Human Reproduction, Vol.28, No.3 pp. 566–577, 2013
Advanced Access publication on January 12, 2013 doi:10.1093/humrep/des447
ORIGINAL ARTICLE Andrology
The role of carbohydrate recognition
during human sperm –egg binding
Gary F. Clark*
Division of Reproductive Medicine and Fertility, and Division of Reproductive and Perinatal Research, Department of Obstetrics, Gynecology
and Women’s Health, University of Missouri School of Medicine, 1 Hospital Drive, Columbia, MO 65212, USA
*Correspondence address. E-mail: [email protected]
Submitted on July 23, 2012; resubmitted on November 19, 2012; accepted on December 4, 2012
study question: What is the role of carbohydrates in the binding of human sperm to the zona pellucida (ZP) and what are potential
implications for pathogenesis?
summary answer: Both lectin-like and protein– protein interactions play an essential role in human gamete interactions.
what is known already: Studies in the mouse and human indicate a role for both lectin-like and protein –protein interactions
during sperm binding to the ZP.
study design, size, duration: Non-systematic literature review.
main results and the role of chance: Ultrasensitive analysis by mass spectrometry of glycans linked to the human ZP has
confirmed that this matrix is coated with a high density of complex type N-glycans terminated with the sialyl-Lewisx (sLex) sequence, the
universal selectin ligand. Selectins are essential for lymphocyte homing, and they participate in the initial binding of circulating leukocytes
to activated endothelium at the sites of infection and tissue injury. Subsequent inhibition studies confirmed that either the sLex tetrasaccharide or neoglycoproteins terminated with this sequence inhibited human sperm– ZP binding by 70% in the hemizona assay. These results
support the hypothesis that both lectin-like and protein –protein interactions play an essential role in human gamete interactions. The
sLex sequence is also a ligand for siglec-9, a lectin-bearing immunoreceptor tyrosine-based inhibitory motif that transmits inhibitory
signals. This siglec is expressed on a wide variety of different types of human leukocytes and lymphocytes. This result is consistent with
the hypothesis that human ZP glycans are also being employed for immune recognition of the egg and the histoincompatible embryo
prior to blastocyst hatching.
limitations, reasons for caution: This field of study is complex and more experimental work is needed to reveal fully the
mechanism of sperm –ZP binding and how it varies between species.
wider implications of the findings: Knowledge about the glycans involved in sperm –egg binding may be relevant to infertility due to fertilization failure and also to the mother’s immune tolerance of the preimplantation embryo.
study funding/competing interest(s): Studies focused on human sperm– egg interactions carried out by the author and
coworkers have been supported by the Life Sciences Mission Enhancement Reproductive Biology Program funded by the State of Missouri, a
Research Board Grant (CB000500) supported by the University of Missouri System and a grant from the Jeffress Memorial Trust of Virginia.
Support from the Breeden-Adams Foundation has also been obtained to investigate potential linkages to tumor evasion. The author has no
conflict of interest to declare.
Key words: fertilization / sperm function / zona pellucida / andrology / assisted reproduction
Introduction
Before 1938, studies on mammalian fertilization were performed exclusively on animal models. Human sperm –egg binding had never
been observed, and the topic of human fertilization was usually only
discussed among scientists and physicians. Between 1938 and 1944,
gynecologist John Rock and his technician Miriam Menkin collected
800 human ova and unsuccessfully attempted to fertilize 138 of
them. However, they were easily able to observe binding between
human sperm and the translucent matrix covering the human egg,
known as the zona pellucida (ZP). Sperm must bind to the ZP and
transit through this matrix to enter the perivitelline space, where
& The Author 2013. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
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Human sperm– egg binding
they fuse with the oocyte. The union of these gametes completes the
cellular recognition events required for natural human fertilization.
In 1944, Menkin allowed the sperm and eggs to stay in contact with
each other for a longer period of time, leading to fertilization and the
generation of 2-cell stage human embryos (Rock and Menkin, 1944).
This achievement created an instant sensation in both scientific
circles and the lay public. Unfortunately, the popular press coined
the term ‘test tube baby’ to describe these embryos. This new terminology fueled public distrust about investigations focused on early
human development that were first evoked by the publication of
Aldous Huxley’s book Brave New World in 1932.
This controversy did not stop determined investigators, who could
clearly see the clinical benefits of IVF for infertile couples. The ex vivo
maturation of a human oocyte was achieved in 1969 (Edwards et al.,
1969). Nine years later, this same investigative group achieved a live
birth after human IVF (Steptoe and Edwards, 1978). They had to overcome many obstacles that few could imagine today. In 2010, Robert
Edwards was awarded the Nobel Prize in physiology or medicine
for his major role in this historic accomplishment (Kirby, 2010).
