo is not required for successful mouse gamete interaction.
This speculation was not addressed in this study.
It would be of interest to investigate the role played by
sulphate in the sperm-zona interaction. For example, a possible
structure of the mouse zona pellucida was proposed by Greve
and Wassarman (1985). This structure has ZP2 and ZP3 closely
juxtaposed within the intact zona. It is possible that sulphate
within the ZP2 glycoprotein is affecting sperm-ZP3 interaction
in an indirect manner. Pronase digested [35S]-sulphate and
[3H]-glucosamine-labelled material co-migrated during gel
filtration experiments. From this observation it was suggested
that the sulphate was attached to the carbohydrate moiety of
this glycoprotein. However, whether tyrosine sulphation was
present or not within these zonae glycoproteins was not
addressed. It has been suggested that [35S]-sulphate incorporation into carbohydrate moieties of glycoproteins can obscure
the presence of tyrosine O-sulphate in these macromolecules
(Huttner, 1982). Recently, it has been observed that tyrosine
sulphation is required for high affinity binding to P-selectin
(Wilkins et al, 1995), while sulphation of carbohydrate moieties of the glycoprotein GlyCAM-1 (the ligand for L-selectin)
is also required for a productive interaction between this
receptor-ligand complex (Rosen and Bertozzi, 1996). The
nature of interaction (if any) that sulphate has with cognate
receptors on the spermatozoa is unclear, e.g. is this interaction
mediated through the anionic nature of the sulphate groups
along the ZP2 glycoprotein and any cationic groups (Arg, Lys
and possibly His residues) located on the spermatozoal receptor
for ZP3? The effect of a high NaCl concentration on the
stability of the sperm-zona pellucida interaction would indicate
whether ionic interactions are required in this binding process.
Sulphation of cell adhesion molecules involved in fertilization in echinoderms has been shown to be critical for the
binding of fucose-containing polysaccharides to spermatozoa
from the sea urchin Strongylocentrotus purpuratus (DeAngelis
and Glabe 1987). Removal of the sulphate moieties from these
polysaccharides destroyed the binding activity of the fucans.
Sperm-fucan interaction could subsequently be restored by
chemical re-sulphation of the fucose-containing polysaccharides.
To study the potential role of sulphation in gamete adhesion
one would try and prevent the incorporation of sulphate into
the molecule of interest (in this case ZP2). Selenate has been
shown to be an inhibitor of sulphation of heparan sulphate in
rabbit endothelial cells (Dietrich et al, 1988) and also an
inhibitor of sulphation of a ligand for P-selectin (also CD62)
in myeloid cell lines THP-1 and HL60 (Aruffo et al, 1991).
It would be of interest to utilize an inhibitor of this kind in
the follicle culture system described by Shimizu et al. (1983).
One could then isolate individual zonae pellucidae and examine
the effect that sulphate depletion has on sperm-zona interaction.
A major drawback of using such an inhibitor in this culture
system is the possibility that non-specific inhibition of the
sulphation of critical cellular macromolecules may lead to
771
N.R-Chapman and C.L.R.Barratt
poor or abnormal growth of the cells in question. Such
inhibition has been demonstrated with the amoeba
Dictyostelium discoideum. Inclusion of sodium selenate in the
growth medium of the D.discoideum arrested vegetative growth
of this organism (Davies and Wheldrake, 1986).
What is the role of the ZP3 polypeptide backbone
in mammalian gamete interaction?
The precise function of the polypeptide backbone of ZP3 in
fertilization is unclear at present. In the mouse it has been
proposed to mediate the aggregation of protein receptors
located within the plasma membrane overlying the acrosome
in spermatozoa subsequent to the initial attachment of spermatozoa via the O-glycosylation of ZP3 (Florman et ai, 1984;
Leyton and Saling, 1989). The protein backbone in humans
(and pigs) is likely to play a more significant role. For example,
several studies have demonstrated that antibodies against the
polypeptide backbone of zona proteins significantly reduce
sperm binding to the zona pellucida in humans (Koyama et ai,
1994). Interestingly, Oehninger et al. (1996), using a specific
peptide antibody to human ZP3, showed that abnormalities in
the polypeptide backbone of the human zona pellucida are a
significant cause of fertilization failure during in-vitro fertilization. Furthermore, in pigs, monoclonal antibodies to peptide
sequences in porcine ZPC (formerly known as ZP3 beta)
significantly block the attachment of boar spermatozoa to the
zona (Gupta et ai, 1995). In contrast, antibodies against mouse
ZP3 do not block spermatozoa binding to the mouse zona
(Vasquez et ai, 1989). We therefore think that the protein
backbone of ZP3 may have a more significant role to play in
spermatozoa binding in the human (and pig) than the mouse.
