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E European Society of Human Reproduction and Embryology
Human Reproduction Update 1999, Vol. 5, No.4 pp. 314–329
Carbohydrate-based interactions on the route of
a spermatozoon to fertilization
Edda Töpfer-Petersen1
Institute of Reproductive Medicine, Veterinary School of Hanover, Germany
Male and female intercommunication along the route which the spermatozoon takes to fertilization utilizes the information
potential of carbohydrates. A hierarchy of carbohydrate-based binding events exists ranging from spermatozoa–oviduct
interaction to primary and secondary binding between spermatozoon and oocyte. Before in-vivo fertilization can occur,
spermatozoa are stored in the caudal part of the isthmus, in tight contact with the epithelium cells lining the oviduct. The
sperm reservoir seems to be created by surface-associated sperm lectins recognizing epithelial glycoconjugates. With the
changing conditions in the oviduct at the time of ovulation, spermatozoa may shed those sperm lectins, creating new
surfaces which allow spermatozoa to be released from the epithelium, complete capacitation and interact with the oocyte in
the appropriate manner. The first contact between both gametes occurs at the spermatozoa–zona pellucida interface. The
‘primary’ binding initiates the acrosomal exocytosis of the spermatozoa, followed by the ‘secondary’ binding of the
acrosome-reacted spermatozoon that in consequence leads to sperm penetration through the zona pellucida. Primary and
secondary binding events are directed by the cooperative interactions of multiple carbohydrate-recognition systems that
may act in a hierarchical and redundant manner. The current perspective will focus on the role of carbohydrate-binding
sperm proteins in the sequence of binding events during fertilization in the pig.
Keywords: gamete recognition/oviduct/spermatozoa/zona pellucida/zona pellucida-binding proteins
TABLE OF CONTENTS
Introduction
Formation of the oviductal sperm reservoir is a
carbohydrate-mediated event
Carbohydrates are the signals for gamete recognition
Zona pellucida-binding proteins
Uncapacitated and capacitated porcine spermatozoa
bind to the zona pellucida in vitro
Is carbohydrate-mediated gamete recognition really
species-specific?
Conclusions
Acknowledgements
References
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Introduction
Fertilization is a fundamental event which involves a highly
coordinated sequence of cellular interactions between the male
and female gamete, that is, between the sperm cell and the egg,
in order to form a diploid zygote and, ultimately, the new
individual. In mammals, fertilization occurs in the female
1Address
reproductive tract. At ejaculation, millions of spermatozoa are
deposited in the female reproductive tract, though only a few
thousand enter the oviduct, a few reach the ampulla at the time
of fertilization, and only one spermatozoon fertilizes the egg.
To guarantee the meeting of the two highly specialized gametes
at the right time, and in the right place, the oviduct and the egg
itself coordinate sperm functions. On reaching the oviduct,
spermatozoa are held back in the reservoir of the lower isthmus
due to binding of the spermatozoa to the epithelium (reviewed
by Hunter, 1988, 1996; Smith, 1998; Suarez, 1998) (Figure 1).
Sperm interactions with the oviductal epithelium appear to
increase the viability of the spermatozoa during storage, and
suppress sperm motility (Smith, 1998; Suarez, 1998). Before
fertilization can occur, however, spermatozoa must enter
a functionally activated or capacitated state and develop a
hyperactivated motility which enables them to respond to the
egg in the appropriate manner (Bedford, 1983). The
capacitation process appears to be coordinated temporally by
the oviductal epithelium in a still unknown fashion. Close to the
time when the egg is ovulated into the ampulla, spermatozoa
start or continue the capacitation process and are released from
for correspondence: Institute of Reproductive Medicine, Veterinary School, Hanover, Bünteweg 15, D-30559 Hanover, Germany.
Tel: 0511 9538520; Fax: 0511 9538504; e-mail: [email protected]
Spermatozoan–egg interactions in fertilization
the oviductal epithelium, whereby the newly developed
hyperactivated motility may help the detachment of
spermatozoa and facilitate their swimming to the site of
fertilization (Hunter, 1996; Suarez, 1996, 1998; Smith, 1998
and references therein). On approaching the oocyte, the
spermatozoon must first be recognized by the oocyte. This
interaction occurs when a spermatozoon first makes contact
with the zona pellucida (ZP), the extracellular coat enveloping
the oocyte. The ZP not only mediates the recognition between
both gametes, but also regulates sperm functions, enabling the
spermatozoon to complete fertilization. Capacitation is a
prerequisite for the subsequent activation of the sperm
transmembrane signalling system(s) by structures of the ZP,
leading to the exocytosis of the sperm acrosome, referred to as
the acrosome reaction. Thereby, the enzymatic equipment of
the acrosome is activated, and is made available to aid sperm
passage through the ZP, finally allowing fusion with the egg
vitelline membrane. After fusion and oocyte activation have
been completed by the spermatozoon, the sperm nucleus
decondenses and delivers the male genome into the egg
cytoplasm, thus marking the start of the programme for
embryonic development. As one consequence of oocyte
activation, the ZP is altered by components released from the
oocyte cortical granules, contributing to the establishment of
the egg-induced block to polyspermy (reviewed by
Yanagimachi, 1994; Storey, 1995). Whereas the capacitation
process is modulated by the oviduct, the ensuing
physiologically significant acrosome reaction is coordinated by
the ZP. It has long been accepted that recognition and initial
binding between spermatozoa and egg involves the binding of
multiple carbohydrate receptors of the sperm cell to the
complementary oligosaccharide chains attached to the ZP
proteins. Recently, sperm binding to the oviduct has also been
shown to involve carbohydrate–protein interactions (Suarez,
1998). Thus, two important regulatory steps on the journey of
the spermatozoon to union with the egg may be initiated by
carbohydrates (Figure 1). In recent years, current knowledge of
the mechanisms of spermatozoa–oocyte interaction has been
excellently reviewed, covering different aspects of the role of
carbohydrates in fertilization (for example Wassarman and
Litscher, 1995; Chapman and Barratt, 1996; Clark et al., 1996;
Snell and White, 1996; Benoff, 1997; Sinowatz et al., 1997;
Tulsiani et al., 1997; McLesky et al., 1998). The current
perspective will therefore focus on the role of ZP-binding
proteins in fertilization under in-vitro conditions in the pig, and
the assumed fate during in-vivo transit to the site of
fertilization.
Formation of the oviductal sperm reservoir is
a carbohydrate-mediated event
In mammals, millions of spermatozoa are stored in the cauda
epididymis until, at ejaculation, they are deposited in the female
reproductive tract. Most of the spermatozoa are lost during their
315
Figure 1. Schematic representation of the carbohydrate-based
interactions on the route of a spermatozoon to fertilization. After
reaching the oviduct, spermatozoa are stored in the oviductal
reservoir created by carbohydrate-based interactions of
spermatozoa and the oviductal epithelium. After capacitation,
spermatozoa are released and swim to the site of fertilization. On
approaching the oocyte, the initial binding and recognition is
mediated by exposed carbohydrate of the oocyte zona pellucida and
complementary carbohydrate/zona pellucida-binding proteins of
the sperm surface.
journey through the female reproductive tract, and only a small
number reach the oviduct. The lower region of the isthmus is
the site where the essential steps of the capacitation process are
initiated, i.e. the sperm plasma membrane changes which are
prerequisite for the onset of the acrosome reaction and motility
hyperactivation. It has been shown across mammalian species
that this region of the oviduct functions as a reservoir in which
the spermatozoa are stored with tight contact to the ciliated
oviductal epithelial cells lining the tract (Hunter, 1981, 1988,
1996; Smith, 1998; Suarez, 1998) (Figure 2A). In contrast to
the observations in many mammalian species, there has been
no conclusive evidence for a distinct oviductal sperm reservoir
in humans. Human spermatozoa do not establish a tight binding
to the homologous oviductal epithelium in vitro (Yeung et al.,
1994; Murray and Smith, 1997). Nonetheless, the viability of
human spermatozoa has been shown to be maintained by
co-culture with oviductal epithelium cells (Yao et al., 1999). To
date, no useful hypothetical model outlining the events of
sperm transport in humans has been described.