Human IVF increased access to human eggs, which was crucial for
understanding the process of human fertilization. The binding of
sperm to the ZP is a piece of this complex puzzle, though certainly
an essential one to comprehend for many practical reasons. A
number of recent developments have brought us closer to understanding the molecular basis of human sperm–egg binding. The
focus of this review is to outline the studies that have led to our
current knowledge about this key adhesion event. Other ramifications
of these studies will also be discussed.
Early experiments focused
on understanding human
sperm – egg binding
When the goal of human IVF was finally realized in 1978, there was
limited knowledge about the composition of the mammalian ZP.
The development of IVF increased access to human eggs that either
failed to bind sperm or undergo subsequent development following
ICSI. Human eggs were also obtained from cadaveric ovaries (Overstreet and Hembree, 1976; Franken et al., 1994). Initially, there
were some concerns about the physiological relevance of the results
obtained from the small numbers of human ZP collected from these
sources. However, an internally controlled assay for human sperm –
ZP binding known as the hemizona assay (HZA) maximized the use
of the available human ZP (Burkman et al., 1988). The results in the
HZA were predictive of male fertility, dispelling concerns related to
the physiological relevance of ZP collected from these sources
(Franken and Oehninger, 2006). Nonetheless, the number of available
eggs remained far less than the amount required to perform useful
biochemical analyses. This problem was due to the relative insensitivity
of the methods available during the early days of human IVF.
In contrast, mouse and porcine ZP were isolated in sufficient quantity for biochemical analyses for the first time in the same year that
human IVF became a reality (Bleil and Wassarman, 1978; Dunbar
et al., 1978). Studies in mammals enabled investigators to easily test
the effects of solubilized ZP preparations and individual ZP glycoproteins on sperm –ZP binding. Previous results obtained in lower marine
567
animal models provided evidence suggesting that sperm–egg binding
relied primarily on carbohydrate recognition. This concept was first
proposed based on the substantial loss of sperm binding that was
observed after mild periodate oxidation of the egg jelly coat covering
the egg in these lower species (Monroy, 1965). This chemical procedure preferentially oxidizes oligosaccharides and polysaccharides at
their vicinal hydroxyl groups, making it highly selective for carbohydrate sequences when employed under carefully controlled conditions
(Woodward et al., 1985). This proposed specificity made the investigation of human sperm –egg binding an even greater challenge, as the
chemical and biophysical methods for analyzing polysaccharides and
oligosaccharides were far less sensitive than the methods for protein
and DNA sequencing.
Greater access to porcine and mouse ZP led many investigators to
focus on investigations in these animal models. The inherent assumption in these studies was that these results could be directly applicable
to the human model. This concept was supported by the overlaps that
were observed during the early events of mammalian fertilization. In
mammals, sperm usually undergo weak binding to their homologous
ZP, resulting in an adhesive interaction known as sperm attachment
(Clark, 2010). The affinity of his interaction gradually increases over
time, until firm binding is established. Acrosomal exocytosis is subsequently induced and sperm move through the ZP, where they enter
the perivitelline space and fertilize the egg cell. Several pertinent
reviews focused on studies in mouse and pig are available, and
should be consulted for additional background in these models
(Clark and Dell, 2006; Hedrick, 2008; Topfer-Petersen et al., 2008;
Wassarman and Litscher, 2008; Clark, 2010; Clark, 2011a,b).
As noted earlier, investigators who desired to study the specifics of
human sperm –egg binding were hampered due to limited access to
human ZP. To obtain preliminary data, these workers decided to take
an approach that had been previously employed by pioneers who
studied lower marine species. These investigators incubated sperm
and eggs with polysaccharides, oligosaccharides and monosaccharides
to determine if they could block sperm –egg binding. It was hoped
that such experiments would provide insight into the carbohydratebinding specificity of putative lectin-like egg-binding proteins (EBPs).
The acrosomal protein bindin was shown to be involved in the attachment of sperm to the vitelline coat of sea urchin eggs (Vacquier and
Moy, 1977). Vacquier and coworkers subsequently demonstrated that
bindin would specifically bind to fucoidan, an algal polysaccharide consisting primarily of fucose sulfate and fucose residues (Glabe et al.,
1982). In this same study, fucoidan was shown to block the bindinmediated agglutination of sea urchin eggs. Fucoidan also inhibited
fertilization in the brown alga Fucus serratus (Bolwell et al., 1979).
The results from these studies persuaded Huang et al. (1982) to investigate the effect of fucoidan on sperm –egg binding in the human and
other mammals. Acrosome-intact human sperm did not bind at all to
the human ZP in the presence of 0.1 or 1 mg/ml fucoidan. Sperm
binding was determined in the presence of fucoidan (0.001, 0.01, 0.1,
0.25, 0.5 and 1 mg/ml) and L-fucose (1 mg/ml or 6.1 mM) in a subsequent study (Oehninger et al., 1990). Fucoidan inhibited binding by
80 –85% at all concentrations tested at or above 0.01 mg/ml
(1027 M). However, binding increased by 33% in the presence of
L-fucose, the major monosaccharide in fucoidan (Oehninger et al.,
1990). These results indicated that both fucoidan and L-fucose interacted with a lectin-like EBP on the plasma membrane of human sperm.
