Download The glycocalyx of the sperm surface

E European Society of Human Reproduction and Embryology
Human Reproduction Update 1999, Vol. 5, No.4 pp. 302–313
The glycocalyx of the sperm surface
Sabine Schröter, Caroline Osterhoff, Wendy McArdle and Richard Ivell1
IHF Institute for Hormone and Fertility Research, University of Hamburg, Grandweg 64, 22529 Hamburg, Germany
The surface of mammalian spermatozoa is covered by a dense coating of carbohydrate-rich molecules forming a
20–60 nm thick glycocalyx. The majority of sugar residues are attached to proteins which are either integrated within the
sperm membrane, or are more or less loosely associated with it. It is estimated that there may be several hundred
different glycoproteins comprising the glycocalyx, some of which are synthesized within the testis. Others, however, are
produced by the epithelia of the efferent ducts, epididymis and possibly other accessory glands, and become associated
with the spermatozoa post-testicularly during transit through, and storage in, the male tract. The acquisition of the
mature glycocalyx is associated with the attainment of full sperm fertilizing ability. Until its complete molecular
structure is elucidated, the complex function of the glycocalyx remains obscure, though it may be related to membrane
maturation and immunoprotection in the female tract, as well as to sperm–zona binding and fertilization.
Key words: cloned human antigens/epididymis/glycoconjugates/sperm maturation/sperm surface
TABLE OF CONTENTS
Introduction
Structure and maturation of the sperm glycocalyx
Origin and regulation of the glycoconjugates on the
sperm surface
Analysis of specific proteins forming part of the human
sperm glycocalyx
Glycosyl-modifying enzymes
The function of the sperm glycocalyx
Conclusion
Acknowledgements
References
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Introduction
The mammalian spermatozoon is a highly specialized
epithelial cell whose main function exists free of its generative
epithelial layer. It undergoes meiotic and mitotic differentiation
within the testis, thereby acquiring a large number of proteins
uniquely expressed in male germ cells. Before leaving the
testis, ~90–95% of the cytoplasm is jettisoned in the form of the
cytoplasmic droplet. The resulting gamete has been subjected
to very rigorous natural selection to design a vehicle capable of
transporting the haploid male nucleus to the oviduct of a
recipient female coincident with the presence there of a mature
oocyte, and to penetrate and fertilize that oocyte. The male
gamete must therefore be specialized to be stored within the
male tract for periods of time varying from several hours to
several weeks; it must be able to penetrate the mucus secretions
1To
whom correspondence should be addressed
of the female reproductive tract and retain and metabolize
sufficient energy to reach the oocyte in the oviduct; and finally
it must be equipped with selective mechanisms to bind and
penetrate the zona pellucida. It then has to fuse with and
traverse the oocyte plasmalemma and undergo nuclear
decondensation and zygote formation. Additionally, it has to be
resilient to the immune defence mechanisms of the female
tract, yet offer selection possibilities to sort good from poor
quality spermatozoa, and be furnished with mechanisms which
would prohibit interspecies fertilization. A number of these
functions are also shared with certain small invading parasites,
such as trypanosomes. It may therefore be helpful also to look
at these organisms to identify which sperm components and
mechanisms may be involved in some of these very special
functions (McConville and Ferguson, 1993).
It has been estimated that the outer membrane of ejaculated
human spermatozoa includes at least 300 different proteins, as
detectable by vectorial labelling of intact spermatozoa with 125I
or biotin (Naaby Hansen et al., 1997). Some of these are
glycosylated and go to form what is generally referred to as the
sperm glycocalyx. Additionally, the glycocalyx comprises
carbohydrate residues linked to lipids and other molecular
structures. Not all such molecules are tightly bound to the sperm
surface membrane. Whereas some component moieties are
intercalated or anchored within the lipid bilayer [e.g.
transmembrane- or glycosylphosphatidylinositol (GPI)-anchored
proteins], others are only superficially associated with the
membrane via polar groups (e.g. protein–protein or lectin–sugar
interactions) or by hydrophobic interaction. The majority (92%)
of extracellular membrane proteins of all cells are glycosylated,
The sperm glycocalyx
as a search in 1996 of the SWISSPROT protein database was
able to show (Gahmberg and Tolvanen, 1996). From
experiments using a variety of different methodologies (e.g.
N-glycosidase F- and neuraminidase digestion, lectin-binding or
periodate oxidation; reviewed in Benoff, 1997) it would appear
that, also for spermatozoa, carbohydrates dominate the
extracellular surface. Taking these observations together, it seems
that almost all interactions between the spermatozoon and its
environment must initially involve an interaction with the sperm
glycocalyx. The aim of this review is to focus on certain aspects
of the structure and function of the sperm glycocalyx with
particular relevance to the human.
Structure and maturation of the sperm
glycocalyx
The sperm glycocalyx represents the interface between the
male gamete and the extracellular milieu. The female gamete is
also encompassed by a heavily glycosylated matrix, the zona
pellucida. However, unlike the oocyte zona pellucida, the
sperm glycocalyx appears to be quite different both in
dimensions and complexity. Whereas the thickness of the zona
pellucida varies from 1–2 µm in marsupials to 16 µm in the pig
(Yanagimachi, 1994), the glycocalyx of the spermatozoon
appears to be considerably thinner. In the guinea pig, electron
microscopic analysis of sectioned sperm heads shows a
heteromorph layer varying in thickness from 20 to 60 nm
apposed to the plasma membrane (Bearer and Friend, 1990).
For comparison, the typical glycocalyx of a human erythrocyte,
as an example of a typical somatic cell, extends only about
10 nm from the plasma membrane, itself being about 7 nm in
thickness (Rademacher et al., 1988). Also, the sperm
glycocalyx differs in complexity from the glycosylated
investment of the oocyte. Only three glycoproteins (ZP1, ZP2
and ZP3; Yanagimachi, 1994) make up the main part of the
zona pellucida, whereas it seems probable that the sperm
glycocalyx comprises somewhere between 50 and 150
different glycoconjugates.
Additionally, unlike the zona pellucida, numerous studies
have shown that the sperm surface is not homogeneous but is
composed of different functional domains, reflecting a
distinctive and regulable spatial distribution of the glycocalyx
components. It is suggested that this microgeographic
heterogeneity and/or compartmentalization is part of the key to
understanding the complex biological functions of the
spermatozoon during its post-testicular existence (Bearer and
Friend, 1990; Yanagimachi, 1994; Moore, 1996).
In contrast to this apparent complexity of the sperm surface,
labelling experiments on intact rat spermatozoa with galactose
oxidase/NaB3H4 show a very high selectivity: in caudal
spermatozoa, only one molecule of 32 kDa seems to be
accessible to the action of the galactose oxidase, which could
not be detected on testicular (Brown et al., 1983) or caput
spermatozoa (Zeheb and Orr, 1984; Jones, 1989). This
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molecule alone is thus supposed to form the major component
of the outer part of the sperm glycocalyx. This 32 kDa molecule
carries a GPI-anchor (Moore et al., 1989) and is
highly glycosylated. Homologous gene products showing
highly similar expression patterns have been found in all
mammals investigated so far, including mouse (Kubota et al.,
1990; Kirchhoff, 1994) and rat (Eccleston et al., 1994;
Kirchhoff, 1994, 1996). Since this molecule appears on the
sperm surface when spermatozoa acquire their fertilizing
capacity, it was called the ‘major maturation antigen’
(Zeheb and Orr, 1984; Moore et al., 1989) or sperm membrane
glycoprotein (SMemG; Eccleston et al., 1994). Spermatozoa
are terminally differentiated cells and appear unable to
synthesize proteins de novo. Kirchhoff and Hale (1996) have
therefore postulated a cell-to-cell transfer of GPI-anchored
proteins from the epithelium of the epididymal duct onto the
sperm surface. During the epididymal passage, the acquisition
of this highly glycosylated antigen seems to be one of the major
events which form the sperm glycocalyx.
The composition of the sperm glycocalyx has been
investigated by a number of authors (Koehler, 1981; Lee and
Wong, 1986; De Cerezo et al., 1996). The majority have made
use of specific lectins, which bind to different types of sugar
side-chain (Cummings, 1994), and thus together provide a
picture of the sugar moieties forming the sperm surface.