Role of the oviductal sperm reservoir
The oviductal reservoir may serve a number of functions
(Suarez, 1998). First, it may contribute to the prevention of
polyspermic fertilization by controlling sperm transport to the
ampulla. Second, during storage the sperm fertilizing capacity
appears to be maintained to span the time between oestrus and
fertilization. Third, the capacitation process is modulated to
synchronize sperm function with the time of ovulation. Since
the capacitated state reduces the life span of spermatozoa, the
need to maintain sperm viability and control capacitation are
mutually associated events. The caudal isthmus appears to be
specialized for these purposes. Very recently, it has been shown
in the pig (Hunter et al., 1998) that the caudal region of the
316 E.Töpfer-Peterson
spermatozoa. Capacitation is controlled by ions, and in
particular Ca2+ plays a critical role at this point. In mouse and
other species, capacitation and the subsequent acrosome
reaction in vitro are accompanied by a biphasic pattern of
internal Ca2+ elevations, in which the first small peak is related
to capacitation and the large influx of Ca2+ triggers acrosomal
exocytosis (reviewed by Fraser, 1995). Under Ca2+-deficient
conditions, spermatozoa fail to complete capacitation,
indicating that Ca2+ has an important directive role in the
progress of capacitation (Fraser, 1995). Sperm survival and
capacitation in the oviduct may therefore be regulated by the
control of Ca2+ uptake by spermatozoa. Equine spermatozoa
attached to homologous oviductal epithelial cells show about
2- to 3-fold lower internal Ca2+ concentrations than freeswimming spermatozoa (Dobrinski et al., 1997). Alterations of
the ionic environment at the time of ovulation (Fraser, 1995)
may promote the transition to the capacitated state; however,
the transmission appears to be dominated by active intervention
of the oviductal epithelium. Binding of spermatozoa to the
apical plasma membrane of the oviductal cells directly
influences the functional state of the spermatozoa by
maintaining low intracellular Ca2+, and in consequence slows
down the rate of capacitation (Murray and Smith, 1997; Smith
and Nothnick, 1997). Inhibition of spermatozoa–oviduct
binding by antibodies raised against the oviductal apical
plasma membranes reduces the controlling effect of the
oviductal cells (Dobrinski et al., 1997). Previously, it was
suggested (Bedford et al., 1983) that capacitation is a
mechanism acquired by the spermatozoa to prevent certain
functions from developing too quickly before fertilization can
occur.
Oviductal carbohydrates and sperm lectins may
be involved in spermatozoa–oviduct binding
Figure 2. Scanning electron micrographs. (A) The porcine
utero-tubal junction and caudal isthmus of the oviduct of the pig, at
10 h after insemination. (B) Capacitated spermatozoa bound to the
zona pellucida of ovarian oocyte of the pig in vitro.
isthmus is more effective in neutralizing seminal plasma
components and stimulating or regulating the capacitation
process than other regions of the Fallopian tube. Although there
are many lines of experimental evidence for specific effects of
the oviductal reservoir on spermatozoa, little is known about
the underlying mechanisms. Binding of the spermatozoa to,
and their release from, the oviductal epithelium appear to be
crucial regulatory steps related to the capacitation state (Smith,
1998). In a number of species including hamster (Smith and
Yanagimachi, 1990), mouse (DeMott et al., 1995), cattle
(Lefebvre and Suarez, 1996) and horse (Dobrinski et al.,
1997), capacitated spermatozoa do not bind, or bind with a less
significant frequency, to the oviduct than do uncapacitated
The first indication that carbohydrate moieties are involved in
spermatozoa–oviduct attachment comes from sugar inhibition
experiments. Fetuin carrying sialylated N- and O-linked glycan
chains effectively inhibits spermatozoa–oviduct binding in
hamster (DeMott et al., 1995), whereas asialofetuin carrying
galactose on the non-reducing terminus of the glycan chains,
fucoidan and ovalbumin have been shown to have less, or no,
effect. Monomeric sialic acid was also able to interfere with the
binding, pointing to the role of sialic acid as the key signal for
spermatozoa–oviduct recognition in hamster. In contrast,
asialofetuin and galactose are the most effective inhibiting
components in horse (Lefebvre et al., 1995) and fucose, in a
α1,4-linkage to the penultimate carbohydrate residue, appears
to be the determining sugar in cattle (Lefebvre et al., 1997;
Suarez et al., 1998). In preliminary studies in pigs using
explants isolated from the isthmic region of the oviduct,
asialofetuin (in the micromolar range) has been shown to
inhibit sperm binding, indicating that oligosaccharides with
terminally located galactose may be critical for forming the
Spermatozoan–egg interactions in fertilization
sperm
reservoir
(R.Gelhaar,
D.Waberski
and
E.Töpfer-Petersen, unpublished result). The sperm surface
(Sinowatz et al., 1989) and the oviductal epithelium
(Raychoudhury et al., 1993) possess a variety of externally
orientated oligosaccharides linked to proteins and lipids that
may serve as recognition signals for the binding events that take
place in the isthmic region of the oviduct. In the context of the
search for the receptors of the ZP carbohydrate ligands, a large
number of carbohydrate-binding sites at the sperm surface
(Sinowatz et al., 1989) and proteins with affinity for the ZP
have been identified in a wide range of species (reviewed by
Töpfer-Petersen et al., 1995; Benoff, 1997; Sinowatz et al.,
1997; McLesky et al., 1998). Recently, carbohydrate-binding
sites with affinity for terminal galactose have been identified in
the tubal epithelium in rabbits, and these allude to the presence
of lectin-like molecules or generally carbohydrate-binding
proteins in the mammalian oviduct (Biermann et al., 1997).
Although both the sperm surface and the oviductal epithelium
possess the tools for the carbohydrate-mediated binding event,
e.g. carbohydrate-ligands and carbohydrate-binding proteins,
most data suggest that the recognition of exposed carbohydrate
structures of the oviductal epithelial cells by lectin-like
molecules associated with the sperm surface is the dominating
mechanism creating the oviductal sperm reservoir. However,
the contribution of other systems cannot be ruled out as yet.
Candidates as oviduct-binding molecules are extrinsic
secretory proteins which associate with the sperm plasma
membrane during epididymal transit and/or ejaculation. In the
hamster, fetuin-binding sites are lost from the acrosomal region
of the sperm head during capacitation. This could be attributed
to the loss of fetuin-binding proteins of 50, 32 and 27.5 kDa
(DeMott et al., 1995). Coincidentally, spermatozoa lose their
ability to bind to the oviductal epithelium. A galactose-binding
protein has been isolated from equine sperm plasma
membranes (Dobrinski et al., 1997), and fucose-binding sites
have been localized at the head of motile bovine spermatozoa.
Furthermore, the spermatozoa–oviduct binding and the
binding of fucose-tagged probes to bovine spermatozoa require
the presence of Ca2+, pointing to the role of a calciumdependent sperm lectin with a specificity for fucose in cattle
(Suarez et al., 1998). In the pig (Figure 2A), candidate proteins
are the spermadhesins which may mediate spermatozoa–
oviduct binding. The spermadhesins form a novel protein
family of 12–14 kDa with a carbohydrate specificity ranging
from galactose in different complex oligosaccharide sequences
to mannose-6-phosphate, or show no carbohydrate binding as
aSFP, the bovine spermadhesin (discussed below).