Presently our group is attempting to produce recombinant
human ZP3 in Escherichia coli. The use of bacteria to express
mammalian zona proteins has been demonstrated by Prasad
et ai (1995). This group demonstrated that it was possible to
express the rabbit 55 kDa protein in E.coli, to purify this
protein and subsequently use it to elicit an immune response
(and hence give a source of polyclonal antiserum in rabbits).
The biological activity of the £.co//-derived 55 kDa protein
was not examined in this study.
The advantages of a bacterial system are that a great deal
of information is available concerning both the genetics and
physiology of E.coli. It is financially attractive relative to other
cell culture systems and, owing to the problems relating to
post-translational modifications and the heterogeneous nature
of glycosylation described above, we believe that this bacterial
system should give a more defined population of ZP3 molecules
that are uniformly non-glycosylated. The latter would thereby
circumvent the need to use relatively harsh measures to
remove the carbohydrate from the polypeptide chain, allowing
experimental analysis of the role of the polypeptide chain in
sperm binding. To ensure that specific binding of ZP3 is
observed spermatozoa must also be exposed to control proteins.
Ideally, these proteins should be of a similar molecular weight
and pi. If £.co/j-derived ZP3 alone binds to human spermatozoa, it would suggest that glycosylation of ZP3 is not vital for
successful human gamete interaction.
772
However, like any system employed to express recombinant
proteins, E.coli has its drawbacks (Marston and Hartley, 1990).
The major disadvantage is that many recombinant proteins are
often expressed as insoluble aggregates, which can interfere
with affinity purification protocols. The second major drawback
of a bacterial expression system is that it is unclear whether
the recombinant protein is folded in a native manner (this is
particularly important if the biological activity of a recombinant
protein is difficult to assay). For example, the cytoplasm of
E.coli does not favour the formation of disulphide bonds (Pollit
and Zalkin, 1983). Human ZP3 polypeptide contains 15 Cys
residues (two of which reside within the putative /V-terminal
signal sequence) (Chamberlin and Dean, 1990). It is possible
that at least some of these are required for intra-molecular
disulphide bond formation to maintain the tertiary structure of
the molecule. To circumvent this difficulty, recombinant proteins can be directed to the periplasmic space of E.coli where
disulphide bond formation usually takes place (Takahara et ai,
1988). Secretion vectors have been developed that take advantage of the natural secretory pathways of bacteria. The cDNA
encoding the desired protein is placed at the 3' end of a
sequence encoding an E.coli secretion signal peptide (such as
pelB or OmpA). It may also be possible to use a strain of
E.coli that permits cytoplasmic disulphide bond formation.
Such a strain has been described by Derman et al. (1993) and
is now commercially available. This strain of E.coli should
facilitate cytoplasmic disulphide bond formation and hence
stimulate the folding of the heterologous protein. However,
the ultimate test of whether or not a recombinant protein is
correctly folded is its biological activity. In the case of human
ZP3, this would involve the determination of specific binding
to spermatozoa and the subsequent initiation of the acrosome
reaction. Our preliminary results show that rhuZP3 produced
in E.coli stimulates a significant increase in phosphorylation
of a 95 kDa membrane protein (possibly ZRK, see Burks
et ai, 1995; Moore, 1995) as assessed by the in-vitro kinase
assay and Western blotting using anti-phosphotyrosine antibodies (Brewis et ai, 1995). However, no induction of the
acrosome reaction has yet been observed (Barratt and
Homby, 1995).
To further examine the role of protein-protein interaction
in human sperm-zona binding we have produced recombinant
human ZP3 using a commercially-available in-vitro transcription and translation system. This is widely used for rapidly
producing and purifying small quantities of protein, and,
in mutagenesis experiments, examining interaction between
proteins (Boyd et al., 1994; Paroush et ai, 1994; Snyder et ai,
1994; King et ai, 1995). In-vitro translated ZP3 is not
glycosylated. Limited proteolytic digestion experiments suggest that the recombinant human ZP3 protein (rhuZP3) has a
similar folding pattern to that of mouse native ZP3 (Whitmarsh
et ai, 1994) thus overcoming the problem of protein folding
which may be apparent in our E.coli. system. In addition,
we have immobilized this rhuZP3 on agarose beads. This
recombinant product is biologically active, as significantly
higher levels of sperm binding to rhuZP3-coated beads were
observed compared with controls, and significant induction of
the acrosome reaction was recorded (Barratt and Hornby,
The role of carbohydrate in sperm-ZP3 adhesion
1995). Thus, we are encouraged to believe that the polypeptide
backbone of human ZP3 does possess some biological activity.