568
Clark
For comparison, these investigators also tested the effect of fucoidan
in the hamster and guinea pig models. Unlike human sperm, acrosomeintact hamster sperm were initially bound to the hamster ZP in the presence of 0.1 or 1 mg/ml fucoidan. However, sperm detached over time,
reducing the number bound by 50and 85% at 30 and 60 min after insemination, respectively (Huang et al., 1982). Fertilization of hamster eggs
also declined from 94% in controls to 70, 8 and 0% in the presence of
0.01, 0.1 and 1 mg/ml of fucoidan, respectively. Fucoidan potently
and immediately inhibited sperm binding to eggs from the guinea pig in
the same range that it inhibited human sperm– ZP binding (Huang
et al., 1982). However, this inhibitory effect was due to the binding of
fucoidan to the inner acrosomal membrane of hamster sperm and not
to the plasma membrane (Huang and Yanagimachi, 1984). These
results supported the concept that fucoidan mediated its inhibitory
effect in the hamster and guinea pig by interacting with the EBPs associated with the inner acrosomal membrane.
These findings provided evidence suggesting that initial human
sperm –egg binding involved lectin-like EBPs with a distinct
carbohydrate-binding specificity compared with other mammals. A
previous cross-species sperm –egg binding analysis confirmed that
human sperm bind to eggs from hominoid species (gibbon, gorillas),
but not to eggs from other sub-hominoids (baboons, rhesus monkeys,
squirrel monkeys) or lower mammalian species (Table I) (Bedford,
1977; Lanzendorf et al., 1992). However, sperm from rabbit, mouse,
hamster, rhesus monkey and squirrel monkey displayed substantial
cross binding to eggs from other mammalian species (Table I)
(Bedford, 1977).
Molecular models for human
sperm –ZP binding
Many types of sugars and adhesion molecules have been implicated in
human gamete binding since the late 1980s. Mannosylated glycans
were initially proposed to be ligands for human EBPs. This specificity
was based on the inhibitory effect of D-mannose (50 mM) and the differential effect of plant lectins on human sperm–egg binding (Mori
et al., 1989; Mori et al., 1993). Human sperm-fertilizing capacity
was also reported to be correlated with the expression of a headspecific mannose receptor (Benoff et al., 1993). These mannose
receptors were visualized on the surface of human sperm by using
fluorescein-isothiocyanate-labeled bovine serum albumin coupled
with D-mannose (FITC-Man-BSA). This neoglycoprotein also induced
acrosomal exocytosis in human sperm (Benoff et al., 1997). A
mannose ligand receptor assay was tested to determine whether it
could be used as a method to predict human fertilization in vitro
(Hershlag et al., 1998); however, no human EBP that recognizes terminal mannose has been identified, nor has any assay been developed
that predicts human sperm –ZP binding based on FITC-Man-BSA
binding.
Exposure of human sperm to a panel of different monosaccharides
at a final concentration of 50 mM prior to the HZA was also employed
in another study to determine the role of carbohydrate recognition in
gamete binding (Miranda et al., 1997). Sperm treated with
N-acetylglucosamine, D-mannose, D-fucose, L-fucose and D-galactose
inhibited binding by 62, 58, 82, 68 and 48% in the HZA, respectively.
Though the concentration of sugars tested was relatively high, they did
not affect other biological activities associated with human sperm.
A number of proteins have been suggested to play a role in mediating human sperm –egg binding. An a-mannosidase is associated with
the plasma membrane of human sperm, and was proposed to play a
major role in gamete interaction (Tulsiani et al., 1990). Saling and coworkers suggested that a specific tyrosine kinase associated with human
sperm was the major EBP that interacts with human ZP3 during fertilization (Burks et al., 1995). However, several questions about the
identity of this kinase were subsequently raised but never addressed
(Tsai and Silver, 1996; Bork, 1996). A galactosyl lectin has also been
localized to the head region of acrosome intact human sperm that is
Table I Binding of mammalian sperm to the ZP of homologous and xenogenic eggs.
Origin of eggs
......................................................................................................................................................................
Human
Gibbon
Gorilla
Baboon
Rhesus
monkey
Squirrel
monkey
Rabbit
Mouse
Hamster
Guinea
pig
.............................................................................................................................................................................................
Origin of sperm
Human
+
+
Gibbon
+
+
Gorilla
+
+
2
2
2
2
2
2
2
Rhesus
monkey
+
+
+
+
+
+
Squirrel
monkey
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Rabbit
+
+
Mouse
+
+
Hamster
+
+
Guinea pig
+
+
+
+
+
+
+/2
+/2
2
+
Key to interaction: (+) active attachment not disrupted by strong fluid currents; (+/2) weak attachment by relatively few sperm; (2) sperm show no tendency to adhere to the ZP.
Blank spaces indicate no data available. Data were adapted from studies performed by Bedford (1977) and Lanzendorf et al. (1992).