Fate of sperm lectins during sperm transport in
pig oviduct
Spermadhesins are secreted by the male genital tract of pig (and
also of cattle and horse), comprising about 80% of total porcine
seminal plasma proteins (reviewed by Töpfer-Petersen et al.,
317
1998). During epididymal transit and at ejaculation,
spermadhesins form, together with other proteins such as serine
proteinase inhibitors and pB1 (a member of the bovine
BSA1–3, and equine HSP1–2 family; Calvete et al., 1997b), a
thick multi-molecular layer, particularly around the sensitive
acrosomal region of the sperm head, thus probably preventing
premature capacitation and the acrosome reaction
(Töpfer-Petersen et al., 1998). At about 1–2 h after mating,
sufficient spermatozoa are stored in the oviductal reservoir
(Figure 2A) to ensure subsequent fertilization (Figure 2A),
thereby leaving most of the seminal plasma components
behind (Hunter, 1996 and references therein). Seminal plasma
appears not to pass through the barrier of the utero-tubal
junction, except for those proteins coating the sperm surface.
This barrier function for seminal plasma was demonstrated by
two-dimensional SDS–PAGE of oviductal fluid collected from
the isthmus of inseminated sows (Einspanier et al., 1996;
Calvete et al., 1997a). After deposition into the female genital
tract, the spermatozoa entering the oviduct have an exposed
surface loaded with a protein coat which may now interact with
the oligosaccharides of the oviductal epithelium. Preliminary
studies demonstrate that seminal plasma and the isolated
spermadhesin fraction are able significantly to inhibit the
binding of the spermatozoa to isthmic explants of the porcine
oviduct in a concentration-dependent manner (D.Waberski,
R.Gelhaar and E.Töpfer-Petersen, unpublished results). The
ability of seminal plasma and its major components to interfere
with oviduct–spermatozoa binding may refer to the
contribution of spermadhesins in establishing the sperm
reservoir in pig by recognizing galactose in complex
oligosaccharides of the oviductal epithelium. During in-vitro
capacitation, most of the protein coat is removed from the
sperm surface. Commencing 1–2 h before ovulation,
spermatozoa are released progressively from the isthmic region
and proceed towards the site of fertilization in tightly controlled
numbers (Hunter, 1996 and references therein). With the
changing conditions in the oviduct at the time of ovulation,
spermatozoa may shed their protein coat sequentially, thus
creating new surface structures which could allow spermatozoa
to be released from the epithelium, complete capacitation and
interact with the ZP in the appropriate manner, leading to
fertilization.
Carbohydrates are the signals for gamete
recognition
The mammalian ZP surrounding the oocyte is a unique, highly
organized three-dimensional matrix that is between 2 and
25 µm thick, and which protects the egg and the
preimplantation embryo from physical damages; in addition,
the ZP modulates sperm function. The penetration of the ZP is a
crucial step in mammalian fertilization. Consequently, an
inability of spermatozoa to pass this extracellular layer
inevitably leads to infertility. Recognition and initial binding
318 E.Töpfer-Peterson
between spermatozoa and the ZP are prerequisites for
subsequent penetration (Figure 2B). The basic mechanism
appears to be conserved throughout evolution from marine
invertebrates to eutherian mammals, and is based on
carbohydrate–protein interactions between the spermatozoa
and the oocyte envelope. In most species, certain exposed
oligosaccharide chains of the oocyte envelope and
complementary carbohydrate-binding proteins on the
spermatozoa–oocyte interface mediate, at least in part, the
initial binding and the recognition between the spermatozoon
and the egg cell (Sinowatz et al., 1997).
Zona pellucida proteins
The ZP matrix is composed of only three glycoproteins, which
build a typical complex fibrillogranular structure by nonco-valent interactions. The ZP proteins are encoded by three
different genes termed according to their decreasing molecular
size, ZPA, ZPB and ZPC (Harris et al., 1994). The amino acid
sequences of ZP glycoproteins in mammals and corresponding
egg envelope proteins in widely divergent species (fish and
amphibians) have been found to be highly conserved during
evolution (Harris et al., 1994; Epifano et al., 1995; Hedrick,
1996). Post-translational modifications and processing of the
protein backbone are species-specific events and result in
heterogeneity of the attached oligosaccharides and in differing
polypeptide chain lengths of the assembling egg envelope
proteins. It appears that the ZP gene products display
a conserved modular structure. A newly defined ZP module, a
large domain spanning 260 amino acids, has been identified
near the C-terminally located putative transmembrane domain;
both of them are shared by all ZP proteins (Bork and Sander,
1992). In addition, a trefoil domain has been proposed in ZPB
located next to the ZP-module (Bork, 1993). The strictly
conserved cysteine residues, implying a similar organization of
disulphide bonds, the same pattern of hydrophobicity, polarity
and turn-forming structures within the ZP module and the
trefoil domain are consistent with a highly conserved threedimensional structure across species.
Whereas in mouse the synthesis of the three ZP proteins is
restricted exclusively to the oocyte (Wassarman and Litscher,
1995; reviewed by Castle and Dean, 1996), in other species
such as pig (Sinowatz et al., 1995; Kölle et al., 1996), cattle
(Kölle et al., 1998; Totzauer et al., 1998), rabbit (Prasad et al.,
1998), cynomolgus monkey (Martinez et al., 1996) and man
(Grootenhuis et al., 1996), follicle cells participate in
assembling the ZP matrix in a development-dependent manner.
In pig and cattle, both ZPB and ZPC are predominantly
expressed in primary follicles by the oocyte. During follicular
development the follicle cells contribute to an increasing extent
to ZP protein synthesis, and in the pig the granulosa cells and
the corona radiata of tertiary and preovulatory follicles take
over ZP protein synthesis almost completely (Sinowatz et al.,
1995; Kölle et al., 1996, 1998). Fertilization appears to
Figure 3. Scanning electron micrograph of the spherical structures
which are formed occasionally by the zona pellucida glycoproteins
of adult sows. After solubilization and storage overnight at 4°C, the
zona pellucida glycoproteins are able to reorganize to a higher
structure. Scale bar = 20 µm.
terminate ZP gene expression in all species. The different
biosynthetic patterns in mouse and in other species such as pig
and cow, may have consequences for the organization of the
three-dimensional ZP matrix in these species. In mouse, the
relatively thin ZP coat is formed by long filaments of
periodically arranged heterodimeric units of mZP2 (ZPA) and
mZP3 (ZPC), randomly cross-linked by mZP1 (ZBA)-dimers
(Wassarman and Mortillo, 1991). However, the mouse model
may not hold for larger animals, and certainly not for the thick
ZP of pigs and cattle (Totzauer et al., 1998; Kölle et al., 1998).
Very recently it has been reported that porcine pZPB and pZPC
in about equimolar ratios (1:1 up to 1:2) reconstitute to
high-mass heteromultimeric complexes, whereas the highly
purified proteins are predominantly monomeric (pZPB) or as
pZPC form smaller aggregates with molecular masses of about
300 and 160 kDa (Yurewicz et al., 1998). Coincidentally with
this report are observations that after heat-solubilization and
storage overnight at 4°C, occasionally the native ZP
glycoproteins collected from adult sows, but not from
prepubertal sows (<100 kg), reorganize to spherical structures
with a smooth surface (Figure 3) (E.Töpfer-Petersen and
F.Sinowatz, unpublished results). The failure to develop the
typical network-like architecture of the intact ZP under these
in-vitro conditions may suggest that self-aggregation of the ZP
glycoproteins is under the control of the synthesizing cells, e.g.
the oocyte and the follicle cells.
