Download Towards the Molecular Basis of Sperm and Egg Interaction during

Cells Tissues Organs 2001;168:36–45
Towards the Molecular Basis of
Sperm and Egg Interaction during
Mammalian Fertilization
Paul M. Wassarman Eveline S. Litscher
Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York, N.Y., USA
Key Words
Sperm W Eggs W Fertilization W Species specificity W mZP3 W
Sperm receptor W Acrosome reaction W Oligosaccharides W
Mutations
Abstract
During the past 2 decades, a number of genes have been
cloned from mammals which encode polypeptides that
participate in the process of fertilization. Among these
are glycoproteins ZP1–3 that constitute the zona pellucida of eggs from mice to human beings. In mice, one of
these glycoproteins, mZP3, acts as a primary sperm
receptor and acrosome reaction-inducer. The evidence
suggests that acrosome-intact sperm recognize and bind
to a specific class of mZP3 oligosaccharides present on
two serine residues (O-linked) located near the carboxyterminus of the polypeptide. Mutagenesis of either of
these residues results in the synthesis of an inactive form
Abbreviations used in this paper
CHO
EC
hZP3
mZP3
ZP
Chinese hamster ovary
embryonal carcinoma
hamster ZP3
mouse ZP3
zona pellucida
ABC
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E-Mail [email protected]
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© 2001 S. Karger AG, Basel
Accessible online at:
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of the receptor. Therefore, mammalian fertilization is a
carbohydrate-mediated event. It is possible that changes
in the structure of these oligosaccharides (e.g., composition, sequence, linkages, modifications, etc.) could account for species-specific binding of sperm to eggs. Stably transfected somatic cells, null mutant animals, and
DNA constructs are now available to test this possibility
both in vivo and in vitro.
Copyright © 2001 S. Karger AG, Basel
Introduction
How do free-swimming sperm and ovulated eggs from
the same species recognize one another and bind to each
other? The answer to these questions is of great interest
since binding of sperm to eggs initiates a pathway that
leads to fertilization of eggs and development of a new
individual of the species. An understanding of the molecular basis of this event could have many practical consequences for human beings. For example, it could lead to
development of novel methods of contraception [Paterson et al., 2000], as well as to novel therapies that correct
infertility. In addition, it could lead to new insights into
cellular recognition, adhesion, and signalling in general
[Villalobo and Gabius, 1998].
In mammals, fertilization begins with the binding of
sperm to the extracellular coat of ovulated eggs, called the
Paul M. Wassarman, PhD, Department of Biochemistry and
Molecular Biology, Mount Sinai School of Medicine
One Gustave L. Levy Place, New York, NY 10029-6574 (USA)
Tel. +1 212 241 8616, Fax +1 212 427 7532
E-Mail [email protected]
1
Fig. 1. Light photomicrograph (Nomarski DIC) of mouse sperm
bound to the ZP of an ovulated mouse egg in vitro.
Fig. 2. Light photomicrograph (dark-field) of mouse sperm, eggs and
embryos at the end of an in vitro binding assay. Sperm are bound to
the ZP of ovulated eggs, but are not bound to the ZP of two-cell
embryos.
2
zona pellucida (ZP) [Gwatkin, 1977; Wassarman, 1987,
1999a; Yanagimachi, 1994; Snell and White, 1996]
(fig. 1). All mammalian eggs have a ZP and its thickness
varies among different species [Sinowatz et al., 2001].
Although the restrictions on binding imposed by the ZP
are not absolute, they provide a relatively high degree of
species-specific fertilization in vitro. Notably, removal of
the ZP from unfertilized mammalian eggs, thereby exposing egg plasma membrane directly to sperm, eliminates
the barrier to interspecific fertilization in vitro for many,
but not all mammals [Yanagimachi, 1977, 1981, 1994].
Furthermore, whereas sperm can bind to the ZP of unfertilized eggs, they fail to bind to the ZP of fertilized eggs or
cleavage-stage embryos (fig. 2). These observations have
led to the idea that the unfertilized egg ZP possesses
sperm receptors to which free-swimming sperm of the
same species can bind. Following fertilization, the receptors are altered in such a way that binding of sperm is
precluded.