Human sperm– egg binding
similar to lectins associated with rat and rabbit sperm (Goluboff et al.,
1995). A b1–4 galactosyltransferase enzyme was reported to be
present in the human sperm plasma membranes where it was
hypothesized to play a role in sperm binding (Huszar et al., 1997).
A recombinant form of b-N-acetylhexosaminidase that was originally
isolated from human sperm plasma membranes was also reported
to block human sperm– ZP binding in the HZA, indicating that it
could act as an EBP (Miranda et al., 2000).
Zonadhesin is a putative EBP that has been implicated in mammalian
gamete binding. This binding protein was originally isolated from solubilized pig sperm membranes, but genes for species-specific forms are
expressed in the human, mouse, rabbit, donkey, stallion and rodents
(Hardy and Garbers, 1994; Wilson et al., 2001; Tardif and Cormier,
2011). Though zonadhesin is located within the acrosome, the possibility exists that it could be transiently externalized on the sperm
plasma membrane, enabling this protein to bind the ZP prior to acrosomal exocytosis. This potential human EBP needs to be tested on a
glycan array to determine whether it possesses lectin-like domains
specific for sLex.
More recently, human sperm proteins that interact with human ZP3
glycoprotein were identified by employing the yeast two-hybrid system
(Naz and Dhandapani, 2010). The highest affinity ZP3-interacting
protein displayed 97% homology with ubiquitin associated protein-2like. Antibodies directed against this protein specifically inhibited
human sperm–ZP binding in the HZA by 83%. However, this potential receptor has not yet been screened to determine whether it has
lectin-like properties.
Multimeric protein complexes have been recently implicated in
human sperm –ZP binding (Redgrove et al., 2011). Complex I consists
of eight subunits that make up the chaperonin containing the TCP-1
complex plus the putative zona recognition molecule known as
ZP-binding protein isoform 2 (ZPBP2). Complex II consists of a
mixture of the a- and b-type proteasome subunits. Antibodies directed against ZPBP2, a3 subunit and b1 subunits significantly reduced
sperm –ZP binding by 42, 57 and 68%, respectively (Redgrove et al.,
2011). However, results obtained in a previous study indicate that
79% of sperm binding on the human ZP rely on carbohydrate recognition, with the remainder due to protein–protein interactions
(Ozgur et al., 1998). It is possible that ZPBP2 and/or these proteasome subunits could be involved in lectin-like interactions, but this
hypothesis has not yet been tested.
Human sperm – egg binding and
the involvement of the universal
selectin ligand sLex
In 1983, fucoidan was shown to potently inhibit the binding of lymphocytes to high endothelial venules, an interaction that is essential for
lymphocyte recirculation (Stoolman and Rosen, 1983). This process
was subsequently found to involve an adhesion molecule known as
L-selectin. What was particularly striking was the observation that
human sperm–egg binding and lymphocyte recirculation were inhibited by fucoidan to a similar extent in the same concentration range
(Stoolman and Rosen, 1983; Oehninger et al., 1990). The sialyl-Lewisx
(sLex) tetrasaccharide was later shown to be the universal selectin
ligand (Table II) (Phillips et al., 1990; Foxall et al., 1992).
569
The existing structural formula for fucoidan in 1990 was inconsistent
with its ability to inhibit selectin-mediated adhesions (Percival and
McDowell, 1967). To address this issue, fucoidan was analyzed by
employing more definitive methods of carbohydrate structural analysis, yielding an average sequence that was a closer structural
analog of the sulfated selectin ligands (Patankar et al., 1993). Based
on the shared effect of fucoidan on lymphocyte and sperm binding,
human sperm –egg binding was proposed to involve a ‘selectin-like’
interaction (Patankar et al., 1993). This overlap was further supported
by the observation that selectins mediate the weak carbohydratebinding interactions that occurred during the tethering and rolling of
leukocyte on activated vascular endothelium (Cummings and
McEver, 2009).
The presence of selectin ligands on the ZP could in theory promote
the binding of immune and inflammatory cells to human eggs. Such
cells could block human sperm–egg binding and perhaps even facilitate the destruction of the egg. Also, if this model was correct, then
logically human sperm should express selectins on their surface.
However, probing human sperm with anti-selectin antibodies yielded
equivocal results. One group reported the presence of L-selectin on
human sperm (Lucas et al., 1995), whereas no selectins were detected
in another study (Clark et al., 1995). These observations led many to
doubt this model for human gamete binding.
The sLex antigen and a close structural analog known as the sialylLewisa (sLea) antigen were detected on the human ZP by immunocytochemistry (Table II) (Lucas et al., 1995), and later confirmed in
another study (Jimenez-Movilla et al., 2004). However, monoclonal
antibodies directed against sLex and sLea did not block human
sperm –ZP binding in one study (Lucas et al., 1995). Instead, a monoclonal antibody directed against the Lewisb antigen reportedly blocked
human sperm –ZP binding in the HZA (Lucas et al., 1994). Human
sperm binding to the ZP was maximally inhibited by 70% by the
sLex tetrasaccharide (Clark et al., 1995).