Whereas the majority of authors have looked at lectin-binding
to ejaculated spermatozoa, some have used this technique to
assess the changes in the glycocalyx during post-testicular
sperm maturation, particularly during transit through the
epididymis (Eddy et al., 1985; Margagee et al., 1988; Kumar
et al., 1990; Bains et al., 1993; Arenas et al., 1996). Much
confusion in the literature has arisen through the use of
differing sperm preparation protocols prior to lectin
application. A particular fixation protocol might expose
epitopes not detectable on native spermatozoa, for example, on
the inner acrosomal membrane, and disrupt the outer structures
of the glycocalyx, thereby removing some sugar epitopes and
exposing others (Haas et al., 1988). Additionally, lectin
specificity can vary with lectin concentration, temperature and
various other technical conditions, and needs to be checked by
the appropriate use of competing sugars (Zeng and Gabius,
1992; Gabriel et al., 1994). A further factor of relevance is the
secondary or tertiary structure of a particular glycan, which
may strongly enhance the interaction with the corresponding
lectin. This can lead to the very high affinities reported for some
lectins (Koehler, 1981). In addition to the possible variability in
lectin detection due to the use of differing protocols, there may
also be considerable species differences in the lectin patterns
reported, and it has been suggested that this could represent part
of the cross-species fertilization barrier. Additionally, lectin
binding may even vary within a species, in, for example,
seasonal breeders (Calvo et al., 1995). Nevertheless,
carbohydrate–lectin interactions of this type are considered to
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S.Schröter et al.
be one of the most important components of cell–cell
recognition (Gahmberg and Tolvanen, 1996).
Because of this inherent variability, attention has often
focused on features which appear to be shared in common
across a wide range of species. Runnebaum et al. (1995) have
described ConA-binding (Concanavalin A; Canavalia
ensiformis lectin) molecules on the acrosomal region of the
sperm plasma membrane which are recognized by a specific
monoclonal antibody across several species. This would imply
that this epitope on the sperm surface is conserved and thus of
physiological importance. Similar ConA-binding has even
been demonstrated on the surface of Drosophila spermatozoa
(Perotti and Pasini, 1995).
A further feature associated with sperm maturation also
shared in common by many species is the general increase in
negative surface charge during maturation (Yanagimachi et al.,
1972; Holt, 1980; Koehler, 1981; Eddy et al., 1985). This
charge increase is probably due to the acquisition of sialic acid
residues or acidic sperm antigens, and can be demonstrated by
the increase in electrophoretic mobility of spermatozoa
following epididymal transit (Bedford, 1963). It also correlates
with a rise in WGA-binding (wheat germ agglutinin, Triticus
vulgaris
lectin;
specific
for
sialic
acid
and
N-acetylglucosamine, GlcNac) on the sperm surface (Arenas
et al., 1996). Indeed, Fierro et al. (1996) showed a strong
WGA-binding over the whole surface of ejaculated human
spermatozoa, with presumably the thick layer of negatively
charged sugars reported by Tsuji and colleagues (Tsuji et al.,
1988; Tsuji, 1995) being responsible for this binding.
Furthermore, Lassalle and Testart (1994) found a
dose-dependent decrease in WGA-binding after digestion of
motile human ejaculated spermatozoa with a neuraminidase
(from Arthrobacter urefaciens) which preferentially cleaves
terminal 2,6-linked sialic acid residues. In the rat, the major
epididymally derived sperm surface proteins D and E are
sialoproteins expressed in the proximal epididymis (Brooks,
1983). Kumar et al. (1990) showed that binding of WGA over
the acrosomal region of the living sperm head increased during
epididymal transit in five different species (rat, mouse, rabbit,
hamster, goat) with a maximum in the corpus region. In the
rhesus monkey, Navaneetham et al. (1996) also showed that
in unfixed epididymal spermatozoa there was an increase in
WGA-binding in the corpus epididymides. The increase
in WGA-binding is probably caused by an increase in GlcNac
and/or sialic acid residues; however, this could be due either to
a local glycosylation reaction on pre-existing substrates on the
sperm membrane, or to the transfer of new sialylated
glycoconjugates to the spermatozoa during transit.
Focusing on detection of lectin within the human
epididymis, which appears to show less pronounced
regionalization compared to other mammals, Arenas et al.
(1996, 1998), using lectin-histochemistry, indicated a
considerable increase in lectin-binding molecules along the
epididymis. In the caudal lumen, epididymal fluid was positive
for all lectins studied and therefore should contain
β-GlcNac/sialic acid (WGA; wheat germ agglutinin, Triticus
vulgaris lectin), α-mannose (ConA; Canavalia ensiformis
lectin), α-fucose (UEA-1; Ulex europaeus lectin), α-GalNAc
(DBA; Dolichos biflorus lectin), β-GalNAc (SBA; soybean
agglutinin; Glycine max lectin) and β-Gal (PNA; peanut
agglutinin, Arachis hypogaea lectin). In contrast, in the efferent
ducts, the lumen was only positive for WGA-reacting
components. Thus the epididymis is a site of high extracellular
glycosyl metabolism, presumably involving the secretory
activity of the principal cells lining the epididymis. However,
whereas some of this activity may involve the spermatozoa in
transit directly, a large part of this may have less to do with the
spermatozoa than with the composition and properties of the
epididymal luminal fluid, where the spermatozoa are retained
prior to ejaculation.
Origin and regulation of the glycoconjugates
on the sperm surface
Very little is known about non-protein glycoconjugates on the
sperm surface, for example, glycolipid components of the
glycocalyx. Most attention has been given to the glycoprotein
components of the sperm membrane. In general, there appear
to be at least three different types of glycoprotein interaction
with the sperm membrane. Some glycoproteins are made
within the testis and have defined transmembrane domains,
with extracellular regions glycosylated on the outer cell
surface. These would include typical hormone-receptor
proteins and similar integral membrane proteins. Whereas the
majority of such proteins will be of spermatozoal origin, it
cannot be excluded that even this class of protein might be
transferred post-testicularly from cells of the male tract through
the agency of prostasome or seminosome apocrine vesicles
(Aumuller et al., 1997).
A second class of glycoproteins may be produced after
completion of spermiogenesis either within the testis (for
example, from Sertoli cells or from epithelial cells of the rete
testis) or post-testicularly in the epididymis or subsequent parts
of the male tract. Such glycoproteins could undergo polar
interactions with integral polar moieties on the sperm
membrane. These might be in the form of protein–protein
interactions or lectin–sugar interactions. Some of these
interactions might be specific and of high affinity, others may
be of relatively low affinity, the glycoproteins being later
released from the sperm surface, for example, during
capacitation (e.g. HE4, see below; or the so-called ‘acrosome
reaction inhibiting glycoprotein’, described by Drisdel et al.,
1995). The spermadhesins also fall into this category
(Töpfer-Petersen, 1999). One of the first indications for this
sort of sperm surface interaction came from the work of Tezon
et al. (1985). These authors showed that washing ejaculated
human spermatozoa with high concentrations of salt removed a
group of proteins which, when used as immunogen to raise
The sperm glycocalyx
305
the outer membrane lipid layer (Kirchhoff, 1996; Kirchhoff
and Hale, 1996).
Analysis of specific proteins forming part of
the human sperm glycocalyx
Figure 1. Denaturing polyacrylamide gel electrophoresis of proteins
eluted in 0.6 M NaCl from fresh human semen (lane 1) or from
semen stored at 4°C overnight in the presence of proteinase
inhibitors (lane 2). Lanes 3–12: lectin blot of the same proteins using
different lectins as indicated, following incubation or not with
N-glycosidase F. (GNA, Galanthus nivalis lectin; SNA, Sambucus
nigra lectin; DSA, Datura stramonium lectin; MAA, Maackia
amurensis lectin; PNA, Arachis hypogaea lectin, peanut agglutinin.)
antibodies, indicated their source to be within the epididymis
rather than within the testis. We have repeated these
experiments and showed that treatment of isotonic
saline-washed, ejaculated human spermatozoa with 0.6 M
NaCl removed a group of ~20 proteins with molecular weights
ranging from 10 from 100 kDa (Figure 1, left panel).
Subsequent incubation of these proteins with lectins showed
that the majority were indeed glycosylated, and that this
glycosylation was largely eliminated by pretreatment with
N-glycosidase F (Figure 1, right panel) to remove the
N-glycosidic side chain from the primary amino acid substrate.
Use of these proteins to raise monoclonal antibodies confirmed
the observations of Tezon et al. (1985) for at least two of the
proteins, i.e. that these originated within the epididymis and not
the testis (McArdle, 1991).