For more than a decade, considerable evidence has
accumulated suggesting that binding of sperm to eggs in
mammals is a carbohydrate-mediated event [Wassarman,
1992; Litscher and Wassarman, 1993; Miller and Shur,
1994; Chapman and Barratt, 1996; Benhoff, 1997; Tulsiani et al., 1997; Sinowatz et al., 1998; Töpfer-Petersen,
1999; Wassarman, 1999a, Prasad et al., 2001]. Apparently, sperm recognize and bind to specific oligosaccharides
associated with a specific egg-ZP glycoprotein [for pig see
also Nakano and Yonezawa, 2001; for human see also
Focarelli et al., 2001; Oehninger, 2001]. Therefore, as in
the binding of bacteria, animal viruses, and other pathogens to their cellular hosts, binding of pollen to the plant
stigma, and sexual agglutination in yeast, binding of
sperm to eggs, for both vertebrate and invertebrate species, is mediated by carbohydrates [the basic principles
are outlined in Solis et al., 2001]. The variety of oligosaccharide structures potentially available to an organism is
staggering [Drickamer and Taylor, 1998], easily providing
a molecular basis for species-specific interactions between
gametes.
Here, we focus on experiments carried out primarily in
our own, as well as in other laboratories, which lead to the
conclusion that mouse sperm recognize and bind to oligosaccharides covalently linked to mouse egg-ZP glycopro-
Mammalian Gamete Interactions
Cells Tissues Organs 2001;168:36–45
37
Fig. 3. Electrophoretic separation of mZP1, mZP2 and mZP3.
Shown are the positions and molecular weights of radiolabelled
mouse ZP glycoproteins following one-dimensional SDS-PAGE under nonreducing conditions and autoradiography. Note the breadth
of the individual bands, reflecting heterogeneous N- and O-linked
glycosylation of unique polypeptides.
tein mZP3. It is proposed that changes in the structure of
these oligosaccharides (e.g., their composition, sequence,
linkage, and/or modification), together with changes in
the egg-binding protein(s) on sperm that recognize mZP3
oligosaccharides, account for species-specific binding of
sperm to eggs in mammals [Wassarman and Litscher,
1995; Wassarman, 1999a]. Null mutant mice and appropriate reagents (e.g., DNA and cDNA constructs and promoters) are now available to test this proposal in vivo.
Mouse Egg ZP
The mouse egg ZP is F6.2 Ìm thick, contains F3.5 ng
of protein, and stains positively for carbohydrate (e.g., by
PAS or fluorescent lectins) [Wassarman, 1988; Dietl,
1989]. It consists of three, relatively acidic glycoproteins,
called mZP1–3, that have apparent molecular weights of
F200, F120 and F83 kD, respectively. The glycoproteins appear as broad bands on SDS-PAGE gels as a consequence of heterogeneous glycosylation of a unique polypeptide (fig. 3). The polypeptides of mZP1–3 have apparent molecular weights of F75 kD (dimer of F150 kD
held together by intermolecular disulfides), F75 kD
(monomer) and F44 kD (monomer), respectively. However, the polypeptides are subjected to proteolytic processing at their amino-terminus (N-terminal ‘signal se-
38
Cells Tissues Organs 2001;168:36–45
quence’) and carboxy-terminus (C-terminal ‘furin cleavage-site’) prior to secretion [Litscher et al., 1999] (fig. 4).
The glycoproteins possess both asparagine (N-)-linked
(complex-type) and serine/threonine (O-)-linked oligosaccharides. The three ZP glycoproteins are synthesized and
secreted exclusively by oocytes during their relatively
short growth phase (F2–3 weeks). During this period, the
ZP increases in thickness as the oocyte increases in diameter from F12 to F80 Ìm. At about the time of ovulation,
synthesis and secretion of ZP glycoproteins cease.
