Molecular Human Reproduction vol.3 no.7 pp.599–637, 1997
Carbohydrates and fertilization: an overview
Susan Benoff1,2,3
1Department
of Obstetrics and Gynecology, North Shore University Hospital, Manhasset, New York and 2Departments of
Obstetrics and Gynecology and Cell Biology, New York University School of Medicine, New York, New York, USA
3To whom correspondence should be addressed at: North Shore University Hospital, 300 Community Drive, Boas–Marks
Biomedical Science Research Center, Room 125, Manhasset, New York 11030, USA
Initial sperm–egg binding in mammals involves recognition of glycosylated proteins of egg zonae by
glycosylated proteins on sperm surfaces. Egg zona protein structure is relatively simple, and has been strongly
conserved. Species specificity must reside in the carbohydrate modifications on the egg surface, and in the
co-ordinated assembly of a unique cohort of sperm proteins at capacitation. Fruitful advances have been
made along four lines. Oligosaccharide structures capable of binding spermatozoa have been dissected by
in-vitro synthesis and binding experiments, informed by the general advance of knowledge of protein
glycosylation processes. Site-specific mutagenesis of zona proteins and their expression in tissue culture
have identified glycosylation sites involved in species-specific sperm binding. Antibody and lectin labelling
studies show a continuing process of remodeling of glycosylated sperm surface epitopes within a set of
stable compartments during epididymal transit and capacitation of spermatozoa. Characterization of sperm–
egg binding proteins from a variety of mammalian species shows that a different set of effectors induce
acrosome reactions in each species, with each set including one or more sugar-recognizing proteins.
Sequencing of some of these effectors suggests that each group may form a supermolecular complex to
induce a species-specific acrosome reaction, with the functional activities distributed in a species limited or
non-limited manner among the individual proteins.
Key words: fertilization/lectins/mammals/oligosaccharides/zona pellucida
Introduction
A cascade of cell–cell interactions is required to achieve
fertilization in mammals. Upon ejaculation and residence in
the female reproductive tract, mammalian spermatozoa then:
(i) bind to oviductal cells; (ii) undergo capacitation; (iii)
respond to chemoattractants; (iv) penetrate the cumulus complex; (v) bind to the zona pellucida; (vi) undergo an acrosome
reaction; (vii) enter into the perivitelline space after passage
through the zona; (viii) bind to the egg plasma membrane;
(ix) fuse with the egg plasma membrane, and (x) be drawn
into the ooplasm and undergo nuclear decondensation and
activate zygote development (Yanagimachi, 1994; Snell and
White, 1996; Myles and Primakoff, 1997). Although the sperm
surface plays an active role in the regulation of this pathway,
it is clear that carbohydrates of one of the three barriers that
the spermatozoa must cross, i.e. the zona pellucida, strongly
contribute to the modulation of steps (v) to (vii).
The zona pellucida, which surrounds the plasma membrane
of mammalian oocytes, is a relatively thick, highly glycosylated
extracellular matrix. This matrix plays at least four important
roles during development: (i) regulation of normal endocrine
profiles during folliculogenesis; (ii) formation of a barrier
to heterospecific fertilization; (iii) formation of a block to
polyspermic fertilization in some mammals; and (iv) protection
of the embryo during preimplantation development (Bronson
and McLaren, 1970; Keenan et al., 1991; Yamagimachi, 1994;
© European Society for Human Reproduction and Embryology
Liu et al., 1996). This review will focus on the contribution
of carbohydrates to the regulation of zona functions (ii) and (iii).
Interactions between carbohydrates on the extracellular
matrix of the egg and glycoproteins on the sperm surface
provide the molecular basis for gamete recognition and adhesion in species as diverse as sea urchins (Garbers, 1989),
ascidians (Rosati, 1985), amphibians (Gerton, 1985), algae
(Callow et al., 1985), mouse (Wassarman, 1989, 1990) and
man (Benoff et al., 1995a). In general, a compulsory order of
initial interactions of mammalian spermatozoa and eggs is
observed: (i) attachment or adhesion; (ii) tight binding, and
(iii) secondary binding (Wassarman, 1987; Wolf, 1996). Attachment is defined as relatively loose, non-specific association of
the sperm head plasma membrane with the zona pellucida.
Attached spermatozoa can be readily dissociated from the zona
by pipetting. Tight binding is the relatively tenacious interaction
between acrosome-intact, capacitated spermatozoa and the
zona pellucida which is species-specific and regulated by
glycoproteins. Spermatozoa which are tightly bound to the zona
can not be removed by vigorous pipetting. The physiological
acrosome reaction occurs after tight binding. Secondary binding
is thought to maintain zona binding by acrosome-reacted
spermatozoa. Herein, the molecules which participate in the
complementary, carbohydrate-based binding processes which
regulate initial specific interactions between mammalian spermatozoa and ovum, and thus fertilization, will be reviewed.
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The first section of this review summarizes the recent
advances in glycobiology that have permitted the dissection
of the mechanisms by which carbohydrates regulate sperm–
zona pellucida binding and induction of the acrosome reaction.
In the second section, the structure of the zona pellucida in a
variety of mammalian species is compared and contrasted. In
this discussion, the role of molecular biological techniques in
the elucidation of both the primary structure of zona glycoproteins and the identification of potential glycosylation sites
which contribute to the formation of the primary sperm binding
site is emphasized. Recent data concerning the structure of the
oligosaccharide chains recognized by receptors on the sperm
surface are also presented. The third section outlines a large
number of studies in which carbohydrate moieties on the
sperm surface have been examined. Changes in glycosylation
of membrane-associated sperm proteins and lipids accompanies
epididymal maturation and the acquisition of sperm fertilizing
potential. Although these are thought to play a role in generation
of a periacrosomal plasma membrane capable of calciumstimulated fusion with the outer acrosomal membranes, the
argument is made that the primary role of sperm surface glycan
structures is as antigenic determinants in natural immunoregulation of gamete function. The fourth section is of particular
interest to me as it presents results of informed research on
the identification of sperm receptors for zona carbohydrate
ligands. Recent data from my laboratory on how expression
of sperm surface carbohydrate binding sites can affect the
outcome of human in-vitro fertilization (IVF) is included in
this review. From the findings presented in this section, it will
become evident that fertilization mechanisms in man cannot
necessarily be equated with those in animal models. The
concluding section summarizes the complex roles of carbohydrates in the sequence of sperm–egg interactions leading to
fertilization and addresses issues that remain to be resolved.
Recent advances in carbohydrate biochemistry
The last 10 years have been witness to major advances in our
understanding of the function of glycoproteins (Lis and Sharon,
1993; Varki, 1993). This is the result of the development of a
variety of methodologies to study the structure and function
of carbohydrate chains (‘glycans’) (see Table I). The addition
of carbohydrates, ‘glycosylation’, is the most common form
of covalent modification of newly synthesized proteins (Kobata,
1992). Glycans, oligosaccharides [composed of 2–10 monosaccharide units (simple sugars)] and polysaccharides (long
linear or branched chains of many monosaccharide units), are
secondary gene products. It must thus be emphasized that,
while the polypeptide backbone sequence of a glycoprotein is
directly correlated with the sequence of bases in DNA, the
addition and structure of carbohydrate chains is cell-, tissueand species-specific (Kinloch et al., 1991; Kobata, 1992; Lis
and Sharon, 1993; Varki, 1993). Biological or functional
diversity is generated by the combination of monosaccharides
present: by their sequence, chain length, branching points,
linkages, anomery (α, β) and/or covalent attachment of modifying groups (sulphate, phosphate, acetyl, methyl). However,
extant data suggest that terminal sequences, unusual structures
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Table I. Advanced methods which have aided in the study of the structure
and function of glycoproteins (Adapted from Noguchi et al., 1992; Lis and
Sharon, 1993; Varki, 1993; Seppo et al., 1995)
Chemical methods
Smith degradation
acetolysis
methylation analysis
Physiochemical methods
mass spectroscopy
1H-nuclear magnetic resonance (NMR)
13C-NMR
Enzymatic methodologies
endoglycosylases to release oligosaccharides en bloc
monosaccharide sequence analysis by sequential
exoglycosidase digestion
Non-enzymatic separations
gel filtration
chromatography
reaction with lectins
Biological systems
mutant mammalian cell lines
specific inhibitors of transferases
competition studies
in-vitro expression of mutant proteins
transgenic animals
‘knockout’ animals
Analytical techniques for the structural study of oligosaccharide chains
compositional analyses
in-vitro synthesis
competition studies
and modifications are most likely to mediate specific biological
roles (Varki, 1993). Further, most glycoproteins contain more
than one glycan, and glycan heterogeneity at a single attachment
site is often observed (e.g. Mori et al., 1991). Intra-species
polymorphisms in glycan structure have been noted (Varki,
1993). As a result, a variety of functions can be ascribed to
the carbohydrate modifications of proteins. The common
features of the varied functions is that they either mediate
specific recognition events (e.g. cell–cell; cell–matrix; immune
responses; binding of noxious agents; facilitators of infectious
and malignant disease) or that they modulate protein function
(e.g. ligand binding; intra- and inter-cellular trafficking;
stability/clearance/turnover; storage depot for biologically
important molecules) (Varki, 1993).
Glycans can be linked to proteins through the nitrogen (‘Nlinked’) of asparagine, through the β-hydroxyl groups (‘Olinked’) of serine and threonine and, to lesser extent, of
tyrosine, 5-hydroxylysine and 4-hydroxyproline, through a
glycosyl-phosphatidylinositol (‘GlPtdIns’) anchor or through
a carbohydrate–peptide bond (Table II). In the latter two
linkages, the carbohydrate chain is attached respectively, to
the carboxyl terminal amino acid of the protein by ethanolamine
phosphate or by covalent linkage of 3-hydroxyl of monomeric
or polymeric ADP-ribose to a variety of amino acids (‘ADPribosylation’). As these do not represent glycosylation in the
strict sense, as a direct glycosidic linkage between protein and
carbohydrate is absent, these novel linkages will not be further
addressed in this review. Finally, sugars also form links nonenzymatically in vivo to the epsilon amino group of lysine.
These adducts further react to form advanced glycation products, which have clinical significance in diabetes. These
modifications are also excluded from this review.
Carbohydrates and fertilization: an overview
Table II. Protein–carbohydrate linkages (adapted from Lis and Sharon,
1993)
Amino acid
Linkage
Carbohydrate residue
Asparagine
N
Serine/Threonine
O
Serine
O
Threonine
5-Hydroxylysine
4-Hydroxyproline
O
O
O
Tyrosine
Carboxy-terminus
Amino Acid (Xaa)
Glutamic acid
Aspartic acid
Arginine
Cysteine
Glucose
N-Acetylglucosamine
N-Acetylgalactosamine
L-Rhamnose
Mannose
N-Acetylglucosamine
N-Acetylgalactosamine
Xylose
Glucose
Galactose
L-Fucose
Galactose
L-Arabinose
Galactose
Glucose
Glycosyl-Phosphatidylinositol
O
Ethanolamine
Phosphate
Covalent
ADP-ribose (monomeric or polymeric)
(3-hydroxyl of Ribose)
N-linked glycans
N-linked glycans share a common biosynthetic pathway
(Kornfeld and Kornfeld, 1985; Elbein, 1991; Goochee
et al., 1991). A pre-synthesized oligosaccharide moiety
(Glc3Man9GlcNAc2; see abbreviations in legend to Figure 1)
is co-translationally transferred from a lipid (dolichyldiphosphate) to the nascent polypeptide chain in the endoplasmic
reticulum. Further processing (trimming and addition) in the
endoplasmic reticulum and Golgi apparatus generates high
mannose (Figure 1A), hybrid or complex (Figure 1B) oligosaccharide structures which contain a common pentasaccharide
core (Kobata, 1992). Potential glycosylation sites along a
polypeptide backbone for N-linked glycans can be identified
from the consensus sequence Asn-Xaa-Ser/Thr (Struck and
Lennarz, 1980). Whether or not a particular asparagine residue
associated with this consensus sequence is glycosylated is
dependent, in part, on protein conformation (Marshall, 1972).
The asparagine must be in an exposed position to be available
for glycosylation, such as in a β-loop.
N-linked glycans modulate the physiochemical properties of
the proteins to which they are attached by altering solubility,
surface charge and adhesive properties. N-linked glycans can
stimulate the immune response by acting as immunodeterminants, as in human blood group antigens (Watkins, 1987), or
by activating the complement system (Sim and Malhotra,
1994). In the context of reproduction, it has been well
demonstrated that carbohydrate moieties on the sperm surface
play an important role in immune infertility (see below), a
problem which affects .10% of all couples seeking fertility
evaluation (Bronson et al., 1984; Isojima, 1989). Plasma membrane antigens provide potential immunogens for the development of contraceptive vaccines (e.g. Anderson and Alexander,
1983; Liang et al., 1994). Likewise, it is not unexpected that
the zona pellucida, a glycosylated structure which is highly
antigenic (e.g. Sacco, 1977; Sacco et al., 1986; Yurewicz et al.,
1987), is now a target for immunocontraception (Paterson
and Aitken, 1990; Epifano and Dean, 1994). In addition,
carbohydrates can participate in protection of the embryo from
maternal rejection. Glycodelin is a glycoprotein synthesized
by the secretory and decidual endometrium, with maximal
expression observed at the time of embryo implantation, which
manifests immunosuppressive activities in in-vitro assays
(Clark et al., 1996b). Glycodelin contains fucosylated glycans
(Dell et al., 1995) which block lymphocyte homing (Yednock
and Rosen, 1989). It has therefore recently been suggested that
the N-linked oligosaccharides of glycodelin, which potentially
could compete or block primary adhesive interactions between
the embryo and maternal leukocytes, contribute to the highly
region-specific immunosuppression of the maternal immune
system to the fetus (Clark et al., 1995, 1996a,b).
The most important role of N-linked glycans, however, is in
the regulation of protein conformation. N-linked glycans aid in
folding of nascent polypeptide chains and stabilization of mature
glycoproteins. As a result, N- linked glycans modulate the biological activity and immunological properties of a wide variety of
enzymes, hormones, receptors and lectins. Three examples are
particularly relevant to a discussion of reproductive biology.
Firstly, glycan structure regulates the expression of functional
human chorionic gonadotrophin (HCG). N-linked glycans facilitate correct disulphide bond formation and thereby assist in the
folding of the β-subunit of HCG; the unglycosylated protein
folds more slowly and is more easily degraded (Feng et al.,
1995). The glycan structure of the α subunit of HCG regulates
its ability to interact with the β subunit to form the mature
hormone (Blithe, 1990). In addition, N-linked glycans on the α
subunit modulate signal transduction, leading to the production
of cAMP and steriodogenesis, after receptor binding (Matzuk
et al., 1989; Sairam, 1989). The native hormone and its deglycosylated analogue apparently interact with a different domains
of the HCG receptor (Ji and Ji, 1990). Secondly, examination of
wild-type and mutant rat luteinizing hormone receptor (LHR)
expressed in insect cells indicates that N-linked oligosaccharide
chains regulate intramolecular folding of the LHR required to
achieve high affinity ligand binding (Zhang et al., 1995). Thirdly,
N-linked glycans regulate the enzymatic activity of plasminogen
activator by modulating the conversion of single chain to double
chain forms and also limit their susceptibility to serum and
tissue-derived protease inhibitors (Wittwer et al., 1989; Wittwer
and Howard, 1990; Howard et al., 1991). Plasminogen activator
is a key enzyme in the proteolytic cascade leading to follicular
rupture (Canipari and Strickland, 1986; Sappino et al., 1989).
O-linked glycans
O-linked glycosylation, in contrast to the N-linked process,
occurs exclusively in the Golgi apparatus. A preformed, lipidcoupled oligosaccharide precursor does not exist. Instead, the
covalent attachment of N-acetylgalactosamine to an acceptor
amino acid is through an α1 linkage in the O-linked glycosylation pathway (Figure 1C). Nucleotide sugars serve as substrates for all steps in O-linked glycan processing. The specific
polypeptide chain structural requirements for the addition of
O-linked oligosaccharides remains to be determined (Sadler,
1984; Carraway and Hull, 1989; Wilson et al., 1991).
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Figure 1. Typical key saccharide structures*. The arrows in the figure represent glycosidic bonds and the numbers correspond to the type of
linkage. (A) N-linked high mannose oligosaccharide (adapted from Elbein, 1991). (B) Consensus sequence of related O-linked and N-linked,
complex type oligosaccharides of porcine zona pellucida glycoproteins (adapted from Mori et al., 1991; Hokke et al., 1994). (C) O-linked
glycan common to proteins expressed by many mammalian somatic cells (adapted from Goochee et al., 1991). (D) O-linked glycan from
mouse ZP3 (adapted from Nagdas et al., 1994). (E) Consensus sequence of blood group-related oligosaccharides that inhibit binding of
mouse spermatozoa to mouse ova (adapted from Litscher et al., 1995). *Asn 5 asparagine; Ser 5 serine; Thr 5 threonine; Fuc 5 fucose;
Gal 5 galactose; Glc 5 glucose; Man 5 mannose; GlcNAc 5 N-acetylglucosamine; GalNac 5 N-acetylgalactosamine; NeuAc 5 N-acetylneuraminic (sialic) acid.
O-linked glycans alone account for the zona’s sperm receptor
activity in the mouse (Florman and Wassarman, 1985) and,
where information on sugar chain composition is available,
other mammals (e.g. Yurewicz et al., 1991).
Carbohydrates as cellular recognition determinants
Both O-linked and N-linked glycans serve as recognition
determinants in targeting proteins to a cellular or tissue
compartment (e.g. Dahms et al., 1989), binding of pathogenic
organisms to animal cells (e.g. Gambaryan et al., 1995), neural
adhesion, including cell interactions during development, modification of synaptic activity and regeneration of nerve connections after damage in the adult (e.g. Schachner and Martini,
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1995), and leukocyte homing to activated platelets or endothelium (Gahmberg et al., 1992; McEver, 1994). Fertilization is
a specialized form of cell–cell recognition.
Although the mouse is the best studied system with regard
to the role of carbohydrates in fertilization mechanisms
(Wassarman, 1989, 1990, 1992; Dean, 1992; Epifano and
Dean, 1994), fragmentary data from other mammalian systems
is also available. As such, this review will touch upon
information gathered from different species as appropriate.
Where possible, correlations between saccharide moieties,
glycan receptors and outcome of human IVF will be examined
in order to help evaluate the precise carbohydrate binding
criteria necessary for human fertility.
Carbohydrates and fertilization: an overview
(Loret de Mola et al., 1996), it will be of interest to determine
how differential alterations in these two layers might affect
female fertility potential. Thus, polarization microscopy can
potentially have an important impact upon clinical practice. In
this review, however, the discussion will focus on the outer
layer of the zona pellucida as it provides the ligands required for
initial sperm binding and induction of the acrosome reaction.
Structure of the zona pellucida
Figure 2. Hamster oocyte imaged with a polarized light
microscope and digital image processing (‘pol-scope’). In the polscope image the zona pellucida is divided into outer and inner
layers, each layer displaying birefringence. The orientation of the
birefringence axis is indicated by the black lines drawn on a regular
grid throughout the image. The measured birefringence axis
indicates the orientation of filament arrays which are likely to
induce the birefringence in the zona. The slow axis of birefringence
(filaments) in the outer layer is oriented tangentially while in the
inner layer the slow axis orientation is radial (Keefe et al., 1996).
(Photograph generously provided by Dr David Keefe, Brown
University School of Medicine, Providence, R.I., and Dr Rudolf
Oldenbourg, Marine Biological Laboratory, Woodshole, MA, USA).
Zona pellucida
Spermatozoa, particularly those of man, do not readily adhere
to the oocytes of foreign species (Austin and Bradin, 1956;
Bedford, 1977; Yanagimachi, 1977; Oehninger et al., 1993).
The zona pellucida, an extracellular glycoprotein matrix surrounding the oocyte, thus provides a barrier to cross fertilization. During evolution, as the oocyte has become smaller, its
extracellular vestments have disproportionally increased in
size, e.g. compare those of monotreme, marsupial and eutherian
mammals (Bedford, 1991). Concomitantly, novel sperm head
features have been developed, including decreased enzymic
content of the acrosome, so that for the spermatozoa to achieve
zona penetration of oocytes of their own species both lysis
and thrust (hyperactivated motility) are required. In some
limited cases where interspecies sperm–egg interactions have
been studied in vitro, however, sperm motility has been sufficient to allow penetration of the zona without prior acrosome
exocytosis (Wakayama et al., 1996). Nevertheless, it is safe to
assume that such obviation of zona pellucida specificity was
largely artefactual as attempts at interspecies breeding are not
likely to occur in vivo.
The zona pellucida of most nonrodent species can be
resolved into a bilaminar structure consisting of an inner
(oocyte–proximal) layer and an outer (oocyte–distal) layer, by
lectin binding (Shalgi et al., 1991; Maymon et al., 1994) and
electron microscopy (Dunbar et al., 1991) and more recently
by non-invasive polarization microscopy (Keefe et al., 1996;
see Figure 2). As abnormal zona pellucida quality (thickness)
may contribute to infertility at the level of embryo implantation
(Cohen et al., 1989, 1992), particularly in the older woman
The mammalian zona pellucida is composed of three secreted
sulphated glycoproteins, zona protein 1 (‘ZP1’), zona protein
2 (‘ZP29) and zona protein 3 (‘ZP39) (Shimizu et al., 1983;
Wassarman, 1988), which form heteropolymer filaments
of ZP2/ZP3 which are cross-linked by ZP1 (Greve and
Wassarman, 1985). Each contains a signal peptide, which
directs their secretion and is absent from the amino terminal
sequence of the mature molecule, and possesses very little αhelical structure. In addition, each polypeptide contains a
highly hydrophobic region at its carboxy terminus which may
modulate the formation of the recognizable structural repeat
(Liang et al., 1990). The importance of this region is discussed below.
Early evidence for conservation of zona pellucida components across species was obtained from the examination of
antigenic epitopes (Sacco, 1977; Sacco et al., 1981). Antihuman ovary sera prepared in rabbits cross-reacted with
isolated pig zona pellucida. Similarly, the cross-reactivity of
anti-pig ovary sera for human zona pellucida was demonstrated
by agar-gel immunodiffusion, by formation of a precipitation
layer on the zona, and by indirect immunofluorescence cytochemistry. The advent of cDNA technology has permitted the
detailed characterization of the structure and sequence of the
genes encoding these proteins (Harris et al., 1994). The high
degree of homology of their DNA and deduced amino acid
sequences in mouse, human, rabbit, cat, dog and pig indicate
the existence of three highly conserved gene families. The
regions which have been most highly conserved are postulated
to be important in maintenance of the three-dimensional
structure of the zona (Epifano and Dean, 1994). It is also
interesting to note that the amino acid sequences of these
proteins are not significantly similar to characterized proteins
from somatic cells. However, as mentioned above, both ZP2
and ZP3 have a very hydrophobic region near their respective
carboxyl termini (Liang et al., 1990; Liang and Dean, 1993a).
The three-dimensional structure of this region (based on the
pattern of cysteine, hydrophobic, polar and/or turn-forming
residues and not on amino acid sequence conservation) appears
to be shared with regions in the extracellular domain of somatic
cell proteins such as uromodulin, βglycan [transforming growth
factor (TGF)-β type III receptor] and the major zymogen
granule membrane protein (Bork and Sander, 1992). It must
be noted that the common biological property of the somatic cell
membrane-bound proteins which contain the shared domain is
that all have been detected in soluble form (Bork and Sander,
1992; Lopez-Casillas et al., 1994). Although the function of
this shared structure remains unknown, it has been postulated
that it may regulate protein stability or interaction of these
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proteins in the pericellular environment (Liang and Dean,
1993a; Lopez-Casillas et al., 1994).
Using [35S]-methionine, [3H]-fucose, specific antibodies, gel
and in-situ hybridization, it has been ascertained that the genes
encoding ZP1, ZP2 and ZP3 are transcribed and translated
during the 2 week oocyte growth phase, where they represent
7–8% of total protein synthesis (Bleil and Wassarman, 1980b;
Salzmann et al., 1983; Ringuette et al., 1986, 1988; Philpott
et al., 1987; Roller et al., 1989; Liang et al., 1990). While
expression of ZP2 and ZP3 is restricted to the oocyte proper,
there is some evidence that ZP1 transcripts may be transiently
expressed in granulosa cells (Lee and Dunbar, 1993). Although
once secreted, these proteins have a long half-life (.100 h;
Bleil and Wassarman, 1980c; Shimizu et al., 1983), zona
pellucida protein synthesis is strictly correlated with mRNA
levels and both decline in fully grown oocytes. ZP2 and ZP3
transcripts in ovulated eggs are 200 nucleotides shorter than
those of growing oocytes, presumably due to deadenylation
(Liang et al., 1990). As a result of both low copy number and
an inability to translate the shortened transcripts, zona pellucida
protein synthesis is not detectable in ovulated eggs.
In the mouse, ZP1, ZP2, and ZP3 exhibit apparent molecular
weights of 185–200 kDa, 120–140 kDa and 83 kDa respectively
(Greve and Wassarman, 1985). As ZP1 is thought to serve
primarily as a structural protein and may not contain N-linked
oligosaccharide side chains (Tulsiani et al., 1992), glycan
modifications have been better studied in ZP2 and ZP3. These
polypeptides contain high percentages of proline, serine and
threonine residues. Although the sequence for ZP2 encodes
seven sites which could direct N-linked glycosylation and
more than 100 amino acid residues that could serve as receptors
for O-linked glycosylation, the core secreted protein contains
six complex-type N-linked glycans and an undetermined number of O-linked glycans (Greve et al., 1982; Liang et al., 1990).
ZP3 contains six potential sites for N-linked glycosylation, of
which 3–4 are used, and, similar to ZP2, an undetermined
number of O-linked sugar side chains attached at .70 potential
sites (Salzmann et al., 1983; Harris et al., 1994). Differences
detected between species with regard to sizes of homologous
zona proteins (e.g. Shabanowitz and O’Rand, 1988a) are
attributed to differences in glycosylation. As a result, human
zona pellucida glycoproteins appear to be of a lower molecular
weight than their mouse counterparts (Liang and Dean, 1993b).
In addition, although only a single ZP3 polypeptide is synthesized in mice, at least two ZP3 isoforms are expressed by pig
and human oocytes (Hedrick and Wardip, 1986; Yurewicz
et al., 1987; Shabanowitz, 1991; Bercegeay et al., 1995).
Both N-linked and O-linked oligosaccharides are added prior
to secretion by synthetic pathways similar to those utilized in
somatic cells (Roller and Wassarman, 1983; Salzmann et al.,
1983; Wassarman, 1988). For N-linked glycans, high mannose
oligosaccharides added to nascent chains are processed to
complex-type moieties in the Golgi. The N-linked glycans of
mouse ZP2 and ZP3 have very similar structures (Noguchi
and Nakano, 1993). The N-linked oligosaccharides of mouse
ZP3 are complex type (bi-, tri-, and tetra-antennary) containing
linear N-acetyllactosamine repeats formed on a fucosylated
trimannosyl core which may or may not contain significant
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amounts of sialic acid (Figure 1B; Mori et al., 1991; Tulsiani
et al., 1992; Nagdas et al., 1994). In the mouse and in the pig,
these are highly heterogeneous in structure (Mori et al., 1991;
Yurewicz et al., 1991; Noguchi et al., 1992; Noguchi and
Nakano, 1993; Hokke et al., 1994). As these sulphated glycoproteins behave as acidic species (Sacco et al., 1981), Yurewicz
et al. (1991) have postulated that their N-linked glycans represent the mammalian analogue of sulphated fucan anionic
polysaccharides found in the jelly coat of sea urchin eggs
(DeAngelis and Glabe, 1987). Asparagine-linked sugar
moieties are, however, not required for secretion (Roller and
Wassarman, 1983). Periodic acid Schiff staining and antibody
and lectin binding studies suggest that additional modifications
in the content and distribution of sugar residues in the secreted
proteins occur following ovulation (Sakai et al., 1988; Kaufman
et al., 1989; Shalgi et al., 1991). As a result of their extracellular
location, ‘structural’, ‘barrier’ and ‘recognition’ functions can
be ascribed to the carbohydrates of the zona pellucida.
Primary role in sperm–egg interactions
A considerable body of evidence indicates that ZP3 serves as
the primary sperm receptor regulating tight binding to the zona
pellucida. For example, purified mouse ZP3 competitively
inhibits binding of mouse spermatozoa to mouse eggs in vitro
(Bleil and Wassarman, 1980a; Florman et al., 1984; Florman
and Wassarman, 1985; Endo et al., 1987; Leyton and Saling,
1989a). ZP1 and ZP2 lack this inhibitory activity. Mouse
spermatozoa bind by their heads to the zona matrix (Drobnis
and Katz, 1991). Similarly, binding of purified mouse ZP3 is
restricted to the plasma membrane overlying the acrosome
(Wassarman et al., 1985b; Bleil and Wassarman, 1986). Only
mouse spermatozoa with intact acrosomes have the capability
of binding to purified zona pellucida proteins from unfertilized
mouse eggs. Consistent with this, binding of purified mouse
ZP3 is restricted to acrosome-intact mouse spermatozoa and
is not observed on spermatozoa which have undergone an
acrosome reaction (Bleil and Wassarman, 1986). As would be
expected, ZP3 purified from mouse embryos fails to bind to
mouse spermatozoa or inhibit sperm–egg binding in vitro. As
a consequence of fertilization, the zona pellucida is modified
such that mouse sperm receptor activity is lost (Bleil and
Wassarman, 1983, 1988).
The link between zona pellucida fine structure and sperm
binding capability has been investigated both biochemically
and by electron microscopy. Superficial pores very similar in
diameter to size of the sperm head have been detected on zona
pellucida of unfertilized human oocytes (Nikas et al., 1994);
these pores may be essential to fertilization. The anterior portion
of the human sperm head has been observed to initially penetrate
the zona pellucida through these pores in the zona surface (Tsuiki
et al., 1986; Familiari et al., 1988). Treatment of human zonae
in vitro which increase the number of these pores is associated
with a dramatic increase in sperm binding (Henkel et al., 1995).
Fertilization, atresia or artifical egg activation with ionophores
results in oocyte cortical granule dehiscence which mediates
chemical modifications of the zona (Gulyas, 1980). The proteolytic activity of the cortical granule exudate (Gwatkin et al.,
1973) may, in part, be responsible for the loss of sperm binding
Carbohydrates and fertilization: an overview
activity and the block to polyspermy. In man, these functional
changes are correlated with changes in the three-dimensional
structure of the zona, from one of numerous elastic ring shaped
hoops with superficial pores to an amorphous mass without pores
(Familiari et al., 1988; Nikas et al., 1994). It has been postulated
that the condensation of the outer layer of the zona pellucida
causes disorientation of sperm binding sites (Familiari et al.,
1988; Shabanowitz and O’Rand, 1988b). The changes in structure associated with fertilization are more dramatic at the level
of the inner zona, and consistent with prior evidence, the cortical
granule release block to polyspermy acts at the level of the inner
zona in mouse (Sathananthan et al., 1982; Sathananthan and
Trounson, 1982, 1985).
The migration of ZP3 on sodium dodecyl sulphate (SDS)–
polyacrylamide gels is unchanged by the cortical granule
reaction (Miller et al., 1992). Loss of ZP3 sperm binding
activity and the contribution of ZP3 to the block to polyspermy
after fertilization has been attributed to the removal of terminal
carbohydrate residues from ZP3-linked oligosaccharide chains
by glycosidases in the cortical granule exudate (Miller et al.,
1992, 1993). In contrast, the electrophoretic mobilities of ZP1
and ZP2 are dramatically altered following fertilization. In the
mouse, ZP2 is modified by limited proteolysis via a proteinase
associated with the cortical granules (Bleil et al., 1981; Moller
and Wassarman, 1989; Moos et al., 1994; Ducibella et al.,
1995). Although preliminary data suggest the human ZP2 may
be similarly modified (Moos et al., 1995), a significant body
of evidence indicates that species specific changes occur in
ZP1 such that ZP1 of human oocytes, but not that of mouse
oocytes, is modified by proteolysis or deglycosylation after
fertilization (Shabanowitz and O’Rand, 1988a; Bercegeay
et al., 1995; Ducibella et al., 1995; Moos et al., 1995).
O-linked oligosaccharide chains regulate sperm
binding
Although the ability of monosaccharides, various complex
moieties and lectins to effectively block tight binding of
spermatozoa to homologous oocytes in vitro has been examined
in a number of mammalian species including man (Oikawa
et al., 1974; Huang et al., 1982; Shur and Hall, 1982b; Ahuja,
1985; Shalgi et al., 1986; Mori et al., 1989; Oehninger et al.,
1990, 1991), the gamete molecules involved in this binding
during fertilization have been best characterized in the mouse.
In mice, four lines of evidence strongly suggest that O-linked
oligosaccharide ligands on the ZP3 polypeptide backbone act
as the primary sperm receptor. Firstly, sperm receptor activity
is unaffected by exposure to elevated temperature, reducing
agents, denaturants or detergents: treatments which destroy
protein-based active sites (Bleil and Wassarman, 1980a;
Wassarman et al., 1985b; Wassarman, 1988). Secondly, extensive digestion with pronase similarly fails to affect sperm
receptor activity (Florman et al., 1984; Leyton and Saling,
1989a). Thirdly, complete deglycosylation with trifluoromethanesulphonic acid or selective removal of O-linked oligosaccharides following mild alkaline hydrolysis abrogates the
ability of ZP3 to inhibit sperm–egg binding (Florman and
Wassarman, 1985). The O-linked oligosaccharides released
by these procedures were demonstrated to bind directly to
spermatozoa and reduce sperm–zona binding. Fourthly, ZP3
treated with endoglycosidase F to selectively remove N-linked
glycans and untreated, control ZP3 were equally effective in
reducing the number of spermatozoa bound to mouse egg
zonae pellucidae in an in-vitro competition binding assay
(Florman and Wassarman, 1985). All of the above data were
obtained using native ZP3 polypeptides. Recently, recombinant
ZP3 (rZP3) has been synthesized in tissue culture cells (Kinloch
et al., 1991; Beebe et al., 1992). When expressed in cell lines
with the capacity to synthesize a wide variety of oligosaccharides, the ligand activity of rZP3 in sperm–zona pellucida
binding competition assays is indistinguishable from that of
the native molecule. These arguments are further supported
by studies indicating that: (i) human rZP3 must be appropriately
glycosylated to be biologically active (Barratt et al., 1994; Van
Duin et al., 1994); (ii) O-linked glycosylation is an important
step in the processing of human rZP3 (Van Duin et al.,
1994); (iii) O-linked oligosaccharide ligands mediate pig sperm
adhesion to ZP3 (Yurewicz et al., 1991; Dostalova et al.,
1995b); and (iv) deglycosylated pig ZP3 lacks sperm receptor
activity and is unable to elicit the production of antibodies
which can block sperm–zona binding in vitro (Berger et al.,
1989a; Sacco et al., 1989).