Spermatozoan–egg interactions in fertilization
Carbohydrate structures of the zona pellucida
Although carbohydrates play a crucial role in gamete
recognition, our knowledge of the oligosaccharide structures of
mammalian ZP glycoproteins is limited. One approach to
characterize the oligosaccharide chains of the ZP is to employ
lectins as tools. Comparative cytochemical studies demonstrate
the species-specific variation in the lectin-binding pattern,
which increases with the evolutionary distance of species
(Skutelsky et al., 1994). Some carbohydrate structures are
found in all species examined, such as mannose and
N-acetylglucosamine, which are usually parts of the core
region of N-linked oligosaccharides, whereas binding sites for
α-galactose-recognizing lectins appear to be typical for the
mouse and rat (Maymon et al., 1994). GalNAcβ1,4Gal
sequences have been localized ultrastructurally in the
innermost region of the intact murine ZP, indicating the
regionalization of oligosaccharide structures within the
three-dimensional structure of the zona matrix (Aviles et al.,
1999). In humans, the occurrence of N-linked glycans of the
bi-antennary complex type and/or the high mannose
(concanavalin A, Con A) as well as of the tri-antennary
core-fucosylated complex type (lentil lectin, LCA) and typical
O-linked oligosaccharide chains have been shown by
lectin-binding. Remarkably, most of the O-linked
Galβ1,3GalNAc sequences appear to be blocked by
α2,3-linked sialic acid (Maymon et al., 1994; Ozgur et al.,
1998).
Recently, the oligosaccharide sequences of the pig ZP
proteins (Noguchi and Nakano, 1992; Noguchi et al., 1992;
Hokke et al., 1994), of part of the mouse mZP2 and mZP3
(Noguchi and Nakano, 1993; Nagdas et al., 1994) and of the
unfractionated cattle ZP glycoproteins have been reported
(Katsuma et al., 1996). The ZP glycoproteins carry a complex
pattern of N- and O-glycosidically linked oligosaccharides.
ZPA is the largest ZP protein in the pig, with six potential
N-glycosylation sites, whereas ZPB and ZPC possess three and
five potential N-glycosylation sites respectively. Carbohydrate
analysis suggested that the ZPB and ZPC proteins contain three
and six additional O-glycosylation sites respectively (Yurewicz
et al., 1991). The N-linked glycans in the pig, cattle and mouse
possess basically the same structures. They belong to the
fucosylated complex-type core structure elongated by nonbranched N-acetyllactosamine chains which in the case of the
acidic glycans have sialic acid at the non-reducing end, and/or
sulphate in the C-6 position of the N-acetylglucosamine
residues of the lactosamine repeats. In the pig, additionally,
sulphated residues have been localized at the C-6 position of
N-acetylglucosamine residues in non-repeated antennae, and at
the C-3 position of reducing terminal N-acetylglucosamine
residues (Mori et al., 1998). However, species differ in the ratio
of di-, tri- and tetra-antennary chains, and in the degree of
sulphation and sialylation. Mouse and bovine ZP glycoproteins
contain mainly acidic tri-and tetra-antennary chains. In both
319
species the sulphate contents are lower than in pig ZP
glycoproteins, and the acidic properties are mainly due to the
content of sialic acid. The most significant difference between
the ZP matrices of these species is the structure of the neutral
N-glycans. The major neutral N-type oligosaccharide in bovine
ZP glycoproteins is a high-mannose-type oligosaccharide,
whereas the major neutral N-glycans in the pig belong to the
di-antennary fucosylated complex-type glycans containing
N-acetyllactosamine chains (Figure 4). In both species, the
neutral oligosaccharides constitute about 25% of the total
carbohydrate portion, whereas in mice the content of neutral
N-glycans is <5%. Terminal N-acetylgalactosamine and
α-galactose residues, which are absent in porcine N-glycans,
are minor components in the mouse and cow. The sulphated
polylactosamine backbone of the O-linked oligosaccharides of
porcine ZP glycoproteins exhibits the same structure as that of
the N-glycans extending from the Galβ1,3GalNAc
disaccharide core. Sialic acid can be linked with the nonreducing termini of the oligosaccharide chain and/or with the
proximal N-acetylgalactosamine residue of the core
disaccharide. Neutral structures terminate the non-reducing
end of the carbohydrate chain with β-galactose and
β-N-acetylglucosamine residues, and as minor components
also with α-galactose and β-N-acetylgalactosamine residues.
The amount of sulphated lactosamine repeats and the degree of
sialylation in both N- and O-glycans contribute to the enormous
heterogeneity of the ZP glycoproteins in all species. Studies on
the attachment sites of individual oligosaccharides, their
distribution within the three-dimensional structure of the intact
ZP network, and their physiological relevance are still in their
infancy, though first attempts have been made (Kudo et al.,
1998). Sugar-mapping of the glycopeptides of pZPB has
revealed that all three potential N-glycosylation sites, Asn203,
Asn220 and Asn333 of the mature pZPB (cDNA-deduced
residues 137–446) carry neutral bi-antennary N-glycans,
whereas only Asn220 is additionally glycosylated with neutral
tri- and tetra-antennary chains. At least one disulphide bond
between the neighbouring cysteine residues Cys224 and
Cys243 has been localized in the N-terminal half of pZPB
(Kudo et al. 1998). Nonetheless, current knowledge of the ZP
oligosaccharide structures in the pig makes this species an ideal
model for studying the underlying molecular mechanisms of
spermatozoa–oocyte recognition.
Carbohydrate structures determining gamete
recognition
In most species, the nature and glycosylation sites of the
individual oligosaccharides and the cognate carbohydratebinding protein that are involved in spermatozoa–zona binding
and recognition are essentially unknown. In the mouse and
hamster, the recognition signals and the ability to induce the
spermatozoa acrosome reaction have been mapped to ZP3, the
gene product of ZPC, whereas in the pig and rabbit
320 E.Töpfer-Peterson
Figure 4. Schematic representation of the biologically active N-linked
oligosaccharide of the zona pellucida glycoproteins in pig and cow. (A)
The major neutral oligosaccharide of pig zona pellucida is a
bi-antennary complex type N-glycan (Noguchi et al., 1992). (B) Only
tri- and tetra-antennary complex type N-glycans of ZPB are able to
inhibit spermatozoa–oocyte binding in the pig (Kudo et al., 1998). (C)
The major neutral N-glycan of high-mannose-type exhibit prominent
inhibitory activity of spermatozoa–egg binding and in-vitro fertilization
(Nakano and Yonezawa, 1998). Gal = galactose; Man = mannose;
Fuc = fucose; GlcNAc = N-acetylglucosamine.
sperm-binding activity is associated with the glycoproteins
coded by the ZPB genes (Yurewicz et al., 1991; Hedrick, 1996;
Prasad et al., 1998). Interestingly in Xenopus laevis, the sperm
receptor (gp69/64) is the homologue of mZP2, the ZPA gene
product (Tian et al., 1999). By analogy with the mouse system,
sperm receptor activity has been also postulated for human ZP3
(ZPC). While recombinant hZP3 possesses the ability to induce
the acrosome reaction (Brewis et al., 1996), no direct evidence
exists for the contribution of human ZP3 in spermatozoa–zona
binding. Transgenic mice expressing human ZP3 restore
fertility. However, as observed in wild-type mice, human
spermatozoa do not bind to the chimeric ZP (Rankin et al.,
1998).
For the mouse, there is general consent that certain O-linked
oligosaccharides clustered at the C-terminal sperm-combining
site of mZP3 are critical to achieve high-affinity binding. On
the contrary, the identity of the sugar moieties which determine
the carbohydrate specificity of the spermatozoa–zona
interaction, i.e. whether terminal α/β-linked galactose,
β1,4-linked N-acetylglucosamine or fucose residues, is open to
debate. The degree of sialylation and sulphation, however,
appears to have no effect on bioactivity (Litscher et al., 1995;
Litscher and Wassarman, 1996; Thaler and Cardullo, 1996; Liu
et al., 1997; Johnston et al., 1998; Shur, 1998). A more
differentiated model for sperm recognition in mouse has been
presented (Johnston et al., 1998). These authors postulate a
high-affinity binding site on spermatozoa recognizing
α3-fucosylated oligosaccharides which also could be occupied
with less affinity by α3-galactosyl-capped carbohydrates, and a
second
low-affinity
binding
site
for
terminal
β-galactose-containing structures. In the case of the pig, the
presentation of the bioactive oligosaccharide chains in
heteromultimeric pZPB/ZPC complexes appears to be
necessary to create high-affinity binding sites for spermatozoa
(Yurewicz et al., 1998). Similar to the mouse system, there is
considerable controversy regarding the structural entity of the
carbohydrate ligand mediating porcine spermatozoa–zona
binding. It has been shown (Yurewicz et al., 1991) that
O-linked glycans attached to pZPB confer the bulk of
sperm-binding activity in the intact ZP. In contrast, others
(Yonezawa et al., 1995a,b; Nakano et al., 1996) have reported
that neutral tri- and tetra-antennary N-glycans (Figure 4) linked
to Asn220 located at the N-terminal region of
endo-β-galactosidase-treated pZPB, but not the bi-antennary
N-glycans and O-glycans of pZPB, play the critical role in
mediating gamete-binding and recognition. As in the mouse,
enzymatic removal of polylactosamine extensions of the Nand O-glycans has been shown not to diminish the biological
activity of pZPB and the ZPB/ZPC complexes (Nakano et al.,
1996; Yurewicz et al., 1998).