The ZP is a very porous extracellular matrix, permeable to large macromolecules (e.g., immunoglobulins) and
small viruses [Gwatkin, 1977]. As expected, ZP glycoproteins are organized in a specific manner within the extracellular coat [Greve and Wassarman, 1985; Wassarman
and Mortillo, 1991; Green, 1997; Wassarman, 1999a;
Sinowatz et al., 2001]. Dimers composed of mZP2 and
mZP3 polymerize into long filaments that constitute the
ZP. This arrangement of mZP2-mZP3 dimers accounts
for a structural periodicity of F140–150 Å seen along ZP
filaments in electron micrographs of undecorated and
IgG-decorated material (fig. 5). mZP1, a dimer of identical polypeptides, serves as the cross-linker between filaments. The regions of ZP glycoproteins that support these
interactions (e.g., between mZP2 and mZP3 and between
mZP1 and mZP2 and/or mZP3) have not been determined as yet.
mZP1–3 are found in the ZP of eggs from virtually all
mammalian species, from mice to human beings [Wassarman, 1999a, b]. In fact, the ZP polypeptides of even the
most distantly related species are very similar to each other (i.e., mouse and human ZP3, 170% identical). Furthermore, the vitelline envelope surrounding eggs from fish,
amphibians and birds also contains glycoproteins whose
polypeptides resemble those of ZP glycoproteins. Thus,
there is a significant evolutionary link between glycoproteins of the nonmammalian egg vitelline envelope and
glycoproteins of the mammalian egg ZP. It is clear that the
site(s) of expression of genes encoding these glycoproteins,
growing oocytes, follicle cells and/or liver, varies from
organism to organism.
A structural role for mZP3 in the ZP is supported by
the phenotype of null mutant mice. Targeted disruption
of the mZP3 gene by homologous recombination in embryonic stem cells has no effect on the phenotype of male
mice, but results in infertility in homozygous null females
[Liu et al., 1996; Rankin et al., 1996]. Ovaries from the
homozygous null females (mZP3 –/– ) contain growing oocytes that completely lack a ZP, while oocytes from heterozygous null females (mZP3+/– ) have a ZP that is about
Wassarman/Litscher
Fig. 4. Primary structure and structural
landmarks of the mZP3 polypeptide. Shown
is the amino acid sequence (single letter amino acid code) of the mZP3 precursor polypeptide (424 amino acids) and the mZP3
mature polypeptide (331 amino acids) after
removal of the amino-terminal ‘signal sequence’ (22 amino acids) and carboxy-terminal peptide (71 amino acids) at the ‘furin
cleavage-site’. Also indicated are the positions of the cysteine residues (P), potential
N-linked glycosylation sites ($), immunoglobulin-like hinge region, the sperm-combining site with O-linked oligosaccharides at
serine-332 and serine-334 (black lines), and
the hydrophobic region (22 amino acids) of
the carboxy-terminal peptide released by
proteolytic cleavage.
one half the thickness (F2.7 Ìm) of the wild-type [Wassarman et al., 1997]. These results are consistent with the
proposed structural role for mZP3, as well as with current
models for ZP structure [Wassarman and Mortillo, 1991;
Wassarman et al., 1996]. Furthermore, they are consistent
with results of experiments in which mZP2 and mZP3
messenger RNA was degraded in the presence of a large
excess of complementary (antisense) oligonucleotide injected into the cytoplasm of isolated growing mouse
oocytes [Tong et al., 1995]. It was found that the absence
of synthesis of either glycoprotein prevented the incorporation of the other glycoprotein into the ZP. It should be
noted that a ZP can be restored to oocytes and eggs from
mZP3 –/– mice by inclusion of a wild-type ZP3 gene (in
this case, encoding human ZP3) as a transgene in these
animals [Rankin et al., 1998].