Evidence has been presented indicating N-linked neutral
carbohydrate chains of porcine ZP3 may also contribute to its
sperm receptor activity (Noguchi et al., 1992). Competition of
sperm–zona binding by such N-linked oligosaccharides is,
however, only observed at or above concentrations equivalent
to 500 zonae pellucidae. The high concentrations required to
effect inhibition of sperm binding suggest that N-linked glycans
play a minor role at best in regulating species-specific gamete
interactions. In addition, sperm binding activity was retained
by porcine ZP3 digested with endo-β-galactosidase, which
removes peripheral sulphated polylactosamines of N-linked
glycans (Sacco et al., 1989). Further, sperm receptor activity
was retained by ZP3 containing truncated O-glycans and by
truncated O-glycans liberated by alkaline-borohydride treatment (Sacco et al., 1989; Yurewicz et al., 1991). Similarly
truncated (trisaccharide) O-linked chains of mouse ZP3 can
inhibit mouse sperm–zona attachment (Nagdas et al., 1994).
The latter findings are consistent with the hypothesis that
sperm binding activities of the zona pellucida can be associated
with internal saccharide core structures (Yurewicz et al., 1991)
as well as with terminal residues (Bleil and Wassarman,
1988). This hypothesis is supported by examination of the
carbohydrate binding affinities of at least one human sperm
surface receptor for zona carbohydrate ligands (see below,
sperm receptors for zona carbohydrate ligands).
The carbohydrate moieties of ZP3 are also important from
a contraceptive standpoint, with the idea of producing reversible
interference of sperm–egg interactions (Epifano and Dean,
1994). The antigenicity of ZP3 is directly related to its
carbohydrate content (Sacco et al., 1986; Yurewicz et al.,
1987). Although the role of carbohydrate moieties as targets
for specific antibody production is addressed in the next
section, it must be noted that in marmosets, baboons and
pigs immununized with zona glycoproteins, glycosylated ZP3
induced immediate production of antibodies whereas only a
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S.Benoff
weak or no response was obtained with deglycosylated ZP3
as immunogen (Paterson and Aitken, 1990; Paterson et al.,
1992). Consistent with these findings, immunization of mice
and rabbits with their cognate native ZP3 results in autoimmune
disease with significant pathological ovarian damage, whereas
immunization with deglycosylated ZP3 does not (Keenan et al.,
1991; Rhim et al., 1992; Lou et al., 1995; Bagavant et al.,
1997). However, although it has been postulated that immunoregulation of gamete function is effected by direct antibody
coating of the zona pellucida which inhibits sperm–zona
binding and/or penetration (East et al., 1985; Millar et al.,
1989; Bagavant et al., 1997), the endocrine changes (elevation
of LH and FSH; inhibition of gonadotrophin-induced progesterone secretion; Skinner et al., 1984; Mahi-Brown et al., 1985;
Keenan et al., 1991) which occurred in animals immunized
with glycosylated ZP3 indicate that inhibition of normal
ovarian function also contributes to the production of the
infertile state (Keenan et al., 1991). A decrease in the number
of primordial follicles, developing follicles and corpora lutea
was observed in association with the endocrine changes. When
deglycosylated ZP3 was used as the zona antigen, normal
endocrine profiles were observed and folliculogenesis was
unaffected (Skinner et al., 1984; Mahi-Brown et al., 1985;
Keenan et al., 1991). Nevertheless, it must be noted that use
of deglycosylated ZP3 as immunogen results in a reduction,
and not a complete loss, of fertility (Millar et al., 1989; Lou
et al., 1995; Bagavant et al., 1997).
In the mouse, oligosaccharide side chains comprise ~50%
of the mass of ZP3 (Salzmann et al., 1983). O-linked glycans
are found attached to both serine and threonine residues
(Florman and Wassarman, 1985). A specific size class of these
glycans, ~4 kDa or equivalent to 20–25 monosaccharide
residues, inhibit sperm–zona binding and also bind directly to
spermatozoa (Florman and Wassarman, 1985), potentially via
α-galactose residues at their non-reducing termini (Bleil and
Wassarman, 1988). That the sperm receptor activity of these
glycans is sensitive to very small changes in carbohydrate
structure is indicated by the fact that activity is lost if the
terminal galactose residue is removed or if its C-6 hydroxyl
is converted to an aldehyde (Bleil and Wassarman, 1988).
The amino terminal and carboxyl terminal domains of ZP3
are separated by a flexible (‘hinge’) region similar to that
of immunoglobulins (Wassarman and Litscher, 1995). The
presence of this hinge region allows proteolytic cleavage of
ZP3 (Rosiere and Wassarman, 1992). Separate analysis of the
bioactivities of the amino and carboxyl halves of the ZP3
molecule indicate that the carboxyl terminal half of ZP3 is
sufficient to inhibit sperm binding to oocytes in vitro and to
induce the acrosome reaction (Rosiere and Wassarman, 1992;
Litscher and Wassarman, 1996). Complete removal of N-linked
oligosaccharides with N-glycanase, release of lactosaminoglycan units from N-linked glycans and removal of sialic acid
from non-reducing termini with neuraminidase had no effect
on the bioactivities of the carboxyl terminal peptide (Litscher
and Wassarman, 1996). Thus, the O-linked glycans responsible
for the sperm binding activity of ZP3 are derived from the
carboxyl terminal half of ZP3 (Rosiere and Wassarman, 1992).
Results from peptide cleavage experiments indicate that the
606
relevant O-linked glycans appear concentrated in a small
region of 16 amino acids (328 to 343; exon 7) containing five
serine residues. Mapping of the mouse ZP3 combining site
for spermatozoa by exon swapping supports these findings
(Kinloch et al., 1995). In addition, site directed mutagenesis
has produced a mutant gene which converted the five serine
residues of exon 7 to non-hydroxyl amino acids. When this
mutant gene (mZP3[ser]) is expressed in vitro, its protein
product is less extensively glycosylated than wild-type mouse
rZP3 and does not exhibit sperm binding or acrosome reaction
inducing activities (Kinloch et al., 1995; Liu et al., 1995). The
amino acid sequence in and around this region is less highly
conserved than other portions of the molecule (Kinloch et al.,
1995), supporting the concept that the regions of the ZP3
molecule which potentiate species specific high affinity interactions with spermatozoa are hydrophilic regions lying on the
surface of the protein which exhibit maximal difference in
sequence between species (Chamberlin and Dean, 1990). Thus,
it is not unexpected that the amino acid sequence of mouse
ZP3 exon 7 is also highly antigenic. Vaccination of mice with
a version of this 16 amino acid peptide synthesized in vitro
elicits the production of antibodies that bind to the zona
pellucida (Millar et al., 1989). These antibodies produce longterm, reversible contraception in female mice. Although antigenic epitopes in this region of mouse ZP3 can not be detected
in hamster, guinea pig, cat or dog ovaries (East et al., 1985),
a comparable domain containing O-linked oligosaccharides
has been identified in the porcine and human homologues of
ZP3 (Yurewicz et al., 1992; Bagavant et al., 1997).
Results from the site directed mutagenesis studies described
above also provide insight into whether all or only some of
the ZP3 molecules in the mouse zona pellucida are inactivated
following the cortical granule reaction at fertilization which
ablates zonae sperm receptor activity. The zona pellucida of
the ovulated mouse egg contains .109 copies of ZP3 (Greve
and Wassarman, 1985; Wassarman and Mortillo, 1991). This
is in great excess of the estimated 50–500 ZP3 molecules
which participate in the initial adhesive event with a spermatozoon (Liu et al., 1995). Thus, it may be postulated that the
composition of the zona pellucida can be substantially altered
without loss of ability to bind spermatozoa. Although there is
a .50% reduction in the number of sperm receptors in the
zona pellucida of mice transfected with mZP3[ser], these
transgenic mice are fertile and produce litters of normal size
(Liu et al., 1995). These findings, when taken together with
the fact that embryo ZP3 exhibits a low level of sperm binding
activity when tested at concentrations 3–5 times higher than
those commonly employed for egg ZP3 (Wassarman et al.,
1985a), argue that complete inactivation of a large percentage
(.65%) of ZP3 following fertilization will inhibit subsequent
sperm–egg binding and polyspermy.
Inhibition of ZP3 protein synthesis in mouse oocytes by injection of anti-sense oligonucleotides to degrade ZP3 mRNA (Tong
et al., 1995) or by targeted disruption of the ZP3 gene (Liu et al.,
1996; Rankin et al., 1996) results in female infertility. Adult
ovaries from females homozygous for the disrupted ZP3 gene
contain growing and fully grown oocytes. Although ZP1 and
ZP2 protein synthesis is unaffected by the anti-sense oligonucle-
Carbohydrates and fertilization: an overview
otides or disruption of the ZP3 gene, all oocytes lack a zona
pellucida. Directed degradation of ZP2 mRNA similarly results
in the absence of the zona (Tong et al., 1995). Collectively, these
data indicate that assembly of the zona pellucida requires the
coordinate synthesis of at least ZP2 and ZP3. However, infertility
in female mice homozygous for the targeted ZP3 gene disruption
is probably not due to the loss of the extracellular carbohydrates
which regulate sperm binding activity. It has been demonstrated
that spontaneously acrosome-reacted mouse spermatozoa can
fertilize zona-free mouse oocytes in vitro (Naito et al., 1992).
Rather, infertility is more likely attributed to adherence of zonafree embryos to the oviductual epithelium which inhibits passage
through the oviduct and subsequent implantation (Bronson and
McLaren, 1970; Modlinski, 1970). It must be stressed, however,
that a naturally occurring genetic defect causing loss of ZP3
expression or absence of the zona pellucida has never been
observed to be an underlying cause of mammalian infertility.
The latter is consistent with data indicating that genetic defects
in glycosylation are rarely observed in intact organisms (Varki,
1993).
O-linked glycan structure is complex
Analysis of the structure of the O-linked oligosaccharide
ligands responsible for sperm receptor activity has been complicated by glycan heterogeneity, which in the hamster occurs
on a poly-N-acetyllactosamine backbone (Yurewicz et al., 1991;
Hokke et al., 1994). The ability to synthesize in vitro multiply
branched oligosaccharide constructs (Seppo et al., 1995) has
helped to define structures that inhibit binding of mouse
spermatozoa to unfertilized eggs in vitro (Litscher et al., 1995).
Constructs tested were rich in N-acetyllactosamine repeats
for three reasons. Firstly, multiple polylactosamine units are
commonly associated with developmentally regulated glycoconjugates (Fukada et al., 1985; Yurewicz et al., 1987) and
mouse zona glycoproteins possess at least two polylactosamine
structures per molecule (Cahova and Draber, 1992). Secondly,
antibodies to these structures inhibit fertilization (Cahova and
Draber, 1992). Thirdly, rZP3 synthesized in mouse embryonal
carcinoma cells known to be particularly rich in poly-Nacetyllactosamine glycans (Muramatsu et al., 1978; Renkonen,
1983) exhibited sperm receptor and acrosome reaction inducing
activities while that produced in other transfected cell lines
with low polylactosamine glycan production did not (Kinloch
et al., 1991),
Preincubation of capacitated mouse spermatozoa with either
blood group β-related linear tri- and penta-saccharides and
biantennary blood group I-related oligosaccharides (Figure 1E)
resulted in a significant inhibition of sperm–egg binding. In
the latter case, increasing chain length also increased the
affinity of the branched oligosaccharide for spermatozoa. These
data are supported by the direct compositional analysis of Olinked oligosaccharides derived from mouse ZP3 (Nagdas
et al., 1994). Treatment of de-N-glycosylated mouse ZP3 with
O-glycanase in the presence of exoglycosidases provides
evidence for the presence of O-linked trisaccharide chains
(Figure 1D), as well as polylactosaminylated glycans (Nagdas
et al., 1994). That glycan size and branching pattern play a
role in the sperm receptor activity of mouse zona ligands
containing terminal α-galactose residues (Litscher et al., 1995)
and that O-linked sugar moieties purified from mouse or pig
ZP3 are 1–3 orders of magnitude less effective than the
native molecule in inhibiting mouse sperm–zona attachment
(Yurewicz et al., 1991; Litscher et al., 1995) are consistent
with the concept that the nature of the clustering regulates the
binding affinity of carbohydrate ligands to their cognate
receptor proteins (Lee et al., 1983; Lee, 1989, 1992). Further,
the binding to the sperm surface of synthetic glycans which
mimic those of blood groups may help to explain why human
spermatozoa accumulate seminal blood group antigens on their
surface (Cooper and Bronson, 1990; Gilbert et al., 1991; see
below, carbohydrates on the sperm surface).
Additional evidence that α-galactose-terminated polylactosamines on the mouse zona pellucida regulate species specific
tight binding of mouse spermatozoa comes from a study of
mouse sperm–erythrocyte interactions (for review see Clark
et al., 1996a). Capacitated mouse spermatozoa bind to rabbit
erythrocytes but not erythrocytes from other species. An intact
acrosome is a requirement for rabbit erythrocyte binding.
Pretreatment of rabbit erythrocytes with α-galactosidase
reduced mouse sperm binding by a maximum of 40% in
comparison with untreated controls. The residual 60% binding
of mouse spermatozoa to α-galactosidase-treated rabbit erythrocytes has been attributed to an interaction with internal
polylactosamine sequences. On rabbit erythrocytes, α-galactosyl residues are primarily associated with branched polylactosamines.
Finally, it must be recognized that the identification of the
specific zona carbohydrate ligands regulating tight binding of
spermatozoa is complicated by the fact that the glycosyl
modifications presumably exhibit intra-individual variation.
Oocytes from the same donor exhibit variations in their sperm
binding capacity (e.g. Fazeli et al., 1993).
Species-specific glycosylation patterns
The size and structural features of the active oligosaccharide
site of ZP3 which regulates sperm binding appears to differ
between species (Noguchi et al., 1992). For example, while
terminal carbohydrate residues apparently form the sperm
receptor site in the mouse zona (Bleil and Wassarman, 1988;
Miller et al., 1992, 1993), larger internal carbohydrate units
regulate sperm binding to pig zonae (Yurewicz et al., 1991).
It is therefore interesting to note that mouse embryonal
carcinoma cells which constitutively express mouse or hamster
ZP3 genes can discriminate between these highly related
polypeptides (Kinloch et al., 1991). Although these cells
secrete a biologically active form of mouse rZP3 which is
glycosylated to an extent similar to that of the native mouse
ZP3, the mature hamster srZP3 failed to exhibit mouse sperm
receptor activity. That hamster rZP3 secreted by mouse cells
fails to inhibit hamster sperm binding to ovulated eggs in vitro
may be due to: (i) the fact that it is glycosylated to a lesser
extent than the authentic protein; (ii) the possibility that the
oligosaccharide structure of the recombinant molecule differs
from that of hamster oocytes, thus altering the sperm binding
site; and/or (iii) the possibility that the oligosaccharide structure
of the recombinant molecule differs from that of hamster
607
S.Benoff
oocytes such that protein folding is altered. In addition, the
lectin binding affinities of zonae pellucidae differ between
species (Shalgi et al., 1991; Maymon et al., 1994). All of these
arguments support the concept of species-specific glycosylation
patterns for ZP3. The majority of evidence that glycosyl
modification of ZP3 is species specific will, however, be
discussed in detail below in regard to the sperm molecules
participating in zona adhesion (see below, sperm receptors for
zona carbohydrate ligands).
Carbohydrates on the sperm surface
The surface of the mature mammalian spermatozoon can be
divided into five domains [anterior head (acrosomal segment),
equatorial segment, posterior head (post-acrosomal segment),
midpiece, and principal piece] that originate during elongation
of round cells and are conserved across species (for review
see Bearer and Friend, 1990). These structural domains are
thought to reflect the presence of ‘functional domains’ involved
in the process of fertilization. Studies employing specific
saccharides in hapten inhibition tests, exoglycosidases to
remove functional saccharides from the cell surface and
inhibitors of glycoprotein synthesis all suggest that a large
variety of oligosaccharides synthesized by the lipid-linked
pathway (N-linkage) are expressed on the sperm surface and
serve as carbohydrate determinants involved in mammalian
fertilization (review, Ahuja, 1985). Therefore, although glycoproteins are involved in intercellular adhesion and information
transfer during spermatogenesis (Lee and Damjanov, 1985;
Malmi et al., 1987; Wollina et al., 1989a,b), the majority of
studies have focused on the role of surface carbohydrates
in the evolution of membrane domains during epididymal
maturation and capacitation post-ejaculation.
Distribution
Fluorescent dye-, peroxidase-, biotin-, ferritin-, colloidal gold-,
and radio-labelled phytoagglutinins (plant ‘lectins’) have been
used as probes to study carbohydrates on the sperm surface
(Edelman and Millette, 1971; Nicolson and Yanagimachi,
1972, 1974; Nicolson et al., 1972; Nicolson, 1974; Koehler,
1981; Kallojoki et al., 1985; Singer et al., 1985; Cross and
Overstreet, 1987; Kumar et al., 1990; Ahluwalia et al., 1990;
Vasquez et al., 1990; Bains et al., 1992; Mladenovic et al.,
1993; Gabriel et al., 1994; Fierro et al., 1996). Surface charge
differences, potentially attributable to the negative charge
associated with terminal sialic acid residues, have been assessed
with colloidal iron hydroxide or by electrophoresis of immobilized live spermatozoa (Nevo et al., 1961; Bedford, 1963;
Cooper and Bedford, 1971; Yanagimachi et al., 1972). To date,
the results of these studies indicate that a wide variety
of saccharide groups are accessible on the surface plasma
membrane, both on the termini and within the cores of
polysaccharide complexes. Their distribution is non-uniform,
e.g. concanavalin A (‘ConA’; specific for D-mannose and Dglucose residues) binding is limited to the region overlying
the acrosome in human, mouse, hamster, guinea pig, rat, rabbit
and ram spermatozoa.
Significant differences exist between species with regard to
608
lectin binding affinities. Most important are observations
indicating reproducible changes in lectin binding affinities
during epididymal maturation and capacitation, e.g. ConA
labelling of the sperm head is decreased while that of wheat
germ agglutinin (‘WGA’; specific for N-acetylglucosamine
and/or sialic acid) is increased. Some of these changes are
correlated with the acquisition of the ability to bind zona
pellucida ligands (Lassalle and Testart, 1994). The overall
loss of lectin binding sites from the periacrosomal plasma
membrane correlates with changes in the distribution of intramembranous particles prior to the acrosome reaction first
described by freeze fracture/electron microscopy (Friend et al.,
1977; Yanagimachi and Suzuki, 1985; Aguas and Pinto da
Silva, 1989; Suzuki and Yanagimachi, 1989). This allows
calcium to induce phase transitions which create disorganized
lipid arrays necessary for the initiation of fusion
(Papahadjopoulos, 1978) of plasma and outer acrosomal membranes at particle-deficient interfaces leading to acrosome
exocytosis.
Membrane contraints restrict diffusion of bound lectins in
the anterior head and the tail regions but not in the posterior
head. This is indicative of sperm membrane compartments
where lipid is immobile and is not free to diffuse laterally as
in most somatic cells (Wolf et al., 1988), and is consistent
with observations indicating that alterations in such lipid
domains are associated with decreased fertility potential of
human spermatozoa (Sinha et al., 1994). The best studied lipid
diffusion barrier is that in the equatorial segment of the
spermatozoon (Arts et al., 1994); as (i) this is the primary
domain of mammalian acrosome-reacted spermatozoa that
harbour in vitro fusogenic capacity for artificial membranes
(Arts et al., 1993) and (ii) it is this domain which initiates
fusion of the spermatozoa with the oolemma (Bedford et al.,
1979; Talbot and Chacon, 1982; Clark and Koehler, 1990;
Yanagimachi, 1994). It is unlikely that transmembrane protein
barriers to lipid diffusion exist in this region. Intramembranous
particles are cleared from the equatorial segment after the
acrosome reaction (Friend et al., 1977; Aguas and Pinto da
Silva, 1989).
The stability of the equatorial segment barrier to lipid
diffusion appears to be dependent on the presence of calcium
ions (Arts et al., 1994). Annexins have been detected in the
equatorial segment of human spermatozoa (Berruti, 1988;
Feinberg et al., 1991). Annexins, which require calcium for
their functioning, have been implicated in the attachment of
cytoskeletal elements to membranes (Creutz, 1992). Thus, the
possibility that annexins contribute to the barrier to lipid
diffusion in the equatorial segment cannot be excluded.
Calcium also regulates the physical state of lipids. Although
calcium can trigger some lipids to partially ‘melt’ (e.g. become
more fluid), it can also trigger the transition of other lipids
from a liquid-crystalline to a gel state (‘freezing’) (Lee, 1977;
Papahadjopoulos, 1978; Cullis and DeKruijff, 1979). Lipid
factors, such as the sperm-specific glycolipid sulphatoxygalactosyl-glycerolipid localized to the equatorial segment, can form
gel-like areas in sperm plasma membranes where molecular
motion is very slow (Wolf et al., 1988; Benoff et al., 1993a;
Gadella, 1993). Regulation of the formation of these gel
Carbohydrates and fertilization: an overview
domains by calcium offers a plausible alternative to annexins
as an equatorial diffusion barrier. In addition, the existence
of glycolipids associated with the sperm plasma membrane
suggests that glycoprotein turnover and remodelling may not
be the exclusive agents regulating sperm surface lectin binding
affinities during epididymal transit and sperm capacitation.
Sperm plasma membrane lipid/sterol ratios change markedly
during capacitating incubations and the extent of spontaneous
and induced acrosome reactions is critically dependent on
these ratios (e.g. Go and Wolf, 1983; Hoshi et al., 1990;
Benoff, 1993; Benoff et al., 1993a,c,d, 1996a). The changes
that occur in membrane composition are specific to a particular
domain of the mature spermatozoa (Bearer and Friend, 1982).
Thus, modification of sulphatoxygalactosylglycerolipid and
similar glycosylated lipids may have a biological role in the
regulation of fertilization processes occurring after induction
of the acrosome reaction.
Type of association
In the study of sperm surface glycans, a distinction needs to
be made between those which are part of integral plasma
membrane proteins and those which are derived from proteins
and lipids which are only peripherally attached. There are
additions to the sperm surface after testicular differentiation
(review, Yanagimachi, 1994). Spermatozoa dramatically alter
their antigenicity during epididymal transit for two reasons.
Firstly, the epididymis secretes a variety of glycoproteins,
some of which bind tightly to spermatozoa. Secondly, active
glycosylation of membrane-integrated components has also
been noted and is thought to be mediated by galactosyltransferase and sialyltransferase in epididymal fluid. Thus, the storage
function of the epididymis may be responsible for the need
for capacitation in order to undergo the zona pellucida-induced
acrosome reaction and to fertilize metaphase II oocytes in vitro
(Yanagimachi, 1994; Visconti et al., 1995a). However, as
epididymal modulation of expression of adhesion molecules,
such as β-integrin and fibronectin, has been observed (Glander
et al., 1994), the glycoproteins added in the epididymis are
thought to help stabilize the sperm plasma membrane and
prevent premature capacitation or acrosome reactions. This is
particularly important because spermatozoa differ significantly
from somatic secretory cells which are able to undergo repeated
cycles of exocytosis. The acrosome reaction can occur but
once and the lifetime of an acrosome-reacted spermatozoa is
extremely limited (Drisdel et al., 1995).
Seminal plasma from many mammalian species also exerts
antifertility activities (Robertson et al., 1971; Eng et al., 1978;
Reddy et al., 1978; Fraser, 1984; Cross, 1996). Glycoproteins
in seminal plasma can readily bind to the sperm plasma
membrane (e.g. Han et al., 1990; Drisdel et al., 1995). This
may help explain why ejaculated spermatozoa from many
species are more difficult to capacitate than spermatozoa from
the cauda epididymis (Yanagimachi, 1994).
At least one of these seminal plasma glycoproteins has been
subjected to a detailed characterization (Drisdel et al., 1995).
A 74 kDa glycoprotein from human seminal plasma specifically
blocks the induction of the human sperm acrosome reaction
by a variety of agonists. The 74 kDa glycoprotein contains
both complex N-linked and O-linked oligosaccharide chains
with high concentrations of galactose, galactosamine, glucosamine and mannose. Biological activity is lost if either the Nlinked or O-linked oligosaccharides are removed. The 74 kDa
protein is either inactivated or detached from the sperm surface
during capacitation. These data suggest that interaction of
this glycoprotein with human spermatozoa is mediated by
carbohydrate-binding proteins on the surface plasma membrane. Further, the carbohydrate composition of the glycans
associated with the 74 kDa protein suggests that sperm surface
proteins which bind this glycoprotein may also be involved in
zona pellucida binding (see below, sperm receptors for zona
carbohydrate ligands).
The peripherally attached glycocalyx is removed during
capacitation in the female tract. Sperm-coating blood groupspecific carbohydrates can be similarly removed in vitro from
ejaculated spermatozoa by Percoll density gradient centrifugation (Gilbert et al., 1991). Surface glycans, in the form of
glycosaminoglycans (GAGs), absorbed from uterine, oviductal
and follicular fluid, in contrast to those from epididymal fluid,
promote sperm capacitation and the acrosome reaction, possibly
by increasing intracellular pH and calcium ion uptake (review,
Miller and Ax, 1990). The GAG content of these fluids is
highest at oestrus (Parrish et al., 1989), consistent with prior
observations on oestrus cycle dependency of capacitation
(Viriyapanich and Bedford, 1981). The ability of GAGs to
promote sperm fertilizing potential is correlated with their
content of sulphate and amino sugars, with heparin having
relatively high potency. Radiolabelled heparin is disproportionately bound to the head of human and other mammalian
spermatozoa in comparison with other membrane compartments. At least nine proteins isolated from bovine sperm
plasma membranes are capable of binding heparin. The most
important of these may be fibronectin and fibronectin-related
peptides. Fibronectin contains the Arg-Gly-Asp sequence
implicated as a ligand in sperm adhesion to oolemmal integrins
(Bronson and Fusi, 1990; Fusi and Bronson, 1992; Schaller
et al., 1993; Fusi et al., 1992, 1993). Anti-fibronectin antibodies
inhibit binding and penetration of zona-free hamster oocytes
by human and hamster spermatozoa. Fibronectin contains
two heparin-binding domains (for review see Hynes, 1985).
Fibronectin has been found overlying the head region of human
spermatozoa, and expression of fibronectin in the equatorial
segment is specifically correlated with normal sperm morphology (Vuento et al., 1984; Glander et al., 1987). The percentage
of human spermatozoa exhibiting surface binding of antifibronectin antibodies is increased in association with capacitation (Fusi and Bronson, 1992). That neither the proportion
of spermatozoa exhibiting anti-fibronectin antibody reactivity
nor the distribution of fibronectin on spermatozoa is altered
by induction of the acrosome reaction with progesterone (Fusi
and Bronson, 1992) suggests that fibronectin is located on the
surface plasma membrane, not the inner acrosomal membrane.
This localization would be consistent with its ability to react
with GAGs during capacitation.
Detection
The most convenient method for detecting sperm glycans is
that of lectin binding. Results from lectin binding studies must,
609
S.Benoff
however, be interpreted with caution for two reasons. Firstly,
lectin specificities are dependent on technical conditions such
as fixation, temperature and concentrations of ligands and/or
competing sugars (Kochibe and Furukawa, 1980; Gallagher,
1984; Hindsgaul and Ballou, 1984; Sato and Muramatsu, 1985;
Yamashita et al., 1985, 1987, 1988). Secondly, the method of
sperm preparation greatly influences analytical outcome, e.g.
whether or not internal carbohydrate residues are exposed by
drying and/or fixation (Bronson et al., 1984; Haas et al.,
1988; Bains et al., 1992). Nevertheless, glycan expression in
extracellular and intracellular compartments, as determined by
lectin binding, can be used to predict the functional state of
human spermatozoa.
Glycan expression may be used as a marker for assessment
of sperm morphology (Gabriel et al., 1994), i.e. by identifying
spermatozoa with normal oval head morphology and an intact
acrosome, requirements for binding and penetration through
the human zona pellucida (Liu and Baker, 1994). Lectin
binding properties can also distinguish subpopulations within
normospermic semen (Singer et al., 1986). Lectin binding
affinities have been observed to be altered in spermatozoa
from infertility patients (Vasquez et al., 1990; Bains et al.,
1992). For example, decreased expression of WGA receptors
has been suggested as one cause of human male infertility (Fei
and Hao, 1990; Zhixing and Yifei, 1990). N-acetylglucosamine
and/or sialic acid receptors for WGA localized to the equatorial
segment and posterior head of normal human spermatozoa have
been postulated to play an important role during fertilization
(Cerezo et al., 1982; Kallojoki et al., 1985; Franken et al.,
1990). The intensity of sperm surface WGA labelling is high
in normal spermatozoa and decreases with increasing degree
of teratozoospermia (Gabriel et al., 1994), suggesting that
either WGA receptors play a quasi-structural role in sperm
surface organization or that the boundaries of diffusible/nondiffusible lipid domains change with morphology.
Peanut agglutinin (‘PNA’) is specific for β-galactosyl residues whereas ConA and Pisum sativum agglutinin (‘PSA’)
are specific for D-mannosyl and D-glucosyl residues. These
lectins can be used to differentiate between human sperm head
compartments. PNA binds to the plasma membrane/outer
acrosomal membrane (Mortimer et al., 1990; Bains et al.,
1992), PSA to acrosome content (Cross et al., 1986) and ConA
to the inner acrosomal membrane (Holden et al., 1990). Thus,
these lectins can be used to assess differences between control
and patient populations with regard to the ability to undergo
induced acrosome loss (Mladenovic et al., 1993). That lectins
with similar monosaccharide specificities, e.g. PSA and ConA,
label different sperm compartments emphasizes the diversity
of sperm glycan structure. Lectin binding is not merely the
result of a stoichiometric relationship to respective monosaccharides, but also reflects the secondary and tertiary structure
of the glycan as a whole (e.g. Lee et al., 1983, 1984; Kery
et al., 1992; Lee, 1992).
Immune infertility
The most important role ascribed to sperm surface carbohydrate
moieties is that of antigens stimulating the production of
naturally occurring anti-sperm antibodies (ASAs). Cell surface
610
carbohydrates are highly antigenic (Sela et al., 1975). Use
of monoclonal antibodies raised against human spermatozoa
demonstrated that different surface antigens are confined to
regional domains similar to those originally described by
electron microscopy (Villarroya and Scholler, 1984, 1986). As
above, however, surface coat antigens which are peripherally
associated with the sperm membrane must be distinguished
from those linked to integral plasma membrane proteins.
Freshly ejaculated and capacitated human spermatozoa have
been reported to react with different types of ASAs (Fusi and
Bronson, 1990; Monroe et al., 1990; Margalioth et al., 1992).
The alterations in antigenicity associated with capacitation are
consistent with changes in sperm surface lectin binding affinities described above and may reflect surface modifications
associated with changes in functional state of spermatozoa.
Loss of peripherally associated surface coating molecules,
immunoglobulin (Ig) M isotypic antibody reactive blood group
antigens and non-blood group seminal antigens reacting with
IgG isotypic antibodies, may to a large part, account for
changes in sperm antibody test results when spermatozoa are
subjected to capacitating conditions (Cooper and Bronson,
1990; Gilbert et al., 1991). Antibodies to blood group antigens
do not appear to influence sperm function.
In contrast, ASAs directed against intrinsic sperm surface
antigens originating in the testis can dramatically reduce the
potential for gamete interaction (Bronson et al., 1984; Esponda
and Bedford, 1985; Patrizio et al., 1992). Conception rates are
markedly reduced in couples where immunity to spermatozoa
is detected (Ayvaliotis et al., 1985; Busacca et al., 1989). Only
a limited number of glycans on lipids (Kurpisz et al., 1989)
and proteins (Primakoff et al., 1990; Snow and Ball, 1992;
D’Cruz et al., 1993; Benoff et al., 1995b) are immunodominant. Their distribution on the sperm surface correlates with
effects of ASAs on sperm function. Results from IVF indicate
that sperm head-directed antibodies may prevent or reduce
fertilization, whereas tail-directed antibodies do not (e.g. Zouari
et al., 1993). Such data are consistent with in-vitro studies
following passive transfer of anti-sperm immunoglobulins to
antibody-free spermatozoa of fertile donors: tail-directed ASAs
are associated with motility loss in the presence of complement
while head-directed ASAs primarily impede zona pellucida
binding or subsequent acrosome loss (e.g. Bronson et al.,
1982a,b; Lansford et al., 1990). The anti-fertility effect of
head-directed ASAs is produced by at least two different
mechanisms: (i) inhibition of membrane cholesterol efflux, an
important part of the capacitation process (Benoff et al., 1993a;
see Figure 3); and (ii) inhibition of progesterone binding to
the non-nuclear progesterone receptor (Benoff et al., 1995b).
Evidence has been presented that immune infertility is the
result of local inflammation which augments natural anticarbohydrate immunities against infectious agents (Witkin and
Toth, 1983; Cooper et al., 1991; Kurpisz and Alexander, 1995).
The same simple sugars terminate bacterial and sperm surface
antigens (Tsang et al., 1987; Cooper et al., 1991; Kurpisz and
Alexander, 1995). Antisperm antibodies from human males
cross-react with bacteria (Tung, 1975). Autoimmunity to
spermatozoa is observed following chronic prostatitis
(Fjallbrant and Nilsson, 1977). Isoimmunity to spermatozoa
Carbohydrates and fertilization: an overview
often develops in women following genital tract infections
(Cunningham et al., 1991).