Conclusions
Clearly, a more detailed characterization of the molecules
involved in initial gamete interaction is warranted. The production of various forms of recombinant zona proteins provides
a significant increase in our understanding of this interaction.
However, to understand further the apparent complexities
of this phenomenon the structures of active forms of the
corresponding molecules must be determined. Only when
crystal structures of ZP3 and its cognate receptors) from
spermatozoa become available will it be possible to determine
with confidence the mechanisms by which these molecules
specifically interact in the process of mammalian gamete
adhesion.
Acknowledgements
The authors would like to thank D.P.Hornby and PJ.Hurd for critical
reading of this manuscript. We are grateful for the financial support
provided by the Infertility Research Trust (Sheffield), and the University of Sheffield and Wellbeing to undertake this work.
References
Aruffo, A., Kolanus, W., Walz, G. et al. (1991) CD62/P-Selectin recognition
of myeloid and tumour cell sulphatides. Cell, 67, 35—44.
Barrett, C.L.R. and Hornby, D.P. (1995) Induction of the acrosome reaction
by rhuZP3. In Fenichel, P. and Parinaud, J. (eds), Human Sperm Acrosome
Reaction. Colloque INSERM Vol. 236. John Libbey Eurotext, pp. 105-122.
Barratt, C.L.R., McCann, C.T., Hornby, D.P et al (1993) Recombinant human
ZP3 expressed in Chinese hamster ovary cells (CHO) is a potent inducer
of the acrosome reaction. Hum. Reprod., 8 (Suppl.), 407.
Bar-Shira Maymon, B., Maymon, R., Ben-Nun, I. et al. (1994) Distribution
of carbohydrates in the zona pellucida of human oocytes. J. Reprod. Fertii,
102, 81-86.
Bleil, J.D. and Wassarman, P.M. (1980) Mammalian sperm—egg interaction.
Identification of a glycoprotein in mouse egg zona pellucida possessing
receptor activity for sperm. Cell, 20, 873-882.
Bleil, J.D. and Wassarman, P.M. (1988) Galactose at the nonreducing terminus
of O-linked oligosaccharides of mouse egg zona pellucida glycoprotein
ZP3 is essential for the glycoprotein's sperm receptor activity. Proc. Natl.
Acad. Sci. USA., 85, 6778-6782.
Boyd, J.M., Malstrom, S., Subramanian, T. et al. (1994) Adenovirus E1B
KDa and Bcl-2 proteins interact with a common set of cellular proteins.
Cell, 79, 341-351.
Brewis, I.A., Chapman, N.R., Barratt, C.L.R et al. (1995) The signal
transduction pathway of the acrosome reaction in human spermatozoa in
response to purified recombinat human ZP3. In Fenichel, P. and Parinaud,
J. (eds), Human Sperm Acrosome Reaction. Colloque INSERM Vol. 236.
John Libbey Eurotext, pp. 426-427.
Burks, DJ., Carballada, R., Moore, H.D.M. et al. (1995) Interaction of a
tyrosine kinase from human sperm with the zona pellucida at fertilisation.
Science, 269, 83-86.
Chamberlin, M.E. and Dean, J. (1990) Human homologue of the mouse sperm
receptor. Proc. Natl. Acad. Sci. USA, 87, 6014-6018.
Creighton, T.E. (ed.) (1993) Proteins, Structures and Molecular Properties.
2nd edn. W.H.Freeman and Company, New York, pp. 437-441.
Davies, SJ. and Wheldrake, J.F. (1986) Sulphation and the vegatative growth
of Dictyostelium discoideum. Eur. J. Biochem., 158, 179-185.
DeAngelis, P.L. and Glabe, C.G. (1987) Polysaccharide structural features
that are critical for the binding of sulphated fucans to bindin, the adhesive
protein from sea urchin sperm. J. Biol. Chem., 262, 13946-13954.
Derman, A.I., Prinz, W.A., Belin, D. and Beckwith, J. (1993) Mutations that
allow disulphide bond formation in the cytoplasm of Escherichia coli.
Science, 262, 1744-1747.