Mouse ZP3 Is a Primary Sperm Receptor
Only acrosome-intact sperm bind to the ovulated
mouse egg ZP. Experimental evidence strongly supports
the conclusion that, during binding of sperm to eggs,
mZP3 serves as a receptor for sperm. For example, of the
three glycoproteins that constitute the ZP, only purified
mZP3 binds exclusively to heads of acrosome-intact
Mammalian Gamete Interactions
Fig. 5. Transmission electron photomicrograph of solubilized mouse
ZP filaments. Shown is an enzyme-solubilized ZP preparation adsorbed to substrate-coated grids and negatively stained. Note the
structural repeat located every 140–150 Å or so along the filaments,
as well as the interconnections between the long filaments.
Cells Tissues Organs 2001;168:36–45
39
Fig. 6. In vitro assays of egg mZP3 and embryo mZP3 sperm recep-
tor activity. Binding of mouse sperm to eggs was examined in the
absence of mZP3 (hatched circle) and in the presence of either purified egg mZP3 ([) or two-cell embryo mZP3 (P) at several concentrations. Note that egg mZP3 is a very effective inhibitor at nanomolar concentrations, whereas embryo mZP3 is inactive as an inhibitor.
sperm and thereby prevents sperm from binding to ovulated eggs in vitro [Bleil and Wassarman, 1980, 1986;
Wassarman, 1990; Mortillo and Wassarman, 1991; Wassarman and Litscher, 1995]. Even at nanomolar concentrations, purified, unfertilized egg mZP3 is a very effective inhibitor of sperm binding in this competition assay
(fig. 6). On the other hand, at similar concentrations,
mZP3 from fertilized eggs or early embryos has no effect
on binding of sperm to eggs in vitro. This is consistent
with the failure of free-swimming sperm to bind to the ZP
of fertilized eggs and preimplantation embryos. It can be
concluded from these and other observations [e.g., Bleil
and Wassarman, 1980; Florman and Wassarman, 1985;
Wassarman, 1990] that, as a consequence of the zona
reaction, mZP3 is altered such that free-swimming sperm
can no longer recognize and bind to the glycoprotein.
Interestingly, the ability of mZP3 to act as a sperm
receptor in vitro is not significantly affected by high temperatures, detergents, denaturants, reducing agents, or
limited proteolysis. Apparently, mZP3 bioactivity is not
dependent on the extent of glycosylation of its polypeptide or on sulfation and sialylation of its oligosaccharides
[Liu et al., 1997]. Even after extensive proteolysis of
mZP3, the small glycopeptides produced retain activity as
a sperm receptor, although higher than normal concentrations are required [Florman et al., 1984; Florman and
40
Cells Tissues Organs 2001;168:36–45
Fig. 7. In vitro assays of binding of sperm to eggs in the presence of
tetraantennary octadecasaccharides. Shown are the structures of the
two oligosaccharides, one terminating in Gal·1 and the other in
GlcNAcß1 (nonreducing terminus), used as potential inhibitors of
binding of mouse sperm to eggs. The two oligosaccharides were
assayed at three different concentrations, from 1 to 10 ÌM. Note that
the oligosaccharide terminating in Gal·1 was a potent inhibitor,
whereas the oligosaccharide terminating in GlcNAcß1 caused only
background levels of inhibition [Litscher et al., 1995].
Wassarman, 1985]. These observations suggest that
mZP3 polypeptide does not play a direct role in sperm
receptor function. However, there is considerable information to suggest that mZP3 oligosaccharides play a
direct role in sperm receptor function. For example,
chemical or enzymatic removal of all mZP3 oligosaccharides results in complete inactivation of the glycoprotein
as a sperm receptor. Furthermore, O-linked oligosaccharides recovered from mZP3 by mild alkaline hydrolysis
under reducing conditions [Florman and Wassarman,
1985; Bleil and Wassarman, 1988; Miller et al., 1992] and
certain O-linked related oligosaccharides [Litscher et al.,
1995; Johnston et al., 1998] inhibit binding of sperm to
eggs in vitro at micromolar concentrations (fig. 7). Results
of some of these studies suggest that galactose, N-acetylglucosamine and/or fucose are essential sugars for sperm
Wassarman/Litscher
Fig. 8. Site-directed mutagenesis of the
mZP3 gene at its sperm-combining-site. a
Schematic diagram of the PGK/mZP3 recombinant gene used to generate stably
transfected EC-mZP3 cell lines. pPGK-1 represents the mouse phosphoglycerate kinase1 promoter region. Restriction enzymes ClaI
and SstII were used to generate linearized
DNA fragments for electroporation of EC
(F9) cells. Arrow indicates the transcriptional start site on the PGK-1 promoter. The
sites of mutagenesis in exon 7 of the mZP3
gene are indicated as amino acids 329–334.