Although no information is available concerning the nature
of the carbohydrate structures recognized by head-directed
ASAs, those recognized by ASAs participating in complementmediated sperm immobilization have been partially characterized using anti-sperm human monoclonal antibodies (Tsuji
et al., 1988; Isojima, 1989). The internally repetitive,
unbranched N-acetyllactosamine (heteropolymers of galactose
and N-acetylglucosamine; blood type I antigen) and sialyl
branched internally repetitive N-acetyllactosamine (sialyl blood
type I antigen) on the surface of ejaculated human spermatozoa
are reactive with this class of ASAs. The ASAs recognize an
internal sequence of the glycans, as terminal substitutions of
galactose with sialic acid have no effect on monoclonal
antibody binding. Nevertheless, sialylation levels appear
critical to overall antibody recognition. If removed, other
ASAs will bind to the glycan.
Biological role in reproduction
The findings described above indicate that the sperm surface
glycans serve mainly as ‘traitors’ in generating an immune
response and an infertile state. Sperm surface glycans and
their remodeling during capacitation also serve in a ‘protective’
or ‘stabilizing’ role, e.g. to prevent premature loss of acrosome
content. It is therefore unlikely that oligosaccharides on the
sperm surface directly participate in species-specific tight
binding to the zona. Thus, sperm surface glycans are of lesser
interest than are sperm plasma membrane receptors for zona
pellucida glycoproteins.
Figure 3. Comparison of the sugar ligand binding affinities of
human, cynomologus macaque and rabbit spermatozoa. Ligand
binding was performed using the calcium-supplemented buffer
system previously described (Benoff et al., 1993b–d). Data are
presented as mean 6 SEM for fresh isolates and duplicate aliquots
subjected to capacitating incubations. (A) The sugar ligand binding
affinities of motile spermatozoa populations from fertile donors (n 5
14) were assessed at time of swim-up and after overnight incubation
at 37°C in 5% CO2 in air in Ham’s F-10 supplemented with 30 mg/ml
delipidated human serum albumin. (B) Cynomologus macaque
spermatozoa (n 5 6) was obtained by rectal electroejaculation under
ketamine anaesthesia by Dr Mary C.Mahony (Jones Institute,
Norfolk, VA, USA). Each ejaculate was collected into TALP–Hepes
medium, diluted to a concentration of ~53106/ml and transported
overnight to NSUH at ambient temperature. Surface sugar ligand
receptor affinities were measured in aliquots of untreated spermatozoa
and in duplicate aliquots after activation for 30 min at 37°C in 5%
CO2 in air in TALP medium supplemented with 1 mM each of
caffeine and dibutyrl cyclic AMP (conditions for monkey IVF;
VandeVoort et al., 1992). (C) Rabbit spermatozoa (n 5 6) were
collected using an artificial vagina. X-FITC-LIGAND binding was
assessed at time of collection and after overnight capacitation in high
salt media (Breitenstein and Schrader, 1996). (Data generously
provided by Dr Steven M.Schrader and Michael Breitenstein, NIOSH,
Cincinnati, OH, USA). α-MAN 5 α-D-mannosylated serum albumin
(Sigma No. A7790); α-GAL 5 α-D-galactosylated serum albumin
(Sigma No. A2420); β-GAL 5 β-D-galactosylated serum albumin
(Sigma No. A8165); α-FUC 5 α-L-fucosylated serum albumin
(Sigma No. A5793); α-GLU 5 α-D-glucosylated serum albumin
(Sigma No. A5543); β-GLU 5 β-D-glucosylated serum albumin
(Sigma No. A7172); β-LAC 5 β-D-lactosylated serum albumin
(Sigma No. A8040). FITC 5 fluorescein isothiocyanate.
Spermatozoa receptors for zona carbohydrate
ligands
The conserved structure and simple composition of the mammalian zona pellucida has facilitated identification of surface
components which interact with spermatozoa. In contrast, it
has been postulated that the sperm receptors for the zona
pellucida have not been conserved and that, in fact, such
variation drives speciation (Snell and White, 1996). However,
the complexities of the patterns of expression of proteins in
the sperm plasma membrane (Gillis et al., 1978; Russell et al.,
1983; Mack et al., 1987; Naaby-Hansen, 1990; Xu et al.,
1994), the regional specificities of sperm protein distributions
(Peterson et al., 1987; Bearer and Friend, 1990) and difficulties
in isolating plasma membrane fractions free of cytoplasmic
contamination (e.g. Snow and Ball, 1992) have made the
identification of components which bind to zona glycoproteins
a daunting task. Even where abnormal sperm plasma membrane
protein expression has been correlated with human male
infertility (e.g. Morgentaler et al., 1990), the large number
of proteins to be studied has precluded identification of
protein function.
At least three different sperm head membrane compartments
interact with unfertilized eggs at different stages of the fertilization process in mice (Mortillo and Wassarman, 1991). In
the mouse, competition analysis has demonstrated selective
binding of acrosome-intact spermatozoa to the zona in the
611
S.Benoff
Table III. Sperm surface proteins with affinity for the extracellular matrix
of the egg*
Species
Sperm receptor
Mouse
p95
Rat
Boar
Guinea pig
Rabbit
Human
Reference
Leyton et al. (1992); Kalab et al.
(1994)
β-1,4-galactosyl transferase Miller et al. (1992)
trypsin-like enzyme
Saling, 1981; Benau and Storey
(1987)
α-D-mannosidase
Cornwall et al. (1991)
SLIP1
Tanphaichitr et al. (1993)
15 kDa protein
Poirier et al. (1986); Aarons
et al. (1991)
sp56
Cheng et al. (1994)
α-D-mannosidase
Tulsiani et al. (1995)
galactose receptor
Abdullah et al. (1991); Mertz
et al. (1995)
APz
Peterson and Hunt (1989);
Peterson et al. (1991)
Zonadhesin (p105/45)
Hardy and Garbers (1994, 1995)
AWN-1 (spermadhesin)
Calvete et al. (1995); Dostalova
et al. (1995b)
L-fucose receptor
Huang et al. (1982)
PH-20
Primakoff et al. (1985)
RSA (54 kDa)
Abdullah et al. (1991)
RSA (Sp17)
O’Rand et al. (1988);
Richardson et al. (1994)
mannose receptor
Benoff et al. (1993b,c, 1995a)
α-D-mannosidase
Tulsiani et al. (1990)
hu9
Burks et al. (1995)
β-1,4-galactosyl transferase Humphreys-Beher and
Blackwell (1989);
Humphreys-Beher et al. (1990)
trypsin-like enzyme
Pillai and Meizel (1991); Llanos
et al. (1993)
15 kDa prtoein
Boettger-Tong et al. (1993)
galactose lectin
Goluboff et al. (1995)
selectin
Lucas et al. (1995); Clark et al.
(1995)
FA-1
Naz and Ahmad (1994)
*Although every attempt was made to ensure that the numerous alternative
candidates have been included in this review, this table may still be
incomplete.
SLIP 5 sulphoglycolipid immobilizing protein; AP 5 adhesion protein;
RSA 5 rabbit sperm antigen; FA 5 fertilization antigen; PH 5 posterior
head antigen.
presence of an excess of acrosome-reacted spermatozoa (Saling
and Storey, 1979). Electron microscopy has demonstrated
that mouse spermatozoa first establish contact with the zona
pellucida via the region of the head plasma membrane overlying
the acrosome (Wassarman, 1990; Drobnis and Katz, 1991).
These findings have been confirmed by binding of radioiodinated or colloidal gold-labelled ZP3 to mouse spermatozoa
(Bleil and Wassarman, 1986; Mortillo and Wassarman, 1991).
Autoradiographic grains were specifically restricted to the
sperm head; none were observed along the midpiece or tail
principal piece. Further, it was established that ZP3 bound to
acrosome-intact, but not acrosome-reacted spermatozoa. Taken
together, these data indicate that the molecules distributed over
the periacrosomal plasma membrane were responsible for the
initial tight binding of spermatozoa to zona. A large body of
data indicates that this group of proteins exhibits speciesrestricted expression (Table III; Figure 3). Spermatozoa that
undergo an acrosome reaction after zona binding remain bound
to oocytes, as a result of secondary binding to ZP2 (Bleil and
Wassarman, 1983, 1986; Bleil et al., 1988). As the acrosome
612
reaction results in the exposure of a new membrane compartment, it is argued that molecules responsible for maintenance
of binding of acrosome-reacted spermatozoa to oocytes are
located on the inner acrosomal membrane. Examination of the
distribution of colloidal gold-labelled ZP2 binding to acrosomeintact and acrosome-reacted mouse spermatozoa by transmission electron microscopy supports this argument (Mortillo and
Wassarman, 1991). Acrosin is the primary candidate for a ZP2
binding protein (Jones, 1989, 1991). Other acrosomal antigens
may also be involved, e.g. HSA-63 (Liu et al., 1992) and SP10 (Wright et al., 1990). Preliminary findings suggest that
secondary binding to ZP2 is not species limited as [125I]labelled hamster zona glycoproteins bind to sperm proacrosin
from boar, ram, bull, rat and mouse (Williams and Jones,
1993) and as the fucoidin ligand for acrosin blocks sperm–
zona binding in a variety of species (Yanagimachi, 1994). In
addition, the acrosome reaction also generates a restricted and
modified segment of the sperm plasma membrane overlying
the equatorial region which is capable of fusion with the
oolemma (Moore and Bedford, 1978; Bedford et al., 1979;
Yanagimachi, 1994). At least three sperm surface molecules
in this final membrane compartment have been postulated to
participate in oolemmal binding and gamete fusion: vitronectin/
fibronectin (Bronson and Fusi, 1990; Fusi and Bronson, 1992;
Schaller et al., 1993), β integrin (Schaller et al., 1993; Klenteris
et al., 1995) and PH-30/‘fertilin’ (Blobel et al., 1992; Myles
et al., 1994). In this review, the discussion will concentrate on
the zona binding proteins expressed on the surface of acrosomeintact spermatozoa.
Six major approaches have been employed in the identification of sperm surface components which exhibit zona binding
activity: (i) immobilized, size-separated sperm proteins were
probed with radiolabelled or biotinylated zona glycoproteins
(e.g. O’Rand et al., 1985; Topfer-Petersen and Henschen, 1987;
Jones, 1989); (ii) sperm membrane components obtained
following detergent solubilization were fractionated by a variety of chromatographic techniques and then tested for their
ability to block sperm–zona binding (e.g. Peterson et al.,
1983; Jonakova et al., 1991); (iii) affinity chromatography of
detergent-extracted spermatozoa by passage through columns
of zona glycoproteins bound to a solid support (e.g. Hanqing
et al., 1991; Hardy and Garbers, 1994); (iv) specific polyclonal
and monoclonal antisera which inhibit sperm–zona binding
(e.g. Peterson and Hunt, 1989); (v) cross-linking of sperm
surface proteins to zona ligands (O’Rand et al., 1988; Bleil
and Wassarman, 1990); and in man, (vi) correlation with the
infertile state (e.g. Benoff et al., 1993b,c). The individual
molecules which have been identified by these techniques have
been divided by species and described in detail below.
Mouse
Five integral plasma membrane components [p95, β-1,4galactosyl transferase, a trypsin-like proteinase, α-D-mannosidase and sulphoglycolipid immobilizing protein (SLIP)1] and
two peripherally associated proteins (15 kDa and sp56) have
been implicated in zona recognition and tight binding by
mouse spermatozoa.
Carbohydrates and fertilization: an overview
p95
A 95 kDa protein, ‘p959, has been identified on the head
surface of motile, acrosome-intact mouse spermatozoa, which
binds to whole mouse zonae or purified mouse ZP3 and also
serves as a tyrosine kinase substrate (Leyton and Saling,
1989b). The anti-phosphotyrosine antibody reactivity of this
protein is increased by interaction with zona glycoproteins
(Leyton and Saling, 1989b). Oligomerization of p95 by
immunoaggregation induces the acrosome reaction (Leyton
et al., 1995). p95 is an autophosphorylating protein (Leyton
et al., 1992). Spermatozoa express voltage-dependent calcium
channels (Florman, 1994; Goodwin et al., 1997; see Figure 10)
and tyrosine phosphorylation regulates ion fluxes through
voltage-dependent calcium channels (Sieglebaum, 1994;
A.Jacob, L.O.Goodwin and S.Benoff, unpublished observations). As inhibition of tyrosine kinase activity inhibits acrosome exocytosis (Leyton et al., 1992), p95 has been identified
as a sperm surface protein which is aggregated by ZP3 (Leyton
and Saling, 1989b; Leyton et al., 1995).
There are conflicting reports as to whether p95 tyrosine
phosphorylation is increased during mouse sperm capacitation
(Leyton and Saling, 1989b; Ducan and Fraser, 1993; Visconti
et al., 1995a,b). Whether or not the capacitation associated
increase in the phosphotyrosine content of p95 is regulated by
a cyclic AMP/protein kinase A signalling pathway is similarly
debated (Duncan and Fraser, 1993; Visconti et al., 1995b).
Leyton et al. (1995) provide no sequence data for the p95
mouse sperm antigen that reacts with anti-phosphotyrosine
antibodies. Therefore, there are conflicting reports in the
literature as to whether p95 is in fact a unique tyrosinephosphorylated form of hexokinase (Kalab et al., 1994). This
leaves open the possibility that there are two phosphotyrosinecontaining proteins that participate in the initial events of
mouse gamete binding.
It is interesting to note that four different isoforms of p95/
hexokinase are transcribed in mouse male germ cells and
spermatozoa (Visconti et al., 1996). At least one of these
isoforms is an integral membrane protein present on the sperm
head and has an extracellular domain. This isoform becomes
phosphorylated on tyrosine residues during spermatogenesis.
This protein is not phosphorylated in spermatozoa from mice
carrying two t haplotypes (Olds-Clarke et al., 1996). Spermatozoa from such mice are also unable to penetrate the mouse
zona pellucida (Johnson et al., 1995). These recent findings
suggest that it is this p95/hexokinase isoform which binds
zona ligands. However, a direct demonstration that p95/
hexokinase binds zona carbohydrates has yet to be presented.
β-1,4-Galactosyltransferase
β-1,4-Galactosyl transferase (GalTase) has been postulated to
be an externally oriented, integral plasma membrane
component of the sperm surface that is necessary for sperm
binding to the zona pellucida (Shur and Hall, 1982b; Shur
and Neely, 1988; Shur, 1991; Miller et al., 1992). GalTase
participates in the synthesis of complex carbohydrates by
specifically transferring galactose from its sugar nucleotide
substrate, UDP-galactose (UDPGal), to N-acetylglucosamine
(GlcNAc), its sugar acceptor substrate. GalTase can transfer
galactose from UDPGal to ZP3, but not ZP1 or ZP2. In the
absence of manganese, however, GalTase is thought to act as
a lectin, not an enzyme. That a lectin with GlcNAc specificity
exists on the mouse sperm surface is supported by the fact
that mouse sperm agglutination of unfixed rabbit erythrocytes
is inhibited by GlcNAc (Kawai et al., 1991).
In its capacity as a lectin, it is thought that GalTase reacts
with terminal β-linked GlcNAc or N-acetyl galactosaime
(GalNAc) residues on the mouse zona pellucida (Benau et al.,
1990). The murine zona pellucida has in fact been demonstrated
to contain O-linked oligosaccharides with terminal β-linked
GlcNAc residues (Nadgas et al., 1994). The latter observation
is of particular importance. Although terminal α-galactose
residues on mouse ZP3 oligosaccharides have been implicated
as being critical in the species-specific binding of mouse
gametes (Bleil and Wassarman, 1988), recent data indicate
that female mice lacking terminal α-galactosyl residues due
to a deficiency in α-glycosyltransferase are fully fertile (Thall
et al., 1995). These suggest that β-linked GlcNAc is a critical
residue in the regulation of tight binding of mouse spermatozoa
to the mouse zona.
In competition studies, both purified GalTase and GalTase
substrates inhibit sperm–zona binding. Agents which perturb
GalTase activity also perturb mouse sperm–egg binding. For
example, UDP-dialdehyde, a substrate analogue, and α-lactalbumin, which stimulates GalTase activity towards glucose,
produce a dose-dependent inhibition of sperm–egg binding. In
addition, UDPGal, which would force GalTAse enzymatic
reaction to completion, and enzymatic pretreatment of the
zonae, either to galactosylate GlcNAc or to hydrolyse terminal
GlcNAc residues, inhibits sperm binding to the zona. Finally,
loss of GlcNAc at fertilization appears responsible for loss of
sperm binding activity by ZP3. Recent data suggest that an
egg cortical granule N-acetylglucosaminidase is released at
fertilization, thereby inactivating the sperm GalTase-binding
site and producing the block to polyspermy (Miller et al., 1993).
GalTase is first observed on primary spermatocytes, where
it functions during adhesion to Sertoli cells (Pratt et al.,
1993). A surface redistribution of immunodetectable GalTase
is observed after meiosis. In freshly ejaculated spermatozoa,
GalTase sugar binding sites are masked by epididymally
derived glycoside ‘decapacitation factors’ which are then shed
during in-vitro capacitation (Shur and Hall, 1982a). GalTase
is then localized by indirect immunofluorescence to the anterior
sperm head plasma membrane overlying the acrosome. Acid
solubilized mouse zonae trigger both induction of the acrosome
reaction and redistribution of GalTase to the lateral surface of
spermatozoa (Lopez and Shur, 1987). That GalTase–zona
binding contributes to the induction of the physiological
acrosome reaction is demonstrated by the facts that: (i)
aggregation of GalTAse on mouse spermatozoa by bivalent
anti-GalTase antibody or by anti-GalTase Fab fragments crosslinked with secondary antibody induces the acrosome reaction
(Macek et al., 1991); and (ii) once redistributed to its new
membrane domain, GalTase is no longer able to bind ZP3
(Miller et al., 1992).
Sperm surface GalTase expression, like phosphorylation of
p95/hexokinase (see above), is regulated by the T/t locus (Shur
613
S.Benoff
and Bennett, 1979). A significant increase in GalTase activity
is observed on t-bearing spermatozoa (Shur and Scully, 1990),
which is the result of elevation of mRNA expression during
meiotic division (Pratt et al., 1993) and is associated with
premature acrosome loss (Brown et al., 1989). That overexpression of GalTase is associated with decreased fertility is directly
demonstrated in transgenic mice (Youakim et al., 1994).
Spermatozoa from these animals exhibited two defects: (i)
increased binding of decapacitation factors, resulting in masking of presumptive zona binding sites, and (ii) premature
acrosome loss, also limiting tight binding to the zona. These
data suggest that an ‘optimal’ level of zona binding sites on
the sperm surface is required to achieve fertilization.
There is presumptive evidence that the cytoplasmic tail of
sperm surface GalTAse is associated with the cytoskeleton.
mRNAs encoding Golgi and cell surface forms of GalTAse
are distinguishable by size (Lopez et al., 1991). Mouse spermatozoa preferentially express the long form of GalTase mRNA
(Pratt et al., 1993), which targets the encoded protein to the
cell surface in somatic cells. In somatic cells, movement of
GalTase on the surface membrane is regulated by its attachment
to the cytoskeleton (Appeddu and Shur, 1994). As cytoskeletal
elements participate in antigen redistribution in association
with sperm maturation and the acrosome reaction in other
species (Feuchter et al., 1986; Ochs et al., 1986; Saxena et al.,
1986a,b), it is reasonable to postulate that translocation of
GalTase on developing spermatids and following the acrosome
reaction is regulated by GalTase–cytoskeletal interactions. The
cytoplasmic domain of GalTase is also linked (through the
cytoskeleton?) to a pertussis toxin-sensitive guanine nucleotide
binding protein (G protein) (Gong et al., 1995). Ligandstimulated aggregation of GalTase results in activation of a G
protein complex containing the Gi α subunit. These observations are consistent with findings indicating that G-protein
activation participates in signal transduction leading to acrosome exocytosis following ZP3 binding and that spermassociated Gi subunits are critical to this second messenger
system (Ward and Kopf, 1993; Ward et al., 1994). Voltagedependent calcium channels, which regulate calcium influx
necessary for acrosome exocytosis (Florman, 1994), are activated by class Gi G-proteins (Ward et al., 1994). Finally,
redistribution of GalTase to the equatorial segment and inner
acrosomal membrane in association with acrosome exocytosis
has been postulated to allow GalTase to play a role in
subsequent binding events with the zona pellucida and/or the
oolemma (Lopez and Shur, 1987).
The relative importance of the above data is somewhat
diminished by two findings. First, a body of data now indicates
that GalTase activity is associated with plasma membranes
derived from bovine, equine and human spermatozoa (Sullivan
et al., 1989; Humphreys-Beher and Blackwell, 1989;
Humphreys-Beher et al., 1990; Fayrer-Hosken et al., 1991).
These data suggest that GalTase may participate in sperm–
zona binding and/or acrosome exocytosis in a variety of
species. Although GalTase exhibits all characteristics expected
of a mouse sperm protein with primary zona recognition
activity, it cannot determine species specificity of expression.
GlcNAc is a ubiquitous component of cell surface glycopro614
teins. GalTase is likely to be a generalized receptor that
functions during initial sperm–egg attachment as part of a
complex with molecules regulating species-specific gamete
binding.
A second and more disturbing finding suggests that the
GlcNAc-specific lectin may not be directly concerned with
sperm–egg interaction. The results of sugar competition experiments examining sperm–egg binding and sperm–erythrocyte
agglutination are not correlated (Kawai et al., 1991). Sialic
acid and asialo-transferrin are considerably more effective than
GlcNAc in blocking species-specific sperm–zona binding. In
addition, the localization of GalTase to the anterior sperm head
may be an artefact of antibody-induced cross-linking (Cardullo
and Wolf, 1995). Using monovalent fluorescent probes specific
for GalTase and the technique of fluorescence recovery after
photobleaching, data have been presented indicating that GalTase resides on the posterior sperm head and migrates to the
anterior region of the sperm head after cross-linking with
polyvalent antibodies. More importantly, this migration does
not lead to the release of acrosin. However, induction of the
acrosome reaction with the ionophore A23187 leads to the
loss of GlcNAc-specific lectin activity from the sperm surface;
it is then found in released membrane vesicles (Kawai et al.,
1991; Cardullo and Wolf, 1995). This suggests that migration
of GalTase may be an early event in the induction of the
acrosome reaction and that binding between the zona and other
sperm surface molecules are required to complete acrosome
exocytosis. An electron microscopic time study has suggested
that inital mouse sperm–zona pellucida contact is random
(Saling et al., 1979). These findings raise the question of
whether the posterior head region of mouse spermatozoon also
participates in tight binding to the mouse zona pellucida,
as previously described for hamster sperm–zona interactions
(Drobnis et al., 1988).
Trypsin-like enzyme
A trypsin-like enzyme on the surface of the plasma membrane
has been postulated to be involved in binding of mouse
spermatozoa to the mouse zona pellucida, as spermatozoon
binding occurs prior to the acrosome reaction (Florman and
Storey, 1982) and trypsin inhibitors competitively block this
binding (Saling, 1981). A unique property of this potential
zona binding site is that it reacts with trypsin inhibitors but
not with trypsin substrates (Benau and Storey, 1987). Although
this trypsin inhibitor binding site is separate from that responsible for GalTase activity, the two zona binding sites are
apparently located in close proximity on the sperm surface
(Benau and Storey, 1988). Binding of ligand at one site elicits
a steric hindrance effect which limits ligand binding at the
other site. The component(s) of this trypsin inhibitor-sensitive
site which mediates zona binding have not been subjected to
detailed biochemical and molecular characterization. Thus, the
possibility remains that this zona binding site is one and the
same with the mouse sperm 15 kDa peripheral protein with
proteinase inhibitor binding capabilities described below. It is
unlikely, however, that this mouse protein is directly related
to trypsin inhibitor binding sites present on the surface of
hamster and human spermatozoa (Dravland et al., 1984; Pillai
Carbohydrates and fertilization: an overview
and Meizel, 1991; Llanos et al., 1993). In these species, trypsin
inhibitors block the acrosome reaction (see below).
α-D-Mannosidase
Given that an α-D-mannosidase activity has been detected on
the surface of rat, mouse, hamster and human spermatozoa
(Tulsiani et al., 1989, 1990), mouse in-vitro sperm–zona binding assays were performed in the presence of D-mannose,
mannose analogues and mannose-containing oligosaccharides
(Cornwall et al., 1991). In all cases a dose-dependent decrease
in the number of spermatozoa bound per egg and in sperm
mannosidase activity was observed. Mannose-containing
ligands were not, however, observed to induce the acrosome
reaction. Whether this enzyme actually participates in the
initial binding of mouse spermatozoa to mouse zonae pellucidae
is thus in doubt for two reasons. First, in the studies of
Cornwall et al. (1991), galactose, the sugar identified as the
mouse sperm receptor on O-linked zona oligosaccharides (Bleil
and Wassarman, 1988), had no effect on sperm–egg binding.
Second, and more importantly, high mannose/hybrid oligosaccharide chains are found on mouse ZP2 and ZP3 as Nlinked, not O-linked, oligosaccharide chains (Tulsiani et al.,
1992). Only O-linked zona oligosaccharides have been convincingly demonstrated to exhibit sperm receptor activity (Florman
and Wassarman, 1985; Rossiere and Wassarman, 1992; Kinloch
et al., 1995).
Sulphoglycolipid immobilizing protein (SLIP)1
SLIP1 is a 68 kDa sulphoglycolipid immobilizing protein
which is observed to be distributed on the mouse sperm head
in two distinct patterns: (i) over the whole acrosomal region
except the convex ridge, and (ii) concentrated in the posterior
acrosomal area which borders on the post-acrosome region
(Tanphaichitr et al., 1992, 1993). SLIP1 is of testicular origin
and binds in vitro to the major sulphoglycolipid found only in
mammalian testes and spermatozoa (Lingwood, 1985). Two
populations of SLIP1 are detected in ejaculated mouse spermatozoa: one which is peripherally attached and readily released
in the absence of detergent and a second which is released
after exposure to Triton X-100, suggesting that it is an integral
membrane component (Tanphaichitr et al., 1993).
Although recognition of ZP3 by SLIP1 has never been
directly demonstrated, in in-vitro competition studies, purified
SLIP1 inhibits mouse sperm–egg binding (Tanphaichitr et al.,
1993). Pre-exposure of mouse eggs to SLIP1 reduces the
number of spermatozoa subsequently bound. Exposure of
spermatozoa to anti-SLIP1 antibodies also decreases the number of spermatozoa bound per egg. These observations are
difficult to reconcile with the fact that SLIP1 is not detected
on the convex ridge of the mouse sperm head (Tanphaichitr
et al., 1993) which participates in the initial binding of spermatozoa to the mouse zona pellucida (Bleil and Wassarman,
1986). Further, bivalent anti-SLIP1 antibodies do not induce
capacitated mouse spermatozoa to undergo acrosome exocytosis. No cDNA or amino acid sequence information is
available, but it is clear from the fact that heat inactivates the
inhibitory activity of SLIP1 that its native conformation plays
an important role in sperm–egg binding. These data indicate
that it is unlikely that SLIP1 is the primary mouse sperm zona
receptor and/or is associated with signal transduction activity.
Rather, it has been suggested that SLIP1 may act synergistically to facilitate zona binding with other sperm proteins
(Tanphaichitr et al., 1993).
15 kDa protein
A 15 kDa component of the mouse sperm apical plasma
membrane has been identified which binds a proteinase inhibitor of seminal plasma origin and, in spermatozoa capacitated
in vitro, participates in binding to homologous zonae (Poirier
et al., 1986). This protein becomes expressed on the sperm
surface during epididymal maturation and is also detected on
the luminal surface of epididymal epithelium (Richardson
et al., 1987), suggesting that the 15 kDa protein becomes
adsorbed to the sperm surface during epididymal transit.
Proteinase inhibitor is apparently bound by spermatozoa at
ejaculation but the level of bound proteinase inhibitor is
significantly reduced following incubation in media which
support sperm capacitation but not in media which do not
support capacitation (Boettger et al., 1989). Zona glycopeptides
and purified seminal plasma inhibitor compete for sperm
binding (Boettger-Tong et al., 1992). Competition studies also
demonstrate that the seminal plasma inhibitor blocks sperm–
zona binding only if added during the first 15 min of coincubation, indicating that the site which binds the proteinase
inhibitor participates in the early events of gamete adhesion
but not in tight binding (Robinson et al., 1987). Supporting
this are observations indicating that immunoaggregation of
bound proteinase inhibitor and translocation to the equatorial
segment induces the acrosome reaction in a manner paralleling
induction of acrosome loss by immunoaggregation of zona
peptides produced by pronase digestion (Aarons et al., 1991).
Induction of the acrosome reaction was observed only in
capacitated spermatozoa; in fresh spermatozoa, aggregation
and translocation were without effect on acrosome status
(Aarons et al., 1992). Although this 15 kDa sperm protein
exhibits many characteristics which would be expected of a
receptor for zona pellucida ligands, no information is available
as to whether or not this protein specifically binds carbohydrates.
sp56
Of all mouse sperm proteins postulated to be involved in the
initial stages of binding to the zona, only sp56 exhibits all
characteristics predicted for a mouse sperm protein with
primary zona recognition activity: (i) highly specific affinity
for mouse ZP3; (ii) high affinity for 3.9 kDa O-linked oligosaccharide with sperm receptor activity (‘functional domain
oligosaccharide’ 5 FD oligo); (iii) binding to galactose affinity
columns, and not N-acetylglucosamine affinity columns; (iv)
presence on the surface of acrosome-intact sperm heads, but
not on acrosome-reacted spermatozoa; (v) distribution on the
convex ridge of the dorsal surface of the sperm head colocalizing with ZP3 binding sites; and (vi) aggregation by FD
oligos (Bleil and Wassarman, 1986, 1988, 1990; Cheng et al.,
1994; Suzuki-Toyota et al., 1995). sp56 can be purified by
cross-linking to zona ligands or by ZP3 affinity chromatography.
Post-translational truncation from 62 to 56 kDa results in
615
S.Benoff
the production of the active zona receptor (Bookbinder et al.,
1995). This truncation results in the deletion of an aminoterminal sequence with high homology to the α subunit of
complement 4B-binding protein, suggesting that sp56 may
belong to a superfamily of protein receptors (Reid et al., 1986;
Cheng et al., 1994; Bookbinder et al., 1995). The purified
native protein is a homomultimer (Mr 110 kDa); each monomer
is stabilized by disulphide bonds. sp56 is a peripheral membrane
protein that can be released from the sperm surface in the
absence of detergents by agents that disrupt hydrogen bonds
(Cheng et al., 1994). This is consistent with the absence of a
transmembrane-spanning domain in the deduced amino acid
sequence of this protein (Bookbinder et al., 1995). cDNA
sequence analysis also fails to reveal a sequence with homology
to the carbohydrate recognition domain of previously characterized galactose lectins (e.g. ‘galectins’, Barondes et al., 1994;
Bookbinder et al., 1995).
Expression of sp56 mRNA is restricted to mouse spermatids.
That sp56 protein epitopes are not detected on guinea pig or
human spermatozoa and that sp56 mRNA is not detected on
Northern blots of testicular mRNA from guinea pig and man
suggests that sp56 participates in species-specific gamete
adhesion in the mouse (Bookbinder et al., 1995).
Rat
Lectin binding studies indicate that the zona pellucida of rat
oocytes is particularly rich in mannose and GlcNAc residues
(Shalgi et al., 1991). Other sugar residues which are in abundance on the rat zona include glucose, α and β galactose,
GalNAc and fucose (Shalgi et al., 1991).
α-D-Mannosidase
Sugar competition for rat sperm–egg binding and pretreatment
of rat oocytes with specific enzymes indicates that sulphated
glycans containing α-methyl-mannoside are critical determinants of the sperm binding site on the rat zona pellucida (Shalgi
et al., 1986). Consistent with these observations, an α-Dmannosidase is found associated with rat sperm plasma membranes (Tulsiani et al., 1989). Sperm mannosidase exhibits
activity mainly towards mannose-containing oligosaccharides.
This activity and its pH optimum of 6.5 distinguishes the
sperm surface enzyme from ‘acidic’ (pH optimum 5 4.4)
α-D-mannosidase of the acrosomal matrix which shows a
preference for synthetic substrates. Rat sperm surface mannosidase is proteolytically processed by trypsin-like enzymes during
epididymal maturation from a 135 kDa inactive precursor to
a 115 kDa enzymatically active mature form (Tulsiani et al.,
1995). It is localized on the periacrosomal region of the sperm
head (Tulsiani et al., 1995), with its catalytic domain externally
oriented (Tulsiani et al., 1989).
Both surface mannosidase activity and zona binding ability
increase as rat spermatozoa move from the caput to cauda
region of the epididymis (Yanagimachi, 1994; Tulsiani, 1995).
Thus, it has been proposed that rat sperm surface mannosidase
participates in tight binding of spermatozoa to the rat zona
pellucida. Whether, however, this novel α-D-mannosidase regulates species-specific gamete binding is in doubt for two
reasons. Firstly, the catalytic domain of this protein is purported
to interact with high-mannose/hybrid oligosaccharide chains
616
from N-linked, not O-linked, glycoproteins (Tulsiani et al.,
1992, 1995). Secondly, surface α-mannosidase activity is also
observed in spermatozoa from mouse and men (Cornwall
et al., 1990; Tulsiani et al., 1990).
Galactosyl receptor
Given that lectins play an important role in cellular adhesion
processes (Sharon and Lis, 1989), a rat testis and sperm surface
calcium-dependent galactosyl receptor (RTG-r), capable of
binding galactose and/or GalNAc, has been postulated to be a
zona binding protein of rat spermatozoa. RTG-r shares antigenic
and sequence homology with a calcium-dependent asialoglycoprotein lectin of liver (Abdullah and Kierszenbaum, 1989;
Abdullah et al., 1991; Mertz et al., 1995). The hypothesis that
RTG-r is involved in rat sperm–zona interaction is supported
by immunolocalization of RTG-r to the dorsal surface of the
sperm head overlying the acrosome and loss of immunoreactivity after induction of the acrosome reaction (Mertz et al.,
1991). It is important to note that no function has been ascribed
to RTG-r. Whether this molecule participates in species-specific
binding of spermatozoa to the rat zona pellucida is in doubt
as galactosyl residues are predominantly found on the inner,
and not the predicted outer layer, of the rat zona pellucida
(Shalgi et al., 1991), and because related molecules with
similar sperm head topography have been detected on rabbit,
human, pig and mouse spermatozoa (Abdullah et al., 1991;
Goluboff et al., 1995).