Dietrich, C.P., Nader, H.B., Buonassisi, V. and Colbum, P. (1988) Inhibition
of synthesis of heparan sulphate by selenate, possible dependence on
sulphation for chain polymerisation. FASEB J., 2, 56-59.
Dwek, R.A. (1995) Glycobiology: towards understanding the function of
sugars. Biochem. Soc. Trans., 23, 1-25.
Elliot, S., Bartlcy, T., Delorme, E. et al. (1994) Structural requirements
for addition of O-linked carbohydrate to recombinant erythropoietin.
Biochemistry, 33, 11237-11245.
Florman, H.M. and Storey, B.T. (1982) Mouse gamete interactions: the zona
pellucida is the site of the acrosome reaction leading to fertilisation in vitro.
Dev. Biol, 9, 121-130.
Florman, H.M., Bechtol, K.B and Wassarman, P.M. (1984) Enzymatic
dissection of the functions of the mouse egg's receptor for sperm. Dev.
Biol., 106, 243-255.
Florman, H. M. and Wassarman, P. M. (1985) O-linked oligosaccharides of
mouse egg ZP3 account for its sperm receptor activity. Cell, 41, 313—324.
Foxall, C , Watson, S.R., Dowbenko, D. et al. (1992) The three members of
the selectin receptor family recognize a common carbohydrate epitope, the
Sialyl Lewis* oligosaccharide J. Cell Biol, 117, 895-902.
Gerken, T.A , Butenhof, KJ. and Shogren, R. (1989) Effects of glycosylation
on the conformation and dynamics of O-hnked glycoproteins. Carbon-13
NMR studies of Ovine submaxillary mucin. Biochemistry. 28, 5536-5543.
Glabe, C.G., Grabel, L.B., Vacquier, V.D. and Rosen, S.D. (1982) Carbohydrate
specificity of sea urchin bindin, a cell surface lectin mediating sperm-egg
adhesion. J. Cell Biol, 94, 123-128.
Graves, B.J., Crowther, R.L., Chandran, C. et al (1994) Insight into Eselectin/ligand interaction from the crystal structure and mutagenesis of the
lec/EGF domains. Nature, 367, 532-538.
Greve, J.M. and Wassarman, P.M. (1985) Mouse egg extracellular coat is a
matrix of interconnected filaments possessing a structural repeat. J. Mol.
Biol, 181, 253-264.
Gupta, S.K., Yurewicz, E.C., Afzalpurkar, A. et al. (1995) Localisation of
epitopes for monoclonal antibodies at the N-terminus of the porcine zona
pellucida glycoprotein pZPC Mol. Reprod. Dev., 42, 220-225.
Huttner, W.B. (1982) Sulphation of tyrosine residues—-a widespread
modification of proteins. Nature, 299, 273—276.
Huttner, W.B (1984) Determinauon and occurrence of tyrosine O-sulphate in
proteins. Meth. Enzymol, 107, 200-223.
Joziasse, D.H., Shaper, J.H., Jabs, E.W. and Shaper, N.L. (1991)
Characterisation of an al-3-galactosyltransferase homologue on human
chromosome 12 that is organised as a processed pseudogene. /. Biol
Chem., 266, 6991-6998.
Kallin, E. (1994) Use of glycosylamines in preparation of oligosaccharide
polyacrylamide copolymers. Meth. Enzymol, 242, 221-226.
Karlsson, K.-A. (1995) Microbial recognition of target-cell glycoconjugates.
Curr. Opin. Struc. Biol, 5, 622-635.
Kiessling, L.L. and Pohl, N.L. (19%) Strength in numbers, non-natural
polyvalent carbohydrate derivatives. Chem. Biol., 3, 71-77.
King, S.R., Ronen-Fuhrmann, T, Timberg, R. et al. (1995) Steroid production
after in vitro transcription, translation, and mitochondrial processing of
protein products of complementary deoxyribonucleic acid for steroidogenic
acute regulatory protein. Endocrinology, 11, 5165—5176.
Kinloch, R.A., Sakai, Y. and Wassarman, P.M. (1995) Mapping the mouse
ZP3 combining site for sperm by exon swapping and site-directed
mutagenesis. Proc. Natl. Acad. Sci. USA, 92, 263-267.
Koyama, K., Hasegawa, A., Inoue, M. et al. (1994) Blocking of human sperm
zona interaction by monoclonal antibodies to a glycoprotein family (ZP4)
of porcine zona pellucida. Biol. Reprod., 45, 727-735.