In each case, the amino acid was changed to
Ala, Val or Gly (i.e., a nonhydroxyl amino
acid). b Summary of site-directed mutagenesis of EC-mZP3. Shown are the amino acid
changes caused by mutations in the region of
the mZP3 gene encoding amino acids 329–
334. Note that only mutations involving serine-332 and serine-334 resulted in synthesis
of an inactive form of the sperm receptor.
binding. Collectively, these observations suggest that species-specific binding of sperm to eggs in mammals is a carbohydrate-mediated event. On the other hand, the identity of sugars on mZP3 recognized by sperm remains an
unresolved and controversial issue.
To locate essential O-linked oligosaccharides on mZP3
polypeptide we utilized limited proteolysis [Rosiere and
Wassarman, 1992; Litscher and Wassarman, 1996a],
exon swapping [Kinloch et al., 1995] and site-directed
mutagenesis [Kinloch et al., 1995; Chen et al., 1998].
Results of such studies suggest that all sperm receptor
activity of mZP3 is associated with the carboxy-terminal
half of the polypeptide and that the essential oligosaccharides are present on just two of five serine residues, serine-
332 and serine-334, in a region of polypeptide near the
carboxy-terminus, a region encoded by exon-7 of the
mZP3 gene (8 exons). For example, mutation of either
serine-332 or serine-334 to a small aliphatic amino acid
results in production of an inactive form of mZP3 in stably transfected cells (fig. 8). Interestingly, of the five serine residues, only these two are conserved from mouse to
human ZP3. In this context, the numerous amino acid
changes neighboring serine-332 and serine-334 that have
occurred during evolution may impose changes in the
structure of O-linked oligosaccharides added to ZP3 and,
thereby, affect species specificity of sperm-egg interaction
[Wassarman and Litscher, 1995].
Mammalian Gamete Interactions
Cells Tissues Organs 2001;168:36–45
41
Fig. 9. In vitro assays of mouse egg mZP3, hamster egg hZP3, EC-
hZP3, and CHO-hZP3 sperm receptor activity. Shown is the percentage inhibition of sperm binding for mouse (m) and hamster (h) eggs
(first letter) and sperm (second letter). Mouse and hamster sperm
were exposed to F100 nM egg mZP3, egg hZP3 or recombinant
hZP3 (secreted by transfected EC or CHO cells and purified), sperm
were incubated with mouse or hamster eggs, and the number of
sperm bound per egg determined. Note that EC-hZP3 and CHOhZP3 are only effective inhibitors of binding of hamster sperm to
mouse and hamster eggs; they do not inhibit binding of mouse sperm
to mouse eggs.
Mouse ZP3 Is an Acrosome Reaction Inducer
The acrosome is a large secretory vesicle that overlies
the nucleus in the apical region of the sperm head [Yanagimachi, 1994; Wassarman, 1999a]. Acrosomal membrane just underlying the plasma membrane is referred to
as ‘outer’ acrosomal membrane and that overlying the
nucleus is referred to as ‘inner’ acrosomal membrane.
Morphologically, the acrosome reaction is seen as multiple fusions between outer acrosomal membrane and plasma membrane at the anterior region of sperm head, extensive formation of hybrid membrane vesicles, and exposure of inner acrosomal membrane and acrosomal contents. Only acrosome-reacted sperm can penetrate the ZP
and fuse with egg plasma membrane.