A third group of proteins, however, appears to undergo a
secondary integration into the sperm plasma membrane. The
best described of these are those furnished with a
glycosylphosphatidylinositol (GPI) anchor structure, such as
CD52 and CD59 (see below). In some way, which is still
unclear, but may involve direct membrane–membrane or
membrane–seminosome interactions, these molecules are
transferred from the site of original synthesis, for example, in
the principal cells of the epididymis, to the sperm plasma
membrane, where the lipophilic GPI-anchor intercalates into
As mentioned above, some proteins that could be removed by
washing ejaculated human spermatozoa with moderately high
salt concentrations were shown to have their origins in the
epididymis. In an alternative approach, proteins secreted from
epididymal epithelial cell cultures, and which were
metabolically labelled with tritiated amino acids or
[35S]methionine, were shown to relocate to co-cultured
spermatozoa (Tezon et al., 1985; Ross et al., 1990; reviewed
for other species also in Cooper, 1986; Moore, 1990). Precise
information on the nature and synthesis of some of the human
glycoproteins associated with the sperm surface and
post-testicular sperm maturation have only emerged following
the systematic molecular cloning of testicularly and
epididymally expressed genes (e.g. PH-20: Lin et al., 1993;
fertilin (PH-30): Gupta et al., 1996; Jury et al., 1997; ARP:
Hayashi et al., 1996; HE2: Osterhoff et al., 1994; HE5:
Kirchhoff, 1996). Although sperm surface glycoproteins have
now been characterized from a number of species, there still
appears to be a high degree of species diversity in expression.
One example is the selenium-dependent glutathione
peroxidase (GPX5) which was originally cloned from the
mouse (Ghyselinck et al., 1991). This gene product is very
variable between species and does not appear to occur in the
human (Beiglböck et al., 1998; Hall et al., 1998). Here, we
concentrate on those expressed in human tissues, and discuss
other species only for comparative purposes. The most
important of these glycoproteins are listed in Table I in order of
their first appearance in the male tract.
PH-20
PH-20 is a sperm glycoprotein of ~64 kDa in the human
(Sabeur et al., 1997) and appears to be highly conserved across
species. It was originally discovered in the guinea pig
(Primakoff et al., 1988), as an integral sperm protein made in
the testis, though it is anchored within the sperm plasmalemma
and in the intra-acrosomal membrane (IAM) via a GPI-anchor.
PH-20 has been shown to relocate during epididymal transit
(Phelps and Myles, 1987). In the testis it is localized over the
entire sperm surface, whereas it moves during passage through
the epididymis to be localized in the posterior head region
(hence PH). There is a further redistribution during the
acrosome reaction when the PH-20 epitopes on the IAM also
become exposed (Myles and Primakoff, 1984; Phelps et al.,
1990). There appears to be some species specificity in regard to
the precise domain localization on the sperm surface; in the
mouse, monkey and human, PH-20 appears initially to be
restricted to the whole head region.
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S.Schröter et al.
The human and cynomolgus monkey PH-20 cDNAs have
been cloned, and share 90% identity with each other and 59%
with the guinea pig PH-20 (Lin et al., 1993). In particular, all 12
cysteine residues are conserved. This general conservation
suggests that in all species this protein has a similar important
function. The N-terminal domain of the molecule contains a
hyaluronidase activity in the species examined, and it is assumed
that this enables the spermatozoa to penetrate the cumulus cell
layer surrounding the oocyte. Additionally, proteolytic
processing of the PH-20 molecule has been reported following
the acrosome reaction and is clearly subsequent to its insertion
into the sperm plasma membrane. The resulting fragments,
however, remain associated via disulphide bridges. It has been
speculated that this processing may be part of the secondary
binding to the zona pellucida, though the mechanisms are still
poorly understood (Myles and Primakoff, 1997).
Fertilin (PH-30)
Fertilin is an α–β heterodimer, which like PH-20, was also
originally described from the guinea pig. Both subunits
represent closely related transmembrane proteins, and appear
to be involved in gamete fusion (Blobel and White, 1992;
reviewed in Myles and Primakoff, 1997). Also like PH-20,
fertilin appears to exhibit a species-specific domain
localization on the sperm surface. Both primate and human
fertilin cDNAs have been cloned from the testis. However, at
least the α-gene appears to be non-functional in the human
(Jury et al., 1997). Fertilin α and β were the first identified
members of the ADAM (a disintegrin and metalloprotease)
family. It is therefore possible that other members of this family
may also be involved in gamete fusion, for example in the
mouse (Cho et al., 1996). Another member of this family has
been cloned from the efferent ducts and caput epididymides of
the monkey, the so-called epididymal apical protein 1 (EAP-1;
Perry et al., 1992). However, this has only been located on the
apical membrane of the epididymal principal cells and not as
yet on the sperm surface. A growing number of ADAMs have
been found in the testis (Wolfsberg et al., 1995), suggesting a
more broad-based function of these heterodimeric
transmembrane glycoproteins.
CD59
The 19 kDa CD59 glycoprotein appears to be present on the
whole sperm surface of ejaculated, capacitated and also
acrosome-reacted spermatozoa, as well as in tissue sections of
the testis, as determined using immunohistochemistry
(Fenichel et al., 1994). This GPI-anchored molecule is widely
distributed on cell surfaces throughout the body, and was
originally found on the membranes of human blood cells
(erythrocytes and leukocytes), and also on endothelial cells and
ductular epithelia (Meri et al., 1991). The CD59 cDNA was
cloned from human lymphocytes by Sawada et al. (1990).
CD59, also known as ‘protectin’, effectively inhibits lysis by
the membrane attack complex of the homologous complement
system (Lachmann, 1991; Meri et al., 1991; Walsh et al.,
1991). Thus, the sperm CD59 glycocalyx antigen is also
suggested to participate in the protection of male gametes
against complement-mediated damage as they travel through
the female genital tract. Furthermore, sperm CD59 is
speculated to be involved in the gamete adhesion process
during fertilization (Fenichel et al., 1994).
Table I. Cloned human glycoproteins which bind sequentially to the plasma membrane (PM) of spermatozoa during their transit through the
male reproductive tract
Name of
cloned
glycoprotein
Origin
(site of first
synthesis)
Size in amino acids (aa)
(+ signal peptide, SP)
Mr of glycoprotein
Type of glycosylation
(consensus sites for
N- and O-glycosylation
Sperm domain
localization
Proposed
functions/comments
Reference
PH-20
testis
455 aa (+35 SP +
19 GPI-anchor SP)
64 kDa
7 N-glycosylation sites
>20 O-glycosyl. sites
ejaculated and capacitated
spermatozoa: entire head
acrosome reacted: IAM→
2 functions: hyaluronidase
activity→cumulus penetration
IAM-anchored PH-20→secondary
binding to zona pellucida
Lin et al. (1993)
CD59
testis
103 aa (+25 SP +
22 GPI-anchor SP)
19 kDa
1 N-glycosylation site
(verified)
9 O-glycosylation sites
whole sperm surface
protection from complement?
Sawada et al. (1990)
SOB1
testis
853 aa
100 kDa
5 N-glycosylation sites
4 O-glycosylation sites
ejaculated spermatozoa:
subequatorial
involved in sperm–oocyte
binding
Lefèvre et al. (1998)
ARP
efferent ducts
230 aa (+19 SP)
30 kDa
1 N-glycosylation site
verified by N-glycanase
F (carbohydrate ≈4 kDa)
postacrosomal on sperm
head
involved in fusion of gametes?
functional counterpart of
rodent AEG
Hayashi et al. (1996)
HE2
caput
epididymidis
81 aa (+22 SP)
1 N-glycosylation site
6 O-glycosylation sites
ejaculated spermatozoa:
acrosomal & equatorial
capacitated spermatozoa:
mainly equatorial
LRE-motif: cell adhesion?
Osterhoff et al. (1994)
HE5
corpus
epididymidis
12 aa (+24 SP +
25 GPI-anchor SP)
1 N-glycosylation site
(verified)
20–25 kDa
whole sperm surface
lesser over chromosome
GPI-anchor: increase in PM
stability? carbohydrates:
immunosuppression?
Kirchhoff et al. (1993)
HE4
cauda
epididymidis
95 aa (+30 SP)
1 N-glycosylation site
8 O-glycosylation sites
epididymis: associated
to sperm surface
lost during capacitation
protease inhibitor?
Kirchhoff et al. (1991)
IAM = intra-acrosomal membrane; AEG = acidic epididymal glycoprotein; LRE = Leu-Arg-Glu; GPI = glycosylphosphatidylinositol.
The sperm glycocalyx
SOB1
SOB1 is a glycoprotein of testicular origin (Boué et al., 1992;
Lefèvre et al., 1998) that is localized on the subequatorial region
of epididymal and ejaculated spermatozoa using the monoclonal
antibody CA6 as probe (Boué et al., 1992, 1995). Western
blotting of extracts of human, macaque and rodent spermatozoa
revealed a size for the SOB1 protein of 100 kDa, agreeing well
with the size predicted from the cloned cDNA (94.8 kDa). The
small difference in size compared to Western blotting is likely to
be due to glycosylation of the sperm antigen (Lefèvre et al.,
1998). Northern blot analysis shows that the testis-specific
expression of SOB1 is conserved among mouse, rat, rabbit, dog
and cat. SOB1 reveals 32% homology with the mouse sperm
fibrous sheath component (Fulcher et al., 1995). The
SOB1-recognizing Fab fragments of CA6 were also shown to
inhibit specifically human sperm binding to hamster and human
oocytes in a dose-dependent manner, suggesting that the SOB1
glycoprotein is involved in oocyte binding (Boué et al., 1995).