Kornfeld, R. and Kornfeld, S. (1985) Assembly of asparagine-linked
oligosaccharides. Ann. Rev. Biochem., 54, 631-664.
Leyton, L. and Saling, P.M. (1989) Evidence that aggregation of mouse sperm
receptors by ZP3 triggers the acrosome reaction. J. Cell. Biol, 108,
2163-2168.
Litscher, E. S., Juntunen, K., Seppo, A.etal.(i995) Oligosaccharide constructs
with defined structures that inhibit binding of mouse sperm to unfertilized
eggs in vitro. Biochemistry, 34, 4662-4669.
Lopez, L.C., Bayna, E.M., Litoff, D. et al. (1985) Receptor function of mouse
sperm surface galactosyltransferase during fertilization. J. Cell Biol., 101,
1501-1510.
Marston, F.A.O. and Hartley, D.L. (1990) Solubilisation of protein aggregates.
Meth. Enzymol, 182, 264-276.
Miller, DJ., Macek, M.B. and Shur, B.D. (1992) Complementarity between
sperm surface p" 1,4-Galactosyltransferase and egg-coat ZP3 mediates spermegg binding. Nature, 357, 589-593.
Moller, C.C., Bleil, J.D., Kinloch, R.A. and Wassarman, P.M. (1990) Structural
773
N.R.Chapman and C.L.R.Barrett
and functional relationships between mouse and hamster zona pellucida
glycoproteins. Dev. Biol, 137, 276-286.
Moore H.D.M. (1995) Modification of sperm membrane antigens during
capacitation. In Fenichel, P. and Parinaud, J. (eds), Human Sperm Acmsome
Reaction. Colloque INSERM Vol. 236. John Libbey Eurotext, pp. 35-44.
Oehninger, S., Acosta, A. and Hodgen, G.D. (1990) Antagonistic and agonistic
properties of saccharide moieties in the hemizona assay. Fenil. Sterii, 53,
143-149.
Oehninger, S., Qaric, G.F., Acosta, A. and Hodgen, G.D. (1991) Nature of
the inhibitory effect of complex saccharide moieties on die tight binding
of human spermatozoa to the human zona pellucida. Fertil. Sterii, 55,
165-169.
Oehninger, S., Hinsch, E., Pfisterer. et al. (1996) Use of a specific zona
pellucida (ZP) protein 3 antiserum as a clinical marker for human ZP
integrity and function. Fertil. Sterii., 65, 139-149.
Parekh, R.B., Dwek, R.A., Thomas, J.R. et al. (1989a) Cell-type specific and
site-specific W-glycosylation of Type I and Type II human tissue plasminogen
activator. Biochemistry, 28, 7644-7662.
Parekh, R.B., Rudd, P.M., Dwek, R.A. and Rademacher, T.W. (1989b) Effects
of JV-glycosylation on in vitro activity of Bowes melanoma and human
colon fibroblast derived tissue plasminogen activator. Biochemistry, 28,
7662-7669.
Paroush, Z., Finley, R.L., Kidd, T, Wainwright, S. M. et al. (1994) Groucho
is required for drosophila neurogenesis, segmentation and sex determination
and interacts directly with hairy-related bHLH proteins. Cell, 79, 805-814.
Patankar, M.S., Oehninger, S., Barnett, T. et al. (1993) A revised structure
for fucoidan may explain some of its biological activities. J. Biol. Chem.,
268,21770-21776.
Pollit, S. and Zalkin, H. (1983) Role of primary structure and di-sulphide
bond formation in p"-Lactamase secretion. /. Bacteriol, 153, 27-32.
Prasad, S.V., Mujtaba, S., Lee, V.H. and Dunbar, B.S. (1995) Immunogenicity
enhancement of recombinant rabbit 55-Kilodalton zona pellucida protein
expressed using the baculovirus expression system. Biol. Reprod,, 52,
1167-1178.
Quiocho, F. A. and Vyas, N. K. (1984) Novel stereospecificity of the Larabinose-binding protein. Nature, 310, 381-386.
Rickert, K.W. and Imperiali, B. (1995) Analysis of the conserved glycosylation
site in the nicotinic acetylcholine receptor, potential roles in complex
assembly. Chem. Biol., 2, 751-759.
Rosen, S.D. and Bertozzi, C.R. (1996) Leukocyte adhesion. Two selectins
converge on sulphate. Curr. Biol., 6, 261-264.