Boar
Confocal laser-scanning microscopy (CLSM) of boar spermatozoa in hemizona assays has recently suggested that a gradient
of sperm binding sites exists on the porcine zona pellucida,
with the number of sperm binding sites decreasing as spermatozoa move from the outside to the inside of the zona (Fazeli
et al., 1997). The same study also indicates that acrosomeintact boar spermatozoa initiate binding to the homologous
zona pellucida in vitro. This is consistent with findings in other
species that lectin binding sites are more abundant on the outer
as compared with the inner side of the zona (Ahuja and
Bolwell, 1983). Prior findings that boar spermatozoa must be
at least partially acrosome reacted to bind to the porcine zona
pellucida (Jones et al., 1988; Yonezawa et al., 1995) can be
explained by the rapidity with which the acrosome reaction is
induced by solubilized zona glycoproteins (Berger et al.,
1989b). Peterson et al. (1980, 1981) were the first to report
that boar spermatozoa bind to homologous zonae via receptors
located on the sperm head plasma membrane. More recently,
the binding of zona glycoproteins to the acrosomal cap
region of the boar sperm surface has been demonstrated by
immunofluorescence microscopy (Hanqing et al., 1991). At
least three classes of sperm proteins (APz, p105/45, and
spermadhesins) have been proposed to be involved in the
initial binding of boar spermatozoa to porcine eggs.
Zona adhesion protein (APz)
Zona adhesion protein (APz) is an Mr 55 kDa integral plasma
membrane protein in boar spermatozoa that does not exhibit
enzymatic activity, or react with antibodies to proacrosin or
bind to fucose, a sugar which binds proacrosin (Petersen and
Carbohydrates and fertilization: an overview
Hunt, 1989; Peterson et al., 1985, 1991). APz is a minor
sperm membrane component, representing ,1% of the total
membrane protein and showing microheterogeneity, which
exhibits high affinity for sulphated polysaccharides. Antigenically active APz is first detected on epididymal spermatozoa and the amount of APz is increased after ejaculation in
association with capacitation. That APz is associated with the
membrane cytoskeleton in boar spermatozoa is consistent with
the hypothesis that actin–plasma membrane protein interactions
play a major role in sperm capacitation (Saxena et al., 1986a,b).
Monovalent antibodies to APz completely block sperm–egg
binding. Competition studies suggest APz zona and antibody
binding sites overlap. Such studies also indicate that APz
specifically binds to ZP3α, the component of the porcine zona
pellucida with sperm binding activity (Sacco et al., 1989;
Yurewicz et al., 1993). Ligand binding is unaffected by GalTase
substrates and inhibitors and anti-proteases suggesting that
APz is a unique protein. APz tends to aggregate on standing
which suggests that hydrophobic interactions are important in
carbohydrate binding and indicates that APz exhibits lectinlike properties. APz appears to be a major plasma membrane
protein regulating tight binding of acrosome-intact boar spermatozoa to porcine ova.
p105/45
p105/45 and p56-62 were isolated after disulphide bond
reduction of sperm membrane protein complexes of respectively, Mr 130–170 and 56 000 kDa. The original complexes
were purified from detergent solubilized membrane proteins
of boar spermatozoa by chromatography over native, particulate
porcine zona pellucida glycoproteins (Hardy and Garbers,
1994). The proteins in these complexes represent only a small
fraction of total membrane protein and bind with high affinity
to the porcine zona pellucida at physiological ionic strength.
p56-62 exhibits only limited species specificity with regard to
zona binding. On the basis of size, p56-62 could represent
GalTase, sp56 or PH-20. Species limited expression of these
proteins has not been reported. In contrast, binding of p105/
45 to heterologous zona (murine, bovine) and Xenopus laevis
oocyte envelopes is not observed. p105/45 is likely to be a
unique protein, as sequences from tryptic peptides do not
match peptide sequences deposited in existing databases.
Although the carbohydrate specificities of p105/45 remain
to be investigated, these data suggest that p105/45 is of
physiological importance.
p105/45 has recently been cloned and renamed ‘zonadhesin’
(Hardy and Garbers, 1995). Analysis of the deduced amino
acid sequence of its precursor molecule indicates a multidomain
structure. A single transmembrane segment separates a short
basic intracellular carboxy terminal segment from a large
extracellular region containing a mucin-like domain and 5
tandem repeated sequences which are homologous to Ddomains of prepro-von Willebrand factor (vWF). Zonadhesin
is apparently synthesized in haploid spermatids and undergoes
proteolytic processing during epididymal transit or sperm
capacitation to generate p105 and p45 as well as to remove
the amino terminal and mucin-like domains. The remaining
vWF D-domains are postulated to mediate covalent oligomeriz-
ation, promoting membrane protein aggregation and induction
of the acrosome reaction, as well as heparin binding. It is
therefore interesting to note that heparin-binding proteins are
thought to act as extrinsic regulatory capacitation factors
that enhance the zona pellucida-induced acrosome reaction
(Florman and First, 1988). Thus, it appears that zonadhesin is
a multifunctional protein.
Spermadhesins
Spermadhesins are a family of four low molecular weight
proteins (AQN-1, AQN- 2, AQN-3, AWN; 111-113 amino
acids), with further diversity regulated by post-translational
modification (AWN-1, AWN-2). Post-translation modification
also regulates zona binding ability; addition of a single
carbohydrate chain blocks their zona pellucida binding sites.
The conformation of spermadhesins is regulated by two
disulphide bonds which modulate the binding of polysulphated
ligands (review, Calvete et al., 1995). Amino acid compositional analyses suggest that spermadhesins may be weakly
related to the sea urchin sperm protein bindin which binds to
the egg vitelline membrane (Jonakova et al., 1991). Spermadhesins are located on the apical portion of the sperm head
plasma membrane overlying the acrosome, consistent with
prior findings on the topography of the porcine egg receptor
on spermatozoa (Yurewicz et al., 1993). They are derived from
secretions of the seminal vesicle epithelium and are added to
the sperm surface at the time of ejaculation. These absorbed
proteins are retained on the sperm surface as a result of their
interaction with a major phospholipid component, phosphorylethanolamine (Dostalova et al., 1995a). The structural determinants for lipid and carbohydrate binding lie on non-overlapping
domains of the molecules. Spermadhesins appear to be multifunctional proteins in that they exhibit both lectin-like binding
of carbohydrates and heparin-binding activity or serine protease
inhibitor binding capabilities (Sanz et al., 1992, 1993; Calvete
et al., 1995). Heparin-binding proteins have been discussed
above and inhibitors of serine proteases are released from
spermatozoa in the female reproductive tract exposing surface
zona binding sites (Robinson et al., 1987). This suggests that
spermadhesins participate both in sperm–zona binding and
earlier events regulating sperm capacitation.
Spermadhesins can be purified by affinity chromatography
on isolated zona glycoproteins (Hanqing et al., 1991). AQN1, -2 and -3 bind fucoidin (Jonakova et al., 1991; Sanz et al.,
1991). AQN-1 also binds acrosin inhibitors (Sanz et al., 1992).
By analogy with the proposed function of acrosin as a ZP2
binding protein (Jones, 1989, 1991), these data suggest that
AQN proteins are active in secondary binding to the zona
pellucida. In contrast, the following characteristics of AWN
suggest that this protein participates in the initial steps of
sperm–egg recognition. AWN-1 synthesized by the rete testis
is the only spermadhesin found on the surface of epididymal
spermatozoa and may be a factor contributing to the fertilizing
ability of such spermatozoa. Additional AWN molecules
become absorbed to the sperm surface on ejaculation and
AWN-1 remains preferentially bound to capacitated spermatozoa when other spermadhesins are released (Dostalova et al.,
1994). AWN-1 can be detected on capacitated, zona-bound
617
S.Benoff
boar spermatozoa. Interaction of capacitated boar spermatozoa
with isolated zona pellucidae can be inhibited by preincubation
of zonae with purified AWN protein. Consistent with findings
in the mouse (Florman and Wassarman, 1985), AWN-1 binds
to a minor fraction of O-linked carbohydrate chains of porcine
zona pellucida glycoproteins (Hirano et al., 1993; Hokke et al.,
1993, 1994; Dostalova et al., 1995b). The basic structure of
the preferred ligand is Gal-GalNAc 6 terminal NeuAc (sialic
acid) (Dostalova et al., 1995b). AWN-1 molecules are present
on boar spermatozoa which bind to zona-encased oocytes
(Dostalova et al., 1995b). That these spermatozoa have the
capability to penetrate the zona suggests, again by analogy
with findings from other species, that AWN-1 participates in
the acrosome reaction. Alternatively, AWN-1 could serve
simply to anchor spermatozoa to the zona. However, AWN-1
is also capable of binding acrosin inhibitors (Sanz et al., 1992),
which suggests that this sperm surface molecule may have
more than one function. Further, that AQN-1 and AWN-1 both
bind acrosin inhibitors suggests that a complex of molecules
is required to effect species specific fertilization.
It is interesting to note that similar low molecular weight
zona binding proteins have been identified in a variety of
species including man (O’Rand et al., 1985). However,
although these proteins share antigenic epitopes with AWN-1,
their topology on the sperm head differs in human and stallion
spermatozoa. Thus, whether or not such proteins play a role
in the binding of human and stallion spermatozoa to their
homologous zonae remains to be determined.
Guinea pig
Both acrosome-intact and acrosome-reacted guinea pig spermatozoa can bind to the homologous zona pellucida
in vitro (Huang et al., 1981; Myles et al., 1987; Myles and
Primakoff, 1997).
Only one of the two candidates for the guinea pig sperm
zona receptor has been demonstrated to bind carbohydrates
and neither candidate shows species restricted expression.
Nevertheless, the changing patterns of protein localization
during spermatogenesis, epididymal maturation and the acrosome reaction have been well studied (Myles and Primakoff,
1984; Cowan et al., 1986, 1987, 1991; Phelps et al., 1988;
Myles and Primakoff, 1997). These studies provide strong
evidence for barriers to protein/lipid diffusion in the guinea
pig sperm plasma membrane. These studies also emphasize
that when a sperm membrane protein is relocated it comes
in contact with different lipids or proteins that could alter
its function.
L-fucose receptor
Both L-fucose and fucoidin, an algal molecule containing
sulphated L-fucose, are very strong inhibitors of binding of
guinea pig spermatozoa to the guinea pig zona pellucida in
comparison with a variety of other monosaccharides (Huang
et al., 1982). Binding of human spermatozoa to the human
zona pellucida and of hamster spermatozoa to the hamster
zona pellucida is also inhibited by fucoidin (Huang et al.,
1982). Therefore, it is likely that fucose is a part of a common
recognition signal regulating binding of mammalian gametes.
Whether or not the hamster L-fucose receptor is a homologue
618
of the fucose-binding human sperm L-selectin molecule (see
below) remains to be determined. As acrosin from a variety
of species binds fucose ligands (Yanagimachi, 1994), an
alternative possibility is that the L-fucose receptor of guinea
pig spermatozoa is acrosin. The latter would be consistent
with data indicating that only acrosome-reacted guinea pig
spermatozoa bind to the zona pellucida (Huang et al., 1984).
Posterior head antigen (PH)-20
Posterior head antigen (PH)-20 is a surface protein which is
inserted into the guinea pig sperm plasma membrane by at least
two separate mechanisms (Cowan et al., 1986). Proteolytic
processing during epididymal maturation and sperm capacitation is required for specific surface localization and formation
of the active protein that comprise two disulphide-linked
fragments (Primakoff et al., 1988; Phelps et al., 1990). In
mature spermatozoa, PH-20 is located both on the sperm
plasma membrane and on the inner acrosomal membrane. PH20 is anchored via a GlPtdIns linkage (Phelps et al., 1988).
A role for PH-20 in guinea pig sperm binding to the egg
zona pellucida was first derived from findings that a monoclonal
antibody to this sperm protein inhibited binding of spermatozoa
of undetermined acrosomal status (Primakoff et al., 1985).
Studies with antibodies to other sperm surface antigens indicated that they failed to elicit this inhibition and that PH-20
may be complexed on the sperm surface with at least one
other protein (Primakoff et al., 1985). PH-20, however, is not
required for the binding of acrosome-intact spermatozoa to the
zona pellucida (Myles et al., 1987). Competition studies with
purified PH-20 indicate that PH-20 inhibits the binding to the
zona pellucida of acrosome-reacted, but not acrosome-intact,
guinea pig spermatozoa (Myles et al., 1987). Therefore, it
must be noted that PH-20 migrates from one surface domain
(posterior head) to another (inner acrosomal membrane) in
association with the acrosome reaction. At the same time,
proteolytic cleavage occurs and the number of PH-20 molecules
on the sperm surface is increased. The latter is consistent with
findings in somatic cells that cell adhesion requires a threshold
density of functional surface molecules (e.g. Doherty et al.,
1990).
No homology is detected between PH-20 and any other
previously identified sperm surface egg binding protein
(Lathrop et al., 1990; Lin et al., 1993). PH-20 has a hyaluronidase activity (Lin et al., 1994) which is homologous to that
found in bee venom (Gmachl and Kreil, 1993). In addition to
zona binding, one suggested function for PH-20 on the posterior
sperm head is thus to enable acrosome-intact spermatozoa to
penetrate the cumulus layer in order to reach the zona pellucida
(Hunnicutt et al., 1996). Cumulus penetration and zona binding
are likely to be independent functions as antibodies which
block sperm–zona binding have no effect on the hyaluronidase
activity of PH-20 and inhibition of hyaluronidase activity with
apigenin has no effect on sperm–zona binding (Hunnicutt
et al., 1996). This suggestion is consistent with the structure
of PH-20. The hyaluronidase activity of PH-20 has been
localized to the N-terminal domain of the protein while the
active site involved in secondary binding to the zona is
localized to the C-terminal end. These domains are separated
Carbohydrates and fertilization: an overview
by proteolytic cleavage in association with the acrosome
reaction (Primakoff et al., 1988; Myles and Primakoff, 1997).
The gene encoding PH-20 is found by Southern blotting to
be present in genomic DNA from mouse, rat, hamster, rabbit,
cow, monkey and man (Lathrop et al., 1990). PH-20 is found
on the plasma membrane of spermatozoa from a variety of
species including man (Lin et al., 1993, 1994). However, in
contrast to the guinea pig, PH-20 is localized to the anterior
or whole head in the mouse, macaque and man (Lin et al.,
1994; Overstreet et al., 1995) and to the tail in the rat (Jones
et al., 1990). Expression of human PH-20 mRNA is restricted
to the testis (Lin et al., 1993). In mouse spermatozoa, PH-20
is expressed as both a soluble form with the acrosomal matrix
which is released during the acrosome reaction and GlPtdInslinked isoform which is present on the surface of acrosomeintact and acrosome-reacted spermatozoa (Thaler and Cardullo,
1995). Similarly, cynomologus macaque spermatozoa exhibits
two forms of PH-20 hyaluronidase (Cherr et al., 1996). In
addition, results from immunolabelling of fixed macaque
spermatozoa indicate that PH-20 is distributed over most of
the head of acrosome-intact spermatozoa and migrates to the
inner acrosomal membrane after the acrosome reaction. This
suggests that this protein plays a role in primary or secondary
binding of macaque spermatozoa to macaque zona (Overstreet
et al., 1995). Thus, it may be hypothesized that PH-20 participates in a complex which regulates the species specificity of
sperm–zona binding, but by itself, is not responsible for species
limited tight binding of gametes.
Rabbit
Rabbit sperm–zona interaction is unusual for two reasons.
Firstly, although as in other species, acrosome-intact rabbit
spermatozoa bind to the rabbit zona pellucida and then acrosome react at the zona surface (Bedford, 1968, 1972), acrosome-reacted spermatozoa recovered from the perivitelline
space can also bind and penetrate additional zonae (Kusan
et al., 1984). Secondly, the numbers of spermatozoa penetrating
the zona and entering the perivitelline space is considerably
higher than that reported for other species, and is sometimes
in the hundreds (Bedford, 1971; Overstreet and Bedford, 1974;
Berrios and Bedford, 1979).
Galactose residues on the rabbit zona pellucida may be
the primary determinant regulating tight binding of rabbit
spermatozoa. Activation of rabbit spermatozoa is associated
with a larger increase in binding for galactose ligands in
comparison with all other sugar ligands tested (see Figure 3C).
Both rabbit sperm surface proteins identified as exhibiting
zona ligand binding affinity specifically bind galactose.
Rabbit sperm autoantigen (54 kDa)
Rabbit sperm autoantigen (RSA) is a membrane glycoprotein
which exhibits lectin-like binding of sulphated, complex carbohydrates with high affinity (O’Rand et al., 1988). Lower
affinity binding of monosaccharides, e.g. galactose, is also
observed. RSA is thought to be a 54 kDa protein which is
capable of zona binding as identified by zona blotting (O’Rand
et al., 1985). In acrosome-intact rabbit spermatozoa, RSA is
localized over the post-acrosomal–equatorial region border
(Esaguy et al., 1988). RSA is relocalized after the acrosome
reaction in large aggregates in the medial aspect of the
equatorial region (Esaguy et al., 1988). High affinity zona
binding is effected by the aggregated form of RSA (O’Rand
et al., 1988). This protein shares antigenic epitopes with the
rat sperm galactose lectin (RTG-r) (Abdullah et al., 1991).
Purified rabbit testis galactose receptor is composed of two
subunits, 54 and 49 kDa (Abdullah et al., 1991). Purified rabbit
testis galactose receptor readily binds heat solubilized rabbit
zonae (Abdullah et al., 1991). These data indicate that the 54
kDa RSA zona binding protein is one component of the rabbit
sperm galactose receptor.
RSA (Sp17)
A 17 kDa rabbit sperm protein (Sp17), which is a member of
the rabbit sperm autoantigen (RSA) family, binds with high
affinity to rabbit zonae pellucidae and sulphated carbohydrates
when in an aggregated state (O’Rand et al., 1988). Native
Sp17, identified on Western blots following cross-linking of
zona glycoproteins, effectively competes with capacitated rat
spermatozoa for zona pellucida binding and Sp17–zona binding
is strongly inhibited by complex sulphated carbohydrates
including fucoidin, dextran sulphate, chondroitin-β sulphate
and dextran. Recombinant Sp17 exhibits similar properties
(Richardson et al., 1994; Yamasaki et al., 1995). These data,
in conjunction with observations that cholesterol-3-sulphate
has only limited effects on binding of Sp17 to rabbit zonae,
indicate that Sp17 functions as a rabbit sperm zona pellucida
binding protein.
The deduced amino acid sequence of Sp17 exhibits three
important homologies with other protein sequences: (i) the
amino terminus is 43% identical to human testis cAMPdependent protein kinase type IIa regulatory subunit; (ii) the
amino terminus also contains residues critical to the formation
of the galactose binding domain of rat hepatic lectin (e.g.
Halberg et al., 1987; Drickamer, 1992); and (iii) there is a
domain which is extremely similar to the calmodulin binding
site of neuromodulin (Richardson et al., 1994). Thus, as
observed in other species, it is likely that the rabbit sperm
receptor for the zona pellucida is a multi-functional, lectinlike protein.
Ultrastructural mapping of fixed rabbit spermatozoa before
and after the acrosome reaction demonstrates that Sp17 is
localized and aligned along with actin under the plasma
membrane (Welch and O’Rand, 1985; Esaguy et al., 1988)
and suggests that cytoskeletal interactions are responsible for
the limited lateral mobility of this antigen (O’Rand, 1982).
A reversible plasma membrane association is possible via
interaction with palmitic acid (Richardson et al., 1994). The
Sp17 polypeptide appears closely associated with the inner
aspect of the rabbit sperm apical plasma membrane and is
only accessible to antibodies after the initiation of the acrosome
reaction (Richardson et al., 1994). For Sp17 to function as a
zona binding protein, it is argued that rabbit spermatozoa begin
the acrosome reaction within the cumulus oophorus as the result
of zona glycoprotein synthesis by granulosa cells (Richardson
et al., 1994) and that acrosome-reacted rabbit spermatozoa can
bind to the zona pellucida (O’Rand and Fisher, 1987). Two
lines of evidence support this argument. Firstly, although zona
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S.Benoff
glycoprotein synthesis differs according to species of origin,
cultured rabbit granulosa cells synthesize zona glycoproteins
(Lee and Dunbar, 1993). Secondly, it has been demonstrated
that fluorescein isothiocyanate (FITC)-labelled, heat-solubilized rabbit zonae bind in patches along the apical ridge of the
rabbit sperm head, consistent with early fenestration, and that
such solubilized zonae will induce the acrosome reaction
(O’Rand and Fisher, 1987).
Homologues for Sp17 can be found in mouse and human
testis (O’Rand et al., 1985; Naz and Ahmad, 1994; Kong et al.,
1995) and immunization of female mice with synthetic peptides
derived from the rabbit Sp17 sequence results in infertility
(O’Rand et al., 1993; O’Rand and Widgren, 1994), raising the
question of whether this protein participates in the regulation
of species-limited binding to the zona pellucida. Here, an
argument can be put forth that the specificity of zona binding
and induction of the acrosome reaction resides in the affinity
of zona binding rather than binding itself (O’Rand and Fisher,
1987). Although rabbit spermatozoa are observed to bind to
pig zonae, solubilized pig zonae will not induce acrosome loss
by capacitated rabbit spermatozoa (O’Rand and Fisher, 1987).
Human
Although both acrosome-intact and acrosome-reacted human
spermatozoa can initiate attachment to the human zona pellucida (Morales et al., 1989), a considerable body of evidence
now indicates that, for fertilization to occur, human spermatozoa must be acrosome-intact when they bind to homologous
zonae and must undergo an acrosome reaction after zona
binding (for review see S.Benoff et al., manuscript submitted).
Sugar competition of sperm–zona binding, lectin binding
specificities of the human zona pellucida, and direct detection
of carbohydrate ligand binding sites on the sperm plasma
membrane have indicated that human spermatozoa express
surface receptors for a variety of simple and complex saccharide
moieties, including fucoidin (Oehninger et al., 1990, 1991),
fucose (Tesarik et al., 1993b; Lucas et al., 1994), fructose
(Mori et al., 1993), GlcNAc (Brandelli et al., 1994), and
mannose (Mori et al., 1989; Oehninger et al., 1991; Benoff
et al., 1993c; Brandelli et al., 1994; Maymon et al., 1994) (see
Figure 3A). Of these, the best studied receptor is that involved
in mannose binding and is the only sugar ligand receptor
whose expression has been directly correlated with naturally
occurring and drug-induced male infertility and with fertilization outcome following conventional IVF insemination (Tesarik
et al., 1991; Benoff et al., 1993b–d, 1994b, 1995a, 1996a;
S.Benoff et al., manuscript submitted; Hershlag et al., 1995,
1996).
Mannose lectin
Ejaculated human spermatozoa may require 3–5 h preincubation before they are capable of binding to native zonae
pellucidae or recombinant ZP3 and undergoing an acrosome
reaction after binding (White et al., 1990; Morales et al., 1994;
Van Duin et al., 1994). This latency in response is consistent
with the time course of appearance of mannose ligand receptors
on the sperm surface during capacitation (Benoff et al., 1993b).
The topographical distribution of mannose receptors on the
620
Figure 4. The topographical distribution of human spermatozoa
surface mannose ligand binding sites and acrosome status are
compared (Benoff et al., 1993b,d; S.Benoff et al., manuscript
submitted). Mannose ligand binding sites are visualized on motile
spermatozoa following reaction with fluorescein-conjugated, α-Dmannosylated serum albumin (Man–FITC–BSA; upper panel).
Acrosome status is assessed following ethanol permeabilization and
reaction with tetramethyl-rhodamine labelled Pisum sativum
agglutinin (RITC–PSA; lower panel). Mannose receptors first
appear over the entire head region of acrosome-intact spermatozoa
during capacitation. Aggregation and translocation to the equatorial/
postacrosome region occurs in association with loss of acrosome
content. Note that the sites in the acrosome content to which
RITC–PSA binds are progressively lost following exocytosis
(Tesarik et al., 1993a).
human sperm surface strongly correlates with acrosome status
(Figure 4; Benoff et al., 1993b,d, 1995c; S.Benoff et al.,
manuscript submitted). Double labelling experiments demonstrate that mannose receptors first appear uniformly distributed
over the acrosome cap. Binding of polyvalent mannosecontaining ligands induces receptor aggregation and translocation to the equatorial/post-acrosome region of the spermatozoa
(S.Benoff et al., manuscript submitted). This translocation is
followed by induction of the acrosome reaction (Benoff et al.,
1993c,d, 1996a). That the time of induction of the acrosome
reaction is markedly decreased when polyvalent mannose
ligands are mixed with D-mannose monosaccharide indicates
that allosteric interactions between mannose carbohydrate
recognition domains within a single mannose receptor contributes to receptor function (S.Benoff et al., manuscript submitted). In addition, mannose receptor apparently forms a complex
with non-nuclear progesterone receptors on the surface of
capacitated human spermatozoa (Benoff et al., 1995b), suggesting that in-vivo progesterone and zona pellucida ligand
binding act in conjunction to produce the physiological acrosome reaction.
The level of non-esterified cholesterol in the sperm plasma
membrane regulates the surface appearance of functional
mannose binding sites (Figure 5; Benoff, 1993; Benoff et al.,
1993c,d). While it is recognized that numerous biochemical
changes occur during sperm capacitation (Benoff, 1993;
Drobnis, 1993; Shi and Roldan, 1995; Zeng et al., 1996), it is
the efflux of cholesterol to external sterol acceptors which
creates a state that thermodynamically favours the insertion of
Carbohydrates and fertilization: an overview
Figure 5. The diagram depicts the four stages in the expression of
mannose lectins (MLs) on the surface of motile human
spermatozoa.
mannose receptors into the plasma membrane from subplasmalemmal stores. Anti-sperm antibodies and calcium ion channel
blockers limit membrane cholesterol efflux and thus inhibit
the appearance of mannose binding sites on the human sperm
surface (Benoff et al., 1993a, 1994b, 1995a; Hershlag et al.,
1996).
In both retrospective, blinded analyses (Benoff et al.,
1993b,c, 1994b, 1995a, 1996a) and for the first time in blinded,
prospective analyses (e.g. see Figure 6; Benoff et al., 1996b;
S.Benoff et al., manuscript submitted; Hershlag et al., 1996),
my laboratory has demonstrated that the fractional increase,
following capacitating incubation, in the percentages of acrosome-intact spermatozoa capable of binding mannose ligands,
such as mannosylated bovine serum albumin (‘Man–FITC–
BSA’); (Figure 7A), and the ability to undergo mannosestimulated acrosome loss are predictive of the rate of fertilization of mature oocytes following insemination in vitro. These
data support the argument that human sperm mannose receptors
contribute to the initial adhesion of spermatozoa to egg. That
mannose-containing ligands on the human zona pellucida are
involved in species-limited tight binding of gametes is further
supported by the failure to detect naturally occurring antisperm antibodies to mannosyl-terminated antigens in the sera
from female immune infertility patients (Cooper and Bronson,
1990; Cooper et al., 1991), as this could result in autoimmune disease.
Human sperm mannose receptors require calcium to bind
tightly to mannose-containing ligands (Benoff et al., 1993b)
and surface expression of mannose receptors is highly
correlated with the level of zinc in seminal plasma (1996b).
Figure 6. Prospective analysis of 70 consecutive males undergoing
an in-vitro fertilization (IVF) cycle. Two semen specimens were
obtained from each patient prior to IVF. Motile spermatozoa
populations were isolated and analyzed for mannose receptor
expression both at the time of isolation and again after 18 h
incubation in Ham’s F-10 1 30 mg/ml human serum albumin at
37°C in 5% CO2. Where sufficient numbers of spermatozoa were
obtained (n 5 28), capacitated spermatozoa were assessed for
mannose-stimulated acrosome loss. Both the increase in percentages
of acrosome-intact spermatozoa capable of binding mannose ligands
on their heads after capacitating incubation and the ability of
capacitated spermatozoa to undergo an acrosome reaction upon
exposure to mannose ligands directly correlate with the rate of
fertilization following conventional IVF inseminations (respectively,
Pearson Product Moment Correlation, r 5 0.571, P ,0.0001, and
t-test, P ,0.0016) (Benoff et al., 1996b; Hershlag et al., 1996;
A.Hershlag and S.Benoff, unpublished observations).
These observations suggest that the mannose receptor is a
lectin, as transition metal ions help to position amino acid
residues and regulate carbohydrate binding in a variety of
plant and animal lectins (Sharon, 1993). My laboratory has
investigated the structure of the human sperm surface mannoseligand binding site through analysis of its effector specificities,
by reaction with specific polyclonal and monoclonal antisera,
and by cDNA sequence analysis (Benoff et al., 1992a,b,
1993a,d–f, 1994a, 1996b; S.Benoff, unpublished observations).
The results from these studies indicate that this putative zona
acceptor shares the amino acid sequence and ligand binding
characteristics of the conserved carbohydrate recognition
domain (CRD) common to a family of membrane-bound and
soluble mannose-specific lectins (Drickmer, 1989; Taylor et al.,
1992). It is therefore unlikely that the CRD of the human
sperm mannose lectin would behave as a ‘self’ antigen.
The contention is supported by observations indicating that
naturally occurring antisperm antibodies in the sera of immune
infertility patients (both men and women) fail to react with
the mannose lectins expressed on the surface of capacitated
human spermatozoa (Benoff et al., 1993a, 1995b).
These data on the structure of the human sperm mannose
lectin also provide a basis for speculation on the nature of the
specific oligosaccharides on the human zona pellucida with
sperm binding activity that are recognized by this marker.
Other C-type mannose lectins have been reported to be ‘corespecific’, recognizing not only terminal sugars but also those
within the core region of an oligosaccharide (Summerfield and
Taylor, 1986; Taylor and Summerfield, 1987; Childs et al.,
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S.Benoff
Figure 7. The topographical distribution of the binding of fluorescein-conjugated polyvalent mannose ligands on the surface of motile
human spermatozoa (A, 3600 original magnification) was compared with that detected by indirect immunofluorescence of anti-myosin (B,
3400 original magnification) and anti-actin (C, 3400 original magnification) antibodies. Antibody labelling was performed following
permeabilization of unfixed human spermatozoa (Benoff et al., 1996a; S.Benoff et al., manuscript submitted). Labelled spermatozoa were
photographed on 400 ASA film with a 50 s exposure time. Mannosylated BSA and anti-cytoskeletal antibodies exhibit similar distributions
over the spermatozoa head: over the acrosome cap (pattern II; large arrow) and limited to the equatorial segment (pattern III: small arrow).
Double labelling with rhodamine-conjugated Pisum sativum agglutinin, which stains acrosome content (Cross et al., 1986), has revealed that
pattern II binding occurs on acrosome-intact human spermatozoa while pattern III is observed on spermatozoa which have undergone an
acrosome reaction (Benoff et al., 1993b,d, 1996a, 1997a; S.Benoff et al., manuscript submitted).
1990). These findings suggest that mannose need not be a
terminal residue of the glycoconjugates of human ZP3 in order
to regulate the initial steps in species specific tight binding of
human gametes.
Analysis of the deduced amino acid sequence of the human
sperm mannose lectin indicates that, as in other species,
this putative zona binding molecule is multifunctional. Its
cytoplasmic tail contains a binding/hydrolysis site for ATP
and two actin binding motifs, all of which exhibit both
antigenic and sequence homology to those found in the globular
head of skeletal muscle myosin (Figure 7B; Benoff et al.,
1994a, 1996a). A superfamily of human sperm plasma membrane proteins containing similar cytoplasmic ‘myosin motors’
was identified during the course of this analysis (Benoff
et al., 1994a, 1996a). This discovery raises the possibility that
directed movement and clearance of these molecules from the
apical plasma membrane prior to the acrosome reaction is
controlled by interaction of their myosin motors with underlying actin (Benoff et al., 1997a; S.Benoff, unpublished observations).
Mannose lectins share characteristics with actin binding
proteins of somatic cells (for review see Isenberg, 1991), in
that their expression on the sperm surface is regulated by
membrane fluidity changes (Benoff, 1993; Benoff et al.,
1993a,c,d, 1994b, 1995a, 1996a). Actin is readily detected in
the unfixed permeabilized human sperm head by indirect
immunofluorescence cytochemistry (Figure 7C; Benoff et al.,
1997a). Other laboratories have reported that cytoskeletal actin
participates in antigen redistribution associated with induction
of the acrosome reaction (Feuchter et al., 1996; Ochs et al.,
1986) and disruption of actin filaments with cytochalasin D
reduces the number of human spermatozoa capable of penetrating zona-free hamster ova (Rogers et al., 1989), an event
requiring prior acrosome loss. Recent findings demonstrate
that human spermatozoa which exhibit reduced expression of
either plasma membrane proteins with anti-myosin antibody
622
reactivity or sperm head actin also exhibit a reduced ability to
undergo spontaneous and induced acrosome loss (Benoff et al.,
1996a, 1997a). These data support the argument that actin–
myosin-like interactions are involved in the rearrangements of
proteins in the human sperm head plasma membrane and
induction of the acrosome reaction.
α-D-Mannosidase
Despite the findings described above, it is also possible that
mannose-based human sperm–egg interactions could theoretically be mediated, at least in part, by carbohydrate binding
enzymes. Another laboratory has described an α-D-mannosidase activity associated with the human sperm plasma membrane
(Tulsiani et al., 1990) which is similar to those of rat and
mouse sperm plasma membrane (Tulsiani et al., 1989; Cornwall
et al., 1991) and can be differentiated from the acidic αmannosidase activity associated with the acrosome (Tulsiani
et al., 1990). The effector specificities of this membrane-bound
mannosidase activity and that of the mannose lectin described
above differ significantly, such that one could not readily be
confused for the other.