Though these results were equivocal, another line of evidence supported the proposal that human sperm– ZP binding involved a
selectin-like interaction. Many glycoproteins and fluids were screened
for their ability to inhibit sperm –ZP binding in the human HZA
(Burkman et al., 1988). The peritoneal fluid from patients with endometriosis decreased human sperm– ZP binding in this assay system,
but fluid from normal patients did not (Coddington et al., 1992).
Testing of the individual glycoproteins isolated from the peritoneal
fluid of endometriosis patients confirmed that the placental protein
14 (PP14) was the inhibitory factor (Oehninger et al., 1995). This
glycoprotein had previously been shown to be a major secreted immunosuppressive factor in the pregnant uterus during implantation,
constituting 4–10% of the total decidual protein (Clark et al., 1996).
These results provided evidence that the immune and the gamete recognition systems overlapped in the types of carbohydrate ligands that
they recognized.
Based on the previous studies that showed the inhibitory effects of
fucoidan and the sLex tetrasaccharide in the HZA, the expectation
was that PP14 expressed terminal sLex sequences on its glycans;
however, this epitope was not detected (Dell et al., 1995). Another
selectin ligand known as the fucosylated lacdiNAc sequence was profusely expressed on PP14 (Table II) (Dell et al., 1995). This sequence is
reportedly a higher affinity selectin ligand than the sLex sequence
(Grinnell et al., 1994). Based on the expression of these carbohydrate
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Clark
Table II Inhibitors and other glycans implicated in human sperm–ZP binding.
Name
Sequence1
References
Evidence2
.............................................................................................................................................................................................
Fucoidan3
Huang et al. (1982); Oehninger
et al. (1990)
Structural analog only
Simple sugars
Mori et al. (1989); Mori et al.
(1993); Miranda et al. (1997)
Weak, not glycan, high
concentrations needed
Sialyl-Lewisx
Clark et al. (1995); Pang et al.
(2011)
Good, direct inhibition in HZA,
present in the ZP
Sialyl-Lewisa
Clark et al. (1995); Pang et al.
(2011)
Poor, not detected by MS
analysis in human ZP
Lewisb
Lucas et al. (1994)
Poor, not detected by MS
analysis in human ZP
Fucosylated
LacdiNAc
Clark et al. (1995); Pang et al.
(2011)
Fair, not present in human ZP
Sialyl-Lewisx –
Lewisx
Pang et al. (2011)
Good, present in human ZP
1
Fuc, fucose; Man, mannose; Gal, galactose; GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine; NeuAc, N-acetylneuraminic acid.
Evidence that these carbohydrate sequences or monosaccharides were involved in human sperm –ZP binding.
Average structure of fucoidan (Patankar et al., 1993).
2
3
sequences on PP14, its name was changed to glycodelin to reflect the
role of its glycans as functional groups responsible for its biological
activities.
Though there was strong inferential evidence that human sperm –
egg binding involved the participation of a selectin ligand, serious
doubts remained about the validity of this model. The definitive experimental pathway to address this issue was to sequence the
glycans associated with the human ZP, and determine whether
glycans terminated with this sequence inhibited binding. However,
each human egg is coated with only 32 ng of ZP glycoprotein (Chiu
et al., 2008). Until recently, the methods of carbohydrate sequencing
were not sufficiently sensitive to analyze human ZP glycans due to the
miniscule amounts of available human ZP.
In 2010, biophysical methods of structural analysis finally emerged
that would enable the sequencing of human ZP glycans. Ultrasensitive
matrix-assisted laser desorption/ionization –time-of-flight (MALDITOF) mass spectrometry (MS) was used in combination with collisionally activated dissociation on a MALDI-TOF-TOF instrument to
analyze the N- and O-glycans linked to human ZP (Pang et al.,
2011). This task required ZP isolated from only 195 human eggs
that had failed fertilization during human IVF. Structural analysis of
the N-glycans indicated that 70% of their antennae were terminated
with the sLex tetrasaccharide or the sLex –Lex heptasaccharide
(Table II). Models for the major biantennary, triantennary and tetraantennary N-glycans are shown in Fig. 1. Minor amounts of sulfated
N-glycans terminated with sulfo-Lex sequences were also detected
Human sperm– egg binding
Figure 1 Formulas for biantennary, triantennary and tetraantennary N-glycans associated with the human ZP. This figure was previously published (Pang et al., 2011).
571
(Pang et al., 2011). The N-glycans were far more abundant than the
O-glycans. However, the sLex sequence was also linked to two
O-glycan sequences (Fig. 2) (Pang et al., 2011). These results indicated
that sLex expressed on both N- and O-glycans could participate in
binding.