ARP
To date, no functional counterpart to the guinea pig fertilin
(PH-30) has been described for the human. However, a potential
candidate could be the human glycoprotein ARP [acidic
epididymal glycoprotein (AEG)-related protein], which has also
been suggested to be involved in the sperm–egg interaction
(Hayashi et al., 1996). ARP is localized on the posterior region of
the sperm head, similar to the localization of the guinea pig
PH-proteins. However, ARP is synthesized post-testicularly by
the epithelium of the efferent ducts, and expression persists in the
caput and corpus regions of the epididymis (Hayashi et al.,
1996). Western blot analysis showed a protein of ~30 kDa (with a
weak secondary band at 26 kDa) to be present throughout the
epididymal lumen, in seminal plasma and on the surface of
washed ejaculated spermatozoa. N-Glycosidase F treatment
reduced the size of the protein by ~4 kDa and implied only a
single sugar side-chain attached to Asn211 close to the
C-terminus of the polypeptide.
ARP is a novel member of the CRISP (cysteine-rich secretory
protein) family (Haendler et al., 1993), which includes a number
of gene products specific to the male genital tract (Kratzschmar
et al., 1996). ARP is also related to another well-characterized
secretory protein, the acidic epididymal glycoprotein (AEG) or
protein DE (reviewed in Brooks, 1987) described in rodents.
Although ARP is not the true homologue of the rodent AEG
(Hayashi et al., 1996), it resembles this protein in as much as it
also binds to the post-acrosomal region of the sperm head.
However, since a true AEG counterpart has not yet been
identified in the human, ARP may prove to be the functional
homologue of the rodent molecule. In rats, AEG is considered to
be involved in sperm–egg fusion: specific antibodies block
fertilization in vivo and in vitro (Cuasnicu et al., 1984; Perez
Martinez et al., 1995). Purified AEG binds to the egg surface and
inhibits sperm–egg fusion in a dose-dependent manner
307
(Rochwerger et al., 1992). After in-vitro and in-vivo capacitation,
AEG migrates from the post-acrosomal to the equatorial region
of the sperm head, the region which first fuses with the oolemma
(Rochwerger and Cuasnicu, 1992).
HE2
HE2 is an abundant gene product from the proximal human
epididymis, comprising 0.2% of the total epididymal mRNA
pool, and was identified by tissue-specific differential
screening of a human epididymal cDNA library (Kirchhoff
et al., 1990; Osterhoff et al., 1994). Both the mRNA and
protein can be detected in the principal cells of the proximal
epididymal epithelium, and immunoreactive epitopes are also
detected on fixed epididymal and ejaculated spermatozoa over
the acrosome and equatorial segment. Following in-vitro
capacitation, the immunoreactivity is restricted to the
equatorial segment (C.Osterhoff, unpublished data). The HE2
mRNA encodes a small polypeptide which after removal of a
signal peptide has 81 amino acids (size ~9 kDa), including a
single N-glycosylation site and six possible sites for
O-glycosylation. Preliminary Western blot analysis of sperm
protein extracts, however, using antibodies raised against
different epitopes, suggests a much larger size of ~37–38 kDa
(C.Osterhoff, unpublished data). Whether this is due to a
considerable glycosylation or to possible complexation with
other molecules, or to dimerization, is still an open question. An
interesting feature of the HE2 molecule is that it appears to be
retained with relatively high affinity by ejaculated
spermatozoa, and yet there are no obvious structural features
which could account for this. There is, however, an LRE-motif
(-Leu-Arg-Glu-) within the primary sequence that has been
implicated in cell adhesion (Hunter et al., 1989; Kawaoka
et al., 1990). This motif is located within a region of the HE2
molecule which shows a weak but significant homology to the
influenza virus haemagglutinin sequence responsible for the
attachment of the virus to cell receptors.
It thus seems possible that HE2 may also be involved in
sperm–egg interaction like fertilin, which has a related TDE
motif and a similar localization on the sperm head. To date,
HE2 has only been identified in the human and recently also in
the chimpanzee (Pan troglodytes) epididymis (Young and
Fröhlich, 1998). In the chimpanzee epididymis, the
homologous HE2 mRNA expression seems to be androgendependent. Homologous molecules have not yet been found in
other mammalian species, thus prohibiting detailed experimental studies on its possible function.
CD52/HE5
When the HE5 gene transcript was first cloned from the human
epididymis (Kirchhoff et al., 1993), it was shown to be identical
in its peptide backbone to the lymphocyte antigen CD52 (Xia
et al., 1991). This peptide backbone is unusual in being
remarkably short (only 12 amino acids) and results from cleavage
308
S.Schröter et al.
of a secretory signal peptide from the N-terminus and a
GPI-anchor signal sequence from the C-terminus of the primary
translation product. The peptide is additionally furnished with a
substantial N-glycosylation attached to an asparagine residue.
Preliminary studies show that the epididymal CD52 has a
different glycosylation from that on lymphocytes (S.Schröter,
unpublished data). Antibody studies showed this molecule to
represent quantitatively the most important glycoprotein on the
surface of ejaculated human spermatozoa. Although initial
studies could find no rodent homologue for this molecule, studies
at the genomic level showed that HE5 is indeed the genomic
homologue of the main maturation-associated, sperm surface
antigen in rats, SMemG (Moore et al., 1989; Eccleston et al.,
1994), and in mice (Kubota et al., 1990; Kirchhoff, 1994).
Non-coding regions and exon–intron structures are highly
homologous at the genomic level. Nevertheless, comparisons of
CD52 peptide sequences revealed that the post-translational
peptide backbone is quite different between species, though all
homologues possess a conserved putative N-glycosylation site,
conserved
displaced
GPI-anchor
signal
peptides,
and conserved displaced signal peptides. Because the CD52
antigen is attached to the sperm surface by intercalation of the
GPI-anchor into the outer leaflet of the sperm plasma membrane,
it can potentially cover the whole of the sperm surface.
HE5/CD52 is expressed relatively late during epididymal transit,
with maximal expression in the distal corpus and cauda regions,
coinciding with the major increase in negative sperm surface
charge alluded to above. Antibody studies confirm that it indeed
covers the whole of the sperm surface, and that this does not
appear to alter following in-vitro capacitation (Osterhoff et al.,
1995; Yeung et al., 1997; S.Schröter, unpublished data).
Molecular modelling of the lymphocyte CD52 molecule (Figure
2) shows that the glycosylation probably makes up ~60% of the
molecule (Xia et al., 1991; Treumann et al., 1995). The sperm
CD52, like the lymphocyte molecule, is also highly glycosylated
(Schröter et al., 1997) and should similarly form a highly flexible
structure on the sperm surface (Figure 2). It is easy to imagine
how a molecule like this, being expressed late during sperm
maturation and in large amounts, could effectively coat the sperm
surface with a highly fluid glycosyl matrix, thus masking other
aspects of the sperm surface which may have been exposed
and/or modified earlier during post-testicular transit.
A comparison of all the known CD52 molecules from five
mammalian species (Figure 3) shows that, although the
dimensions of the sperm glycocalyx may be altered, the principal
features of the CD52 molecule, with a putative N-terminal
N-glycosylation site upon a flexible GPI-anchored peptide
backbone remain unchanged. The sequence comparisons of the
homologous cDNAs have revealed a conserved consensus site
for N-glycosylation in all examined species. This strongly
suggests that the in-vivo product is in fact N-glycosylated in all
mammals. In the case of the human sperm CD52 counterpart, the
existence of an extensive N-glycosylation site has been
convincingly demonstrated (Schröter et al., 1997). Also, Zeheb
and Orr (1984) have found that the rat CD52 molecule binds to a
ConA–agarose column, specific for N-glycosidic mannose-rich
chains. Whereas the human sperm CD52 appears to possess no
sites for O-glycosylation (Xia et al., 1993), the rat CD52 is
probably highly O-glycosylated (Eccleston et al., 1994). For the
CD52 homologues from other species, there is still no direct
experimental information on the degree of O- or N-glycosylation.