Sacco, A.G., Yurcwicz, E.C., Subramanian, M.G. and Matzat, P.D. (1989)
Porcine zona pellucida, association of sperm receptor activity with the aglycoprotein component of the Mr = 55,000 family. Biol. Reprod., 41,
523-532.
Sareneva, T, Pirhonen, J., Cantell, K. et al. (1994) Role of A'-glycosylation
in the synthesis, dimerisation and secretion of human interferon-y. Biochem.
/ , 303,831-840.
Shakin-Eshleman, S.H., Spitalnik, S.L. and Kasturi, L. (1996) The amino acid
at the X position of an Asn-X-Ser sequon is an important determinant of
/^-linked core-glycosylation efficiency. J. Biol. Chem., 271, 6363-6366.
Shimizu, S., Tsuji, M. and Dean, J. (1983) In vitro biosynthesis of three
sulphated glycoproteins of murine zonae pellucidae by oocytes grown in
follicle culture. /. Biol. Chem., 258, 5858-5863.
Shur, B.D. and Neely, C.A. (1988) Plasma membrane association, purification
and partial characterisation of mouse sperm pi,4-Galactosyltransferase.
J. Biol. Chem., 263, 17706-17714.
Spaltenstein, A. and Whitesides, G.M. (1991) Polyacrylamides bearing pendant
a-sialoside groups strongly inhibit agglutination of erythrocytes by influenza
virus. J. Am. Chem. Soc., 113, 686-687.
Snyder, P.M., McDonald. FJ.. Stokes, J.B. et al. (1994) Membrane topology
of the amiloride-sensitive epithelial sodium channel. J. Biol. Chem., 269,
24379-24383.
Takahara, M., Sagai, H., Inouye, S. and Inouye, M. (1988) Secretion of human
superoxide dismutase in Escherichia coll Bio/Technology, 6, 195-198.
Thall. A.D., Mali), P. and Lowe, J.B. (1995) Oocyte Galal,3Gal epitopes
implicated in sperm adhesion to the zona glycoprotein ZP3 are not required
for fertilization in the mouse. J. Biol. Chem., 270, 21437-21440.
Vasquez, M., Phillips, D.M. and Wassarman, P.M. (1989) Interaction of mouse
sperm with purified sperm receptors covalently linked to silica beads.
J. Cell Sci., 92, 713-722.
Van Duin, M., Ploman, J.E.M., De Breet, I.T.M. et al. (1994) Recombinant
human zona pellucida protein ZP3 produced by Chinese hamster ovary cells
induces the human sperm acrosome reaction and promotes sperm-egg
fusion. Biol Reprod., 51, 607-617.
Weigel, PH., Schnaar, R.L., Kuhlenschmidt, M.S. et al. (1979) Adhesion of
hepatocytes to immobilised sugars. A direshold phenomenon. J. Biol.
Chem., 254, 10830-10838.
Wilkins, P.P., Moore, K.L., McEver, R.P. and Cummings, R.D. (1995) Tyrosine
sulphation of P-selectin glycoprotein ligand-1 is required for high affinity
binding to P-selectin. J. Biol. Chem., 270, 22677-22680.
Wilson, I.B.H., Gavel, Y. and Von Heijne, G. (1991) Amino acid distributions
around O-linked glycosylation sites. Biochem. J., 275, 529-534.
Wittwer, AJ., Howard, S.C., Nelson, R. et al (1989a) Cell-type specific and
site-specific /V-glycosylation of Type I and Type II human tissue plasminogen
activator. Biochemistry, 28, 7644-7662.
Wittwer, AJ., Howard, S.C., Can, L.S. etal. (1989b) Effects of /V-glycosylation
on in vitro activity of Bowes melanoma and human colon fibroblast derived
tissue plasminogen activator. Biochemistry, 28, 7662-7669.
Whitmaish, AJ., Barratt, C.L.R., Moore, H.D.M. and Hornby, D.P. (1994)
Structural features of recombinant human zona pellucida proteins ZP2 and
ZP3. Hum. Reprod. 9 (Abstr. Book), no. 134.
Yonezawa, N., Aoki, H., Hatanaka, Y. and Nakano, M. (1995) Involvement
of W-linked carbohydrate chains of pig zona pellucida in sperm-egg binding.
Eur. J. Biochem., 233, 35-41.
Yurewicz, E C , Pack, B.A. and Sacco, AG. (1991) Isolation, composition
and biological activity of sugar chains of porcine oocyte zona pellucida
55K glycoproteins. Mol. Reprod. Dev., 30, 126-134.
Received on June 14, 1996; accepted on August 20, 1996