This enzyme has only been studied in freshly isolated human
spermatozoa. Despite generalizations from data obtained in
the mouse (see above), there is no direct evidence that the
sperm surface-associated α-mannosidase regulates interactions
between human gametes. In fact, an argument against an
important role for this enzyme in human sperm–zona binding
rests in the fact that only 5% of its total activity is found
tightly associated with sperm plasma membrane fractions
(Tulsiani et al., 1990).
hu9
A 95 kDa antigen is localized to the equatorial segment
of acrosome-intact human spermatozoa and shares antigenic
epitopes with hamster spermatozoa (Moore et al., 1987). A
monoclonal antibody, mAB 97.25, that reacts with this antigen
prevents human sperm binding to the zona pellucida of salt-
Carbohydrates and fertilization: an overview
stored human oocytes (Moore et al., 1987). It is possible that
this antigen is the 94 6 3 kDa autophosphorylating protein of
human spermatozoa which has been independently identified
(Naz et al., 1991a) and which may be identical with fertilization
antigen-2 (FA-2) of human spermatozoa (Naz et al., 1993).
Both the 94 6 3 kDa antigen and FA-2 have been localized
to the acrosomal region of the human sperm head using indirect
immunofluorescence with an anti-phosphotyrosine monoclonal
antibody. Phosphorylation of this protein is increased during
human sperm capacitation (Naz et al., 1991a; Luconi et al.,
1996; Emiliozzi and Fenichel, 1997) and is further stimulated
by exposure to zona pellucida glycoproteins (Naz et al., 1991a).
Binding of this 94 6 3 kDa protein to ZP3 apparently involves
its phosphotyrosine residues, as it can be eluted from ZP3 in
the presence of excess o-phospho-L-tyrosine (Naz and Ahmad,
1994). The 95 kDa/94 6 3 kDa/FA-2 protein described above
may have been independently identified as hu9 (Burks et al.,
1995). hu9 is a potential human sperm homologue of mouse
spermatozoa p95 (see above). cDNA sequencing indicates that
hu9 is a transmembrane protein which exhibits no homology
with hexokinase. Indirect immunofluorescence analysis with
mAB 97.25 (Moore et al., 1987; see above) localizes hu9 to
the surface in the acrosomal region of capacitated human
spermatozoa (Burks et al., 1995) and autophosphorylation of
hu9 is stimulated by capacitation and by exposure to human
ZP3, characteristics which are shared with the 94 6 3 kDa
human sperm protein described above. In competition studies,
synthetic peptides to its extracellular region inhibited human
sperm–zona pellucida binding. These data suggest that p95 is
an unlikely candidate for a receptor regulating species specific
tight binding of gametes.
The possibility exists, however, that hu9 is part of a complex
which regulates sperm–zona interaction and/or subsequent
signal transduction. Particularly relevant to this argument is
the finding from cDNA sequence analysis that hu9 contains
an intracellular tyrosine kinase domain that can participate
in both autophosphorylation and transphosphorylation events
(Burks et al., 1995). With regard to the latter, my laboratory
has demonstrated that mannose ligand binding is associated
with an increase in tyrosine phosphorylation on proteins in the
human sperm head (Figures 8 and 9; Benoff et al., 1995c). It
is likely that activation of a tyrosine kinase (Bielfeld et al.,
1994) is ultimately responsible for calcium influx through
voltage-dependent calcium channels in the post-acrosome
region of the human sperm head (Figure 10; Goodwin et al.,
1997; A.Jacob, L.O.Goodwin, N.B.Leeds and S.Benoff, unpublished observations) and the acrosome reaction. In men with
iatrogenic infertility associated with the therapeutic administration of calcium ion channel blockers, mannose-stimulated
tyrosine phosphorylation is markedly reduced (Figure 8; Benoff
et al., 1995c). That mannose receptor aggregation and translocation precedes induction of this tyrosine kinase activity is
demonstrated in the same infertile population by the fact that
receptor movement can be observed in the absence of an
acrosome reaction (Benoff et al., 1994b, 1995a). The human
sperm mannose lectin is not itself phosphorylated during
capacitation or the acrosome reaction and exhibits no sequence
homology to catalytic domains from identified phosphotyrosine
Figure 8. Examination of the affinity of motile human spermatozoa
populations for anti-phosphotyrosine monoclonal antibodies
following previously described protocols (Naz et al., 1991a). Briefly,
motile spermatozoa populations were isolated by swim- up and
divided in half. One half was used to measure phosphotyrosine
accumulation in the fresh isolate (CONTROL). The other half was
incubated 18 h under spermatozoa capacitating conditions (Ham’s F10 1 30 mg/ml human serum albumin, 37°C, 5% CO2; 5
INCUBATED). Half of each control and incubated aliquot was
exposed to 100 µg/m mannosylated bovine serum albumin (BSA) in
the presence of 75 mM D-mannose to induce acrosome exocytosis
(Benoff et al., 1993c,d, 1995c, 1996a; S.Benoff et al., manuscript
submitted) (1MANNOSE) and the other half was exposed to
phospate-buffered saline. Spermatozoa were then air-dried onto glass
microscope slides, fixed by immersion in 100% ethanol and then
stained with anti-phosphotyrosine monoclonal antibody clone 1G2.
Spermatozoa bound monoclonal was detected with fluorescein
isothiocyanate-conjugated sheep anti-mouse immunoglobulin G. For
each specimen analysed, .300 spermatozoa were scored by visual
inspection at 3400 original magnification for anti-phosphotyrosine
binding in the head versus tail regions. (A) The percentages of motile
spermatozoa binding anti-phosphotyrosine monoclonal antibodies on
their heads was quantitated in specimens from five fertile donors.
There was a significance difference between the four conditions
examined (repeated measures analysis of variance, P ,0.0001). In all
cases, a specific increase in spermatozoa head labelling is observed
following mannose exposure (CONTROL versus INCUBATED, not
significant; CONTROL versus INCUBATED/1MANNOSE, P
,0.004; INCUBATED versus INCUBATED/1MANNOSE, P
,0.0038) (A.Jacob and S.Benoff, unpublished observations). (B) In
spermatozoa from men taking nifedipine, a Ca21 channel blocker
medication, signal transduction in response to mannose treatment is
markedly reduced (A.Jacob and S.Benoff, unpublished observations).
This reduction can be attributed to the inhibition of surface mannose
receptor expression resulting from drug-related increases in
membrane cholesterol content (Benoff et al., 1994,1995a; Hershlag
et al., 1995).
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S.Benoff
Figure 9. Proteins in the head of capacitated human spermatozoa from fertile donors are phosphorylated on tyrosine residues following
exposure to model zona ligands, polyvalent mannosylated bovine serum albumin (BSA) plus mannose monosaccharide. Phosphotyrosine
residues were detected by immunocytochemical staining as described in the legend to Figure 8. Labelled spermatozoa were viewed at 3600
original magnification and photographed at 31500 on 400 ASA film with an exposure time of 50 s. (A) Phase-contrast image of unfixed,
capacitated human spermatozoa. (B) Examination of the same field with UV-epifluorescence illumination shows that binding of antiphosphotyrosine monoclonal antibodies occurs over the spermatozoa head in two distinct topographical distributions: over the acrosome cap
(small arrow) or limited to the equatorial segment (large arrow) (A.Jacob and S.Benoff, unpublished observations).
kinases (S.Benoff, unpublished observations) raising the possibility that either this lectin is directly complexed with hu9
on the sperm surface or that cross-talk between mannose
lectins and hu9 occurs via the cytoskeleton. In the mouse,
cytoskeletal-associated sperm membrane proteins become
phosphorylated during capacitation and induction of the acrosome reaction (Visconti et al., 1996).
β-1,4-galactosyltransferase
Whether or not human spermatozoa express a GalTase activity
has been previously debated (Tulsiani et al., 1990). However,
recent data suggest that a human sperm associated GalTase,
with biochemical characteristics similar to those of mouse
sperm GalTase, segregates with the plasma membrane when
spermatozoa are vortex fractionated (Huszar et al., 1997). Its
role in human sperm–zona pellucida binding has been assessed
only indirectly (Huszar et al., 1997). Surprisingly, immature
spermatozoa containing residual cytoplasm and which lack
zona binding ability overexpress GalTase when compared with
mature spermatozoa. Nevertheless, these findings still do not
rule out a more generalized role for GalTase in human sperm–
egg interactions. In man, expression of a GalTase allelic variant
is observed in a population of human males whose spermatozoa
fail to penetrate zona-free hamster ova (Humphreys-Beher and
Blackwell, 1989), presumably due to the failure to undergo
the acrosome reaction. Antibodies to sperm surface GalTase
may play a role in the aetiology of human male immune
infertility (Humphreys-Beher et al., 1990). Thus, human sperm
surface GalTase may be part of a recognition system common
to mammalian gametes.
Trypsin-like enzyme
The human sperm surface, like that of the mouse, is capable
of specific binding of trypsin inhibitors. However, in contrast
to findings in the mouse, trypsin inhibitors have no effect on
human sperm–zona binding (Llanos et al., 1993). Rather, these
agents block the zona- and follicular fluid-induced acrosome
reactions by preventing the increase in intracellular calcium
624
required for acrosome exocytosis (Pillai and Meizel, 1991;
Llanos et al., 1993). Therefore, a possible role for such trypsin
inhibitor binding sites in human sperm–egg interactions lies
in regulation of signal transduction after zona binding.
15 kDa
A human sperm homologue of the mouse spermatozoa 15 kDa
protein may exist. A 20 kDa component of the human sperm
head plasma membrane apparently recognizes the murine
trypsin–acrosin inhibitor and immunoaggregation of bound
inhibitor can induce the acrosome reaction in capacitated
human spermatozoa (Boettger-Tong et al., 1993). However,
the ability of this protein to block human sperm–zona binding
in a hemizona assay has not been tested and no reports have
appeared documenting defective 15 kDa protein expression in
human male infertility. Therefore, the role of this protein in
tight binding of human spermatozoa to the human zona
pellucida is questionable.
Galactose lectin
Human testis and spermatozoa express a 50 kDa protein which
is antigenically related to galactosyl lectin found on rat
(RTG-r) and rabbit spermatozoa (Goluboff et al., 1995). This
lectin is postulated to be testis-specific as Northern blotting
experiments indicate that expression of the RNA encoding the
human homologue of RTG-r is restricted to the human testis
(Goluboff et al., 1995). This galactosyl receptor is localized to
the head plasma membranes overlying the acrosome (Goluboff
et al., 1995). Its role in tight binding of human spermatozoa
to the human zona pellucida and/or induction of the acrosome
reaction after zona binding has not been directly assessed.
Thus, there is no compelling evidence that this 50 kDa protein
plays an important role in the species specific interaction of
human gametes.
The human sperm galactose receptor shares at least two
biochemical characteristics with the mannose lectin described
above and with selectins (see below): (i) a calcium-dependency
of carbohydrate ligand binding; and (ii) a requirement for non-
Carbohydrates and fertilization: an overview
Figure 10. L-type calcium channels are detected in the postacrosome region of the spermatozoa head following reaction of
unfixed, Triton-permeabilized motile human spermatozoa with
monoclonal antibody IIF7, specific for the α1 subunit of the rabbit
skeletal muscle calcium channel (Leung et al., 1987; a generous
gift of Dr Kevin Campbell, University of Iowa, Iowa, USA)
(Goodwin et al., 1997). Labelled spermatozoa were viewed at 3600
original magnification and photographed on 400 ASA film with an
exposure time of 50 s for the fluorescein image. (Upper panel)
Phase-contrast image. (Lower panel) Corresponding indirect
immunocytochemical labelling patterns detected using UVepifluorescence optics.
ionic detergents for release from sperm membranes (Goluboff
et al., 1995). Mannose ligand binding by mannose lectins can
be partially inhibited by high concentrations of D-galactose
(Summerfield and Taylor, 1986). A few simple amino acid
changes in the carbohydrate recognition domain of mannose
lectins result in the acquisition of high affinity galactose
binding (Drickamer, 1992; Iobst and Drickamer, 1994b). In
the absence of amino acid sequence data, the possibility that
the mannose lectin has been misidentified as a galactose lectin
cannot be ruled out.
L-selectin
Solubilized human zona pellucida glycoproteins compete with
a fucosylated–BSA ligand for binding sites on the human
sperm surface, suggesting that human spermatozoa possess
specific receptors for fucose (Tesarik et al., 1993b). Using
fucosylated–BSA conjugated to a fluorescent tag, these receptors were localized to the acrosome region of the head of
acrosome-intact spermatozoa and the percentages of spermatozoa capable of binding this ligand increased in association
with capacitation (Tesarik et al., 1993b). As immunoreactive
acrosin is observed on the sperm plasma membrane early in
the induction of the acrosome reaction (Tesarik et al., 1988),
it was initially suggested that this fucose receptor could be
acrosin (Tesarik et al., 1993b). However, the involvement of
fucose receptors unrelated to acrosin could not be excluded.
Further studies have established that fucose-containing
ligands, such as fucoidin, block both human sperm–zona
binding and the zona-induced acrosome reaction (Oehninger
et al., 1990, 1991; Mahoney et al., 1991). Fucoidin is a
structural analogue of both the sulphated carbohydrate ligands
that bind to selectins (Patankar et al., 1993) and blood grouprelated antigens found on the surface of the human zona
pellucida (Lucas et al., 1994). It is therefore not surprising
that glycodelin (5 human placental protein 14), which contains
oligosacchride chains that can function as selectin ligands
(Dell et al., 1993; Varki, 1994), and purified blood group
antigens terminating in fucose, reduce the capacity for tight
binding of human spermatozoa to the human zona (Oehninger
et al., 1995; Clark et al., 1995, 1996a,b). Consistent with
this data, monoclonal anti-L-selectin (‘leukocyte selectin’)
antibodies have identified a 90 kDa human sperm protein
which is localized to the sperm head (Lucas et al., 1995).
Pretreatment of spermatozoa with these antibodies blocks tight
binding of human spermatozoa to the zona pellucida under
hemizona conditions (Lucas et al., 1995). Whether this Lselectin-related protein regulates species specific gamete interactions may still be questioned as L-fucose is found on the
zona pellucida from a variety of species. Likewise, fucose
receptors have been described on spermatozoa from other
mammalian species.
L-selectins represent one of three groups of related receptors
that play a role in the regulation of inflammation and the
immune response (McEver, 1994). In all cases, ligand binding
is calcium-dependent and the result of an amino-terminal motif
related to the carbohydrate recognition domain (CRD) of
mannose-binding proteins (Erbe et al., 1992; McEver, 1994).
Both selectins and mannose lectins recognize ‘clustered saccharide patches’ (Varki, 1994; Taylor et al., 1992; S.Benoff
et al., manuscript submitted). Mannose lectins are also capable
of binding fucose (Summerfield and Taylor, 1986; Taylor and
Summerfield, 1987; Childs et al., 1990; Taylor et al., 1992;
Iobst et al., 1994b). Thus, the mannose lectin may have been
misidentified as a selectin.
The role of sperm surface L-selectin-related protein in human
male infertility has never been directly studied.
Fertilization antigen-1
Fertilization antigen-1 (FA-1) is an antigen that is implicated
in the generation of involuntary human immunoinfertility (Naz,
1987a). FA-1 has been localized on the human sperm surface
using monoclonal antibodies (Naz et al., 1992). All spermato625
S.Benoff
zoa exhibit antibody binding on their midpiece and tail regions.
Additionally, 20–40% of spermatozoa exhibit antibody binding
on the post-acrosome region of the head. This same antibody
blocks human sperm–zona pellucida binding but has no effect
on sperm movement characteristics or on the acrosome reaction.
FA-1 exists in the form of a monomer of 23 kDa and/or a
dimer of 51 kDa (Naz, 1988). The 51 kDa form exhibits
autophosphorylating activity in an in-vitro kinase assay. As
observed for the 94 6 3 kDa human sperm antigen described
above, the phosphotyrosine residues of FA-1 appear to regulate
binding of human zona glycoproteins (Naz and Ahmad, 1994).
The sulphhydryl groups of FA-1 may also be important in
sperm–zona binding (Naz and Ahmad, 1994).
Whether or not FA-1 regulates the tight binding of human
gametes is in doubt for several reasons. Firstly, it has not been
demonstrated whether FA-1 binds to carbohydrate or protein
ligands of the zona. Secondly, human FA-1 readily binds
porcine ZP3 and bioneutralizes the boar sperm binding activity
of the porcine zona pellucida (Naz et al., 1991b). Thirdly,
antibodies to human FA-1 inhibit the binding of acrosomeintact mouse and rabbit spermatozoa to their homologous
zonae (Naz et al., 1984; Naz, 1987b, 1990). Fourthly, antibodies
to FA-1 markedly reduce the number of human spermatozoa
fusing with zona-free hamster ova (Naz et al., 1992). Thus,
the exact role of FA-1 in human (mammalian?) fertilization
remains to be defined.
Common features of sperm surface zona binding
proteins
Although it is clear that the sugar binding affinities of the
sperm plasma membrane differ among species (Table III;
Figure 3), the results obtained from the studies described above
indicate that sperm surface zona binding proteins have five
features in common. Firstly, irrespective of whether a candidate
zona receptor is an integral sperm plasma membrane protein
or is only peripherally attached, it is distributed on the sperm
head over the acrosome cap. Secondly, the number of candidate
zona receptor molecules found in this region is increased in
association with sperm capacitation. Thirdly, such molecules
exhibit lectin-like binding of carbohydrates: they are not
immunoglobulins, they bind ligands in a non-enzymatic fashion, and they exhibit a preference for polyvalent ligands but
may or may not agglutinate erythrocytes. Fourthly, candidate
zona receptors which are integral membrane proteins exhibit
an association with the sperm cytoskeleton. Fifthly, ligand or
antibody binding leads to aggregation and clearance from the
acrosome cap region. This movement is associated with signal
transduction, potentially via tyrosine phosphorylation, and
initiation of the acrosome reaction.
Conclusions
Cell–cell recognition by mammalian gametes clearly involves
oligosaccharide modification of conserved proteins. The binding of spermatozoa to egg is a biphasic process of non-specific,
loose attachment followed by species-specific tight binding.
Species specificity of gamete adhesion may involve derivatization of zonae (both in terms of glycan composition and glycan
626
three-dimensional structure), expression of lectins or enzymes
on sperm plasma membranes, or possibly both. Whichever
occurs, initial recognition of an egg by spermatozoa appears
to initiate a train of changes in the sperm head: aggregation
of apical sperm head membrane proteins, translocation of some
of these (and possibly other) proteins to the equatorial region
of the sperm head, and induction of a calcium influx leading
to an acrosome reaction. The chemical effectors of these events
are not well characterized. Limited proteolysis of these proteins
may be involved in the generation of functional zona binding
sites on the sperm surface. Interactions with cytoskeletal actin
may facilitate surface protein movement. Tyrosine phosphorylation occurs following zona binding in all species studied
thus far. There is some evidence that G-proteins and cAMP
are involved in early secondary signalling events initiated by
ZP3 binding.
Is sperm cytoskeleton involved in the events between the
initial adhesion of spermatozoa to egg and secondary binding
of spermatozoa to ZP2? Speculation on this question is based
on results from immunocytochemistry and a cell-free system
in which polymerization and depolymerization of actin was
observed (Welch and O’Rand, 1985; Saxena et al., 1986b;
Peterson et al., 1990; Spungin et al., 1995; Benoff et al., 1996b;
Visonti et al., 1996). Sperm capacitation requires changes in
intracellular pH, calcium content and plasma membrane protein
expression (including zona binding proteins) and efflux of
cholesterol from the plasma membrane. A time-dependent
increase in polymerized actin associated with plasma membrane
proteins is also observed. The initial zona ligand binding event
activates protein clearance from the periacrosomal plasma
membrane, G-protein signalling, with subsequent phosphorylation of sperm proteins including phospholipase C (PLC). In a
manner similar to that observed in somatic cells, phosphorylation may induce a conformational change in PLC so that it may
now be transferred from the cytosol to the actin cytoskeleton.
Calcium influx would simultaneously occur. Under conditions
of increased calcium, PLC activity would prime membranes
for fusion by breakdown of phospholipids to produce the
fusogenic compound diacylglycerol and additionally liberate
actin-severing proteins. The actin severing-proteins would then
induce actin depolymerization, thereby removing the barrier
to membrane fusion. Specific fusion of the plasma and outer
acrosomal membranes would occur in the presence of local
elevations in calcium. Following acrosome exocytosis, sperm
proteins such as acrosin could effect secondary, tight binding
to the zona pellucida. This provides an explanation of how
protein clearance and membrane fusion occur in response to an
initial recognition event. This hypothesis is eminently testable.
A relatively large number of sperm surface proteins have
been implicated in these initial egg recognition events. Over
20 have been reported for the six mammalian species reviewed
here. Their characterizations are still very incomplete, and
some may be shown to be functionally equivalent by future
experiments, or to have multiple functionalities not presently
recognized. However, the data available to date does not
support the supposition that a single sperm protein, however
modified, can be responsible for the various species-specific
recognition events of all mammals. No single protein itself
Carbohydrates and fertilization: an overview
possesses all the functionalities required to induce the acrosome
reaction. Up to five proteins per species, distinguishable by
molecular size on Western gels, have been shown to block
fertilization or zona binding, or to induce acrosome reactions.
More likely than a single protein is a hetero-molecular complex
of proteins which recognizes the zona in a species-specific
manner and induces a co-operative signal for an acrosome
reaction. Multiple proteins recognizing different zona carbohydrate structures fixed in a defined spatial relationship to
each other by the zona protein framework must provide a
wider alphabet for species specificity than can a single lectin
or enzyme. Blocking of zona binding by an antibody would
disrupt the tertiary structure of such a complex, and would
indicate only that the protein possessing the epitope targeted
forms part of this complex. Induction of an acrosome reaction
by cross-linking of proteins would occur irrespective of whether
specific cross-linked proteins were involved in the specieslimited recognition event itself, or in some early internal
signalling step within the complex. Both types of experiments
must be interpreted cautiously. Lectin activities on the sperm
head have been demonstrated in several species and are
commonly implicated in somatic cell–cell recognition. These
appear likely candidates for the regulation of the initial sperm–
egg recognition events, if only because no other function
within such a complex could easily be attributed to them.
Lectins found in some species are clearly extracellular proteins,
and therefore cannot induce intracellular signals without the
intervention of transmembrane proteins with intracytoplasmic
activities. Such transmembrane proteins have been reported in
these same species. Lectins of other species have membranespanning regions and may be able to signal directly with their
cytoplasmic tails, but may not be able to create all secondary
messages necessary to induce acrosome reactions.
Cloning and site-specific mutagenesis experiments in the
next few years will identify the functional domains of the
sperm surface proteins currently believed to be involved in
early egg recognition events. It will be interesting to see if a
common group of functionalities can be characterized which
all mammalian species possess, and if these functionalities are
associated with analogous or distinctly different polypeptide
chains in different species. However, as fertilization in vivo
occurs in the specialized milieu created in the ampulla of the
Fallopian tube, we must be aware of the signals the natural
environment can induce in spermatozoa, and the sperm surface
remodelling it can cause as we draw conclusions as to the
mechanisms of early stages in fertilization.
Acknowledgments
Supported in part by National Institutes of Health Grant No. ES
06100. Appreciation is expressed to Ian R.Hurley, George W.Cooper,
and J.Michael Bedford for stimulating discussions and critical reading
of the manuscript, to David L.Rosenfeld, Gerald M.Scholl, and Avner
Hershlag, for contribution of clinical IVF results, to Asha Jacob and
Leslie O.Goodwin for contribution of unpublished findings on the
role of tyrosine phosphorylation in the activation of human sperm Ltype voltage-dependent calcium channels, to Francine S.Mandel
for statistical consultations, to Steven M.Schrader and Michael
J.Breitenstein for contribution of data concerning the sugar binding
affinities of rabbit spermatozoa, to Mary C.Mahony for the gift of
macaque spermatozoa, to David L.Keefe and Rudolf Oldenbourg for
contribution of polarized light micrograph of the hamster zona
pellucida, and to Michele Barcia and Stephanie Canaras for technical
assistance.
References
Aarons, D., Boettger-Tong, H., Holt, G. and Poirier, G.R. (1991) Acrosome
reaction induced by immunoaggregation of a proteinase inhibitor bound to
the murine sperm head. Mol. Reprod. Dev., 30, 258–264.
Aarons, D., Battle, T., Boettger-Tong, H. and Poirier, G.R. (1992) Role of
monoclonal antibody J-23 in inducing acrosome reactions in capacitated
mouse spermatozoa. J. Reprod. Fertil., 96, 49–59.
Abdullah, M. and Kierszenbaum, A.L. (1989) Identification of rat testis
galactosyl receptor using antibodies to liver asialoglycoprotein receptor:
purification and localization on surfaces of spermatogenic cells and sperm.
J. Cell Biol., 108, 367–375.
Abdullah, M., Widgren, E.E. and O’Rand, M.G. (1991) A mammalian sperm
lectin related to rat hepatocyte lectin-2/3. Mol. Cell. Biochem., 103, 155–161.
Aguas, A.P. and Pinto da Silva, P. (1989) Bimodal redistribution of surface
transmembrane glycoproteins during Ca21-dependent secretion (acrosome
reaction) in boar spermatozoa. J. Cell Sci., 93, 467–479.
Ahluwalia, B., Farshori, P., Jamuar, M. et al. (1990) Specific localization of
lectins in boar and bull spermatozoa. J. Submicrosc. Cytol. Pathol., 22,
53–62.
Ahuja, K.K. (1985) Carbohydrate determinants involved in mammalian
fertilization. Am. J. Anat., 174, 207–223.
Ahuja, K.K. and Bolwell, G.P. (1983) Probable asymmetry in the organization
of components of the hamster zona pellucida. J. Reprod. Fertil., 69, 49–55.
Anderson, D.J. and Alexander, N.J. (1983) A new look at antifertility vaccines.
Fertil. Steril., 40, 557–571.
Appeddu, P.A. and Shur, B.D. (1994) Molecular analysis of cell surface beta1,4-galactosyltransferase function during cell migration. Proc. Natl. Acad.
Sci. USA, 91, 2095–2099.
Arts, E.G.J.M., Kuiken, J., Jager, S. and Hoekstra, D. (1993) Fusion of
artificial membranes with mammalian spermatozoa. Specific involvement
of the equatorial segment after the acrosome reaction. Eur. J. Biochem.,
217, 1001–1009.
Arts, E.G.J.M., Jager, S. and Hoekstra, D. (1994) Evidence for the existence
of lipid-diffusion barriers in the equatorial segment of human sperm.
Biochem. J., 304, 211–218.
Austin, C.R. and Bradin, A.W.H. (1956) Early reactions of the rodent egg to
spermatozoa penetration. J. Exp. Biol., 33, 358–365.
Ayvaliotis, B., Bronson, R., Rosenfeld, D. and Cooper, G. (1985) Conception
rates in couples where autoimmunity to sperm is detected. Fertil. Steril.,
43, 739–742.
Bagavant, H., Fusi, F.M., Baisch, J. et al. (1997) Immunogenicity and
contraceptive potential of a human zona pellucida 3 peptide vaccine. Biol.
Reprod., 56, 754–770.
Bains, H.K., Sehgal, S. and Bawa, S.R. (1992) Human sperm surface mapping
with lectins. Acta Anat., 145, 207–211.
Barondes, S.H., Castronovo, V., Cooper, D.N.W. et al. (1994) Galectins: a
family of animal β-galactose-binding lectins. Cell, 76, 597–598.
Barratt, C.L.R., Whitmarsh, A., Hornby, D.P. et al. (1994) Glycosylation of
human recombinant ZP3 is necessary to induce the human acrosome
reaction. [Abstr. no. 033] Hum. Reprod., 9 (Suppl.).
Bearer, E.L. and Friend, D.S. (1982) Modifications of anionic-lipid domains
preceding membrane fusion in guinea pig. J. Cell Biol., 92, 604–615.
Bearer, E.L. and Friend, D.S. (1990) Morphology of mammalian sperm
membranes during differentiation, maturation, and capacitation. J. Electr.
Microsc. Tech., 16, 281–297.
Bedford, J.M. (1963) Changes in the electrophoretic properties of rabbit
spermatozoa during passage through the epididymis. Nature, 200, 1178–
1180.
Bedford, J.M. (1968) Ultrastructural changes in the sperm head during
fertilization in the rabbit. Am. J. Anat., 123, 329–357.
Bedford, J.M. (1971) Techniques and criteria used in the study of fertilization.
In Daniel, J.C. Jr. (ed.), Methods in Mammalian Embryology. W.H.Freeman
and Co, San Francisco, pp. 39–63.
Bedford, J.M. (1972) An electron microscopic study of sperm penetration into
the rabbit egg after natural mating. Am. J. Anat., 133, 213–253.
Bedford, J.M. (1977) Sperm/egg interaction: the specificity of human
spermatozoa. Anat. Rec., 188, 477–487.
627
S.Benoff
Bedford, J.M. (1991) The coevolution of mammalian gametes. In Dunbar,
B.S. and O’Rand, M.G. (eds.), A Comparative Overview of Mammalian
Fertilization. Plenum Press, New York, pp. 3–35.
Bedford, J.M., Moore, H.D.M. and Franklin, L.E. (1979) Signficance of the
equatorial segment of the acrosome of the spermatozoon in eutherian
mammals. Exp. Cell Res., 119, 119–126.
Beebe, S.J., Leyton, L., Burks, D. et al. (1992) Recombinant mouse ZP3
inhibits sperm binding and induces the acrosome reaction. Dev. Biol., 151,
48–54.
Benau, D.A. and Storey, B.T. (1987) Characterization of the mouse sperm
plasma membrane zona-binding site sensitive to trypsin inhibitors. Biol.
Reprod., 36, 282–292.
Benau, D.A. and Storey, B.T. (1988) Relationship between two types of mouse
sperm surface sites that mediate binding of sperm to the zona pellucida.
Biol. Reprod., 39, 235–244.
Benau, D.A., McGuire, E.J. and Storey, B.T. (1990) Further characterization
of the mouse sperm surface zona-binding site with galactosyltransferase
activity. Mol. Reprod. Dev., 25, 393–399.
Benoff, S. (1993) Preliminaries to fertilization. The role of cholesterol during
capacitation of human spermatozoa. Hum. Reprod., 8, 2001–2008.
Benoff, S., Cooper, G., Arnold, L. et al. (1992a) Sperm surface mannosespecific lectin: a molecular marker for capacitation events. [Abstr. no. 435]
In Scientific Program and Abstracts of the 39th Annual Meeting of the
Society for Gynecologic Investigation, p.326.
Benoff, S., Rushbrook, J.I., Hurley, I. et al. (1992b) Demonstration of
homology between testis-specific mannose-acceptor – a putative zona
recognition lectin and the human macrophage mannose-receptor lectin.
[Abstr. no. O26] In Scientific Program and Abstracts of the 48th Annual
Meeting of the American Fertility Society, p. S12.
Benoff, S., Cooper, G.W., Hurley, I. et al. (1993a) Antisperm antibody binding
to human sperm inhibits capacitation induced changes in the levels of
plasma membrane sterols. Am. J. Reprod. Immunol., 30, 113–130.
Benoff, S., Cooper, G.W., Hurley, I. et al. (1993b) Human sperm fertilizing
potential in vitro is correlated with differential expression of a head-specific
mannose-ligand receptor. Fertil. Steril., 59, 854–862.
Benoff, S., Hurley, I., Cooper, G.W. et al. (1993c) Fertilization potential
in vitro is correlated with head-specific mannose-ligand receptor expression,
acrosome status and membrane cholesterol content. Hum. Reprod., 8,
2155–2166.
Benoff, S., Hurley, I., Cooper, G.W. et al. (1993d) Head-specific mannoseligand receptor expression in human spermatozoa is dependent on
capacitation-associated membrane cholesterol loss. Hum. Reprod., 8,
2141–2154.
Benoff, S., Rushbrook, J.I., Hurley, I. et al. (1993e) Investigations into the
structure of a putative zona recognition molecule on the surface of
capacitated human sperm -α mannose receptor lectin. [Abstr. no. S220] In
Scientific Program and Abstracts of the 40th Annual Meeting of the Society
for Gynecologic Investigation, p. 178.
Benoff, S., Rushbrook, J.I., Hurley, I. et al. (1993f) Human sperm surface
putative zona recognition molecules are mannose-receptor lectins. [Abstr.
no. 7] In Program and Abstracts of the 18th Annual Meeting of the
American Society of Andrology, p. P-24.
Benoff, S., Barcia, M., Rushbrook, J.I. et al. (1994a) Evidence for a myosin
motor regulating movement of receptor proteins in the human sperm plasma
membrane. [Abstr. no. O-001] In Scientific Program and Abstracts of the
50th Annual Meeting of the American Fertility Society, p. S1.
Benoff, S., Cooper, G.W., Hurley, I. et al. (1994b) The effect of calcium ion
channel blockers on sperm fertilization potential. Fertil. Steril., 62, 606–617.
Benoff, S., Cooper, G.W., Hurley, I.R. and Hershlag, A. (1995a) Calcium-ion
channel blockers and sperm fertilization. Assist. Reprod. Rev., 5, 2–13.
Benoff, S., Rushbrook, J.I., Hurley, I.R. et al. (1995b) Co-expression of
mannose-ligand and non-nuclear progesterone receptors on motile human
sperm identifies an acrosome-reaction inducible subpopulation. Am.
J. Reprod. Immunol., 34, 100–115.
Benoff, S., Hurley, I.R., Mandel, F.S. et al. (1995c) Use of mannose ligands
in IVF screens to mimic zona pellucida-induced acrosome reactions and
phosphotyrosine kinase activation. [Abstr. no. 0-078] In Scientific Program
and Abstracts of the 51st Annual Meeting of the American Society for
Reproductive Medicine, p. S38–S39.
Benoff, S., Hurley, I.R., Mandel, F.S. et al. (1995d) Reproductive hazards
assay: the effect of acute exposure to metal ions on human sperm mannose
receptor expression. [Abstr. no. 0-001] In Scientific Program and Abstracts
of the 51st Annual Meeting of the American Society for Reproductive
Medicine, p. S1.
Benoff, S., Barcia, M, Hurley, I.R. et al. (1996a) Classifications of male factor
628
infertility relevant to IVF insemination strategies using mannose ligands,
acrosome status and anti-cytoskeletal antibodies. Hum. Reprod., 11,
1905–1911.