HE4
HE4 is an example of a protein with a relatively loose
association with the sperm glycocalyx. The cloned cDNA
sequence of this human epididymal protein predicts a small (95
amino acid) polypeptide with two whey-acidic-protein (WAP)
domains represented by a duplicated pattern of four cysteine
residues (Seemuller et al., 1986; Kirchhoff et al., 1991). It also
includes a single N-glycosylation site. Antibodies raised
against the biotechnologically produced protein confirm the
in-situ hybridization analysis of the mRNA, i.e. that this
molecule is produced by the epithelial cells in low to moderate
amounts throughout the epididymis, though with highest levels
in the distal corpus and cauda. The antibodies also show that
HE4 is associated with freshly ejaculated spermatozoa, but
during incubation in appropriate capacitation medium, the HE4
protein diffuses away from the gametes, indicating only a low
affinity binding. This is particularly noticeable during in-vitro
capacitation, when the HE4 immunoreactivity is rapidly lost
from the sperm surface (Osterhoff et al., 1995). These
properties are very similar to those expected of a decapacitation
factor. However, the WAP-domain structure of HE4 also links
it to a group of secretory protease inhibitors found in the female
and the male reproductive tract in some species (Seemuller
et al., 1986; Coronel and Lardy, 1992). While this is not
evidence for protease inhibitor interactions, it does imply that
HE4 may be involved in other specific protein–protein
interactions at the sperm surface. HE4 appears to have
homologues in the epididymides of most mammalian species,
at either the immunohistochemical or RNA hybridization
levels; cross-reactive products have been identified in the
human, dog, pig, horse and bull (Kirchhoff et al., 1990, 1991;
Uhlenbruck et al., 1993; Ellerbrock et al., 1994).
Glycosyl-modifying enzymes
In this review we do not focus on the enzymatic action of
glycosyl-modifying enzymes, as there are excellent recent
reviews elsewhere covering this topic in more detail (Benoff,
1997; Tulsiani et al., 1997). Tulsiani et al. (1998) summarize
their results from in-vitro studies in the rat model on the role of
epididymal luminal fluid glycosyltransferases (as synthetic
enzymes, mainly sialyl- and fucosyltransferase) and
glycosidases (as hydrolytic enzymes), together comprising
>80% of total enzyme activities in the epididymal luminal
fluid. They showed that after incubation of live caput
The sperm glycocalyx
Figure 2. Dynamic molecular modelling of one glycoform of the lymp
examined on the basis of a Metropolis Monte Carlo simulation (court
tion of the peptide backbone (green) with its N-glycosylation (light
glycosylphosphatidylinositol (GPI)-anchor has been omitted in the c
sible positions of the sialic acids which terminate the arms of the tetra
are highly flexible and can only be interpreted as dynamic structures
spermatozoa with [14C]fucose, the labelled fucose was
incorporated into an endogenous 85 kDa sperm glycoprotein,
thus providing an example for the action of a synthetic enzyme.
In-vitro activity of a hydrolytic enzyme was shown for a
purified β-D-galactosidase from epididymal luminal fluid
acting on a 135–150 kDa antigen from live caput but not caudal
spermatozoa as substrate. This antigen was suggested to be
degalactosylated during the incubation with the pure enzyme,
indirectly shown by the decrease in PNA binding. This lectin
has a high affinity for the terminal galactosyl residues of
Galβ(1–3)GalNAc. These in-vitro results imply a role for
glycoprotein-modifying enzymes in the modification of rat
sperm plasma membrane glycoproteins during epididymal
maturation, though the in-vivo function of glycosyl-modifying
enzymes remains to be demonstrated.
A soluble galactosyltransferase has also been described from
human seminal plasma (Ross et al., 1993). This highly active
45 kDa component was detected in the caput epididymal
309
Figure 3. Schematic representation of the glycosylphosphatidylinositol
(GPI)-anchored HE5/CD52 antigen in comparison with its
corresponding species homologues. The GPI-anchored molecules are
inserted into the outer leaflet of the sperm plasma membrane bilayer on
the sperm surface. The proportions of the various components have
been drawn to scale, following the calculations of Rademacher et al.
(1988) and Jentoft (1990). The symbols for N- and O-glycosylation
sites are filled in if their existence has been experimentally proven (see
text). The schematic drawing of the glycosylation is based on a typical
complex type N-glycan.
intraluminal fluid, and is therefore another potential candidate
involved in the modification of the human sperm glycocalyx
during epididymal maturation.
Soluble glycosyl-modifying enzymes of the luminal fluid,
acting within the male reproductive tract, may not be the only
enzymes responsible for the changes of the sperm surface
glyco-architecture. A number of intrinsic sperm surface
components also appear to show enzymatic activity (reviewed by
Tulsiani et al., 1997), or at least bind to glycosidic structures on
the zona pellucida. These have been described for several
mammalian species, including the mouse and human, and
include an α-D-mannosidase, mannose-binding protein, and a
selectin-like molecule. Since carbohydrate-binding molecules are
among those proposed as receptors for the zona pellucida
310
S.Schröter et al.
(Tulsiani et al., 1997), these glycosyl-modifying enzymes,
whether soluble or sperm-bound, could encourage sperm
maturation and enable sperm glycoconjugates to interact with
their complementary molecules on the zona pellucida during
fertilization.
The function of the sperm glycocalyx
N-Glycans form a prominent part of the sperm glycocalyx, and
probably contribute greatly to its physicochemical properties.
One of the most striking features of N-glycans is their
extraordinary flexibility, with a very high degree of rotation and
movement possible about each glycosidic bond. Additionally,
there is usually considerable microheterogeneity; on the same
backbone molecule N-glycans may vary markedly in chain
length, branching or monosaccharide composition. Analysis of
such a glycan structure in solution can be modelled by the
application of a MMC (Metropolis Monte Carlo) simulation
(Figure 2; courtesy of Kay Boettcher) to just one of the diverse
carbohydrate structures of the lymphocyte CD52: a
tetra-antennary polylactosamine-containing N-glycan with
terminal sialic acids, as proposed by Treumann et al. (1995).
The coloured dots show all possible positions for the terminal
sialic acids, and clearly demonstrate the mobility and flexibility
of this structure. Statistically, such a mobility would also
enhance the probability of entering a conformation which
could be recognized by lectin-like structures which, themselves
being proteins, are much less flexible. Thus it is important to
note that glycosylation is four-dimensional, rather than
three-dimensional, with the added dimension (time) enhancing
recognition by specific, receptor-type molecules.
It has been suggested that specific oligosaccharide moieties of
certain glycoproteins may be immunosuppressive (El Ouagari
et al., 1995; Clark et al., 1996a,b), though mechanisms as to how
this may work are far from clear. However, carbohydrates can
also definitely be immunogenic, as for example, in the
AB(H)O-system of blood factors, and certainly spermatozoa are
able to raise a relatively high antibody titre, (as seen in
vasectomized men). Important in this context is the fact that a
T-cell-dependent immune response against conjugated
carbohydrates is only possible against molecules that still possess
a peptide moiety, since only this part of the molecule can be
processed by antigen-presenting cells (Austyn, 1989). Equally,
however, monoclonal antibodies raised against sperm surface
membranes tend to be directed preferentially against
carbohydrate moieties (Isojima et al., 1990; Kameda et al., 1991,
1992; Kurpisz and Alexander, 1995; Tsuji, 1995). Thus it is this
part that appears to be ’seen’ by the antibodies.
The regionalization of sperm maturation provides another
possible clue to the role of the glycocalyx. It is evident that all
such membrane modifications do not occur within the testis,
nor simultaneously in any one region of the male tract. It is
particularly noticeable that there is a geographic hierarchy in
the modification of the sperm membrane, with most notably the
major sperm surface antigen, CD52, being added last,
following other epididymal interactions. Given the high degree
of carbohydrate mobility discussed above, it seems logical to
consider that the addition of this antigen is likely to occlude
later interactions between luminal molecules and other proteins
on the sperm surface. Such interactions are probably easier
prior to the advent of CD52. This molecule and similar antigens
may thus also serve to protect the sperm surface from
non-specific pre-emptive interactions in the female tract, prior
to specific surface changes occurring at fertilization.
Another, less obvious, role could be in influencing the
biophysical properties of the sperm membrane. Possibly more
than most other cell types, the properties of the sperm outer
membrane are very important with regard to all the functions
and interactions of the male gamete, e.g. capacitation, sperm
motility, sperm survival in the male and female tracts, the
acrosome reaction, zona binding and penetration, and gamete
fusion. This has been underscored by the numerous studies
which involve the change in membrane fluidity and membrane
cholesterol content during sperm maturation (Jones, 1989;
Bearer and Friend, 1990), and most recently by the dramatic
effects of free radicals and the oxidative status of the sperm
membrane (reviewed in Aitken, 1995). In general, lipid
oxidation and factors causing a reduction of membrane fluidity
lead to a loss of sperm function. It therefore seems likely that
molecules intercalating into the sperm membrane, such as
glycolipids or GPI-anchored glycoproteins, could substantially
influence local membrane properties, including ion flux across
the membrane, and possibly also motility parameters.