It is known that there are many different inducers of
the acrosome reaction [Yanagimachi, 1994]. However, it
is now generally accepted that ZP3 is the natural agonist
that initiates the acrosome reaction upon binding of acrosome-intact sperm to the ZP [Bleil and Wassarman, 1983;
Wassarman, 1995; Darszon et al., 1996; Wassarman and
Florman, 1997; Florman et al., 1998; Nixon et al., 2001].
Criteria now are available that permit one to distinguish
between the, so-called, ‘spontaneous’ acrosome reaction
42
Cells Tissues Organs 2001;168:36–45
and the ZP3-induced acrosome reaction (e.g., sensitivity
to pertussis toxin). While purified mZP3 and large mZP3
glycopeptides induce sperm to acrosome-react in vitro,
small mZP3 glycopeptides and purified mZP3 O-linked
oligosaccharides bind to sperm and inhibit their binding
to eggs, but do not induce the acrosome reaction [Wassarman, 1988, 1990]. In the latter context, it has been
reported that IgG cross-linking of small mZP3 glycopeptides bound to sperm can induce the sperm to acrosomereact [Leyton and Saling, 1989] and that aggregation of
ß-1,4-galactosyltransferase on the sperm surface by mZP3
leads to activation of a G protein complex [Gong et al.,
1995]. These findings suggest that induction of the acrosome reaction by ZP3 probably will turn out to be dependent on multivalent interactions at the sperm surface.
Mouse and Hamster ZP3 Expressed in
Transfected Cells
It is clear that mouse sperm bind to hamster eggs and
hamster sperm bind to mouse eggs [Gwatkin, 1977;
Schmell and Gulyas, 1980; Cherr et al., 1986; Moller et
al., 1990]. Consequently, it was not surprising to find that
mouse ZP3 (mZP3; F83 kD Mr) binds to hamster sperm
and that hamster ZP3 (hZP3; F56 kD Mr) binds to mouse
sperm [Moller et al., 1990]. Although the molecular
weights of the mature glycoproteins are significantly different from each other, their polypeptides are very similar
to each other [F83%; Kinloch et al., 1990]. These observations suggest that the oligosaccharides on mZP3 and
hZP3 must have some common structural features that
are recognized by mouse and hamster sperm.
In view of the above, genomic clones of mZP3 [Kinloch et al., 1988] and hZP3 [Kinloch et al., 1990] were
placed under control of the phosphoglycerate kinase-1
(pgk-1) promoter and were expressed in several lines of
stably transfected embryonal carcinoma (EC) and Chinese hamster ovary (CHO) cells [Kinloch et al., 1991;
Litscher and Wassarman, 1996b]. These cells synthesized
and secreted relatively large amounts of recombinant ZP3
into the culture medium. Sperm receptor assays of the
purified glycoproteins revealed some unexpected results.
Although EC-hZP3 and CHO-hZP3 were receptors for
hamster sperm, unlike egg hZP3, they were not receptors
for mouse sperm (fig. 9). Binding of EC-hZP3 and CHOhZP3 was completely restricted to hamster sperm.
The surprising behavior of recombinant EC-hZP3 and
CHO-hZP3 is of interest for several reasons. For example,
it supports the conclusion that ZP3 oligosaccharides, rath-
Wassarman/Litscher
er than polypeptide, are recognized by free-swimming
sperm when they bind to the ZP of ovulated eggs. The
hZP3 polypeptide synthesized by EC and CHO cells is
identical to that synthesized by oocytes of mice carrying
hZP3 as a transgene [Kinloch et al., 1992]. Yet EC-hZP3
and CHO-hZP3 are bioactive with hamster, but not
mouse sperm, whereas hZP3 synthesized by transgenic
mice, like hamster egg hZP3, is active with mouse sperm.