Benoff, S., Hurley, I.R., Barcia, M. et al. (1996b) Seminal plasma (SP) zinc
(Zn) levels can predict IVF fertilization outcome: regulation of acrosome
loss through an actin cytoskeleton. [Abstr. no. 0-083] In Scientific Program
and Abstracts of the 52nd Annual Meeting of the American Society for
Reproductive Medicine, p.S42–S43.
Benoff, S., Hurley, I.R., Barcia, M. et al. (1997a) A potential role for cadmium
in the etiology of varicocele-associated infertility. Fertil. Steril., 67,
331–342.
Bercegeay, S., Jean, M., Lucas, H. and Barriere, P. (1995) Composition of
human zona pellucida as revealed by SDS–PAGE after silver staining. Mol.
Reprod. Dev., 41, 355–359.
Berger, T., Davis, A., Wardrip, N.J. and Hedrick, J.L. (1989a) Sperm binding
to the pig zona pellucida and inhibition by solubilized components of the
zona pellucida. J. Reprod. Fertil., 86, 559–565.
Berger, T., Turner, K.O., Meizel, S. and Hedrick, J.L. (1989b) Zona pellucidainduced acrosome reaction in boar sperm. Biol. Reprod., 40, 414–442.
Berrios, M. and Bedford, J.M. (1979) Oocyte maturation: aberrant post-fusion
responses of the rabbit primary oocyte to penetrating spermatozoa. J. Cell
Sci., 39, 1–12.
Berruti, G. (1988) Calpactin-like proteins in human spermatozoa. Exp. Cell
Res., 179, 374–384.
Bielfeld, P., Faridi, A., Zaneveld, L.J. and DeJonge, C.J. (1994) The zona
pellucida-induced acrosome reaction of human spermatozoa is mediated by
protein kinases. Fertil. Steril., 61, 536–541.
Bleil, J.D. and Wassarman, P.M. (1980a) Mammalian sperm–egg interaction:
identification of a glycoprotein in mouse egg zonae pellucidae possessing
receptor activity for sperm. Cell, 20, 873–882.
Bleil, J.D. and Wassarman, P.M. (1980b) Synthesis of zona pellucida proteins
by denuded and follicle-enclosed mouse oocytes during culture in vitro.
Proc. Natl. Acad. Sci. USA, 77, 1029–1033.
Bleil, J.D. and Wassarman, P.M. (1980c) Structure and function of the zona
pellucida: identification and characterization of the proteins of the mouse
oocyte’s zona pellucida. Dev. Biol., 76, 185–202.
Bleil, J.D. and Wassarman, P.M. (1983) Sperm–egg interactions in the mouse:
sequence of events and induction of the acrosome by a zona pellucida
glycoprotein. Dev. Biol., 95, 317–324.
Bleil, J.D., Beall, C. and Wassarman, P.M. (1981) Mammalian sperm–egg
interaction: fertilization of mouse eggs triggers modification of the major
zona pellucida glycoprotein, ZP2. Dev. Biol., 86, 189–197.
Bleil, J.D. and Wassarman, P.M. (1986) Autoradiographic visualization of the
mouse egg’s sperm receptor bound to sperm. J. Cell Biol., 102, 1363–1371.
Bleil, J.D. and Wassarman, P.M. (1988) Galactose at the nonreducing terminus
of O-linked oligosaccharides of mouse egg zona pellucida glycoprotein
ZP3 is essential for the glycoprotein’s sperm receptor activity. Proc. Natl.
Acad. Sci. USA, 85, 6778–6782.
Bleil, J.D., Greve, J.M. and Wassarman, P.M. (1988) Identification of a
secondary sperm receptor in the mouse egg zona pellucida: role in
maintenance of binding of acrosome-reacted sperm to eggs. Dev. Biol.,
128, 376–385.
Bleil, J.D. and Wassarman, P.M. (1990) Identification of a ZP3-binding protein
on acrosome-intact mouse sperm by photoaffinity crosslinking. Proc. Natl.
Acad. Sci. USA, 87, 5563–5567.
Blithe, D.L. (1990) N-Linked oligosaccharides on free alpha interfere with its
ability to combine with human chorionic gonadotropin-beta subunit. J. Biol.
Chem., 265, 21951–21956.
Blobel, C.P., Wofsberg, T.G., Turck, C.W. et al. (1992) A potential fusion
peptide and an integrin ligand binding domain in a protein active in sperm–
egg fusion. Nature, 356, 248–252.
Boettger, H., Richardson, R., Free, D. et al. (1989) Effect of in vitro incubation
on a zona binding site found on murine spermatozoa. J. Exp. Zool., 249,
90–98.
Boettger-Tong, H., Aarons, D., Biegler, B. et al. (1992) Competition between
zonae pellucidae and a proteinase inhibitor for sperm binding. Biol. Reprod.,
47, 716–722.
Boettger-Tong, H.L., Aarons, D.J., Biegler, B.E. et al. (1993) Binding of a
murine protease inhibitor to the acrosome region of the human sperm head.
Mol. Reprod. Dev., 36, 346–353.
Bookbinder, L.H., Cheng, A. and Bleil, J.D. (1995) Tissue- and speciesspecific expression of sp56, a mouse sperm fertilization protein. Science,
269, 86–89.
Bork, P. and Sander, C. (1992) A large domain common to sperm receptors
(Zp2 and Zp3) and TGF-β type III receptor. FEBS Letts., 300, 237–240
Carbohydrates and fertilization: an overview
Brandelli, A., Miranda, P.V. and Tezón, J.G. (1994) Participation of
glycosylated residues in the human sperm acrosome reaction: possible role
of N-acetylglucosaminidase. Biochim. Biophys. Acta, 1220, 299–304.
Breitenstein, M.J. and Schrader, S.M. (1996) Mannose binding assay in Dutch
belted rabbits. [Abstr. no. 111] In Program and abstracts of the 21st Annual
Meeting of the American Society of Andrology, p. P-50.
Bronson, R.A. and McLaren, A. (1970) Transfer to the mouse oviduct of eggs
with and without the zona pellucida. J. Reprod. Fertil., 22, 129–137.
Bronson, R.A., Cooper, G.W. and Rosenfeld, D.L. (1984) Sperm antibodies:
their role in infertility. Fertil. Steril., 42, 171–183.
Bronson, R.A., Cooper, G.W. and Rosenfeld, D.L. (1982a) Correlation between
regional specificity of antisperm antibodies to the spermatozoan surface
and complement-mediated sperm immobilization. Am. J. Reprod. Immunol.,
2, 222–224.
Bronson, R.A., Cooper, G.W. and Rosenfeld, D.L. (1982b) Sperm-specific
isoantibodies and autoantibodies inhibit binding of human sperm to the
human zona pellucida. Fertil. Steril., 38, 724–729.
Bronson, R., Cooper, G., Rosenfeld, D. and Witkin, S. (1984) Detection of
spontaneously occurring sperm-directed antibodies in infertile couples by
immunobead binding and enzyme-linked immunosorbent assay. Ann. N.Y.
Acad. Sci., 438, 504–507.
Bronson, R.A. and Fusi, F. (1990) Sperm–oolemmal interaction: role of the
Arg-Gly-Asp (RGD) adhesion peptide. Fertil. Steril., 54, 527–529.
Brown, J., Cebra-Thomas, J.A., Bleil, J.D. et al. (1989) A premature acrosome
reaction is programmed by mouse t haplotypes during sperm differentiation
and could play a role in transmission ratio distortion. Development, 106,
769–773.
Burks, D.J., Carballada, R., Moore, H.D.M. and Saling, P.M. (1995) Interaction
of a tyrosine kinase from human sperm with the zona pellucida at
fertilization. Science, 269, 83–86.
Busacca, M., Fusi, F., Brigante, C. et al. (1989) Evaluation of antisperm
antibodies in infertile couples with immunobead test: prevalence and
prognostic value. Acta Eur. Fertil., 20, 77–82.
Cahova, M. and Draber, P. (1992) Inhibition of fertilization by a monoclonal
antibody recognizing the oligosaccharide sequence GalNAcB1-4GalB1-4
on the mouse zona pellucida. J. Reprod. Immunol., 21, 214–256.
Callow, J.A., Callow, M.E. and Evans, L.V. (1985). Fertilization in Fucus. In
Metz, B. and Monroy, A. (eds), Biology of Fertilization. Vol. 2. Academic
Press, New York, pp. 389–408.
Calvete, J.J., Sanz, L., Dostalova, Z. and Topfer-Petersen, E. (1995)
Spermadhesins: sperm-coating proteins involved in capacitation and zona
pellucida binding. Fertilität, 11, 35–40.
Canipari, R. and Strickland, S. (1986) Studies on the hormonal regulation of
plasminogen activator production in the rat ovary. Endocrinology, 118,
1652–1659.
Cardullo, R.A. and Wolf, D.E. (1995) Distribution and dynamics of mouse
sperm surface galactosyltransferase: implications for mammalian
fertilization. Biochemistry, 34, 10027–10035.
Carraway, K.L. and Hull, S.R. (1989) O-Glycosylation pathway for mucintype glycoproteins. BioEssays, 10, 117–121.
Cerezo, J.M.S., Beuno, M.I., Skowronski, B. and Cerezo, A.S. (1982)
Immunohistochemical localization of concanavalin A and wheat germ lectin
receptors in the normal human spermatozoa. Am. J. Reprod. Immunol., 2,
246–249.
Chamberlin, M.E. and Dean, J. (1990) Human homolog of the mouse sperm
receptor. Proc. Natl. Acad. Sci. USA, 87, 6014–6018.
Cheng, A., Le, T., Palacios, M. et al. (1994) Spermatozoa–egg recognition in
the mouse: characterization of sp56, a sperm protein having specific affinity
for ZP3. J. Cell Biol., 125, 867–878.
Cherr, G.N., Meyers, S.A., Yudin, A.I. et al. (1996) The PH-20 protein in
cynomologus macaque spermatozoa: identification of two different forms
of hyaluronidase activity. Dev. Biol., 175, 142–153.
Childs, R.A., Feizi, T. and Yuen, C.-T. (1990) Differential recognition of
core and terminal portions of oligosaccharide ligands by carbohydraterecognition domains of two mannose-binding proteins. J. Biol. Chem., 265,
20770–20777.
Clark, G.F., Patankar, M.S., Hinsch, K.D. and Oehninger, S. (1995) New
concepts in human sperm–zona pellucida interaction. Hum. Reprod., 10
(Suppl. 1), 31–37.
Clark, G.F., Oehninger, S. and Seppälä, M. (1996a) Role for glycoconjugates
in cellular communication in the human reproductive system. Mol. Hum.
Reprod., 2, 513–517.
Clark, G.F., Oehninger, S., Patankar, M.S. et al. (1996b) A role for
glycoconjugates in human development: the human feto–embryonic defense
system hypothesis. Hum. Reprod., 11, 467–473.
Clark, J.M. and Koehler, J.K. (1990) Observations of hamster sperm–egg
fusion in freeze-fracture replicas including the use of filipin as a sterol
marker. Mol. Reprod. Dev., 27, 351–365.
Cohen, J., Inge, K.L., Suzman, M. et al. (1989) Videocinematography of
fresh and cryopreserved embryos: a retrospective analysis of embryonic
morphology and implantation. Fertil. Steril., 52, 820–827.
Cohen, J., Alikani, M., Towbridge, J. and Rosenwaks, Z. (1992) Implantation
enhancement by selective assisted hatching using zona drilling of human
embryos with poor prognosis. Hum. Reprod., 7, 685–691.
Cooper, G.W. and Bedford, J.M. (1971) Acquisition of surface charge by
the plasma membrane of mammalian spermatozoa during epididymal
maturation. Anat. Rec., 169, 300–301.
Cooper, G.W. and Bronson, R.A. (1990) Characteriziation of humoral
antibodies reactive with spermatozoa, N-acetyl galactosamine, and a putative
blood group antigen in seminal plasma. Fertil. Steril., 53, 888–891.
Cooper, G.W., Zhu, J.Z. and Rosenfeld, D.L. (1991) Relationship between
antisperm IgG’s in female sera and galactosyl and mannosyl terminated
antigens in the human sperm surface. [Abstr. No. O-143] In Scientific
Program and Abstracts of the 47th Annual Meeting of the American
Fertility Society, p. S61.
Cornwall, G.A., Tulsiani, D.R.P. and Orbegin-Crist, M.-C. (1991) Inhibition
of the mouse sperm surface α-D-mannosidase inhibits sperm–egg binding
in vitro. Biol. Reprod., 44, 913–921.
Cowan, A.E., Primakoff, P. and Myles, D.G. (1986) Sperm exocytosis increases
the amount of PH-20 antigen on the surface of guinea pig sperm. J. Cell
Biol., 103, 1289–1297.
Cowan, A.E., Myles, D.G. and Koppel, D.E. (1987) Lateral diffusion of PH20 protein on guinea pig sperm: evidence that barriers to diffusion maintain
plasma membrane domains in mammalian spermatozoa. J. Cell Biol., 104,
917–923.
Cowan, A.E., Myles, D.G. and Koppel, D.E. (1991) Migration of the guinea
pig sperm membrane protein PH-20 from one localized surface domain to
another does not occur by a simple diffusion-trapping mechanism. Dev.
Biol., 144, 189–198.
Cross, N.L. (1996) Human seminal plasma prevents sperm from becoming
acrosomally responsive to the agonist, progesterone: cholesterol is the
major inhibitor. Biol. Reprod., 54, 138–145.
Cross, N.L. and Overstreet, J.W. (1987) Glycoconjugates of the human sperm
surface: distribution and alterations that accompany capacitation. Gamete
Res., 16, 23–35.
Cross, N.L., Morales, P., Overstreet, J.W. and Hanson, W.F. (1986) Two
simple methods for detecting acrosome-reacted human sperm. Gamete Res.,
15, 213–226.
Cruetz, C.E. (1992) The annexins and exocytosis. Science, 258, 924–931.
Cullis, P.R. and DeKruijff, B. (1979) Lipid polymorphism and the functional
roles of lipids in biological membranes. Biochim. Biophys. Acta, 559,
399–420.
Cunningham, D.S., Fulgham, D.L., Rayl, D.L. et al. (1991) Antisperm
antibodies to sperm surface antigens in women with genital tract infection.
Am. J. Obstet. Gynecol., 164, 791–796.
Dahms, N.M., Lobel, P. and Kornfeld, S. (1989) Mannose-6-phosphate
receptors and lysosomal enzyme targeting. J. Biol. Chem., 264, 12115–
12118.
D’Cruz, O.J., Haas, G.G. Jr. and Lambert, H. (1993) Heterogeneity of
human sperm surface antigens identified by indirect immunoprecipitation
of antisperm antibody bound to biotinylated sperm. J. Immunol., 151,
1062–1074.
Dean, J. (1992) Biology of mammalian fertilization: role of the zona pellucida.
J. Clin. Invest., 89, 1055–1059.
DeAngelis, P.L. and Glabe, C.G. (1987) Polysaccharide structural features
that are critical for the binding of sulfated fucans to bindin, the adhesive
protein from sea urchin sperm. J. Biol. Chem., 262, 13946–13942.
Dell, A., Morris, H.R., Easton, R.L. et al. (1995) Structural analysis of the
oligosaccharides derived from glycodelin, a human glycoprotein with
potent immunosuppressive and contraceptive activities. J. Biol. Chem., 270,
24116–24126.
Doherty, P., Fruns, M. Seaton, P. et al. (1990) A threshold effect of the major
isoforms of NCAM on neurite outgrowth. Nature, 343, 464–466.
Dostalova, Z., Calvete, J.J., Sanz, L. and Topfer-Petersen, E. (1994)
Quantitation of boar sperm adhesins in accessory sex gland fluids and on
the surface of epididymal, ejaculated and capaciated spermatozoa. Biochim.
Biophys. Acta, 1200, 48–54.
Dostalova, Z., Calvete, J.J. and Topfer-Petersen, E. (1995a) Interaction of
non-aggregated boar AWN-1 and AQN-3 with phospholipid matrices. A
629
S.Benoff
model for coating of spermadhesions to the sperm surface. Biol. Chem.
Hoppe-Seyler, 376, 237–242.
Dostalova, Z., Calvete, J.J., Sanz, L. and Topfer-Petersen, E. (1995b) Boar
sperm adhesion AWN-1. Oligosaccharide and zona pellucida binding
characteristics. Eur. J. Biochem., 230, 329–336.
Dravland, J.E., Llanos, M.N., Munn, R.J. and Meizel, S. (1984) Evidence for
the involvement of a sperm trypsinlike enzyme in the membrane events of
the hamster sperm acrosome reaction. J. Exp. Zool., 232, 117–128.
Drickamer, K. (1989) Multiple subfamilies of carbohydrate recognition
domains in animal lectins. CIBA Foundation Symp., 145, 45–58.
Drickamer, K. (1992) Engineering galactose-binding activity into a C-type
mannose-binding protein. Nature, 360, 183–186.
Drisdel, R.C., Mack, S.R., Anderson, R.A. and Zaneveld, L.J.D. (1995)
Purification and partial characterization of acrosome reaction inhibiting
glycoprotein from human seminal plasma. Biol. Reprod., 53, 201–208.
Drobnis, E.Z. and Katz, D.F. (1991) Videomicroscopy of mammalian
fertilization. In Wassarman, P.M. (ed.), Elements of Mammalian
Fertilization. Vol. 1.CRC Press Inc, Boca Raton, USA, pp. 269–300.
Drobnis, E.Z. (1993) Capacitation and the acrosome reaction. In Scialli, A.R.
and Zinaman, M.J. (eds), Reproductive Toxicology and Infertility. McGrawHill Inc, New York, pp. 77–132.
Drobnis, E.Z., Yudin, A.I., Cherr, G.N. and Katz, D.F. (1988) Hamster sperm
penetration of the zona pellucida: kinematic analysis and mechanical
implications. Dev. Biol., 130, 311–323.
Ducibella, T., Dubey, A., Gross, V. et al. (1995) A zona biochemical change
and spontaneous cortical granule loss in eggs that fail to fertilize in in vitro
fertilization. Fertil. Steril., 64, 1154–1161.
Dunbar, B.S., Prasad, S.V. and Timmons, T.M. (1991) Comparative structure
and function of mammalian zonae pellucidae. In Dunbar, B.S. and O’Rand,
M.G. (eds), A Comparative Overview of Mammalian Fertilization. Plenum
Press, New York, pp. 97–114.
Duncan, A.E. and Fraser, L.R. (1993) Cyclic AMP-dependent phosphorylation
of epididymal mouse sperm proteins during capacitation in vitro:
identification of an Mr 95000 phosphotyrosine-containing protein. J. Reprod.
Fertil., 97, 287–299.
East, I.J., Gulyas, B.J. and Dean, J. (1985) Monoclonal antibodies to the
murine zona pellucida protein with sperm receptor activity: effects on
fertilization and early development. Dev. Biol., 109, 268–273.
Edelman, G.M. and Millette, C.F. (1971) Molecular probes of spermatozoon
structures. Proc. Natl. Acad. Sci. USA, 68, 2436–2440.
Elbein, A.D. (1991) The role of N-linked oligosaccharides in glycoprotein
function. Trends Biotech., 9, 346–352.
Endo, Y., Mattei, P., Kopf, G.S. and Schultz, R.M. (1987) Effects of a phorbol
ester on mouse eggs: dissociation of sperm receptor activity from acrosome
reaction-inducing activity of the mouse zona pellucida protein, ZP3. Dev.
Biol., 123, 574–577.
Emiliozzi, C. and Fenichel, P. (1997) Protein phosphorylation is associated
with capacitation of human sperm in vitro but is not sufficient for its
completion. Biol. Reprod., 56, 674–679.
Eng, L.A. and Oliphant, G. (1978) Rabbit sperm reversible decapacitation by
membrane stabilization with a highly purified glycoprotein from seminal
plasma. Biol. Reprod., 19, 1083–1094.
Epifano, O. and Dean, J. (1994) Biology and structure of the zona pellucida:
a target for immunocontraception. Reprod. Fertil. Dev., 6, 319–330.
Erbe, D.V., Wolitzky, B.A., Presta, L.G. et al. (1992) Identification of an Eselectin region critical for carbohydrate recognition and cell adhesion.
J. Cell Biol., 119, 215–227.
Esaguy, N., Welch, J.E. and O’Rand, M.G. (1988) Ultrastructural mapping of
sperm plasma membrane autoantigen before and after the acrosome reaction.
Gamete Res., 19, 387–399.
Esponda, P. and Bedford, J.M. (1985) Epididymal fluid macromolecules do
not act as auto- or alloantigens. J. Androl., 6, 359–364.
Familiari, G., Nottola, S.A., Micara, G. et al. (1988) Is the sperm–binding
capability of the zona pellucida linked to its surface structure? A scanning
electron microscopic study of human in vitro fertilization. J. In Vitro Fertil.
Embryo Transfer, 5, 134–143.
Fayrer-Hosken, R.A., Caudle, A.B. and Shur, B.D. (1991) Galactosyltransferase
activity is restricted to the plasma membranes of equine and bovine sperm.
Mol. Reprod. Dev., 28, 74–84.
Fazeli, A.R., Stenweg, W., Bevers, M.M. et al. (1993) Development of a
sperm zona pellucida binding assay for bull semen. Vet. Rec., 132, 14–16.
Fazeli, A., Hage, W.J., Cheng, F.-P. et al. (1997) Acrosome-intact boar
spermatozoa initiate binding to the homologous zona pellucida in vitro.
Biol. Reprod., 56, 430–438.
630
Fei, W. and Hao, X. (1990) Wheat germ agglutinin (WGA) receptors on
human sperm membrane and male infertility. Arch. Androl., 24, 97.
Feinberg, J.M., Rainteau, D.P., Kaetzel, M.A. et al. (1991) Differential
localization of annexins in ram germ cells: a biochemical and
immunocytochemical study. J. Histochem. Cytochem., 39, 955–963.
Feng, W., Matzuk, M.M., Mountjoy, K. et al. (1995) The asparagine-linked
oligosaccharides of the human chorionic gonadotropin B subunit facilitate
correct disulfide bond pairing. J. Biol. Chem., 270, 11851–11859.
Feuchter, F.A., Tabet, A., Green, M. and Gresham, D. (1986) Exposure and
translocation of cytoskeletal-linked surface glycoproteins during
capacitation and the acrosome reaction in human sperm. Anat. Rec., 214, 37a.
Fierro, R., Foliguet, B., Grignon, G. et al. (1996) Lectin-binding sites on
human sperm during acrosome reaction: modifications judged by electron
microscopy/flow cytometry. Arch. Androl., 36, 187–196.
Fjallbrant, B. and Nilsson, S. (1977) Decrease of sperm antibody titre in
males, and conception after treatment of chronic prostatitis. Int. J. Fertil.,
22, 255–261.
Florman, H.M. (1994) Sequential focal and global elevations of sperm
intracellular Ca21 are initiated by the zona pellucida during acrosomal
exocytosis. Dev. Biol., 165, 152–164.
Florman, H.M. and Storey, B.T. (1982) Mouse gamete interactions: the zona
pellucida is the site of the acrosome reaction leading to fertilization in vitro.
Dev. Biol., 91, 121–130.
Florman, H.M. and Wassarman, P.M. (1985) O-Linked oligosaccharides of
mouse egg ZP3 account for its sperm receptor activity. Cell, 41, 313–324.
Florman, H.M. and First, N.L. (1988) Regulation of acrosomal exocytosis:
the zona pellucida-induced acrosome reaction of bovine spermatozoa is
controlled by extrinsic positive regulatory elements. Dev. Biol., 128,
464–473.
Florman, H.M., Bechtol, K.B. and Wassarman, P.M. (1984) Enzymatic
dissection of the functions of the mouse egg’s receptor for sperm. Dev.
Biol., 106, 243–255.
Franken, D.R., Kruger, T.F., Menkveld, R. et al. (1990) Hemizona assay and
teratozoospermia: increasing sperm insemination concentrations to enhance
zona pellucida binding. Fertil. Steril., 54, 497–503.
Fraser, L.R. (1984) Mouse sperm capacitation in vitro involves loss of a
surface-associated inhibitory component. J. Reprod. Fertil., 72, 373–384.
Friend, D.S., Orci, L., Perrelet, A. and Yanagimachi, R. (1977) Membrane
particle changes attending the acrosome reaction in guinea pig spermatozoa.
J. Cell Biol., 74, 561–577.
Fukada, M.N., Dell, A., Oates, J.E. and Fukada, M. (1985) Embryonal
lactosaminoglycan. J. Biol. Chem., 260, 6623–6631.
Fusi, F. and Bronson, R.A. (1990) Effects of incubation time in serum and
capacitation on spermatozoal reactivity with antisperm antibodies. Fertil.
Steril., 54, 887–893.
Fusi, F.M. and Bronson, R.A. (1992) Sperm surface fibronectin expression
following capacitation. J. Androl., 13, 28–35.
Fusi, F.M., Vignali, M., Busacca, M. and Bronson, R.A. (1992) Evidence for
an integrin cell adhesion receptor on the oolemma of unfertilized human
oocytes. Mol. Reprod. Dev., 31, 215–222.
Fusi, F.M., Vignali, M., Gailit, J. and Bronson, R.A. (1993) Mammalian
oocytes exhibit specific recognition of the RGD (Asp-Gly-Arg) tripeptide
and express oolemmal integrins. Mol. Reprod. Dev., 36, 212–219.
Gabriel, L.K., Franken, D.R., van der Horst, G. et al. (1994) Wheat germ
agglutinin receptors on human sperm membranes and sperm morphology.
Andrologia, 26, 5–8.
Gadella, B.M., Colenbrander, B., Van Golde, L.M. and Lopes-Cardozo, M.
(1993) Boar seminal vesicles secrete arylsulfatases into seminal plasma:
evidence that desulfation of seminolipid occurs only after ejaculation. Biol.
Reprod., 48, 483–489.
Gahmberg, C.G., Kotovuori, P. and Tontti, E. (1992) Cell surface carbohydrate
in cell adhesion. Sperm cells and leukocytes bind to their target cells
through specific oligosaccharide ligands. Acta Pathol. Microbiol. Immunol.
Scand., 100 (Suppl. 27), 39–52.
Gallagher, J.T. (1984) Carbohydrate-binding properties of lectins: a possible
approach to lectin nomenclature and classification. Biosci. Rep., 4, 621–632.
Gambaryan, A.S., Piskarev, V.E., Yamskov, I.A. et al. (1995) Human influenza
virus recognition of sialyloligosaccharides. FEBS Lett., 366, 57–60.
Garbers, D.L. (1989) Molecular basis of fertilization. Ann. Rev. Biochem., 58,
719–742.
Gerton, G.L. (1985) Biochemical studies of the envelope transformations in
Xenopus laevis eggs. In Hedrick, J.L. (ed.), The Molecular and Cellular
Biology of Fertilization. Plenum Press, New York, pp. 133–150.
Gilbert, B.R., Cooper, G.W. and Rosenfeld, D.L. (1991) Removal of sperm
Carbohydrates and fertilization: an overview
coating antigens by Percoll gradient centrifugation. [Abstr. no. O-126] In
Scientific Program and Abstracts of the 47th Annual Meeting of the
American Fertility Society, p. S54.
Gillis, G., Peterson, R., Russell, L. et al. (1978) Isolation and characterization
of membrane vesicles from human and boar spermatozoa: methods using
nitrogen cavitation and ionophore induced vesiculation. Prep. Biochem., 8,
363–378.
Glander, H.J., Herrman, K. and Haustein, U.F. (1987) The equatorial fibronectin
band (EFB) in human spermatozoa–a diagnostic help for male fertility?
Andrologia, 19, 456–459.
Glander, H.J., Schaller, J. and Dethloff, J. (1994) Influence of the epididymis
on integrins and matrix proteins of human spermatozoa. Acta Chir. Hung.,
34, 257–261.
Gmachl, M. and Kreil, G. (1993) Bee venom hyaluronidase is homologous to
a membrane protein of mammalian sperm. Proc. Natl. Acad. Sci. USA, 90,
3569–3573.
Go, K.J. and Wolf, D.P. (1983) The role of sterols in sperm capacitation. Adv.
Lipid Res., 20, 317–330.
Goluboff, E.T., Mertz, J.R., Tres, L.L. and Kierszenbaum, A.L. (1995)
Galactosyl receptor in human testis and sperm is antigenically related to
the minor C-type (Ca(21)-dependent) lectin variant of human and rat liver.
Mol. Reprod. Dev., 40, 460–466.
Gong, X., Dubois, D.H., Miller, D.J. and Shur, B.D. (1995) Activation of a
G protein complex by aggregation of beta-1,4,galactosyltransferase on the
surface of sperm. Science, 269, 1718–1721.
Goochee, C.F., Gramer, M.J., Andersen, D.C. et al. (1991) The oligosaccharides
of glycoproteins: bioprocess factors affecting oligosaccharide structure and
their effect on glycoprotein properties. Biotechnology, 9, 1347–1355.
Goodwin, L.O., Leeds, N.B., Hurley, I. et al. (1997) Isolation and
characterization of the primary structure of testis-specific L-type calcium
channel: implications for contraception. Mol. Hum. Reprod., 3, 255–268.
Greve, J.M. and Wassarman, P.M. (1985) Mouse extracellular coat is a matrix
of interconnected filaments possessing a structural repeat. J. Mol. Biol.,
181, 253–264.
Greve, J.M., Salzmann, G.S., Roller, R.J. and Wassarman, P.M. (1982)
Biosynthesis of the major zona pellucida glycoprotein secreted by oocytes
during mammalian oogenesis. Cell, 31, 749–759.
Gulyas, B.J. (1980) Cortical granules of mammalian eggs. Int. Rev. Cytol.,
63, 357–392.
Gwatkin, R.B.L., Williams, D.T., Hartmann, J.F. and Kniazuk, M. (1973) The
zona reaction of hamster and mouse eggs: production in vitro by a trypsinlike protease from cortical granules. J. Reprod. Fertil., 32, 259–265.
Haas, G.G.Jr., DeBault, L.E., D’Cruz, O. and Shuey, R. (1988) The effect of
fixatives and/or air-drying on the plasma and acrosomal membranes of
human sperm. Fertil. Steril., 50, 487–492.
Halberg, D.L., Wager, R.E., Farrell, D.C. et al. (1987) Major and minor forms
of the rat liver asialoglycoprotein receptor are independent galactosebinding proteins. J. Biol. Chem., 262, 9828–9838.
Han, H.L., Mack, S.R., DeJonge, C. and Zaneveld, L.J.D. (1990) Inhibition
of the human sperm acrosome reaction by a high molecular weight factor
from human seminal plasma. Fertil. Steril., 54, 1177–1179.
Han, K.K. and Martinage, A. (1993) Post-translational chemical modifications
of proteins. III. Current developments in analytical procedures for
identification and quantitation of post-translational chemically modified
amino acid(s) and its derivatives. Int. J. Biochem., 25, 957–970.
Hanqing, M., Tai-Ying, Y. and Zhao-Wen, S. (1991) Isolation, characterization,
and localization of the zona pellucida binding proteins of boar sperm. Mol.
Reprod. Dev., 28, 124–130.
Hardy, D.M. and Garbers, D.L. (1994) Species-specific binding of sperm
proteins to the extracellular matrix (zona pellucida) of the egg. J. Biol.
Chem., 269, 19000–19004.
Hardy, D.M. and Garbers, D.L. (1995) A sperm membrane protein that binds
in a species-specific manner to the egg extracellular matrix is homologous
to von Willebrand factor. J. Biol. Chem., 270, 26025–26028.
Harris, J.D., Hibler, D.W., Fonteno, G.K. et al. (1994) Cloning and
characteriziation of zona pellucida genes and cDNAs from a variety of
mammalian species: the ZPA, ZPB and ZPC gene families. DNA Sequence,
4, 361–393.
Hedrick, J.L. and Wardrip, N.J. (1986) Isolation of the zona pellucida and
purification of its glycoprotein families from pig oocytes. Anal. Biochem.,
157, 63–70.
Henkel, R., Cooper, S., Kaskar, K. et al. (1995) Influence of elevated pH
levels on structural and functional characteristics of the human zona
pellucida: functional morphological aspects. J. Assist. Reprod. Genet., 12,
644–649.
Hershlag, A., Cooper, G.W. and Benoff, S. (1995) Pregnancy following
discontinuation of a calcium channel blocker in the male partner. Hum.
Reprod., 10, 599–606.
Hershlag, A., Scholl, G., Rosenfeld, D.L. et al. (1996) Mannose ligand receptor
assay as a test to predict fertilization in vitro: a prospective study. [Abstr.
no. O-079] In Scientific Program and Abstracts of the 52nd Annual Meeting
of the American Society for Reproductive Medicine, pp. S40–S41.
Hindsgaul, O. and Ballou, C.E. (1984) Affinity purification of mycobacterial
polymethyl polysaccharides and a study of polysaccharide-lipid interactions
by 1H NMR. Biochemistry, 23, 577–584.
Hirano, T., Takasaki, S., Hedrick, J.L. et al. (1993) O-linked neutral sugar
chains of porcine zona pellucida glycoproteins. Eur. J. Biochem., 214,
763–769.
Hokke, C.H., Damm, J.B.L., Kamerling, J.P. and Vliegenthart, J.F.G. (1993)
Structure of three acidic O-linked carbohydrate chains of porcine zona
pellucida glycoproteins. FEBS Lett., 329, 29–34.
Hokke, C.H., Damm, J.B.L., Penninkhof, B. et al. (1994) Structure of the Olinked carbohydrate chains of porcine zona pellucida glycoproteins. Eur.
J. Biochem., 221, 491–512.
Holden, C.A., Hyne, R.V., Sathananthan, A.H. and Trounson, A.O. (1990)
Assessment of the human sperm acrosome reaction using Concanavalin A
lectin. Mol. Reprod. Dev., 25, 247–257.
Hoshi, K., Aita, T., Yanagida, K. et al. (1990) Variation in the cholesterol/
phospholipid ratio in human spermatozoa and its relationship with
capacitation. Hum. Reprod., 5, 71–75.