A further biophysical property of a dense hydrophilic
covering of the sperm cells could be to prevent the spermatozoa
from non-specific interactions with proteins—acting as a
natural repellent. Similar non-adhesive glycocalyx-like
surfaces were artificially constructed by Holland et al. (1998),
who coated graphite with oligosaccharide surfactant polymers
possessing dextran and alkanoyl side-chains. These polymers
were shown to assemble on the graphite surface via their
hydrophobic domains, while the dextran side-chains protruded
into the aqueous phase and created a glycocalyx-like surface.
This surface was effective in suppressing protein adsorption
(from human plasma protein solution).
Finally, there is a striking superficial resemblance between the
glycocalyx of the sperm surface and that characterized on the
surface of some successful protozoan parasites. Trypanosoma
brucei also uses highly glycosylated GPI-anchored molecules
(VSG, variant surface glycoprotein) to protect itself within the
host bloodstream (Ferguson et al., 1994). This GPI-anchored
glycocalyx allows a high packing density of glycosylated
molecules, which by being variant offer protection against the
host immune system and protect underlying membrane
molecules which might offer a target for host resistance. Other
GPI-anchored proteins, like the 35/60 kDa antigen from
Trypanosoma cruzi, are involved in specific host–parasite
interactions (and finally appear to be involved in infectivity, i.e.
The sperm glycocalyx
specific membrane–membrane interaction (Ferguson et al.,
1994). A major difference, however, is the induced variability of
trypanosome glycoproteins, which are able to switch structure
once an immune reaction has occurred, thus making the parasites
effectively invisible to the host immune system. Nevertheless, the
resemblance of the sperm surface to that of the protozoan parasite
in terms of structure (GPI-anchored molecules, dense sugar
surface) and function (protection, binding specificity, hierarchical
interactions with host cells) could be very informative about the
mechanisms involved in glycocalyx function.
Conclusion
The spermatozoon has been likened to a spaceship carrying a
very important genetic load across a vast distance of hostile
space, to deliver it in perfect condition to its target planet. Like
the outer hull of that spaceship, the sperm glycocalyx has to be
protective against a variety of potentially inimical influences,
and yet contain complex functions involved in informational
exchange, motility control, docking and delivery of its
contents. In the past few years we have begun to understand the
shape and form of some of the components of the sperm
glycocalyx, including for some molecules full primary
structure. Only when the complete structures of most of the
sperm surface components are known, however, will we begin
to understand the mechanisms underlying these complex
functions and their pathologies manifest as male infertility.
Acknowledgements
We should particularly like to thank Professor Freimut
Leidenberger for continuous support and encouragement of our
research into post-testicular sperm maturation. Very special thanks
are due also to Dr Christiane Kirchhoff for very helpful discussion
and advice, and to Kay Boettcher for the Metropolis Monte Carlo
simulation. Much of our research reported here was supported by
generous grants from the Deutsche Forschungsgemeinschaft
(Iv7/4–6 and Ki317/5) and from the German Federal Ministry for
Education, Research and Technology as part of a concerted project
’Fertilitätsstörungen’ (01Ky9103).
References
Aitken, R.J. (1995) Free radicals, lipid peroxidation and sperm function.
Reprod. Fertil. Dev., 7, 659–668.
Arenas, M.I., de Miguel, M.P., Bethencourt, F.R. et al. (1996) Lectin
histochemistry in the human epididymis. J. Reprod. Fertil., 106,
313–320.
Arenas, M.I., Madrid, J.F., Bethencourt, F.R. et al. (1998) Identification of
N- and O-linked oligosaccharides in the human epididymis.
J. Histochem. Cytochem., 46, 1185–1188.
Aumuller, G., Renneberg, H., Schiemann, P.J. et al. (1997) The role of
apocrine released proteins in the post-testicular regulation of human
sperm function. Adv. Exp. Med. Biol., 424, 193–219.
Austyn, J.M. (1989) Antigen processing and immunostimulation. In Male,
D. (ed.), Antigen-Presenting Cells. IRL Press at Oxford University
Press, Oxford, pp. 46–58.
311
Bains, H.K., Pabst, M.A. and Bawa, S.R. (1993) Changes in the lectin
binding sites on the testicular, epididymal, vas, and ejaculated
spermatozoon surface of dog. Andrologia, 25, 19–24.
Bearer, E.L. and Friend, D.S. (1990) Morphology of mammalian sperm
membranes during differentiation, maturation, and capacitation.
J. Electron 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.
Beiglböck, A., Pera, I., Ellerbrock, K. and Kirchhoff, C. (1998) Dog
epididymis-specific mRNA encoding secretory glutathione
peroxidase-like protein. J. Reprod. Fertil., 112, 357–367.
Benoff, S. (1997) Carbohydrates and fertilization: an overview. Mol. Hum.
Reprod., 3, 599–637.
Blobel, C.P. and White, J.M. (1992) Structure, function and evolutionary
relationship of proteins containing a disintegrin domain. Curr. Opin.
Cell Biol., 5, 760–765.
Boué, F., Lassalle, B., Duquenne, C. et al. (1992) Human sperm proteins
from testicular and epididymal origin that participate in fertilization:
modulation of sperm binding to zona-free hamster oocytes, using
monoclonal antibodies. Mol. Reprod. Dev., 33, 470–480.
Boué, F., Duquenne, C., Lassalle, B. et al. (1995) FLB1, a human protein
of epididymal origin that is involved in the sperm–oocyte recognition
process. Biol. Reprod., 52, 267–278.
Brooks, D.E. (1983) Influence of incubation conditions, tunicamycin and
castration on incorporation of [3H]mannose and [3H]fucose into rat
epididymal glycoproteins in vitro. J. Reprod. Fertil., 67, 97–105.
Brooks, D.E. (1987) Androgen-regulated epididymal secretory proteins
associated with post-testicular sperm development. Ann. N.Y. Acad.
Sci., 513, 179–194.
Brown, C.R., von Glos, K.I. and Jones, R. (1983) Changes in plasma
membrane glycoproteins of rat spermatozoa during maturation in the
epididymis. J. Cell Biol., 96, 256–264.
Calvo, A., Pastor, L.M., Horn, R. and Pallares, J. (1995) Histochemical
study of glycoconjugates in the epididymis of the hamster
(Mesocricetus auratus). Histochem. J., 27, 670–680.
Cho, C., Primakoff, P., White, J.M. and Myles, D.G. (1996) Chromosomal
assignment of four testis-expressed mouse genes from a new family of
transmembrane proteins (ADAMs) involved in cell–cell adhesion and
fusion. Genomics, 34, 413–417.
Clark, G.F., Oehninger, S., Patankar, M.S. et al. (1996a) A role for
glycoconjugates in human development: the human feto-embryonic
defence hypothesis. Hum. Reprod., 11, 467–473.
Clark, G.F., Oehninger, S. and Seppälä, M. (1996b) Role for
glycoconjugates in cellular communication in the human reproductive
system. Mol. Hum. Reprod., 2, 513–517.
Cooper, T.G. (1986) The Epididymis, Sperm Maturation and Fertilisation.
Springer Verlag, Berlin.
Coronel, C.E. and Lardy, H.A. (1992) Functional properties of caltrin
proteins from seminal vesicle of the guinea pig. Mol. Reprod. Dev., 33,
74–80.
Cuasnicu, P.S., Gonzalez Echeverria, F., Piazza, A.D. et al. (1984)
Antibodies against epididymal glycoproteins block fertilizing ability
in rat. J. Reprod. Fertil., 72, 467–471.
Cummings, R.D. (1994) Use of lectins in analysis of glycoconjugates.
Methods Enzymol., 230, 66–86.
de Cerezo, J.M., Marquinez, A.C., Sarchi, M.I. and Cerezo, A.S. (1996)
Fucosylated glycoconjugates of the human spermatozoon.
Comparison of the domains of these glycoconjugates with the
alpha-fucosyl binding sites, and with lactosaminic glycoconjugates
and beta-D-galactosyl binding site domains. Biocell, 20, 11–20.
Drisdel, R.C., Mack, S.R., Anderson, R.A. and Zaneveld, L.J. (1995)
Purification and partial characterization of acrosome reaction
inhibiting glycoprotein from human seminal plasma. Biol. Reprod.,
53, 201–208.
Eccleston, E.D., White, T.W., Howard, J.B., and Hamilton, D.W. (1994)
Characterization of a cell surface glycoprotein associated with
maturation of rat spermatozoa. Mol. Reprod. Dev., 37, 110–119.
Eddy, E.M., Vernon, R.B. and Muller, C.H. (1985) Immunodissection of
sperm surface modifications during epididymal maturation. Am. J.
Anat., 174, 225–237.
312
S.Schröter et al.