Recently, data have become available regarding the
bioactive carbohydrate structure in the bovine system. The
bovine ZP also consists of three glycoproteins, homologous to
pZPA, pZPB and pZPC as shown by Western blotting with
anti-porcine ZPB and anti-porcine ZPC, peptide mapping, and
N-terminal sequencing (Topper et al., 1997). The major neutral
N-glycans of high-mannose-type having five mannose residues
and the isolated bZPB exhibit prominent inhibitory activity of
spermatozoa–egg binding and in-vitro fertilization (Figure 4).
The sperm-binding capacity of fertilized eggs is diminished,
indicating that the structure and/or the conformation of the
sperm-binding site of the ZP changes during fertilization
(Nakano et al., 1996; Nakano and Yonezawa, 1998).
Studies in the human system are restricted by the limited
availability of biological material, e.g. zona pellucidae,
impeding detailed structural carbohydrate analysis. A series of
monosaccharides (fucose, mannose, galactose and
N-acetylglucosamine residues) has been found to inhibit sperm
binding (Miranda et al., 1997). Conflict between the results of
different researchers (Oehninger et al., 1990) has been linked
(Miranda, 1998) to the uncontrolled capacitated states of the
spermatozoa used in experiments. Complex glycoconjugates
bearing selectin-like ligands, e.g. Sialyl-Lewisx(a) and
fucosylated and sialylated unusual GalNAcβ1,4GlcNAc
(lacdiNAc)-antennae of N-glycans, as in glycodelin A, are
potent inhibitors, leading to the hypothesis that in humans a
selectin-like mechanism may be a determinant for gamete
recognition (reviewed by Oehninger et al., 1998). Chemical
modifications affecting particularly the terminal sialic acid
Spermatozoan–egg interactions in fertilization
residues interfere partly with sperm binding (30–40%
inhibition), whereas enzymatic release of sialic acid moieties
(documented by the lectin from Maackia amurensis (MAA)
recognizing α2,3-linked sialic acid) and the release of the bulky
lactosamine repeats by endo-β-galactosidase significantly
increase the binding (3- and 7-fold respectively), probably due
to the demasking of potential binding sites (Ozgur et al., 1998).
Furthermore, recognition and binding of clustered mannose or
N-acetylglucosamine residues of the corresponding
neoglycoproteins appears to induce the acrosome reaction in
human spermatozoa, apparently in the proper manner
(Brandelli et al., 1996; Jacob et al., 1998). As postulated for the
mouse, so also in human, more than one binding siterecognizing carbohydrate with low (sialic acid, selectin-like?)
and high affinities (mannose, N-acetylglucosamine?) at the
sperm surface may act in concert or in a certain sequence with
the multiple oligosaccharides of the ZP.
All these data demonstrate that the acquisition of sperm
receptor activity may be a consequence of at least partly
species-specific, and perhaps tissue-specific glycosylation and
oligosaccharide processing of the distinct ZP glycoproteins,
and that the biologically active carbohydrate structures are not
necessarily linked with the homologous zona proteins.
Furthermore, recognition and binding between spermatozoa
and egg appears to be the result of the cooperation of a
multimeric receptor system (Shur, 1998).
Zona pellucida-binding proteins
Among the bulk of ZP-binding proteins (ZBP) that have been
described to date, only a few are characterized biochemically
and structurally. Sperm proteins known to have specific
carbohydrate-binding
sites
include
mouse
β1,4
galactosyltransferase and sp56, rabbit sp17, guinea pig PH-20,
and a human mannose-binding protein (MBP) (Benoff, 1997;
Sinowatz et al., 1997; McLesky et al., 1998; Shur, 1998).
Rabbit sp17 and the human MBP share some common features
with the expanding group of Ca2+-dependent (C-type) lectins
(Drickamer, 1993) containing some of the invariant amino
acids of the C-type carbohydrate-recognition domain (CRD).
Sp17 has been proposed to belong to the subfamily of lectins
with specificity to galactose (Richardson et al., 1994) and the
cDNA of human sperm MBP has been described to encode
for a protein with seven putative mannose-like binding CRD
(for details, see Benoff, 1997). The carbohydrate specificity
and the binding domains of spermadhesins and proacrosin have
been studied most intensively by determining the
three-dimensional structure and protein modelling (see below),
whereas other ZBP such as the zona receptor kinase (ZRK/hu9)
and two porcine ZBP, P47 and zonadhesin, have been
characterized structurally although the carbohydrate-binding
has not yet been determined (Hardy and Garbers, 1995; Ensslin
et al., 1998; for review see McLesky et al., 1998). ZBP are
localized in different compartments of the sperm head.
321
Spermadhesins (Sinowatz et al., 1997; Töpfer-Petersen et al.,
1998) and the newly described P47 (Ensslin et al., 1998) are
associated peripherally to the sperm surface, whereas mouse
galactosyltransferase (Gong et al., 1995), ZRK (McLesky
et al., 1998) and porcine zonadhesin (Hardy and Garbers,
1995) are a membrane-spanning proteins. ZBP could also be
integrated into the membrane and exposed to the surface at the
time when capacitation and/or the acrosome reaction are
proceeding (rabbit sp17, Richardson et al., 1994; human MBP,
Benoff et al., 1993) or could be part of the acrosomal matrix, as
demonstrated for porcine acrosin (Töpfer-Petersen et al.,
1990), guinea pig AM67 and its mouse orthologue sp56 (Foster
et al., 1997).
Carbohydrate-binding proteins appear to be made available
sequentially during the progress of the fertilization process and
may act in oviductal binding, as primary and secondary
receptors, or may be of still unknown function. The inadequate
attribution of those carbohydrate/ZBP to their distinct roles in
the sequence of binding events in vivo characterizes the
problems in that research field and demonstrates the limitation
of our in-vitro test systems.
Zona pellucida-binding proteins in the pig
Spermadhesins
The spermadhesin family (Figure 5) has its greatest diversity of
members in the pig. AQN-1, AQN-3, AWN (named after the
first three N-terminal amino acids), PSP-I and PSP-II, its
glycosylated (AWN and AQN-3) and N-terminally acetylated
isoforms (AWN) are major secretion products of the seminal
vesicle and comprise the bulk of the proteins in seminal fluid.