Howard, S.C., Wittwer, A.J. and Welply, J.K. (1991) Oligosaccharides at each
glycosylation site make structure-dependent contributions to biological
properties of human tissue plasminogen activator. Glycobiology, 1, 4175–
4180
Huang, T.T.F., Ohzu, E. and Yanagimachi, R. (1982) Evidence suggesting that
L-fucose is part of the recognition signal for sperm–zona pellucida
attachment in mammals. Gamete Res., 5, 355–361.
Huang, T.T.F., Fleming, A.D. and Yanagimachi, R. (1984) Only acrosomereacted spermatozoa can bind to and penetrate zona pellucida: a study of
the guinea pig. J. Exp. Zool., 217, 287–290.
Humphreys-Beher, M.G. and Blackwell, R.E. (1989) Identification of a
deoxyribonucleic acid allelic variant for β1,4 galactosyltransferase
expression associated with male sperm binding/penetration infertility. Am.
J. Obstet. Gynecol., 160, 1160–1165.
Humphreys-Beher, M.G., Garrison, P.W. and Blackwell, R.E. (1990) Detection
of antigalactosyltransferase antibodies in plasma from patients with
antisperm antibodies. Fertil. Steril., 54, 133–137.
Hunnicutt, G.R., Primakoff, P. and Myles, D.G. (1996) Sperm surface protein
PH-20 is bifunctional: one activity is a hyaluronidase and a second, distinct
activity is required for secondary sperm–zona binding. Biol. Reprod., 55,
80–86.
Huszar, G., Sbracia, M., Vigue, L. et al. (1997) Sperm plasma membrane
remodeling during spermiogenetic maturation in men: relationship among
plasma membrane β1,4-galactosyltransferase, cytoplasmic creatine
phosphokinase, and creatine phosphokinase isoform ratios. Biol. Reprod.,
56, 1020–1024.
Hynes, R. (1985) Molecular biology of fibronectin. Ann. Rev. Cell Biol., 1,
67–90.
Iobst, S.T., Wormald, MR., Weis, W.I. et al. (1994a) Binding of sugar ligands
to Ca21-dependent animal lectins. I. Analysis of mannose binding by sitedirected mutagenesis and NMR. J. Biol. Chem., 269, 15505–15511.
Iobst, S.T. and Drickamer, K. (1994b) Binding of sugar ligands to Ca(21)dependent animal lectins. II. generation of high-affinity galactose binding
by site-directed mutagenesis. J. Biol. Chem., 269, 15512–15519.
Isenberg, G. (1991) Actin binding proteins – lipid interactions. J. Muscle Res.
Cell Motil., 12, 136–144.
Isojima,S. (1989) Characterization of epitopes of seminal plasma antigens
stimulating human monoclonal antibody sperm–immobilizing antibodies: a
personal review. Reprod. Fertil. Dev., 1, 193–204.
Ji,I. and Ji,T.H. (1990) Differential interactions of human choriogonadotropin
and its antagonistic aglycosylated analog with their receptor. Proc. Natl.
Acad. Sci. USA, 87, 4396–4400.
Jonakova, V., Sanz, L., Calvete, J.J. et al. (1991) Isolation and biochemical
characterization of a zona pellucida-binding glycoprotein of boar sperm.
FEBS Lett., 280, 183–186.
Jones, R. (1989) Identification of carbohydrate binding proteins in mammalian
spermatozoa (human, bull, boar, ram, stallion and hamster) using
[125I]fucoidin and [125I]neoglycoprotein probes. Hum. Reprod., 4, 550–557.
Jones, R. (1991) Interaction of zona pellucida glycoproteins, sulphated
631
S.Benoff
carbohydrates and synthetic polymers with proacrosin, the putative eggbinding protein from mammalian spermatozoa. Development, 111, 1155–
1163.
Jones, R., Brown, C.R. and Lancaster, R.T. (1988) Carbohydrate-binding
properties of boar sperm proacrosin and assessment of its role in sperm–
egg recognition and adhesion during fertilization. Development, 102,
781–792.
Jones, R., Shalgi, R., Hoyland, J. and Phillips, D.M. (1990) Topographical
rearrangement of a plasma membrane antigen during capacitation of rat
spermatozoa in vitro. Dev. Biol., 139, 349–362.
Johnson, L.R., Pilder, S.H., Bailey, J. and Olds-Clarke, P. (1995) Sperm from
mice carrying one or two t haplotypes are deficient in investment and
oocyte penetration. Dev. Biol., 168, 138–149.
Kalab, P., Visconti, P., Leclerc, P. and Kopf, G.S. (1994) p95, the major
phosphotyrosine-containing protein in mouse spermatozoa, is a hexokinase
with unique properties. J. Biol. Chem., 269, 3810–3817.
Kallojoki, M., Malmi, R., Virtanen, I. and Suominen, J. (1985) Glycoconjugates
of human sperm surface. A study with fluorescent lectins and Lens culinaris
agglutination affinity chromatography. Cell Biol. Int. Rep., 9, 151–164.
Kaufman, M.H., Fowler, R.E., Barratt, E. and McDougall, R.D. (1989)
Ultrastructural and histochemical changes in the murine zona pellucida
during the final stages of oocyte maturation prior to ovulation. Gamete
Res., 24, 35–48.
Kawai, Y., Shimaji, H., Hama, T. and Mayumi, T. (1991) Studies on mouse
sperm lectin: the existence and the participation in sperm–egg binding.
J. Pharmacobiol-Dyn., 14, 244–249.
Keefe, D., Tran, P. and Oldenberg, R. (1996) Polarization microscopy and
digital image processing improves non-invasive imaging of oocytes and
identifies bilaminar structure of the zona pellucida. J. Soc. Gynecol. Invest.,
3 (Suppl.), 182A.
Keenan, J.A., Sacco, A.G., Subramanian, M.G. et al. (1991) Endocrine
response in rabbits immunized with native versus deglycosylated procine
zona pellucida. Biol. Reprod., 44, 150–156.
Kery, V., Krepinsky, J.J.F., Warren, C.D. et al. (1992) Ligand recognition by
purified human mannose receptor. Arch. Biochem. Biophys., 298, 49–55.
Kinloch, R.A., Mortillo, S., Stewart, C.L. and Wassarman, P.M. (1991)
Embryonal carcinoma cells transfected with ZP3 genes differentially
glycosylate similar polypeptides and secrete active mouse sperm receptor.
J. Cell Biol., 115, 655–664.
Kinloch, R.A., Sakai, Y. and Wassarman, P.M. (1995) Mapping the mouse
ZP3 combining site for sperm by exon mapping and site-directed
mutagenesis. Proc. Natl. Acad. Sci. USA, 92, 263–267.
Klentzeris, L.D., Fishel, S., McDermott, H. et al. (1995) Positive correlation
between expression of beta-1 integrin cell adhesion molecules and fertilizing
ability of human spermatozoa in vitro. Mol. Hum. Reprod., 1, see Hum.
Reprod. 10, 728–33.
Kobata, A. (1992) Structures and functions of the sugar chains of glycoproteins.
Eur. J. Biochem., 209, 483–501.
Kochibe, N. and Furukawa, K. (1980) Purification and properties of a
novel fucose-specific hemagglutinin of Aleuria aurantia. Biochemistry, 19,
2841–2846.
Koehler, J.K. (1981) Lectins as probes of the spermatozoon surface. Arch.
Androl., 6, 197–217.
Kong, M., Richardson, R.T., Widgren, E.E. and O’Rand, MG. (1995) Sequence
and localization of the mouse sperm autoantigenic protein, Sp17. Biol.
Reprod., 53, 579–590.
Kornfeld, R. and Kornfeld, S. (1985) Assembly of asparagine-linked
oligosaccharides. Ann. Rev. Biochem., 54, 631–664.
Kumar, G.P., Laloraya, M., Agrawal, P. and Laloraya, M.M. (1990) The
involvement of surface sugars of mammalian spermatozoa in epididymal
maturation and in vitro sperm–zona recognition. Andrologia, 22, 184–194.
Kurpisz, M. and Alexander, N.J. (1995) Carbohydrate moieties on the sperm
surface: physiological relevance. Fertil. Steril., 63, 158–165.
Kurpisz, M., Clark, G.F., Mahony, M. et al. (1989) Mouse monoclonal
antibodies against human sperm: evidence for immunodominant
glycosylated antigenic sites. Clin. Exp. Immunol., 78, 250–255.
Kusan, F.B., Fleming, A.D. and Seidel, G.E. (1984) Successful fertilization
in vitro of fresh intact oocytes by perivitelline (acrosome reacted)
spermatozoa of the rabbit. Fertil. Steril., 41, 766–770.
Lansford, B., Haas, G.G. Jr., Debault, L.E. and Wolf, D.P. (1990) Effect of
sperm-associated antibodies on the acrosomal status of human sperm.
J. Androl., 11, 532–538.
Lassalle, B. and Testart, J. (1994) Human zona pellucida recognition associated
with removal of sialic acid from human sperm surface. J. Reprod. Fertil.,
101, 703–711.
632
Lathrop, W.F., Carmichael, E.P., Myles, D.G. and Primakoff, P. (1990) cDNA
cloning reveals the molecular structure of a sperm surface protein, PH-20,
involved in sperm–egg adhesion and the wide distribution of its gene
among mammals. J. Cell Biol., 111, 2939–2949.
Lee, A.G. (1977) Lipid phase transitions and phase diagrams. I. Lipid phase
transitions. Biochim. Biophys. Acta, 472, 237–281.
Lee, M.C. and Damjanov, I. (1985) Lectin binding sites on human sperm and
spermatogenic cells. Anat. Rec., 212, 282–297.
Lee, R.T., Lin, P. and Lee, Y.C. (1984) New synthetic cluster ligands
for galactose/N-acetylgalactosamine-specific lectin of mammalian liver.
Biochemistry, 23, 4255–4261.
Lee, V.H. and Dunbar, B.S. (1993) Developmental expression of the rabbit
55-kDa zona pellucida protein and messenger RNA in ovarian follicles.
Dev. Biol., 155, 371–382.
Lee, Y.C. (1989) Binding modes of mammalian hepatic Gal/GalNAc receptors.
CIBA Foundation Symp., 145, 80–93.
Lee, Y.C. (1992) Biochemistry of carbohydrate–protein interaction. FASEB J.,
6, 3193–3200.
Lee, Y.C., Townsend, R.R., Hardy, M.R. et al. (1983) Binding of synthetic
oligosaccharides to the hepatic Gal/GalNAc lectin. Dependence on fine
structural features. J. Biol. Chem., 258, 199–202.
Leung, A.T., Imagawa, T. and Campbell, K.P. (1987) Structural characterization
of the 1,4 dihydropyridine receptor of the voltage-dependent Ca21 channel
from rabbit skeletal muscle. Evidence for two distinct high molecular
weight subunits. J. Biol. Chem., 262, 7943–7946.
Leyton, L. and Saling, P. (1989a) Evidence that aggregation of mouse sperm
receptors by ZP3 triggers the acrosome reaction. J. Cell Biol., 108,
2163–2168.
Leyton, L. and Saling, P. (1989b) 95 kDa sperm proteins bind ZP3 and serve
as tyrosine kinase substrates in response to zona binding. Cell, 57,
1123–1130.
Leyton, L., LeGuen, P., Bunch, D. and Saling, P. (1992) Regulation of mouse
gamete interaction by a sperm tyrosine kinase. Proc. Natl. Acad. Sci. USA,
89, 11692–11695.
Leyton, L., Tomes, C. and Saling, P. (1995) LL95 monoclonal antibody
mimics functional effects of ZP3 on mouse sperm: evidence that the antigen
recognized is not hexokinase. Mol. Reprod. Dev., 42, 347–358.
Liang, L.-F., Chamow, S.M. and Dean, J. (1990) Oocyte-specific expression
of mouse Zp-2: developmental regulation of the zona pellucida genes. Mol.
Cell. Biol., 10, 1507–1515.
Liang, L.-F. and Dean, J. (1993a) Conservation of mammalian secondary
sperm receptor genes enables the promoter of the human gene to function
in mouse oocytes. Dev. Biol., 156, 399–408.
Liang, L.-F. and Dean, J. (1993b) Oocyte development: molecular biology of
the zona pellucida. Vitam. Horm., 47, 115–159.
Liang, Z.G., O’Hern, P.A., Yavetz, B. et al. (1994) Human cDNAs identified
by sera from infertile patients: a molecular biological approach to
immunocontraceptive development. Reprod. Fertil. Dev., 6, 297–305.
Lin, Y., Kimmel, L.H., Myles, D.G. and Primakoff, P. (1993) Molecular
cloning of the human and monkey sperm surface protein PH-20. Proc.
Natl. Acad. Sci. USA, 90, 10071–10075.
Lin, Y., Mahan, K., Lathrop, W.F. et al. (1994) A hyaluronidase activity of
the sperm plasma membrane protein PH-20 enables sperm to penetrate the
cumulus cell layer surrounding the egg. J. Cell Biol., 125, 1157–1163.
Lingwood, C.A. (1985) Protein–glycolipid interactions during spermatogenesis: binding of specific germ cell proteins to sulfatoxygalactosylacylalkylglycerol, the major glycolipid of mammalian male germ
cells. Can. J. Biochem. Cell. Biol., 63, 1077–1085.
Lis, H. and Sharon, N. (1993) Protein glycosylation. Structural and functional
aspects. Eur. J. Biochem., 218, 1–27.
Litscher, E.S., Juntunen, K., Seppo, A. et al. (1995) Oligosaccharide constructs
with defined structures that inhibit binding of mouse sperm to unfertilized
egg in vitro. Biochemistry, 34, 4662–4669.
Litscher, E.S. and Wassarman, P.M. (1996) Characterization of a mouse ZP3derived glycopeptide, gp55, that exhibits sperm receptor and acrosome
reaction-inducing activity in vitro. Biochemistry, 35, 3980–3985.
Liu, C., Litscher, E.S. and Wassarman, P.M. (1995) Transgenic mice with
reduced numbers of functional sperm receptors on their eggs reproduce
normally. Mol. Biol. Cell, 6, 577–585.
Liu, C., Litscher, E.S., Mortillo, S. et al. (1996) Targeted disruption of the
mZP3 gene results in production of eggs lacking a zona pellucida and
infertility in female mice. Proc. Natl. Acad. Sci. USA, 93, 5431–5436.
Liu, D.Y. and Baker, H.W.G. (1994) Acrosome status and morphology of
human spermatozoa bound to the zona pellucida and oolemma determined
using oocytes that failed to fertilize in vitro. Hum. Reprod., 9, 673–679.
Carbohydrates and fertilization: an overview
Liu, M.S., Abersold, R., Fann, C.-H. and Lee, C.-Y.G. (1992) Molecular and
developmental studies of sperm acrosome antigen recognized by HS-63
monoclonal antibody. Biol. Reprod., 46, 937–948.
Llanos, M., Vigil, P., Salgado, A.M. and Morales, P. (1993) Inhibition of the
acrosome reaction by trypsin inhibitors and prevention of penetration of
spermatozoa through the human zona pellucida. J. Reprod. Fertil., 97,
173–178.
Lopez, L.C. and Shur, B.D. (1987) Redistribution of mouse sperm surface
galactosyltransferase after the acrosome reaction. J. Cell Biol., 105,
1663–1670.
Lopez, L.C., Youakim, A., Evans, S.C. and Shur, B.D. (1991) Evidence for
molecular distinction between Golgi and cell surface forms of B1,4galactosyltransferse. J. Biol. Chem., 266, 15984–15991.
Lopez-Casillas, F., Payne, H.M., Andres, J.L. and Massague, J. (1994)
Betaglycan can act as a dual modulator of TGF-β access to signaling
receptors: mapping of ligand binding and GAG attachment sites. J. Cell
Biol., 124, 557–568.
Loret de Mola, J.R., Garside, W.T., Bucci, J. et al. (1996) Age and hormonal
environment affect the thickness of the human zona pellucida. [Abstr. no.
O-036] In Scientific Program and Abstracts of the 52nd Annual Meeting
of the Americian Society for Reproductive Medicine, p. S19.
Lou, Y., Ang, J., Thai, H. et al. (1995) A ZP3 peptide vaccine induces
antibody and reversible infertility without ovarian pathology. J. Immunol.,
155, 2515–2720.
Lucas, H., Bercegeay, S., LePendu, J. et al. (1994) A fucose-containing epitope
involved in gamete interaction on the human zona pellucida. Hum. Reprod.,
9, 1532–1538.
Lucas, H., Le Pendu, J., Harb, J. et al. (1995) Identification of a spermatozoa
L-selectin and of 2 potential zona pellucida ligands. C.R. Acad. Sci. Paris,
Life Sci., 318, 795–801.
Luconi, M., Krausz, C., Forti, G. and Baldi, E. (1996) Extracellular calcium
negatively modulates tyrosine phosphorylation and tyrosine kinase activity
during capacitation of human spermatozoa. Biol. Reprod., 55, 207–216.
Macek, M.B., Lopez, L.C. and Shur, B.D. (1991) Aggregation of β-1,4galactosyltransferase on mouse sperm induces the acrosome reaction. Dev.
Biol., 147, 440–444.
Mack, S.R., Everingham, J. and Zaneveld, L.J.D. (1986) Isolation and partial
characterization of the plasma membrane from human spermatozoa. J. Exp.
Zool., 240, 127–136.
Mack, S.R., Zaneveld, L.J.D., Peterson, R.N. et al. (1987) Characterization of
human sperm plasma membrane: glycolipids and polypeptides. J. Exp.
Zool., 243, 339–346.
Mahi-Brown, C.A., Yanagimachi, R., Hoffman, J.C. and Huang, T.T.F. (1985)
Fertility control in the bitch by active immunization with porcine zona
pellucida: use of different adjuvants and patterns of estradiol and
progesterone levels in estrous cycles. Biol. Reprod., 32, 761–772.
Mahony, M.C., Oehninger, S., Clark, G.F. et al. (1991) Fucoidin inhibits
the zona pellucida-induced acrosome reaction in human spermatozoa.
Contraception, 44, 657–665.
Malmi, R., Kallojoki, M. and Suominen, J. (1987) Distribution of
glycoconjugates in human testis. A histochemical study using fluoresceinand rhodamine-conjugated lectins. Andrologia, 19, 322–332.
Margalioth, E.J., Cooper, G.W., Taney, F.H. et al. (1992) Capacitated sperm
cells react with different types of antisperm antibodies than fresh ejaculated
sperm. Fertil. Steril., 57, 393–398.
Marshall, R.D. (1972) Glycoproteins. Ann. Rev. Biochem., 41, 673–702.
Matzuk, M.M., Keene, J.L. and Boime, I. (1989) Site specificity of the
chorionic gonadotropin N-linked oligosaccharides in signal transduction.
J. Biol. Chem., 264, 2409–2414.
Maymon, B.B.-S., Maymon, R., Ben-Nun, I. et al. (1994) Distribution of
carbohydrates in the zona pellucida of human oocytes. J. Reprod. Fertil.,
102, 81–86.
McEver, R.P. (1994) Selectins. Curr. Opin. Immunol., 6, 75–84.
Mertz, J.R., Banda, P.W. annd Kierszenbaum, A.L. (1995) Rat sperm galactosyl
receptor: purification and identification by polyclonal antibodies raised
against multiple antigen peptides. Mol. Reprod. Dev., 41, 374–383.
Millar, S.E., Chamow, S.M., Baur, A.W. et al. (1989) Vaccination with a
synthetic zona pellucida peptide produces long-term contraception in female
mice. Science, 246, 935–938.
Miller, D.J. and Ax, R.L. (1990) Carbohydrates and fertilization. Mol. Reprod.
Dev., 26, 184–198.
Miller, D.J., Macek, M.B. and Shur, B.D. (1992) Complementarity between
sperm surface β-1,4-galactosyltransferase and egg-coat ZP3 mediates
sperm–egg binding. Nature, 357, 589–593.
Miller, D.J., Gong, X., Decker, G. and Shur, B.D. (1993) Egg cortical
granule N-acetylglucosaminidase is required for the mouse zona block to
polyspermy. J. Cell Biol., 123, 1431–1440.
Mladenovic, I., Hajdukovic, L., Genbacev, O. et al. (1993) Lectin binding as
a biological test in vitro for the prediction of functional activity of human
spermatozoa. Hum. Reprod., 8, 258–265.
Modlinski, J.A. (1970) The role of the zona pellucida in the development of
mouse eggs in vivo. J. Embryol. Exp. Morphol., 23, 539–547.
Moller, C.C. and Wassarman, P.M. (1989) Characterization of a proteinase
that cleaves zona pellucida glycoprotein ZP2 following activation of mouse
eggs. Dev. Biol., 132, 103–112.
Monroe, J.R., Altenbern, D.C. and Mathur, S. (1990) Changes in sperm
antibody test results when spermatozoa are subjected to capacitating
conditions. Fertil. Steril., 54, 1114–1120.
Moore, H.D.M. and Bedford, J.M. (1978) Ultrastructure of the equatorial
segment of hamster spermatozoa during penetration of oocytes.
J. Ultrastruct. Res., 62, 110–117.
Moore, H.D.M., Hartman, T.D., Bye, A.P. et al. (1987) Monoclonal antibody
against a sperm antigen Mr 95000 inhibits attachment of human spermatozoa
to the zona pellucida. J. Reprod. Immunol., 11, 157–166.
Moos, J., Kalab, P., Kopf, G.S. and Schiltz, R.M. (1994) Rapid, nonradioactive,
and quantitative method to analyze zona pellucida modifications in single
mouse eggs. Mol. Reprod. Dev., 38, 91–93.
Moos, J., Faundes, D., Kopf, G.S. and Schultz, R.M. (1995) Composition of
the human zona pellucida and modifications following fertilization. Mol.
Hum. Reprod., 1, see Hum. Reprod., 10, 2467–2471.
Morales, P., Cross, N.L. Overstreet, J.W. and Hanson, F.W. (1989) Acrosome
intact and acrosome-reacted human sperm can initiate binding to the zona
pellucida. Dev. Biol., 133, 385–392.
Morales, P., Vigil, P., Franke, D.R. et al. (1994) Sperm–zona interaction:
studies on the kinetics of zona pellucida binding and acrosome reaction of
human spermatozoa. Andrologia, 26, 131–137.
Morgentaler, A., Schopperle, W.M., Crocker, R.H. and DeWolf, W.C. (1990)
Protein differences between normal and oligospermic human sperm
demonstrated by two-dimensional gel electrophoresis. Fertil. Steril., 54,
902–905.
Mori, E., Takasaki, S., Hedrick, J.L. et al. (1991) Neutral oligosaccharide
structures linked to asparagines of porcine zona pellucida glycoproteins.
Biochemistry, 30, 2078–2087.
Mori, K., Daitoh, T., Irahara, M. et al. (1989) Significance of D-mannose as
a sperm receptor site on the zona pellucida in human fertilization. Am
J. Obstet. Gynecol., 161, 207–211.
Mori, K., Daitoh, T., Kamada, M. et al. (1993) Blocking of fertilization by
carbohydrates. Hum. Reprod., 8, 1729–1732.
Mortillo, S. and Wassarman, P.M. (1991) Differential binding of gold-labeled
zona pellucida glycoproteins mZP2 and mZP3 to mouse sperm membrane
compartments. Development, 113, 141–149.
Mortimer, D., Curtis, E.F. and Camenzind, A.R. (1990) Combined use of
fluorescent peanut agglutinin and Hoechst 33258 to monitor the acrosomal
status and vitality of human spermatozoa. Hum. Reprod., 5, 99–103.
Muramatsu, T., Gachelin, G., Nicolas, J.F. et al. (1978) Carbohydrate structure
and cell differentiation: unique properties of fucosyl-glycopeptides from
embryonal carcinoma cells. Proc. Natl. Acad. Sci. USA, 75, 2315–2319.
Myles, D.G. Hyatt, H. and Primakoff, P. (1987) Binding of both acrosomeintact and acrosome-reacted guinea pig sperm to the zona pellucida during
in vitro fertilization. Dev. Biol., 121, 559–567.
Myles, D.G., Kimmel, L.H., Blobel, C.P. et al. (1994) Identification of a
binding site in the disintegrin domain of fertilin required for sperm–egg
fusion. Proc. Natl. Acad. Sci. USA, 91, 4195–4198.
Myles, D.G. and Primakoff, P. (1984) Localized surface antigens of guinea
pig sperm migrate to new regions prior to fertilization. J. Cell Biol., 99,
1634–1641.
Myles, D.G. and Primakoff, P. (1997) Why did the sperm cross the cumulus?
To get to the oocyte. Functions of the sperm surface proteins PH-20 and
fertilin in arriving at, and fusing with, the egg. Biol. Reprod., 56, 320–327.
Naaby-Hansen, S. (1990) Electrophoretic map of acidic and neutral human
spermatozoal proteins. J. Reprod. Immunol., 17, 167–185.
Nagdas, S.K., Araki, Y., Chayko, C.A. et al. (1994) O-Linked trisaccharide
and N-linked polyacetyllactosaminyl glycans are present on mouse ZP2
and ZP3. Biol. Reprod., 51, 262–272.
Naito, K. Toyoda, Y. and Yanagimachi, R. (1992) Production of normal mice
from oocytes fertilized and developed without zonae pellucidae. Hum.
Reprod., 7, 281–285.
Naz, R.K. (1987a) Involvement of the fertilization antigen (FA-1) in involuntary
immunoinfertility in humans. J. Clin. Invest., 80, 1375–1383.
633
S.Benoff
Naz, R.K. (1987b) The fertilization antigen (FA-1) causes a reduction of
fertility in actively immunized female rabbits. J. Reprod. Immunol., 11,
117–133.
Naz, R.K. (1988) The fertilization antigen (FA-1): applications in immunocontraception and infertility in humans. Am. J. Reprod. Immunol., 16, 21–27.
Naz, R.K. (1990) Sperm surface antigens involved in mammalian fertilization.
Curr. Opin. Immunol., 2, 48–51.
Naz, R.K. and Ahmad, K. (1994) Molecular identities of human sperm proteins
that bind human zona pellucida: nature of sperm–zona interaction, tyrosine
kinase activity, and involvement of FA-1. Mol. Reprod. Dev., 39, 397–408.
Naz, R.K., Ahmad, K. and Kumar, R. (1991a) Role of membrane phosphotyrosine proteins in human spermatozoal function. J. Cell Sci., 99, 157–165.
Naz, R.K., Sacco, A.G. and Yurewicz, E.C. (1991b) Human spermatozoal FA1 binds with ZP3 of porcine zona pellucida. J. Reprod. Immunol., 20, 43–58.
Naz, R.K., Brazil, C. and Overstreet, J.W. (1992) Effects of antibodies to
sperm surface fertilization antigen-1 on human sperm–zona pellcuida
interaction. Fertil. Steril., 57, 1304–1310.
Naz, R.K., Alexander, N.J., Isahakia, M. and Hamilton, M.S. (1984)
Monoclonal antibody against human germ cell glycoprotein that inhibits
fertilization. Science, 225, 342–344.
Naz, R.K., Morte, C., Garcia-Framis, V. et al. (1993) Characterization of
sperm-specific monoclonal antibody and isolation of 95-kilodalton
fertilization antigen-2 from human sperm. Biol. Reprod., 49, 1236–1244.
Nevo, A.C., Michaeli, I. and Schindler, H. (1961) Electrophoretic properties
of bull and of rabbit spermatozoa. Exp. Cell Res., 23, 69–83.
Nicolson, G.L. (1974) The interactions of lectins with animal cell surfaces.
Int. Rev. Cytol., 39, 89–190.
Nicolson, G.L. and Yanagimachi, R. (1972) Terminal saccharides on sperm
plasma membranes: identification by specific agglutinins. Science, 177,
276–279.
Nicolson, G.L. and Yanagimachi, R. (1974) Mobility and the restriction of
mobility of plasma membrane lectin-binding components. Science, 184,
1294–1296.
Nicolson, G., Lacorbiere, M. and Yanagimachi, R. (1972) Quantitative
determination of plant agglutinin membrane sites on mammalian
spermatozoa. Proc. Soc. Exp. Biol. Med., 141, 661–663.
Nikas, G., Paraschos, T., Psychoyos, A. and Handyside, A.H. (1994) The
zona reaction in human oocytes seen with scanning electron microscopy.
Hum. Reprod., 9, 2135–2138.
Noguchi, S., Hatanaka, Y., Tobita, T. and Nakano, M. (1992) Structural
analysis of the N-linked carbohydrate chains of the 55-kDa glycoprotein
family (PZP3) from porcine zona pellucida. Eur. J. Biochem., 204, 1089–
1100.
Noguchi, S. and Nakano, M. (1993) Structural characterization of the Nlinked carbohydrate chains from mouse zona pellucida glycoproteins ZP2
and ZP3. Biochim. Biophys. Acta, 1158, 217–226.
Ochs, D., Wolf, D.P. and Ochs, R.L. (1986) Intermediate filament proteins in
human sperm heads. Exp. Cell Res., 167, 495–504.
Oehninger, S., Acosta, A. and Hodgen, G.D. (1990) Antagonistic and agonistic
properties of saccharide moieties in the hemizona assay. Fertil. Steril., 53,
143–149.
Oehninger, S., Clark, G.F., Acosta, A.A. and Hodgen, G.D. (1991) Nature of
the inhibitory effect of complex saccharide moieties on the tight binding
of human spermatozoa to the human zona pellucida. Fertil. Steril., 55,
165–169.
Oehninger, S., Mahony, M.C., Swanson, J.R. and Hodgen, G.D. (1993) The
specificity of human spermatozoa/zona pellucida interaction under hemizona
assay conditions. Mol. Reprod. Dev., 35, 57–61.
Oehninger, S., Coddington, C.C., Hodgen, G.D. and Seppälä, M. (1995)
Factors affecting fertilization: endometrial placental protein 14 reduces the
capacity of human spermatozoa to bind to the human zona pellucida. Fertil.
Steril., 63, 377–383.
Oikawa, T., Nicolson, G.L. and Yanagimachi, R. (1974) Inhibition of hamster
fertilization by phytoagglutinins. Exp. Cell Res., 83, 239–246.
Olds-Clarke, P., Pilder, P.H., Visconit, P.E. et al. (1996) Sperm from mice
carrying two t haplotypes do not possess a tyrosine phosphorylated form
of hexokinase. Mol. Reprod. Dev., 43, 94–104.
O’Rand, M.G. (1982) Modification of the sperm membrane during capacitation.
Ann. N.Y. Acad. Sci., 383, 392–402.
O’Rand, M.G. and Fisher, S.J. (1987) Localization of zona pellucida binding
sites on rabbit spermatozoa and induction of the acrosome reaction by
solublized zonae. Dev. Biol., 119, 551–559.
O’Rand,M.G. and Widgren, E.E. (1994) Identification of sperm antigen targets
634
for immunocontraception: B-cell epitope analysis of Sp17. Reprod. Fertil.
Dev., 6, 289–296.
O’Rand, M.G., Matthews, J.E., Welch, J.E. and Fisher, S.J. (1985) Identification
of zona binding proteins of rabbit, pig, human and mouse spermatozoa on
nitrocellulose blots. J. Exp. Zool., 235, 423–428.
O’Rand, M.G., Widgren, E.E. and Fisher, S.J. (1988) Characterization of the
rabbit sperm membrane autoantigen, RSA, as a lectin-like zona binding
protein. Dev. Biol., 129, 231–240.
O’Rand, M.G., Beavers, J., Widgren, E.E. and Tung, K.S. (1993) Inhibition
of fertility in female mice by immunization with a B-cell epitope, the
synthetic sperm peptide, P10G. J. Reprod. Immunol., 25, 89–102.
Overstreet, J.W. and Bedford, J.M. (1974) Comparison of the penetrability of
the egg vestements of follicular oocytes, unfertilized and fertilized ova of
the rabbit. Dev. Biol., 41, 185–192.
Overstreet, J.W., Lin, Y., Yudin, A.I. et al. (1995) Location of the PH-20 protein
on acrosome-intact and acrosome-reacted spermatozoa of cynomologus
macaques. Biol. Reprod., 52, 105–114.
Papahadjopoulos, D. (1978) Calcium-induced phase changes and fusion in
natural and model membranes. In Poste, G. and Nicolson, G.L. (eds),
Membrane Fusion. Elsevier/North-Holland Biomedical Press, Amsterdam,
pp. 765–790.
Parrish, J.J., Susko-Parrish, J.L., Handrow, R.R. et al. (1989) Capacitation of
bovine spermatozoa by oviductal fluid. Biol. Reprod., 40, 1020–1025.
Patankar, M.S., Oehninger, S., Barnett, T. et al. (1993) A revised structure for
fucoidin may explain some of its biological activities. J. Biol. Chem., 268,
21770–21776.
Paterson, M. and Aitken, R.J. (1990) Development of vaccines targeting the
zona pellucida. Curr. Opin. Immunol., 2, 753–747.
Paterson, M., Koothan, P.T., Morris, K.D. et al. (1992) Analysis of the
contraceptive potential of antibodies against native and deglycosylated
porcine ZP3 in vivo and in vitro. Biol. Reprod., 46, 523–534.
Patrizio, P., Bronson, R., Silber, S.J. et al. (1992) Testicular origin of
immunobead-reacting antigens in human sperm. Fertil. Steril., 57, 183–186.
Peterson, R.N. Russell, L.D., Bundman, D. and Freund, M. (1980) Sperm–
egg interactions: evidence for boar sperm plasma membrane receptors for
porcine zona pellucida. Science, 207, 73–74.
Peterson, R.N., Russell, L.D., Bundman, D. et al. (1981) The interaction of
living boar sperm and sperm plasma membrane vesicles with the porcine
zona pellucida. Dev. Biol., 84, 144–156.
Peterson, R.N., Russell, L.D., Hunt, W. et al. (1983) Characterization of boar
sperm plasma membranes by two-dimensional PAGE and isolation of
specific groups of polypepetides by anion exchange chromatography and
lectin affinity chromatography. J. Androl., 4, 71–81.
Peterson, R.N., Henry, L., Hunt, W. et al. (1985) Further characterization of
boar sperm plasma membrane proteins with affinity for the porcine zona
pellucida. Gamete Res., 12, 91–100.
Peterson, R.N., Gillott, M., Hunt, W. and Russell, L.D. (1987) Organization
of the boar spermatozoan plasma membrane: Evidence for separate domains
(subdomains) of integral membrane proteins in the plasma membrane
overlying the principal segment. J. Cell Sci., 88, 343–349.