Ellerbrock, K., Pera, I., Hartung, S. and Ivell, R. (1994) Gene expression in
the dog epididymis: a model for human epididymal function. Int. J.
Androl., 17, 314–323.
El Ouagari, K., Teissiers, J. and Benoist, H. (1995) Glycophorin A protects
K562 cells from natural killer cell attack. J. Biol. Chem., 270,
26970–26975.
Fenichel, P., Cervoni, F., Hofmann, P. et al. (1994) Expression of the
complement regulatory protein CD59 on human spermatozoa:
characterization and role in gametic interaction. Mol. Reprod. Dev.,
38, 338–346.
Ferguson, M.A., Brimacombe, J.S., Cottaz, S. et al. (1994)
Glycosyl-phosphatidylinositol molecules of the parasite and the host.
Parasitology, 108, S45–S54.
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.
Fulcher, K.D., Mori, C., Welch, J.E. et al. (1995) Characterization of Fsc1
cDNA for a mouse sperm fibrous sheath component. Biol. Reprod., 52,
41–49.
Gabriel, L.K., Franken, D.R., van der Horst, G. and Kruger, T.F. (1994)
Localization of wheat germ agglutinin lectin receptors on human
sperm by fluorescence microscopy: utilization of different fixatives.
Arch. Androl., 33, 77–85.
Gahmberg, C.G. and Tolvanen, M. (1996) Why mammalian cell surface
proteins are glycoproteins. Trends Biochem. Sci., 21, 308–311.
Ghyselinck, N.B., Jimenez, C. and Dufaure, J.P. (1991) Sequence
homology of androgen-regulated epididymal proteins with
glutathione peroxidase in mice. J. Reprod. Fertil., 93, 461–466.
Gupta, S.K., Alves, K., Palladino, L.O. et al. (1996) Molecular cloning of
the human fertilin beta subunit. Biochem. Biophys. Res. Commun.,
224, 318–326.
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.
Haendler, B., Kratzschmar, J., Theuring, F. and Schleuning, W.D. (1993)
Transcripts for cysteine-rich secretory protein-1 (CRISP-1; DE/AEG)
and the novel related CRISP-3 are expressed under androgen control
in the mouse salivary gland. Endocrinology, 133, 192–198.
Hall, L., Williams, K., Perry, A.C.F. et al. (1998) The majority of human
glutathione peroxidase type 5 (GPX5) transcripts are incorrectly
spliced: implications for the role of GPX5 in the male reproductive
tract. Biochem. J., 333, 5–9.
Hayashi, M., Fujimoto, S. and Takano, H. (1996) Characterization of a
human glycoprotein with a potential role in sperm-egg fusion: cDNA
cloning, immunohistochemical localization, and chromosomal
assignment of the gene (AEGL1). Genomics, 32, 367–374.
Holland, N.B., Yongxing, Q., Ruegsegger, M. and Marchant, R.E. (1998)
Biomimetic engineering of non-adhesive glycocalyx-like surfaces
using oligosaccharide surfactant polymers. Nature, 392, 799–801.
Holt, W.V. (1980) Surface-bound sialic acid on ram and bull spermatozoa:
deposition during epididymal transit and stability during washing.
Biol. Reprod., 23, 847–857.
Hunter, D.D., Porter, B.E., Bulock, J.W. et al. (1989) Primary sequence of
a motor neuron-selective adhesive site in the synaptic basal lamina
protein S-laminin. Cell, 59, 905–913.
Isojima, S., Tsuji, Y. and Kameda, K. (1990) Coating antigens of human
seminal plasma involved in fertilization. In Alexander, N., Griffin,
D., Spieler, J.M. and Waites, G.M.H. (eds), Gamete Interaction:
Prospects for Immunocontraception. Wiley-Liss, New York, pp.
155–174.
Jentoft, N. (1990) Why are proteins O-glycosylated ? Trends Biochem.
Sci., 15, 291–294.
Jones, R.C. (1989) Membrane remodelling during sperm maturation in the
epididymis. Oxford Rev. Reprod. Biol., 11, 285–337.
Jury, J.A., Frayne, J. and Hall, L. (1997) The human fertilin alpha gene is
non-functional: implications for its proposed role in fertilization.
Biochem. J., 321, 577–581.
Kameda, K., Takada, Y., Hasegawa, A. et al. (1991) Sperm immobilizing
and fertilization-blocking monoclonal antibody 2C6 to human
seminal plasma antigen and characterization of the antigen epitope
corresponding to the monoclonal antibody. J. Reprod. Immunol., 20,
27–41.
Kameda, K., Tsuji, Y., Koyama, K. and Isojima, S. (1992) Comparative
studies of the antigens recognized by sperm-immobilizing
monoclonal antibodies. Biol. Reprod., 46, 349–357.
Kawaoka, Y., Yamnikova, S., Chambers, T.M. et al. (1990) Molecular
characterization of a new hemagglutinin, subtype H14, of influenza A
virus. Virology, 179, 759–767.
Kirchhoff, C. (1994) A major messenger ribonucleic acid of the rodent
epididymis encodes a small glycosylphosphatidylinositol-anchored
lymphocyte surface antigen. Biol. Reprod., 50, 896–902.
Kirchhoff, C. (1996) CD52 is the ‘major maturation-associated’ sperm
membrane antigen. Mol. Hum. Reprod., 2, 9–17.
Kirchhoff, C. and Hale, G. (1996) Cell-to-cell transfer of
glycosylphosphatidylinositol-anchored membrane proteins during
sperm maturation. Mol. Hum. Reprod., 2, 177–184.
Kirchhoff, C., Osterhoff, C., Habben, I. and Ivell, R. (1990) Cloning and
analysis of mRNAs expressed specifically in the human epididymis.
Int. J. Androl., 13, 155–167.
Kirchhoff, C., Habben, I., Ivell, R. and Krull, N. (1991) A major human
epididymis-specific cDNA encodes a protein with sequence
homology to extracellular proteinase inhibitors. Biol. Reprod., 45,
350–357.
Kirchhoff, C., Krull, N., Pera, I. and Ivell, R. (1993) A major mRNA of the
human epididymal principal cells, HE5, encodes the leucocyte
differentiation CDw52 antigen peptide backbone. Mol. Reprod. Dev.,
34, 8–15.
Koehler, J.K. (1981) Lectins as probes of the spermatozoon surface. Arch.
Androl., 6, 197–217.
Kratzschmar, J., Haendler, B., Eberspaecher, U. et al. (1996) The human
cysteine-rich secretory protein (CRISP) family. Primary structure and
tissue distribution of CRISP-1, CRISP-2 and CRISP-3. Eur. J.
Biochem., 236, 827–836.
Kubota, H., Okazaki, H., Onuma, M. et al. (1990) Identification and gene
cloning of a new phosphatidyl-linked antigen expressed on mature
lymphocytes. J. Immunol., 145, 3924–3931.
Kumar, G.P., Laloraya, M. and Agrawal, P. (1990) The involvement of
surface sugars of mammalian spermatozoa in epididymal maturation
and in-vitro sperm-zona recognition. Andrologia, 2, 184–194.
Kurpisz, M. and Alexander, N.J. (1995) Carbohydrate moieties on sperm
surface: physiological relevance. Fertil. Steril., 63, 158–165.
Lachmann, P.J. (1991) The control of homologous lysis. Immunol. Today,
12, 312–315.
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.
Lee, C.Y. and Wong, E. (1986) Developmental studies of sperm surface
antigens using sperm-specific monoclonal antibodies. J. Reprod.
Immunol., 9, 275–287.
Lefèvre, A., Duquenne, C. and Finaz, C. (1998) SOB1, a human gene
coding for a sperm protein with potential oocyte binding activity. 10th
European Testis Workshop, Capri, 1998, Abstr. F1.
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.
Margagee, S.F., Kunze, E. and Hammerstedt, R.H. (1988) Changes in
lectin-binding features of ram sperm surfaces associated with
epididymal maturation and ejaculation. Biol. Reprod., 38, 667–685.
McArdle, W.L. (1991) Regulation of Epididymal Cell Function In Vitro
and Characterisation of In Vivo Sperm Binding Proteins of Likely
Epididymal Origin. PhD thesis, Hatfield University, UK.
McConville, M.J. and Ferguson, M.A.J. (1993) The structure, biosynthesis
and function of glycosylated phosphatidylinositols in the parasitic
protozoa and higher eukaryotes. Biochem. J., 294, 305–324.
Meri, S., Waldmann, H. and Lachmann, P.J. (1991) Distribution of
protectin (CD59), a complement membrane attack inhibitor, in normal
human tissues. Lab. Invest., 65, 532–537.