By using reverse transcriptase–polymerase chain reaction
(RT–PCR) and histochemical methods, they are also found to
be expressed along the epididymal tract, although AWN is the
only spermadhesin that binds tightly to epididymal
spermatozoa. Spermadhesins are multifunctional proteins
exhibiting a spectrum of ligand-binding affinities, e.g.
carbohydrates/ZP glycoproteins, sulphated glycosaminoglycans, serine proteinase inhibitors, and o-phosphorylethanolamine. Although spermadhesins share more than 60%
amino acid sequence identity, they are not functionally
equivalent, each exhibiting a different set of affinities,
modulated by the actual state of the protein, e.g. glycosylation
and aggregation. Only non-aggregated monomeric AWN and
AQN-3 show specific binding capacity toward phospholipids,
glycosylation of AWN and AQN-3 abolishes the carbohydrate
affinity, and the heparin-binding affinity is dependent on the
formation of a PSPI/PSPII heterodimer (Töpfer-Petersen and
Calvete, 1996; Sinowatz et al., 1997; Töpfer-Petersen et al.,
1998). The most thoroughly studied feature of the
spermadhesins is their carbohydrate-binding capacity. They
bind to the ZP glycoproteins in a cation-independent manner
with a Kd in the low micromolar range. AQN-1, AQN-3 and
AWN display similar binding affinities for glycoproteins
322 E.Töpfer-Peterson
Figure 5. Schematic representation of the major carbohydrate/zona pellucida binding proteins of porcine spermatozoa. Spermadhesins belong to
the CUB-family; inset: the three-dimensional structure consists of five parallel and antiparallel β-strands (Romero et al., 1997). P47 is a mosaic
protein with two tandemly organized N-terminal EGF-domains and two large C-terminal domains displaying similarity to the C1 and C2 region of
blood-coagulating factors V and VIII (Ensslin et al., 1998). Zonadhesin contains five D-domains homologous to the von Willebrand factor, a
mucin-like domain located next to the N-terminal region and a C-terminal transmembrane domain (Hardy and Garbers, 1995). Proacrosin is serine
proteinase zymogen consisting of a light and a heavy chain. N-terminal processing by a single proteolytic clip between Arg23 and Val24 and
C-terminal processing of the proline-rich region activates the mature and stable enzyme (Töpfer-Petersen et al., 1990).
Figure 6. Schematic representation of the binding of uncapacitated and capacitated spermatozoa to the zona pellucida in the pig. Uncapacitated
spermatozoa bind to oligosaccharides of the zona pellucida via the spermadhesins coating the sperm surface. After capacitation, most of the
spermadhesins are released, exposing new zona pellucida binding proteins such as the processed zonadhesin and other receptor proteins which
in concert are now able to interact with the zona pellucida, leading to the acrosome reaction.
carrying Galβ1,3GalNAc and Galβ1,4 GlcNAc sequences in
O- and N-linked oligosaccharides. Thereby, AWN, AQN-1 and
AQN-3 differ slightly in their ability to recognize Galβ1,4
GlcNAc sequences in bi- (AQN-1) or tri/tetra-antennary
Spermatozoan–egg interactions in fertilization
(AQN-3, AWN) N-glycans. N-acetylneuraminic acid (α2,3/6)
linked with the penultimate galactose residue may increase
(AWN) or decrease (AQN-1) affinity. AWN recognizes
Galβ1,3 GalNAc sequences about 50 times better than the
N-acetyllactosamine unit in N-glycans (Dostalova et al., 1995;
Calvete et al., 1996). In contrast, PSPII shows affinity to
mannose-6-phosphate. By protein modelling on the basis of the
crystal structure of the PSPI/PSPII heterodimer (see below),
Arg43 of the PSPII molecule has been suggested to play a
pivotal role in mannose-6-phosphate binding (Solis et al.,
1998). Furthermore, glycosylation at Asn50 of AWN and
AQN-3 interferes with their carbohydrate-binding ability,
suggesting that the CRD is located around the conserved
asparagine residue in position 50. The amino acid sequences do
not show any discernible similarity to known CRD. However,
they do belong to a family of 16 functionally diverse proteins,
all of them sharing the CUB-domain within a modular structure
(Bork and Beckmann, 1993). Spermadhesins, spanning
110–133 amino acids, form a subgroup with a single
CUB-domain architecture. The crystal structure of the
non-covalent heterodimeric PSPI/PSPII complex has been
solved (Romero et al., 1997). The overall structure of the
subunits consists of a β-sandwich comprising two sheets, each
containing five parallel and antiparallel β-strands (Figure 6,
inset). The conformation of the CUB-domain appears to be
highly conserved in other spermadhesin molecules: also the
bovine spermadhesin, aSFP, shows the same overall structure
(Romero et al., 1997). The PSPI/PSPII complex is the first
ZBP to be described in its three-dimensional structure which
additionally reveals the architecture of the CUB-domain.
The similar, though discrete, carbohydrate specificity as well
as their fine modulation dependent on the actual state of the
proteins, e.g. glycosylation and aggregation, as well as their
distribution during different functional stages in vitro, imply
that the spermadhesins are involved in the early events of
fertilization. It has been shown that in vitro AWN is present on
the sperm cell bound to the ZP, and that the bound sperm
population was able to undergo the acrosome reaction
(Figure 6) (Töpfer-Petersen and Calvete, 1996). However,
whether spermatozoa that reach the site of fertilization will
actually carry measurable amounts of AWN and AQN-3
molecules bound to the surface, and thus serve as primary
receptors in vivo, is critical in assigning a biological role for
spermadhesins in gamete interaction.
P47, a novel ZBP
P47 (Figure 5) is another tightly bound peripheral sperm
membrane protein of 47 kDa that has been identified by affinity
chromatography on immobilized ZP glycoproteins (Ensslin
et al., 1998). P47 proteins disclose a mosaic structure organized
by two tandemly arranged N-terminal epidermal growth factor
(EGF)-like domains and two large C-terminal domains
displaying similarity to the C1 and C2 regions of
blood-coagulating factors V and VIII. The second EGF-like
323
domain displays an RGD tripeptide, a motif found in a number
of integrin receptor ligands (Eble and Kühn, 1997). The
C-terminal domains may function as anchors to the lipid
bilayer of the sperm surface, as has been shown for the C2
domain of factor VIII encompassing a peptide with an
α-helical structure that is supposed to bind to anionic
phospholipids (Gilbert and Baleja, 1995). The amino acid
sequence of this region appears to be highly conserved within
the C-terminus of P47. P47 is first detected in testicular
spermatozoa located at the apical rim of the acrosomal region.
Its distribution varies only slightly during epididymal transit,
though under in-vitro capacitation conditions it shows a
granular appearance over the entire acrosome when tested by
indirect immunfluorescence. Traces of P47 could still be
detected at the acrosomal region of spermatozoa bound to the
ZP and starting the acrosome reaction. P47 is able to bind
biotinylated ZP glycoproteins, although it has not been tested
whether it interacts with the carbohydrates or with the
polypeptide backbone of the ZP proteins.
P47 proteins belong to the MFGM-protein family, secretory
proteins of the mammary gland with a still unknown function
(Stubbs et al., 1990; Aoki et al., 1995; Anderson et al., 1997),
and is homologous to rat O-acetyl ganglioside synthase (AGS;
Ogura et al., 1996). P47 is not exclusive to the pig, but has been
characterized by cDNA-sequencing in the testis of mouse,
cattle, horse and human (Ensslin et al., 1998; M.Gentzel and
E.Töpfer-Petersen, unpublished results), and also in the
epididymis, mammary gland, uterus and several other bovine
and porcine tissues, exhibiting an amino acid sequence identity
of 60–100%.
The role of P47-like protein in fertilization is highly
speculative. The ZP affinity may implicate a role in primary
binding, whereas the exposed RGD-sequence in the second
EGF-domain may interact with integrin receptors on the sperm
membrane or with an egg membrane integrin or serve as a
ligand for EGF-receptors, and thus contribute to signalling
events in fertilization. P47 is completely released during the
in-vitro-induced acrosome reaction (T.Läkamp, M.Gentzel and
E.Töpfer-Petersen, unpublished results), which may argue
against a role in spermatozoa–oolemma interaction.