To further test the hypothesis that human sperm –egg binding
required the participation of this carbohydrate sequence, the effect
of sLex-based glycoconjugates on human sperm –ZP binding in the
human HZA was analyzed. The sLex tetrasaccharide inhibited
human sperm –ZP binding in the HZA by 63% at 0.5 mM [hemizona
index (HZI) ¼ 36.8 + 5.1] (Pang et al., 2011). A bovine serum
albumin (BSA) based neoglycoprotein conjugated with 12-14 sLex
sequences blocked binding by 69% at 2 mM (HZI ¼ 30.9 + 3.4)
(Pang et al., 2011). In contrast, human ZP3 inhibited binding by 65%
at 25 nM (HZI ¼ 35.1 + 4.5) (Chiu et al., 2008). Human ZP4 inhibited
sperm binding by 57% at the same concentration (HZI ¼ 42.8 + 6.9).
Human ZP3 and ZP4 obtained from human ZP did not affect human
sperm –ZP binding after the removal of their N-glycans (Chiu et al.,
2008). These inhibition studies demonstrated that human ZP3 is
2000-fold more active inhibitor of sperm –ZP binding than the
sLex tetrasaccharide (Chiu et al., 2008). Similarly, ZP3 and ZP4 isolated from human ZP are 50- to 80-fold more active as inhibitors
of binding in the HZA than the sLex-BSA neoglycoprotein.
It is currently unclear if selectins are expressed on human sperm.
Nonetheless, substantial evidence has also implicated selectinmediated adhesions in early human implantation. Fisher and coworkers previously showed that carbohydrate ligands for L-selectin are
highly up-regulated on the human uterine epithelium during the
luteal but not proliferative phase of the menstrual cycle (Genbacev
et al., 2003). These investigators also observed a complementary increase in the expression of L-selectin on human trophoblasts. Trophoblasts were readily bound to beads coated with a well-defined
carbohydrate ligand for L-selectin, known as the 6-sulfo-sLex sequence
(Genbacev et al., 2003). In addition, trophoblasts were bound to
human uterine epithelial cells isolated during the luteal phase, but
not during the proliferative phase.
The role of the human ZP
protein component in mediating
gamete binding
Figure 2 O-glycans associated with the human ZP. The sialylLewisx (sLex) sequences are in dashed boxes. The symbols for
the monosaccharides are the same as in Fig. 1, except for
N-acetylgalactosamine (yellow squares).
The primary focus of this review is on carbohydrate-dependent interactions involving human ZP glycoproteins. Nonetheless, the role
played by the protein component of ZP glycoproteins must also be
considered. The human ZP is composed of four glycoproteins, designated ZP1, ZP2, ZP3 and ZP4 (Lefièvre et al., 2004; Conner et al.,
2005). Among the mammalian species tested, only the human,
other primates and rat express four ZP glycoproteins (Claw and
Swanson, 2012). The concept that human gamete binding involves
both lectin-like and protein –protein interactions was presented in a
previous report (Ozgur et al., 1998). Consistent with this hypothesis,
recombinant bacterial human ZP proteins lacking any glycosylation
readily bind to human sperm, as do glycosylated forms synthesized
in insect baculovirus systems (Gupta et al., 2009; Chirinos et al.,
2009; Gupta et al., 2012). This type of plasticity is certainly not
new, but was readily predictable based on previous results (Clark
572
et al., 1997). For example, sea urchin bindin interacts with two different egg surface proteins during fertilization, a 350-kDa glycoprotein
and EBR1. Bindin recognizes the 350-kDa egg protein via its sulfated
O-linked oligosaccharides and EBR1 via its repeated protein sequence
molecules (Vacquier, 2012). This complementation could easily
provide the level of species-specificity for sea urchin sperm binding
that is required when many species are present in the same ecological
niche.
An excellent review focused on the evolutionary relationships of the
extracellular barriers of both vertebrate and invertebrate eggs was recently published (Claw and Swanson, 2012). The major conclusion
presented by these authors is that the structure and the function of
egg coats have remained relatively conserved over time, but the constituents are rapidly evolving. Evidence supporting this statement was
presented recently when it was shown that the functional domains of a
molluscan (abalone) sperm receptor protein and a yeast-mating
protein express the same 3D fold as the ZP proteins that mediate
gamete binding in humans (Swanson et al., 2011). This relationship
was established in the absence of any overt amino acid similarity in
the egg envelope proteins. It is likely that the egg envelope proteins
adopt a particular structure for the presentation of glycans that optimizes for initial sperm –egg binding. This difference could explain
why human ZP3 and ZP4 are much more effective inhibitors of
human sperm–ZP binding than the sLex neoglycoprotein (Chiu
et al., 2008; Pang et al., 2011).
The supramolecular complex
model for mouse and human
sperm –egg binding
The hypothesis that mammalian sperm –egg binding relies on a combination of both lectin-like and protein –protein interactions is not universally accepted. A major model for binding in the mouse and the
human is based on the premise that the major ZP glycoproteins
form a supramolecular complex that binds sperm (Dean, 2004).