Moore, D., White, T.W. and Ensrud, K.M. (1989) The major maturation
glycoprotein on rat cauda epididymal sperm surface is linked to the
The sperm glycocalyx
membrane via phosphatidylinositol. Biochem. Biophys. Res.
Commun., 160, 460–468.
Moore, H.D.M. (1990) Development of sperm–egg recognition processes
in mammals. J. Reprod. Fertil., Suppl. 42, 71–78.
Moore, H.D.M. (1996) The influence of the epididymis on human and
animal sperm maturation and storage. Hum. Reprod., 11, 103–110.
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., Flickinger, C.J. and Herr, J.C. (1997) Two-dimensional
gel electrophoretic analysis of vectorially labeled surface proteins of
human spermatozoa. Biol. Reprod., 56, 771–787.
Navaneetham, D., Sivashanmugam, P. and Rajalakshmi, M. (1996)
Changes in binding of lectins to epididymal, ejaculated, and
capacitated spermatozoa of the rhesus monkey. Anat. Rec., 245,
500–508.
Osterhoff, C., Kirchhoff, C., Krull, N. and Ivell, R. (1994) Molecular
cloning and characterization of a novel human sperm antigen (HE2)
specifically expressed in the proximal epididymis. Biol. Reprod., 50,
516–525.
Osterhoff, C., Kirchhoff, C. and Ivell, R. (1995) Fate of two novel human
sperm antigens HE4 and HE5 during in vitro capacitation. Colloque
INSERM, 236, 393–394.
Perez Martinez, S., Conesa, D. and Cuasnicu, P.S. (1995) Potential
contraceptive use of epididymal proteins: evidence for the
participation of specific antibodies against rat epididymal protein DE
in male and female fertility inhibition. J. Reprod. Immunol., 29,
31–45.
Perotti, M.E. and Pasini, M.E. (1995) Glycoconjugates of the surface of the
spermatozoa of Drosophila melanogaster: a qualitative and
quantitative study. J. Exp. Zool., 272, 311–318.
Perry, A.C., Jones, R., Barker, P.J. and Hall, L. (1992) A mammalian
epididymal protein with remarkable sequence similarity to snake
venom haemorrhagic peptides. Biochem. J., 286, 671–675.
Phelps, B.M. and Myles, D.G. (1987) The guinea pig sperm plasma
membrane protein, PH-20, reaches the surface via two transport
pathways and becomes localized to a domain after an initial uniform
distribution. Dev. Biol., 123, 63–72.
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.
Primakoff, P., Lathrop, W., Woolman, L. et al. (1988) Fully effective
contraception in male and female guinea pigs immunized with the
sperm protein PH-20. Nature, 335, 543–546.
Rademacher, T.W., Parekh, R.B. and Dwek, R.A. (1988) Glycobiology.
Annu. Rev. Biochem., 57, 785–838.
Rochwerger, L. and Cuasnicu, P.S. (1992) Redistribution of a rat sperm
epididymal glycoprotein after in vitro and in vivo capacitation. Mol.
Reprod. Dev., 31, 34–41.
Rochwerger, L., Cohen, D.J. and Cuasnicu, P.S. (1992) Mammalian
sperm–egg fusion: the rat egg has complementary sites for a sperm
protein that mediates gamete fusion. Dev. Biol., 153, 83–90.
Ross, P., Kan, F.W., Antaki, P. et al. (1990) Protein synthesis and secretion
in the human epididymis and immunoreactivity with sperm
antibodies. Mol. Reprod. Dev., 26, 12–23.
Ross, P., Vigneault, N., Provencher, S. et al. (1993) Partial characterization
of galactosyltransferase in human seminal plasma and its distribution
in the human epididymis. J. Reprod. Fertil., 98, 129–137.
Runnebaum, I.B., Schill, W.B. and Töpfer Petersen, E. (1995)
ConA-binding proteins of the sperm surface are conserved through
evolution and in sperm maturation. Andrologia, 27, 81–90.
Sabeur, K., Cherr, G.N., Yudin, A.I. et al. (1997) The PH-20 protein in
human spermatozoa. J. Androl., 18, 151–158.
Sawada, R., Ohashi, K., Anaguchi, H. et al. (1990) Isolation and
expression of the full-length cDNA encoding CD59 antigen of human
lymphocytes. DNA Cell Biol., 9, 213–220.
313
Schröter, S., Kirchhoff, C., Yeung, C.H. et al. (1997) Purification and
structural analysis of sperm CD52, a GPI-anchored membrane
protein. Adv. Exp. Med. Biol., 424, 233–234.
Seemuller, U., Arnhold, M., Fritz, H. et al. (1986) The acid-stable
proteinase inhibitor of human mucous secretions (HUSI-I,
antileukoprotease). Complete amino acid sequence as revealed by
protein and cDNA sequencing and structural homology to whey
proteins and Red Sea turtle proteinase inhibitor. FEBS Lett., 199,
43–48.
Tezon, J.G., Ramella, E., Cameo, M.S. et al. (1985) Immunochemical
localization of secretory antigens in the human epididymis and their
association with spermatozoa. Biol. Reprod., 32, 591–597.
Töpfer-Petersen, E. (1999) Carbohydrate-based interactions on the route
of a spermatozoon to fertilization. Hum. Reprod. Update, 5, 314–329.
Treumann, A., Lifely, M.R., Schneider, P. and Ferguson, M.A. (1995)
Primary structure of CD52. J. Biol. Chem., 270, 6088–6099.
Tsuji, Y. (1995) Carbohydrate antigens recognized by anti-sperm
antibodies. In Kurpisz, M. and Fernandez, F. (eds), Immunology of
Human Reproduction, M. Bios Scientific Publishers, Oxford, UK, pp.
23–32.
Tsuji, Y., Clausen, H., Nudelman, E. et al. (1988) Human sperm
carbohydrate antigens defined by an antisperm monoclonal antibody
derived from an infertile woman bearing antisperm antibodies in her
serum. J. Exp. Med., 168, 343–356.
Tulsiani, D.R.P., Orgebin-Christ, M.C. and Skudlarek, M.D. (1998) Role
of luminal fluid glycosyltransferases and glycosidases in the
modification of rat sperm plasma membrane glycoproteins during
epididymal maturation. J. Reprod. Fertil., Suppl. 53, 85–97.
Tulsiani, D.R.P., Yoshida-Komiya, H. and Araki, Y. (1997) Mammalian
fertilization: a carbohydrate-mediated event. Biol. Reprod., 57,
487–494.
Uhlenbruck, F., Sinowatz, F., Amselgruber, W. et al. (1993)
Tissue-specific gene expression as an indicator of epididymis-specific
functional status in the boar, bull and stallion. Int. J. Androl., 16,
53–61.
Walsh, L.A., Tone, M. and Waldmann, H. (1991) Transfection of human
CD59 complementary DNA into rat cells confers resistance to human
complement. Eur. J. Immunol., 21, 847–850.
Wolfsberg, T.G., Straight, P.D., Gerena, R.L. et al. (1995) ADAM, a
widely distributed and developmentally regulated gene family
encoding membrane proteins with a disintegrin and metalloprotease
domain. Dev. Biol., 169, 378–383.
Xia, M.Q., Tone, M., Packman, L. et al. (1991) Characterization of the
CAMPATH-1 (CDw52) antigen: biochemical analysis and cDNA
cloning reveal an unusually small peptide backbone. Eur. J. Immunol.,
21, 1677–1684.
Xia, M.Q., Hale, G., Lifely, M.R. et al. (1993) Structure of the
CAMPATH-1 antigen, a glycosylphosphatidylinositol-anchored
glycoprotein which is an exceptionally good target for complement
lysis. Biochem. J., 293, 633–640.
Yanagimachi, R. (1994) Mammalian Fertilization. In Knobil, E. and Neill,
J.D. (eds), The Physiology of Reproduction. Raven Press, 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, 497–519.
Yeung, C.H., Schröter, S., Wagenfeld, A. et al. (1997) Interaction of the
human epididymal protein CD52 (HE5) with epididymal spermatozoa
from men and cynomolgus monkeys. Mol. Reprod. Dev., 48, 267–275.
Young, L.G. and Fröhlich, O. (1998) HE2/EP2, an androgen-dependent
protein of the chimpanzee (Pan troglodytes) epididymis. J. Reprod.
Fertil., Suppl. 53, 215–220.
Zeheb, R. and Orr, G.A. (1984) Characterization of maturation-associated
glycoprotein on the plasma membrane of rat caudal epididymal sperm.
J. Biol. Chem., 259, 839–848.
Zeng, F.Y. and Gabius, H.J. (1992) Sialic acid-binding proteins:
characterization, biological function and application. Z. Naturforsch.
Ser. C, 47, 641–53.
Received on July 20, 1998; accepted on March 23, 1999