Zonadhesin
Porcine zonadhesin (Figure 5) has been shown to bind in a
species-specific manner to the homologous ZP. The
zonadhesin cDNA, cloned from pig testis, predicts a large
protein with a modular structure containing five D-domains
homologous to the von Willebrand factor, a mucin-like domain
located next to the N-terminal region, and a C-terminal putative
transmembrane domain (Hardy and Garbers, 1995). The
expression of the zonadhesin gene is restricted to the testis,
where it is translated primarily in haploid spermatids. The
cDNA codes for a precursor protein which may be processed
during epididymal transit and/or capacitation to generate the
mature protein by removing the N-terminal and mucin-like
324 E.Töpfer-Peterson
domains. After capacitation, the functional protein appears to
contain two disulphide-bonded subunits, the N-terminal p45
and the p105 moieties. It is still unknown whether the
zonadhesin molecule recognizes the ZP carbohydrates, or is
directed against the polypeptide core of the ZP glycoproteins.
The authors postulate that zonadhesin may recognize the
sulphated oligosaccharide structure of the ZP via the
heparin-binding sequences of the D-domains, thus contributing
to species-specific recognition and subsequent signalling
events. Recently, mouse tectorins—modular matrix proteins
of the inner ear—have been found to display an arrangement of
D-domains similar to that of zonadhesin, as well as a
C-terminally located ZP module (Legand et al., 1997). It is
interesting that in the mouse, the zonadhesin gene has been
localized close to the Zp3 (ZPC) gene on chromosome 5 (Gao
et al., 1997).
Proacrosin
Pro/acrosin (Figure 5) was one of the first sperm proteins to be
biochemically and, in part, structurally characterized
(Töpfer-Petersen et al., 1990). Acrosin is released as a
consequence of the acrosome reaction and was considered to
be essential for sperm penetration through the ZP matrix. It is
the major ZBP of boar spermatozoa, interacting in a
non-enzymatic manner with the ZP glycoproteins. In intact
ejaculated spermatozoa, acrosin is stored in the acrosome as an
inactive 53/55 kDa precursor molecule that is activated by a
single proteolytic clip between Arg23 and Val24, accompanied
by C-terminal processing to the stable 38 kDa protein.
Activation and processing of acrosin occurs concomitantly
with the acrosome reaction, and appears to be regulated on and
by the ZP (Töpfer-Petersen and Cechova, 1990). About 5% of
the latent proteolytic activity in boar spermatozoa is due to a
33 kDa zymogen presenting the same N-terminal amino acid
sequence of 34 amino acid residues as in the proacrosin
molecule (Töpfer-Petersen et al., 1990). Both the long and the
short zymogen forms, as well as the active molecules show
affinity toward the ZP. Limited autoproteolysis of pro/acrosin
has led to the isolation of a 15 kDa N-terminal fragment still
exhibiting about 66% of the ZP-binding activity
(Töpfer-Petersen et al., 1990). Acrosin–ZP binding follows a
polysulphate-recognition mechanism by which clustered basic
amino acids on the pro/acrosin molecule interact with the
sulphate groups within the lactosamine repeats of N- and
O-glycans of the ZP. However, the binding is not restricted to
ZP proteins but also occurs with other polysulphated glycans
(Jones, 1991). Using recombinant techniques and site-directed
mutagenesis (Jansen et al., 1995, 1998), binding has been
mapped to the basic amino acids (particularly Arg250, Lys251,
Arg253) within the 180 amino acids starting at Gly93, with
essential contributions also being made by the basic amino
acids His47, Arg50 and Arg51. Protein modelling on the basis
of thrombin and chymotrypsin indicates that the essential basic
amino acids appear to be located in the loops which are
arranged near the active site. Studies on rabbit proacrosin led to
similar results, demonstrating that the N-terminal region,
containing three clustered Arg47, Arg50 and Arg51 mediates
the sulphate binding (Richardson and O’Rand, 1996). After the
acrosome reaction has been initiated, proacrosin may serve as a
secondary receptor by temporarily anchoring acrosomereacted spermatozoa to the ZP. The polysulphate-binding
domains appear to be involved not only in secondary binding,
but also in the proacrosin–acrosin conversion (which is
induced by ZP glycoproteins and other polysulphated glycans),
as has been shown for porcine and human pro/acrosin
(Töpfer-Petersen and Cechova, 1990; Moreno et al., 1998).
Upon binding, the activation and processing of the enzyme is
regulated by the ZP, resulting in the release of the enzyme and
the spermatozoa. This fits into the proposed cyclic model of
zona penetration (alternating cycles of binding, digestion and
release reactions together with the forward motility is required
to achieve penetration) which was first described in 1986
(O’Rand et al., 1986). The observation that proacrosin
knock-out mice are fertile, though spermatozoa are slow in
penetrating the ZP (Adham et al., 1997), may point to the
important role of redundant systems involved in fertilization.
Uncapacitated and capacitated porcine
spermatozoa bind to the zona pellucida in vitro
The oviductal epithelium and the ZP may share identical
carbohydrate sequences, as could be shown by their
lectin-binding pattern (Raychoudhury et al., 1993; Skutelsky
et al., 1994). Under the conditions of different experimental
approaches in vitro which have been widely used to identify
ZBP (e.g. Western blotting, cross-linking studies, ZP-affinity
chromatography and in-vitro inhibition assays), the sperm ZBP
covering the surface of the uncapacitated spermatozoa may
recognize carbohydrate structures of the ZP glycoproteins,
although in the in-vivo situation they would rarely meet the ZP
of the ovulated oocyte. In the pig, quantitative studies of
spermatozoa–zona binding and penetration is seriously
affected by the tendency of boar spermatozoa to bind to the ZP
under non-capacitating conditions, and additionally by the
considerable variation between semen samples (Petersen et al.,
1984). Binding ability develops rapidly in either noncapacitating or capacitating conditions, while the penetration of
the ZP appears to be a slow event and only occurs under
conditions supporting capacitation (Harrison, 1997).
Furthermore, it was reported (Harrison, 1997) that the
proportion of zona-bound spermatozoa which are actually able
to penetrate the ZP is much less than the proportion which has
undergone capacitational changes in terms of CTC-staining,
alterations of lectin-binding patterns (Harrison, 1997) and the
recently observed increase of ZP binding sites (Harkema et al.,
1998). By flow cytometry studies using solubilized
fluorescence-labelled ZP glycoproteins it could be shown that,
under non-capacitating conditions a low, but distinct number of
Spermatozoan–egg interactions in fertilization
ZP binding sites are localized at the plasma membrane
overlying the acrosomal region which is involved in
spermatozoa–zona binding. The zona-binding ability increases
by incubation in media supporting capacitation with a
maximum after 1–2 h in live, acrosome-intact cells. The
complete omission of Ca2+ suppresses the increase of
zona-binding ability, indicating that the development of ZP
binding sites is related to capacitation (Harkema et al., 1998).
Altogether, in the pig the number of zona-bound spermatozoa
does not necessarily correlate with the possible ‘fertilizing’
subpopulation of spermatozoa that are able to respond
immediately to the ZP. Moreover, boar spermatozoa appear to
be able to bind to the ZP at different stages of capacitation,
which may explain the temporal variation in developing
penetration ability. Spermadhesins, predominantly forming the
protein coat in ejaculated washed spermatozoa, appear to
account for the binding of uncapacitated spermatozoa to intact
ZP in vitro (Figure 6). As described above, their ligand-binding
ability is influenced by the degree of glycosylation and
aggregation. Moreover, serine proteinase inhibitors secreted by
the seminal vesicle have been shown to bind specifically to
members of the spermadhesin family, thereby partly masking
the carbohydrate-binding domains (Sanz et al., 1992b; Calvete
et al., 1993). The composition of the protein coat differs in
individual ejaculates, and thus may influence zona-binding of
the uncapacitated spermatozoa and also the progress of
capacitation. Under capacitating conditions the spermadhesin
aggregates and other bound substances, e.g. serine proteinases,
may now be sequentially removed. This is accompanied by the
observed increase of zona-binding ability that is due to the
concomitant unmasking of other membrane-bound ZBP, e.g.