It also points out once again that two very similar polypeptides can be differentially glycosylated by transfected
cells. These results suggest that addition of O-linked oligosaccharides to nascent hZP3 is not determined by polypeptide structure alone. In this context, although it has
been demonstrated that the rate of addition of N-acetylgalactosamine to nascent glycoproteins is influenced by
the sequence in which glycosylated serine and threonine
residues are located [Elhammer et al., 1993; Nehrke et al.,
1996], as yet no consensus sequence for O-linked glycosylation has been reported [Wilson et al., 1991; Gooley and
Williams, 1994]. A likely conclusion is that the kinds and
distribution of glycosyltransferases present in the cell
greatly influence the structure of O-linked oligosaccharides attached to ZP3. This, in turn, could affect the
bioactivity of the glycoprotein.
Final Comments
There is great diversity when it comes to the mechanism of species-specific fertilization. For example, some
nonmammalian eggs lack an extracellular coat (e.g.,
nematode eggs) while others have a vitelline envelope and
a jelly coat (e.g., echinoderm and amphibian eggs). In
some species sperm enter eggs at a particular site (micropyle) without the need for an acrosome reaction (e.g.,
nematodes), whereas in others sperm undergo the acrosome reaction upon binding to the jelly coat and then bind
in a species-specific manner to the vitelline envelope (e.g.,
echinoderms and amphibians) [see also Focarelli et al.,
2001]. In the latter context, one might think of the ZP,
which is unique to all mammalian eggs, as an amalgamation of the jelly coat and vitelline envelope of some nonmammalian eggs. The ZP is the site of both species-specific sperm receptors and an inducer of the acrosome reaction.
Here, we related what we have learned about the functions of ZP glycoprotein mZP3 during fertilization, with
special emphasis on the role of oligosaccharides in mZP3
function. For example, (1) mZP3 is a structural glycoprotein absolutely required for ZP assembly during oogene-
Mammalian Gamete Interactions
sis, (2) mZP3 is a sperm receptor that supports binding of
free-swimming sperm to unfertilized eggs, and (3) mZP3
is an acrosome reaction inducer for sperm bound to the
ZP of unfertilized eggs. At least the sperm receptor and
acrosome reaction-inducing activities of mZP3 depend
on the glycoprotein’s oligosaccharides. In mice, these oligosaccharides apparently are located on two serine residues located near the carboxy-terminus of the mature
polypeptide (fig. 4). This portion of the polypeptide has
undergone significant changes during evolution and it is
possible that these changes result in alternative oligosaccharide structures for ZP3 from different mammalian
species. The cell’s rules for O-linked glycosylation of nascent proteins will have to be determined in order to
answer key questions about ZP3 oligosaccharides.
Although we know a great deal about mZP3, many
more questions remain. A pressing issue concerns the
nature of the protein associated with plasma membrane
surrounding the sperm head that recognizes and binds to
mZP3 oligosaccharides (so-called, egg-binding protein).
This area of research, while extremely productive, to date
is inconclusive [Wassarman, 1999a; Jansen et al., 2001;
Nixon et al., 2001]. A second issue concerns the nature of
the ZP3 oligosaccharides that are recognized by egg-binding proteins. We know very little about the structure of
these oligosaccharides (e.g., composition, sequence and
linkages) or their sites on ZP3 polypeptides from different
species of mammalian eggs [Nakano and Yonezawa,
2001; Oehninger, 2001]. Can these oligosaccharides account for the species-specific binding of sperm that is
observed experimentally? Finally, it remains to work out
the mechanism for inactivation of mZP3 as a sperm
receptor following fertilization. Preliminary evidence suggests that inactivation can be attributed to enzymatic
destruction of essential mZP3 oligosaccharides (unpubl.
results). We hope that answers to these and other questions will be forthcoming and that such progress will be of
some benefit to clinicians involved in the manipulation of
human reproduction.
Acknowledgment
Many important contributions have been made by past and
present members of our laboratory to research on mammalian fertilization. We are very grateful to all of them. Over the years, much of
the research was supported by the National Institute of Child Health
and Human Development (currently by HD35105) and by Hoffmann-La Roche, Inc.
Cells Tissues Organs 2001;168:36–45
43
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