Peterson, R.N. and Hunt, W.P. (1989) Identification, isolation, and properties
of a plasma membrane protein involved in the adhesion of boar sperm to
the porcine zona pellucida. Gamete Res., 23, 103–118.
Peterson, R.N., Bozzola, J.J., Hunt, W.P. and Darabi, A. (1990) Characterization
of membrane-associated actin in boar spermatozoa. J. Exp. Zool., 253,
202–214.
Peterson, R.N., Campbell, P., Hunt, W.P. and Bozzola, J.J. (1991) Twodimensional polyacrylamide gel electrophoresis characterization of APz, a
sperm protein involved in zona binding in the pig and evidence for its
binding to specific zona glycoproteins. Mol. Reprod. Dev., 28, 260–271.
Phelps, B.M., Primakoff, P., Koppel, D.E. et al. (1988) Restricted lateral
diffusion of PH-20, a PI-anchored sperm membrane protein. Science, 240,
1780–1782.
Phelps, B.M., Koppel, D.E., Primakoff, P. and Myles, D.G. (1990) Evidence
that proteolysis of the surface is an initial step in the mechanism of
formation of sperm cell surface domains. J. Cell Biol., 111, 1839–1847.
Philpott, C.C., Ringuette, M.J. and Dean, J. (1987) Oocyte-specific expression
and developmental regulation of ZP3, the sperm receptor of the mouse
zona pellucida. Dev. Biol., 121, 568–575.
Pillai, M.C. and Meizel, S. (1991) Trypsin inhibitors prevent the progesteroneinitiated increase in intracellular calcium required for the human sperm
acrosome reaction. J. Exp. Zool., 258, 384–393.
Poirier, G.R., Robinson, R., Richardson, R. et al. (1986) Evidence for a
binding site on the sperm plasma membrane which recognizes the murine
zona pellucida. Gamete Res., 14, 235–243.
Carbohydrates and fertilization: an overview
Pratt, S.A., Scully, N.F. and Shur, B.D. (1993) Cell surface beta 1,4
galactosyltransferase on primary spermatocytes facilitates their initial
adhesion to Sertoli cells in vitro. Biol. Reprod., 49, 470–482.
Primakoff, P., Hyatt, H. and Myles, D.G. (1985) A role for the migrating
sperm surface antigen PH-20 in guinea pig spermatozoa binding to the egg
zona pellucida. J. Cell Biol., 101, 2239–2244.
Primakoff, P., Cowan, A., Hyatt, H. et al. (1988) Purification of the guinea
pig sperm PH-20 antigen and detection of a site-specific endoproteolytic
cleavage activity in sperm preparations that cleaves PH-20 into two
disulfide-linked fragments. Biol. Reprod., 38, 921–934.
Primakoff, P., Lathrop, W. and Bronson, R. (1990) Identification of human
sperm surface glycoproteins recognized by autoantisera from immune
infertile men, women, and vasectomized men. Biol. Reprod., 42, 929–942.
Rankin, T., Familari, M., Lee, E. et al. (1996) Mice homozygous for an
insertional mutation in the Zp3 gene lack a zona pellucida and are infertile.
Development, 122, 2903–2910.
Reddy, J.M., Stark, R.A. and Zaneveld, L.J.D. (1978) A high molecular weight
antifertility factor from human seminal plasma. J. Reprod. Fertil., 57,
437–466.
Reid, K.B.M., Bentley, D.R., Campbell, R.D. et al. (1986) Complement system
proteins which interact with C3b or C4b: a superfamily of structurally
related proteins. Immunol. Today, 7, 230–234.
Renkonen, O. (1983) Polysaccharides of embryonal carcinoma cells of line
PC 13. Biochem. Soc. Tran., 11, 265–267.
Rhim, S.H., Millar, S.E., Robey, F. et al. (1992) Autoimmune disease of the
ovary induced by a ZP3 peptide from the mouse zona pellucida. J. Clin.
Invest., 89, 28–35.
Richardson, R., Buckingham, T., Boettger, H. and Poirier, G.R. (1987) A
monoclonal antibody to a zona pellucida-proteinase inhibitor binding
component on murine spermatozoa. J. Reprod. Immunol., 11, 101–116.
Richardson, R.T., Yamasaki, N. and O’Rand, M.G. (1994) Sequence of a
rabbit sperm zona pellucida binding protein and localization during the
acrosome reaction. Dev. Biol., 165, 688–701.
Ringuette, M.J., Sobieski, D.A., Chamow, S.M. and Dean, J. (1986) Oocytespecific gene expression: molecular characterization of a cDNA coding for
ZP-3, the sperm receptor of the mouse zona pellucida. Proc. Natl. Acad.
Sci. USA, 83, 4341–4345.
Ringuette, M.J., Chamberlin, M.E., Baur, A.W. et al. (1988) Molecular analysis
of cDNA coding for ZP3, a sperm binding protein of the mouse zona
pellucida. Dev. Biol., 127, 287–295.
Robertson, R.T., Bhalla, V.K. and Williams, W.L. (1971) Purification and the
peptide nature of decapacitation factor. Biochem. Biophys. Res. Commun.,
45, 1331–1336.
Robinson, R., Richardson, R., Hinds, K. et al. (1987) Features of a seminal
plasma proteinase inhibitor-zona pellucida binding component on murine
spermatozoa. Gamete Res., 16, 217–228.
Rogers, B.J., Bastias, C., Coulson, R.L. and Russell, L.D. (1989) Cytochalasin
D inhibits penetration of hamster eggs by guinea pig and human
spermatozoa. J. Androl., 10, 275–282.
Roller, R.J. and Wassarman, P.M. (1983) Role of asparagine-linked
oligosaccharides in secretion of glycoproteins of the mouse egg’s
extracellular coat. J. Biol. Chem., 258, 13243–13249.
Roller, R.J., Kinloch, R.A., Hiraoka, B.Y. et al. (1989) Gene expression during
mammalian oogenesis and early embryogenesis: quantification of three
messenger RNAs abundant in fully grown mouse oocytes. Development,
106, 251–261.
Rosati, F. (1985) Sperm–egg interactions in Ascidians. In Metz, C.B. and
Monroy, A. (eds), Biology of Fertilization. Vol. 2. Academic Press, New
York, pp. 361–388.
Rosiere, T.K. and Wassarman, P.M. (1992) Identification of a region of mouse
zona pellucida glycoprotein mZP3 that possesses sperm receptor activity.
Dev. Biol., 154, 309–317.
Russell, L.D., Peterson, R.N., Russell, T.A. and Hunt, W.P. (1983)
Electrophoretic map of boar sperm plasma membrane polypeptides and
localization and fractionation of specific polypeptide subclasses. Biol.
Reprod., 28, 393–413.
Sacco, A.G. (1977) Antigenic cross reactivity between human and pig zona
pellucida. Biol. Reprod., 16, 164–173.
Sacco, A.G., Yurewicz, E.C., Subramanian, M.G. and DeMayo, F.J. (1981)
Zona pellucida composition: Species cross-reactivity and contraceptive
potential of antiserum to a purified pig zona antigen (PPZA). Biol. Reprod.,
25, 997–1008.
Sacco, A.G., Yurewicz, E.C. and Subramanian, M.G. (1986) Carbohydrate
influences the immunogenic and antigenic characteristics of the ZP3
macromolecule (Mr 55000) of the pig zona pellucida. J. Reprod. Fertil.,
76, 575–586.
Sacco, A.G., Yurewicz, E.C., Subramanian, M.G. and Matzat, P.D. (1989)
Porcine zona pellucida: Association of sperm receptor activity with the
alpha glycoprotein component of the Mr 5 55,000 family. Biol. Reprod.,
41, 523–532.
Sadler, J.E. (1984) Biosynthesis of glycoproteins: formation of O-linked
oligosaccharides. In Ginsburg, V. and Robbins, P.W. (eds), Biology of
Carbohydrates. John Wiley and Sons, New York, pp. 199–288.
Sairam, M.R. (1989) Role of carbohydrates in glycoprotein hormone signal
transduction. FASEB J., 3, 1915–1926.
Sakai, Y., Araki, Y., Yamashita, T. et al. (1988) Inhibition of in vitro fertilization
by a monoclonal antibody reacting with the zona pellucida of the oviductal
egg but not that of the ovarian egg of the golden hamster. J. Reprod.
Immunol., 14, 177–190.
Saling, P.M. (1981) Involvement of trypsin-like activity in binding of mouse
spermatozoa to zonae pellucidae. Proc. Natl. Acad. Sci. USA, 78, 6231–6235.
Saling, P.M. and Storey, B.T. (1979) Mouse gamete interactions during
fertilization in vitro. J. Cell Biol., 83, 544–555.
Saling, P.M., Sowinski, J. and Storey, B.T. (1979) An ultrastructural study
of epididymal mouse spermatozoa binding to zonae pellucidae in vitro:
sequential relationship to the acrosome reaction. J. Exp. Zool., 209, 229–238.
Salzmann, G.S., Greve, J.M., Roller, R.J. and Wassarman, P.M. (1983)
Biosynthesis of the sperm receptor during oogenesis in the mouse. EMBO
J., 2, 1451–1456.
Sanz, L., Clavete, J.J., Mann, K. et al. (1991) The amino acid sequence of
AQN-3, a carbohydrate-binding protein isolated from boar sperm. FEBS
Lett., 291, 33–36.
Sanz, L., Calvete, J.J., Jonakova, Z. and Topfer-Petersen, E. (1992) Boar
spermadhesins AQN-1 and AWN are sperm-associated acrosin inhibitor
acceptor proteins. FEBS Lett., 300, 63–66.
Sanz, L., Calvete, J.J., Mann, K. et al. (1993) Isolation and biochemical
characterization of heparin-binding proteins from boar sperm seminal
plasma: a dual role for spermadhesins in fertilization. Mol. Reprod. Dev.,
35, 37–43.
Sappino, A.P., Huarte, J., Belin, D. and Vassalli, J.D. (1989) Plasminogen
activators in tissue remodeling and invasion: mRNA localization in mouse
ovaries and implanting embryos. J. Cell Biol., 109, 2471–2479.
Sato, M. and Muramatsu, T. (1985) Reactivity of five N-acetylgalactosaminerecognizing lectins with preimplantation embryos, early postimplanatation
embryos, and teratocarcinoma cells of the mouse. Differentiation, 29, 29–38.
Sathananthan, A.H. and Trounson, A.O. (1982) Ultrastructure of cortical
granule release and zona interaction in monospermic and polyspermic
human ova fertilized in vitro. Gamete Res., 6, 225–234.
Sathananthan, A.H. and Trounson, A.O. (1985) The human pronuclear ovum:
fine structure of monospermic and polyspermic fertilization in vitro. Gamete
Res., 12, 385–398.
Sathananthan, A.H., Trounson, A.O., Wood, C. and Leeton, J.F. (1982)
Ultrastructural observations on the penetration of human sperm the zona
pellucida of the human egg in vitro. J. Androl., 3, 356–364.
Saxena, N., Peteron, R.N., Sharif, S. et al. (1986a) Changes in the organization
of surface antigens during in-vitro capacitation of boar spermatozoa as
detected by monoclonal antibodies. J. Reprod. Fertil., 78, 601–614.
Saxena, N., Peterson, R.N., Saxena, N.K. and Russell, L.D. (1986b)
Microfilaments appear in boar spermatozoa during capacitation. J. Exp.
Zool., 239, 423–427.
Schachner,M. and Martini, R. (1995) Glycans and the modulation of neuralrecognition molecule function. Trends Neurosci., 18, 183–191.
Schaller, J., Glander, H.J. and Dethloff, J. (1993) Evidence of β1 integrins and
fibronectin on spermatogenic cells in human testis. Hum. Reprod., 8,
1873–1878.
Sela, B.-A., Wang, J.L. and Edelman, G.M. (1975) Antibodies reactive with
cell surface carbohydrates. Proc. Natl. Acad. Sci. USA, 72, 1127–1131.
Sensi, M., Pricci, F., Andreani, D. and Di Mario, U. (1991) Advanced nonenzymatic glycation products (AGE): their relevance to aging and the
pathogenesis of late diabetic complications. Diabetes Res., 16, 1–9.
Seppo, A., Penttila, L., Niemela, R. et al. (1995) Enzymatic synthesis of
octadecameric saccharides of multiply branched blood group I-type, carrying
four distal a1,3-galactose or B1,3-GlcNAc residues. Biochemistry, 34,
4655–4661.
Shabanowitz, R.B. (1991) Mouse antibodies to human zona pellucida: evidence
that human Zp3 is strongly immunogenic and contains two distinct isomer
chains. Biol. Reprod., 43, 260–270.
Shabanowitz, R.B. and O’Rand, M.G. (1988a) Characterization of the human
635
S.Benoff
zona pellucida from fertilized and unfertilized eggs. J. Reprod. Fertil., 82,
151–161.
Shabanowitz, R.B. and O’Rand, M.B. (1988b) Molecular changes in the
human zona pellucida associated with fertilization and human sperm–zona
interactions. Ann. N.Y. Acad. Sci., 541, 621–632.
Shalgi, R., Matityahu, A. and Nebel, L. (1986) The role of carbohydrates in
sperm–egg interactions in rats. Biol. Reprod., 34, 446–452.
Shalgi, R., Maymon, R., Bar-Shira, B. et al. (1991) Distribution of lectin
receptor sites in the zona pellucida of follicular and ovulated rat oocytes.
Mol. Reprod. Dev., 29, 365–372.
Sharon, N. (1993) Lectin–carbohydrate complexes of plants and animals: an
atomic view. Trends Biochem. Sci., 18, 221–226.
Sharon, N. and Lis, H. (1989) Lectins as cell recognition molecules. Science,
246, 227–246.
Shi, Q.-X. and Roldan, E.R.S. (1995) Bicarbonate/CO2 is not required for
zona pellucida or progesterone-induced acrosomal exocytosis of mouse
spermatozoa but is essential for capacitation. Biol. Reprod., 52, 540–546.
Shimizu, S., Tsuji, M. and Dean, J. (1983) In vitro biosynthesis of three
sulfated glycoproteins of murine zonae pellucidae by oocytes grown in
follicle culture. J. Biol. Chem., 258, 5858–5863.
Shur, B.D. (1991) Cell surface β-1,4galactosyltransferase: twenty years later.
Glycobiology, 1, 563–575.
Shur, B.D. and Bennett, D. (1979) A specific defect in galactosyltransferase
regulation on sperm bearing mutant alleles of the T/t locus. Dev. Biol., 71,
243–259.
Shur, B.D.and Hall, N.G. (1982a) Sperm surface galactosyltransferase activities
during in vitro capacitation. J. Cell Biol., 95, 567–573.
Shur, B.D. and Hall, N.G. (1982b) A role for mouse sperm surface galactosyltransferase in sperm binding to the egg zona pellucida. J. Cell Biol., 95,
574–579.
Shur, B.D. and Neely, C.A. (1988) Plasma membrane association, purification,
and partial characterization of mouse sperm surface β1,4 galactosyltransferase. J. Biol. Chem., 263, 17706–17714.
Shur, B.D. and Scully, N.F. (1990) Elevated galactosyltransferase activity on
t-bearing sperm segregates with T/t-complex distorter locus. Genet. Res.,
55, 177–181.
Siegelbaum, S.A. (1994) Ion channel control by tyrosine phosphorylation.
Curr. Biol., 4, 242–245.
Singer, R., Sagiv, M., Allaouf, D. et al. (1986) Separation of normozoospermic
human spermatozoa into a subpopulation by selective agglutination with
peanut agglutinin. Andrologia, 18, 17–24.
Singer, S.L., Lambert, H., Cross, N.L. and Overstreet, J.W. (1985) Alteration
of the human sperm surface during in vitro capacitation as assessed by
lectin-induced agglutination. Gamete Res., 12, 291–299.
Sim, R.B. and Malhotra, R. (1994) Interactions of carbohydrates and lectins
with complement. Biochem. Soc. Transact., 22, 106–111.
Sinha, S., Kumar, G.P. and Laloraya, M. (1994) Abnormal physical architecture
of the lipophilic domains of human sperm membrane in oligospermia: a
logical cause for low fertility profiles. Biochem. Biophys. Res. Commun.,
198, 266–273.
Skinner, S.M., Mills, T., Kirchick, H.J. and Dunbar, B.S. (1984) Immunization
with zona pellucida proteins results in abnormal ovarian follicular
differentiation and inhibition of gonadotropin-induced steroid secretion.
Endocrinology, 115, 2418–2432.
Snell, W.J. and White, J.M. (1996) The molecules of mammalian fertilization.
Cell, 85, 629–637.
Snow, K. and Ball, G.D. (1992) Characteriziation of human sperm antigens
and antisperm antibodies in infertile patients. Fertil. Steril., 58, 1011–1019.
Spungin, B., Margalit, I. and Breitbart, H. (1995) Sperm exocytosis
reconstructed in a cell-free system: evidence for the involvement of
phospholipase C and actin filaments in membrane fusion. J. Cell Sci., 108,
2525–2535.
Struck, D.K. and Lennarz, W.J. (1980) The function of saccharide-lipids in
synthesis of glycoproteins. In Lennarz, W.J. (ed.), The Biochemistry of
Glycoproteins and Proteoglycans. Plenum Press, New York, pp. 35–84.
Sullivan,R., Roose,P. and Berube,B. (1989) Immunodetectable galactosyltransferase is associated only with human spermatozoa of high buoyant
density. Biochem. Biophys. Res. Commun., 162, 184–188.
Summerfield, J.A. and Taylor, M.E. (1986) Mannose-binding proteins in
human serum: identification of mannose-specific immunoglobulins and a
calcium-dependent lectin, of broader carbohydrate specificity, secreted by
hepatocytes. Biochim. Biophys. Acta, 883, 197–203.
Suzuki, F. and Yanagimachi, R. (1989) Changes in the distribution of
intramembranous particles and filipin-reactive membrane sterols during
636
in vitro capacitation of golden hamster spermatozoa. Gamete Res., 23,
335–347.
Suzuki-Toyota, F., Maekawa, M., Cheng, A. and Bleil, J.D. (1995) Immunocolloidal gold labeled surface replica, and its application to detect sp56,
the egg recognition and binding protein, on the mouse spermatozoon.
J. Electron Microsc., 44, 135–139.
Talbot, P. and Chacon, R.S. (1982) Ultrastructural observations on binding
and membrane fusion between human sperm and zona pellucida-free
hamster oocytes. Fertil. Steril., 37, 240–248.
Tanphaichitr, N., Tayabali, A., Gradil, C. et al. (1992) Role for a germ
cell-specific sulfolipid-immobilizing protein (SLIP1) in mouse in vivo
fertilization. Mol. Reprod. Dev., 32, 17–22.
Tanphaichitr, N., Smith, J., Mongkolsirikieart, S. et al. (1993) Role of a
gamete-specific sulfoglycolipid immobilizing protein on mouse sperm–egg
binding. Dev. Biol., 156, 164–175.
Taylor, M.E. and Summerfield, J.A. (1987) Carbohydrate-binding proteins of
human serum: isolation of two mannose/fucose-specific lectins. Biochim.
Biophys. Acta, 915, 60–67.
Taylor, M.E., Bezouska, K. and Drickamer, K. (1992) Contribution to ligand
binding by multiple carbohydrate recognition domains in the macrophage
mannose receptor. J. Biol. Chem., 267, 1719–1726.
Tesarik, J., Drahoud, J. and Peknicova, J. (1988) Subcellular immunochemical
localization of acrosin in human spermatozoa during the acrosome reaction
and zona pellucida penetration. Fertil. Steril., 50, 133–141.
Tesarik, J., Mendoza, C. and Carreras, A. (1991) Expression of D-mannose
binding sites on human spermatozoa: comparison of fertile donors and
infertile patients. Fertil. Steril., 56, 113–118.
Tesarik, J., Mendoza, C. and Carreras, A. (1993a) Fast acrosome reaction
measure: a highly sensitive method for evaulating stimulus-induced
acrosome reaction. Fertil. Steril., 59, 424–430.
Tesarik, J., Mendoza, C., Ramirez, J.P. and Moos, J. (1993b) Solubilized
human zona pellucida competes with a fucosylated neoglycoprotein for
binding sites on the human sperm surface. Fertil. Steril., 60, 344–350.
Thaler, C.D. and Cardullo, R.A. (1995) Biochemical characterization of a
glycosylphosphatidylinositol-linked hyaluronidase on mouse sperm.
Biochemistry, 34, 7788–7795.
Thall, A.D., Malt, P. and Lowe, J.B. (1995) Occyte Gal alpha 1,3 Gal epitopes
implicated in sperm adhesion to the zona pellucida glycoprotein ZP3 are
not required for fertilization in the mouse. J. Biol. Chem., 270, 21437–21440.
Topfer-Petersen, E. and Henschen, A. (1987) Acrosin shows zona and fucose
binding, novel properties for a serine protease. FEBS Lett., 226, 38–42.
Tsang, R.S., Chan, K.H., Chau, P.Y. et al. (1987) A murine monoclonal antibody
specific for the outer core oligosaccharide of Salmonella lipopolysaccharide.
Infect. Immunol., 55, 211–216.
Tsuiji, A., Hoshiai, H., Takahashi, S. et al. (1986) Sperm–egg interaction
observed by scanning electron microscopy. Arch. Androl., 16, 35–47.
Tsuji, Y., Clausen, H., Nudelman, E. et al. (1988) Human sperm carbohydrate
antigens defined by antisperm human monoclonal antibody derived from
an infertile women bearing antisperm antibodies in her serum. J. Exp. Med.,
168, 343–356.
Tulsiani, D.R., Skudlarek, M.D. and Orgebin-Crist, M.C. (1989) Novel alphaD-mannosidase of rat sperm plasma membranes: characterization and
potential role in sperm–egg interactions. J. Cell. Biol., 109, 1257–1267.
Tulsiani, D.R., Skudlarek, M.D. and Orgebin-Crist, M.C. (1990) Human
sperm plasma membranes possess alpha-D-mannosidase activity but no
galactosyltransferase activity. Biol. Reprod., 42, 843–858.
Tulsiani, D.R.P., Nagdas, S.K., Cornwall, G.A. and Orgebin-Crist, M.-C.
(1992) Evidence for the presence of high mannose/hybrid oligosaccharide
chain(s) on the mouse ZP2 and ZP3. Biol. Reprod., 46, 93–100.
Tulsiani, D.R.P., NagDas, S.K., Skudlarek, M.D. and Orgebin-Crist, M.C.
(1995) Rat sperm plasma membrane mannosidase: localization and evidence
for proteolytic processing during epididymal maturation. Dev. Biol., 167,
584–595.
Tung, K.S.K. (1975) Human sperm antigens and antisperm antibodies. I.
Studies on vasectomy patients. Clin. Exp. Immunol., 20, 93–104.
VandeVoort, C.A., Tollner, T.L. and Overstreet, J.W. (1992) Sperm–zona
pellucida interaction in cynomologus and rhesus macaques. J. Androl., 13,
428–432.
Van Duin, M., Polman, J.E.M., De Breet, I.T.M. et al. (1994) Recombinant
human zona pellucida protein ZP3 produced by Chinese hamster ovary
cells induces the human sperm acrosome reaction and promotes sperm–
egg fusion. Biol. Reprod., 51, 607–617.
Varki, A. (1993) Biological roles of oligosaccharides: all of the theories are
correct. Glycobiology, 3, 97–130.
Carbohydrates and fertilization: an overview
Varki, A. (1994) Selectin ligands. Proc. Natl. Acad. Sci. USA, 91, 7390–7397.
Vasquez, J.M., Margargee, S.F., Kunze, E. and Hammerstedt, R.H. (1990)
Lectin and heparin-binding features of human spermatozoa as analyzed by
flow cytometry. Am. J. Obstet. Gynecol., 163, 2006–2012.
Villarroya, S. and Scholler, R. (1984) Localization of human sperm antigens
with monoclonal antibodies. Path. Biol. Paris, 32, 832–834.
Villarroya, S. and Scholler,R. (1986) Regional heterogeneity of human
spermatozoa detected with monoclonal antibodies. J. Reprod. Fertil., 76,
435–447.
Viriyapanich, P. and Bedford, J.M. (1981) Sperm capacitation in the Fallopian
tube of the hamster and its suppression by endocrine factors. J. Exp. Zool.,
217, 403–407.
Visconti, P.E., Bailey, J.L., Moore, G.D. et al. (1995a) Capacitation of mouse
spermatozoa. I. Correlation between the capacitation state and protein
tyrosine phosphorylation. Development, 121, 1129–1137.
Visconti, P.E., Moore, G.D., Bailey, J.L. et al. (1995b) Capacitation of mouse
spermatozoa. II. Protein tyrosine phosphorylation and capacitation are
regulated by a cAMP-dependent pathway. Development, 121, 1139–1150.
Visconti, P.E., Olds-Clarke, P., Moss, S.B. et al. (1996) Properties and
localization of a tyrosine kinase phosphorylated form of hexokinase in
mouse sperm. Mol. Reprod. Dev., 43, 82–93.
Vuento, M., Kuusela, P., Virkki, M. and Koskimies, A. (1984) Characterization
of fibronectin on human spermatozoa. Hoppe-Seylers Z. Physiol. Chem.,
365, 757–762.
Wakayama, T., Ogura, A., Suto, J. et al. (1996) Penetration by field vole
spermatozoa of mouse and hamster zonae pellucidae without acrosome
reaction. J. Reprod. Fertil., 107, 97–102.
Ward, C.R. and Kopf, G.S. (1993) Molecular events mediating sperm
activation. Dev. Biol., 158, 9–34.
Ward, C.R., Storey, B.T. and Kopf, G.S. (1994) Selective activation of Gi1
and Gi2 in mouse sperm by the zona pellucida, the egg’s extracellular
matrix. J. Biol. Chem., 269, 13254–13258.
Wassarman, P.M. (1987) The biology and chemistry of fertilization. Science,
235, 553–560.
Wassarman, P.M. (1988) Zona pellucida glycoproteins. Ann. Rev. Biochem.,
57, 415–442.
Wassarman, P.M. (1989) Role of carbohydrates in receptor-mediated
fertilization in mammals. CIBA Foundation Symp., 145, 135–149.
Wassarman, P.M. (1990) Profile of a mammalian sperm receptor. Development,
108, 1–17.
Wassarman, P.M. (1992) Mouse gamete adhesion molecules. Biol. Reprod.,
46, 186–191.
Wassarman, P.M., Bleil, J., Florman, H. et al. (1985a) The mouse egg’s
receptor for sperm: what is it and how does it work? Cold Spring Harbor
Symp. Quant. Biol., 50, 11–19.
Wassarman, P.M., Florman, H.M. and Greve, J.M. (1985b) Receptor mediated
sperm–egg interactions in mammals. In Metz, C.B. and Monroy, A. (eds),
Biology of Fertilization. Vol. 2. Academic Press, New York, pp. 341–360.
Wassarman, P.M. and Mortillo, S. (1991) Structure of the mouse egg
extracellular coat, the zona pellucida. Int. Rev. Cytol., 130, 85–109.
Wassarman, P.M. and Litscher, E.S. (1995) Sperm–egg recognition mechanisms
in mammals. Curr. Top. Dev. Biol., 30, 1–19.
Watkins, W.M. (1987) Biochemical genetics of blood group antigens: retrospect
and prospect. Biochem. Soc. Transact., 15, 620–624.
Welch, J.E. and O’Rand, M.G. (1985) Identification and distribution of actin
in spermatogenic cells and spermatozoa of the rabbit. Dev. Biol., 109,
411–417.
White, D.R., Phillips, D.M. and Bedford, J.M. (1990) Factors affecting the
acrosome reaction in human spermatozoa. J. Reprod. Fertil., 90, 71–80.
Williams, R.M. and Jones, R. (1993) Specificity of binding of zona pellucida
glycoproteins to sperm proacrosin and related proteins. J. Exp. Zool., 266,
65–73.
Wilson, I.B.H., Gavel, Y. and Von Heijne, G. (1991) Amino acid distributions
around O-linked glycosylation sites. Biochem. J., 275, 529–534.
Witkin, S.S. and Toth, A. (1983) Relationship between genital tract infections,
sperm antibodies in seminal fluid, and infertility. Fertil. Steril., 40, 805–808.
Wittwer, A.J. and Howard, S.C. (1990) Glycosylation at Asn-184 inhibits
conversion of single-chain to two-chain tissue-type plasminogen activator
by plasmin. Biochemistry, 29, 4175–4180.
Wittwer, A.J., Howard, S.C., Carr, L.S. et al. (1989) Effects of N-glycosylation
on in vitro activity of Bowes melanoma and human colon fibroblast derived
tissue plasminogen activator. Biochemistry, 28, 7662–7669.
Wolf, D.E., Lipscomb, A.C. and Maynard, V.M. (1988) Causes of nondiffusing
lipid in the plasma membrane of mammalian spermatozoa. Biochemistry,
27, 860–865.
Wolf, D.P. (1996) Sperm–egg interaction. In Centola, G.M. and Ginsburg,
K.A. (eds), Evaluation and Treatment of the Infertile Male. Cambridge
University Press, Cambridge, UK, pp. 6–18.
Wollina, U., Schrieber, G., Zollman, C. et al. (1989a) Lectin- binding sites in
normal human testis. Andrologia, 21, 127–130.
Wollina, U., Schreiber, G., Zollman, C. and Hipler, C. (1989b) Testicular
lectin histochemistry in the Sertoli-Cell-only-Syndrome. Andrologia, 21,
555–558.
Wright, R.M., John, E., Klotz, K. et al. (1990) Cloning and sequencing of
cDNAs encoding for the human intra-acrosome antigen SP-10. Biol.
Reprod., 42, 693–701.
Xu, C., Rigney, D.R. and Anderson, D.J. (1994) Two-dimensional
electrophoretic profile of human sperm membrane proteins. J. Androl., 15,
595–602.
Yamasaki, N., Richardson, R.T. and O’Rand, M.G. (1995) Expression of the
rabbit sperm protein Sp17 in COS cells and interaction of recombinant
Sp17 with the rabbit zona pellucida. Mol. Reprod. Dev., 40, 48–55.
Yamashita, K., Kochibe, N., Ohkura, T. et al. (1985) Fractionation of Lfucose-containing oligosaccharides on immobilized Aleuria aurantia lectin.
J. Biol. Chem., 260, 4688–4693.
Yamashita, K., Totani, K., Ohkura, T. et al. (1987) Carbohydrate binding
properties of complex-type oligosaccharides on immobilized Datura
stramonium lectin. J. Biol. Chem., 262, 1602–1607.
Yamashita, K., Umetsu, K., Suzuki, T. et al. (1988) Carbohydrate binding
specificity of immobilized Allomyrina dichotoma lectin II. J. Biol. Chem.,
263, 17482–17489.
Yanagimachi, R. (1977) Specificity of sperm–egg interaction. In Edidin, M
and Johnson, M.H. (eds), Immunobiology of Gametes. Cambridge University
Press, Cambridge, UK, pp. 187–207.
Yanagimachi, R. (1994) Mammalian fertilization. In Knobil, E. and Neill,
J.D. (eds), The Physiology of Reproduction. 2nd edn. Raven Press Ltd,
New York, pp. 189–317.
Yanagimachi, R, Noda, Y.D., Fujimoto, M. and Nicolson, G.L. (1972) The
distribution of negative surface charges on mammalian spermatozoa. Am.
J. Anat., 135, 497–520.
Yanagimachi, R. and Suzuki, F. (1985) A further study of lysolecithinmediated acrosome reaction of guinea pig spermatozoa. Gamete Res., 11,
29–40.
Yednock, T.A. and Rosen, S.D. (1989) Lymphocyte homing. Adv. Immunol.,
44, 313–378.
Yonezawa, N., Hatanaka, Y., Takeyama, H. and Nakano, M. (1995) Binding
of pig sperm receptor in the zona pellucida to the boar sperm acrosome.
J. Reprod. Fertil., 103, 1–8.
Youakim, A., Hathaway, H.J., Miller, D.J. et al. (1994) Overexpressing sperm
surface β1,4-galactosyltransferase in transgenic mice affects multiple
aspects of sperm–egg interactions. J. Cell Biol., 126, 1573–1583.
Yurewicz, E.C., Sacco, A.G. and Subramanian, M.G. (1987) Structural
characterization of the Mr 5 55,000 antigen (ZP3) of porcine oocyte zona
pellucida. J. Biol. Chem., 262, 564–571.
Yurewicz, E.C., Pack, B.A. and Sacco, A.G. (1991) Isolation, composition,
and biological activity of sugar chains of porcine oocyte zona pellucida
55K glycoproteins. Mol. Reprod. Dev., 30, 126–134.
Yurewicz, E.C., Pack, B.A. and Sacco, A.G. (1992) Porcine oocyte zona
pellucida Mr 55,000 glycoproteins: identification of O-glycosylated
domains. Mol. Reprod. Dev., 33, 182–188.
Yurewicz, E.C., Pack, B.A., Armant, D.R. and Sacco, A.G. (1993) Porcine
zona pellucida ZP3α glycoprotein mediates binding of the biotin-labeled
Mr 55,000 family (ZP3) to boar sperm membrane vesicles. Mol. Reprod.
Dev., 36, 382–389.
Zeng, Y., Oberdorf, J.A. and Florman, H.M. (1996) pH regulation in mouse
sperm: identification of Na1-,Cl–, and HCO3-dependent and
arylaminobenzoate-dependent regulatory mechanisms and characterization
of their roles in sperm capacitation. Dev. Biol., 173, 510–520.
Zhang, R., Cai, H., Fatima, N. et al. (1995) Functional glycosylation sites of
the rat luteinizing hormone receptor required for ligand binding. J. Biol.
Chem., 270, 21722–21728.
Zhixing, P. and Yifei, W. (1990) Quantification and localization of wheat
germ agglutinin receptor on human sperm membrane and male fertility and
infertility. Arch. Androl., 24, 103.
Zouari, R., DeAlmeida, M., Rodrigues, D. and Jouannet, P. (1993) Localization
of antibodies on spermatozoa and sperm movement characteristics are good
predictors of in vitro fertilization success in cases of male autoimmune
infertility. Fertil. Steril., 59, 606–612.
Received on September 12, 1996; accepted on May 15, 1997
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