This complex of glycoproteins becomes non-permissive for binding
when mouse ZP2 is cleaved by a specific protease (ovastacin) after
fertilization (Burkart et al., 2012). Under physiological conditions,
mouse sperm do not bind to 2-cell stage embryos from wild-type
mice. ZP2mut mice have a mutated cleavage site for ovastacin, and
are resistant to this protease. Two-cell stage embryos from ZP2mut
mice continue to bind mouse sperm (Gahlay et al., 2010).
This latter observation seemingly precludes any role for carbohydrate recognition involving mouse ZP3 in mediating murine sperm –
egg binding. However, there are potential explanations for why
mouse sperm continue to bind to 2-cell stage embryos from ZP2mut
mice. In the classical model for murine gamete binding, mouse ZP3
was modified following fertilization to an inactive form (ZP3f) in
2-cell stage embryos that could not bind mouse sperm (Bleil and
Wassarman, 1980). The possibility exists that mouse ZP2 must be
cleaved before the ZP3 ZP3f conversion can occur. However, a
molecular difference between mouse ZP3 and ZP3f has never been
reported.
Another possibility is that mouse ZP2 and ZP3 must form a heterodimer in the ZP matrix to present carbohydrate sequences in an appropriate spatial orientation to bind to a lectin-like EBP. Data
Clark
obtained in the pig model supports this possibility. The porcine ZP
consists of three glycoproteins (ZP2, ZP3 and ZP4). Boar sperm
bind to a heterodimer of native ZP3 and ZP4, but not to any individual
porcine ZP glycoprotein (Yurewicz et al., 1998). In a recent study, recombinant forms of porcine ZP3 and ZP4 (rZP3, rZP4) were synthesized in an insect baculovirus-Sf9 cell expression system and tested as
heterodimers in combination with native porcine ZP glycoproteins
(nZP3, nZP4) to determine the potential role of glycosylation in
sperm binding (Yonezawa et al., 2012). Heterodimers of rZP3/rZP4
or nZP3/rZP4 were unable to bind to boar sperm, but a heterodimer
of rZP3/nZP4 was readily bound to these gametes. The major difference between the recombinant and native ZP3 is their oligosaccharide
sequences. The authors of this report concluded that nZP4 but not
nZP3 is required for the binding activity of the nZP3/nZP4 heterodimer. This finding indicated that the carbohydrate sequences associated with nZP4 mediate sperm binding, but only when presented
in heterodimeric form with rZP3 or nZP3 (Yonezawa et al., 2012).
This same model could apply in the murine ZP. Mouse ZP2 and
ZP3 in the context of the ZP could form a heterodimer that must
remain intact to mediate the sperm– ZP binding events necessary
for fertilization. In this scenario, intact mouse sperm bind to mouse
ZP3 via a lectin-like interaction. Acrosomal exocytosis is triggered,
and the acrosome-reacted sperm binds to ZP2. The sperm moves
through the mouse ZP into the perivitelline space, binds to the egg
plasma membrane and fuses with the egg cell. This fusion triggers
the cortical reaction, and ovastacin is released. Ovastacin clips ZP2
at a specific site, generating two products that are held together by
a disulfide linkage. This specific proteolysis would simultaneously inactivate the ability of mouse ZP2 to bind acrosome-reacted sperm
and disrupt the ZP2-ZP3 heterodimer necessary for the presentation
of the carbohydrate sequences on mouse ZP3 that mediate binding.
This pathway would completely block the binding of mouse sperm
to 2-cell stage embryos. However, in ZP2mut mouse eggs, ovastacin
cannot cleave mouse ZP2. Both ZP2mut and the ZP2mut-ZP3 heterocomplex would remain intact after fertilization, enabling 2-cell stage
mouse embryos to continue to bind sperm. The difference between
the mouse and pig models is that purified porcine ZP4 cannot block
porcine sperm –egg binding, whereas purified mouse ZP3 efficiently
inhibits murine sperm– egg binding in vitro (Bleil and Wassarman,
1980; Yonezawa et al., 2012).
Either of these two models would be consistent with evidence
obtained from several laborataries that supports a major role for
glycan recognition in murine sperm– egg binding (Clark and Dell,
2006; Clark, 2010). For example, complete inactivation of complex
and hybrid type N-glycosylation in mouse oocytes reduces sperm –
egg binding by 81% compared with this interaction involving eggs isolated from wild-type mice (Shi et al., 2004; Hoodbhoy et al., 2005).
Results obtained by several groups indicate that 80% of mouse
sperm bind to the murine ZP via lectin-like interactions, which is consistent with the decrease in binding that is observed when complex
and hybrid-type glycosylation are inactivated in mouse oocytes
(Clark and Dell, 2006; Clark, 2010).