AWN, AQN-3 and P47 and/or processing to the functional
protein of integral zona-binding proteins such as zonadhesin
(Figure 6). The sperm surface that actually responds to the ZP
by activation of the signal transduction pathways and which
leads to the acrosome reaction, may or may not carry
membrane phospholipid-bound AWN and AQN-3.
Regarding the behaviour of boar spermatozoa in vitro, the
controversial finding concerning the biologically active
oligosaccharide involved in spermatozoa–zona binding should
be interpreted with care. Porcine ZP glycoproteins carry minor
O-linked Neu5Acα2,3/Galβ1,3GalNAc tri- and disaccharides
and sialylated bi-, tri- and tetra-antennary N-glycans with
Galβ1,4GlcNAc sequences which may serve as ligands for the
lectin-like spermadhesins. In fact, it could be shown that
in vitro the spermadhesins recognize oligosaccharide structures
on the three-dimensional network of intact zona pellucidae.
AWN-coupled beads bind to the exposed oligosaccharide
chains of ovarian oocytes (Figure 7) and AWN allowed to bind
to intact ZP prevents both the beads and spermatozoa (Sanz
et al., 1992a) from binding. The success of inhibition
experiments with isolated oligosaccharides or glycopeptides of
the ZP glycoproteins, e.g. O-glycans or N-glycans, may be
dependent on the actual sperm subpopulation bound to
325
Figure 7. Scanning electron micrograph of the ovarian oocyte of the
pig. Nucleosil C18 beads of 3 µm are loaded with the isolated AWN
are able to interact with the zona pellucida. The binding could be
inhibited by preincubation of the oocyte with AWN, and is inhibited
competitively with the glycopeptides of fetuin (not shown). Bovine
serum albumin (BSA)-loaded beads are not able to bind to the zona
pellucida (not shown).
the zonae pellucidae, and may be due to the inhibition of the
binding of uncapacitated spermatozoa or of spermatozoa in
various stages of capacitation. The undefined sperm population
in terms of capacitational changes, which bind to zonae
pellucidae in vitro, may be the reason for the apparently
contradictory results that have been reported regarding the
bioactivity of various carbohydrate structures.
Is carbohydrate-mediated gamete recognition
really species-specific?
Species-specificity in mammals has been mostly linked to the
carbohydrate-based recognition events at the spermatozoa–ZP
interface. However, cross-binding has been observed between
a variety of mammals. From mouse up to primates,
spermatozoa are able to bind to human oocytes, though the
reverse binding of human spermatozoa to oocytes of other
species is restricted to near-related primates (Oehninger et al.,
1993). It was reported recently (Wessa et al., 1999) that equine
spermatozoa are able not only to bind and to penetrate the ZP of
bovine ovarian eggs, but also to enter the egg’s cytoplasm.
Capacitated porcine spermatozoa bind and have been found by
electron microscopy to be mostly acrosome-reacted, though
they do not pass bovine zonae pellucidae. The ZP exposes a
variety of oligosaccharides containing basically similar
structural elements in different species. Variable sperm lectins
326 E.Töpfer-Peterson
may recognize those structures with or without consequence
for sperm activation and fertilization. Thus, heterospecific
incompatibility could occur at several steps in the fertilization
cascade, e.g. spermatozoa–zona binding, induction of
acrosome reaction and zona penetration, formation of the male
pronucleus, and fusion of the male and female pronuclei to
restore diploidy. The term ‘species-selectivity’ was created
(Vacquier et al., 1995) to describe the situation in the sea
urchin. Here, heterospecific gamete mixtures yielded the
development of heterospecific zygotes varying from 0% to
100%. In the opinion of these researchers, ‘species-specificity’
means 100% species-exclusive and 0% heterospecific
fertilization. These reflections may be also valid for gamete
recognition in mammals.
Conclusions
Carbohydrates linked to proteins and lipids exercise a wide
variety of functions and influences on the core molecule, e.g.
protein folding, stabilization of conformation, targeting and
sorting, as well as simple protection. One of the most
fascinating features of complex sugar moieties, however, is
their coding potential. The diversity of linkage sites and
branching turn carbohydrates into ideal information-bearing
molecules, having a vast informational potential within a short
sequence, superior to that of other biological polymers such as
DNA or proteins (Laine, 1997). The carbohydrate-encoded
information must be deciphered by a protein with a
complementary structure or CRD, e.g. glycosidases,
glycosyltransferases, antibodies and lectins which transform
the information into biological responses. In a wide variety of
biological systems, cells and molecules use the
information-storing potential of the sugar code for
communication, e.g. recognition, adhesion and signalling
events (Gabius, 1997).
Thus, it is not surprising that along the route to fertilization,
mammalian spermatozoa communicate with the female
environment partly using the sugar codes. Carbohydrate/ZBP
of the spermatozoa appear to be made available sequentially
during the fertilization process. This suggests that there
exists a hierarchy of binding events, ranging from
spermatozoa–oviductal binding to primary and secondary
binding between spermatozoa and oocytes. The binding of
spermatozoa to the oviductal epithelium to create the sperm
reservoir also appears to involve carbohydrates, although until
now the role of the carbohydrates in transmitting oviductal
signals to the spermatozoon is not clear. Moreover, the
processes occurring in the oviduct are not species-specific
events in as much as capacitation can occur in a heterologous
oviduct, as well as under certain conditions in vitro.
Nonetheless, the temporal and spatial regulation of sperm
capacitation by the oviduct may be one way for male and
female to communicate to ensure fertilization.
However, the first contact between both gametes is the
critical step in fertilization. Sperm binding to the ZP is directed
by cooperative interactions of multiple complementary
receptor systems which may act together in a hierarchical and
redundant manner to guarantee survival of the species. The
observations from studies with knock-out mice imply the
existence of redundant systems rather than of single essential
biological functions in fertilization. It has been demonstrated
that the initial molecular interaction may involve low- and
high-affinity components suggesting, therefore, a hierarchy of
binding events at the spermatozoa–zona interface (Thaler and
Cardullo, 1996). The interaction between the spermatozoa and
the oocyte occurs at the surface of the three-dimensional ZP
structure which is assembled by a defined array of polypeptide
chains and arrangement of their oligosaccharides. In order to
achieve high-affinity binding between spermatozoa and
oocyte, a critical density of the different biologically active
carbohydrates must be presented within the supramolecular
architecture of the ZP matrix. It is a matter of speculation
whether the combination of presented carbohydrates at
the sperm-binding site of intact ZP may vary to some extent.
The multivalent ligands which bind to the protein counterparts
at the sperm surface, probably lead to the correct assembly of
the proteins within the plane of the plasma membrane which is
necessary to activate the signalling systems leading to
acrosomal exocytosis. High affinity and multivalency may be
the conditions for initiating the signal transduction pathway.
This may explain why oligosaccharides or small glycopeptides
are able to interfere with binding, but do not induce
the acrosome reaction. Another question relates to whether the
polypeptide backbone contributes to the induction of
the acrosome reaction solely by clustering oligosaccharides or
by direct protein–protein interaction. Recombinant ZP proteins
undecorated with carbohydrates have been shown to activate
acrosomal exocytosis in spermatozoa, thus emphasizing a role
of the protein backbone in spermatozoa–zona interaction
(Chapman et al., 1998). Although there is now a vast amount of
information available on the events which occur when
spermatozoa and oocyte meet, the linking pieces of the puzzle
are still missing.
Acknowledgements
Our own studies were generously supported by the Deutsche
Forschungsgemeinschaft. The author wishes to thank
Dr Schwartz (Institute of Anatomy, University of Göttingen)
for performing the electron microscopy. Drs Rump
(Hannover), F.Sinowatz (Munich) and R.Ivell (Hamburg) are
gratefully acknowledged for helpful discussions and critical
reading of the manuscript, and Mrs E.Podajsky and Mrs
Ch.Hettel for preparation of the photographs and figures.
Spermatozoan–egg interactions in fertilization
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Received on October 14, 1998; accepted on April 21, 1999