Dean and coworkers have previously generated mice that express
human instead of mouse ZP glycoproteins (Rankin et al., 1998;
Yauger et al., 2011; Rankin et al., 2003). These investigators recently
reported that human sperm bind to eggs from mice that express
human ZP2 instead of mouse ZP2, but not until after 3 h of
Human sperm– egg binding
co-incubation (Baibakov et al., 2012). In this paper, Dean and coworkers cited another publication that they claimed showed evidence that
the capacitation of human sperm requires a 4 h incubation (Plachot
et al., 1986). However, Plachot and colleagues reported in this
study that the capacitation of human sperm requires only 45 min,
and that fertilization of human eggs is readily observed after only
1.75 h (1 h capacitation followed by 45 min of contact time). Substantial binding of human sperm to the human ZP is also observed in the
human HZA before 3 h (1 h of capacitation followed by 2 h of contact
time) (Franken et al., 1989). The possibility exists that human sperm
could undergo spontaneous acrosomal exocytosis after 3 h of exposure to huZP2 mouse eggs under these assay conditions. If this reaction
is stimulated under these circumstances, then acrosome-reacted
sperm could bind to huZP2 via protein– protein interactions by
employing the same pathway that was originally proposed in the classical model for murine sperm –egg binding (Bleil et al., 1988).
Dean and coworkers also reported that human sperm did not bind
to either huZP3 or huZP4 mouse eggs in their recent study (Baibakov
et al., 2012). However, this result would be expected if carbohydrate
recognition was primarily responsible for the binding activity of these
two glycoproteins. Yeung and coworkers reported that human ZP3
and ZP4 inhibit human sperm– egg binding in the HZA via the expression of their N-glycans (Chiu et al., 2008). However, a major concern
with this study is that these ZP glycoproteins were isolated under denaturing conditions, which could affect the folding of their protein
backbones and their biological activities. Mouse eggs do not express
terminal sLex sequences on either their N- or O-glycans (Noguchi
and Nakano, 1993; Easton et al., 2000; Dell et al., 2003). Neither
huZP3 nor huZP4 mouse eggs would be expected to bind human
sperm if glycans terminated with sLex are essential carbohydrate
ligands for this interaction, as proposed in a recent study (Pang
et al., 2011).
In summary, the recent evidence obtained by Dean and coworkers
certainly does not preclude the possibility that lectin-like interactions
play a major role in human sperm –ZP binding. However, the arguments presented in this case are quite complicated, and will require
more experimentation to sort through. The primary goal is to determine exactly which of these models for human gamete binding best fits
all the available experimental results.
Other implications of sLex
expression on the human ZP
As discussed previously, the concept that a selectin ligand like sLex
could mediate human gamete binding could be viewed as counterintuitive. Such ligands would be expected to promote the binding of
immune and inflammatory cells to human eggs, either blocking
sperm binding and/or facilitating immune destruction of the human
egg. Nonetheless, sLex expression could also be beneficial for the
mother if viewed in the physiological context. The presence of activated immune cells in the Fallopian tube or uterus would indicate
that either an active infection or inflammatory state exists in these
organs. It would not be beneficial for either the mother or the developing human for a pregnancy to be initiated under such pathological
circumstances. If the human female became pregnant under these
conditions, major immune modulators such as glycodelin-A and
573
CA125 would become highly up-regulated in the decidua during
early implantation (Dalton et al., 1995). The expression of these
factors during an active infection could promote pathogenesis and
threaten maternal fertility, health and survival. However, activated leukocytes and lymphocytes could bind to the human ZP and block fertilization, thus preventing the initiation of pregnancy. This event would
conserve maternal resources until the uterus and the fallopian tubes
were free from infection and inflammation. In summary, sLex expression on the ZP could act as a ‘sensing system’ that prevents pregnancies in the female reproductive system when pathological conditions
exist. However, considerable experimentation must be performed
to confirm this tentative hypothesis.
Peter Medawar originally posed a thoughtful question that illuminated a major immunological paradox in pregnant eutherians that
exists to this day: ‘how does the pregnant mother nourish within
itself for many weeks and months a fetus that is antigenically a
foreign body?’ (Medawar, 1953). It has not been established when
healthy human embryos begin to express foreign paternal major histocompatibility (MHC) antigens, because such experiments are prohibited. However, MHC antigens are readily detected at the 8-cell
stage in the mouse (Ewoldsen et al., 1987). Mouse embryos are
lysed by cytotoxic T lymphocytes directed against paternal MHC
molecules, but only after they have been denuded of their ZP
(Ewoldsen et al., 1987). This result confirms that the murine ZP protects the mouse embryo from cytolytic responses, but exactly how
this protection is mediated is unclear.
The glycosylation of human ZP glycoproteins indicates that this
matrix could be involved in immune cell signaling. Siglecs (sialic acid
binding immunoglobulin-like lectins) are molecules that express an
immunoreceptor tyrosine-based inhibitory motif (ITIM) that transmits
an inhibitory signal to immune cells (Avril et al., 2004). This motif is
critical for the inhibitory signaling that occurs following the binding
of siglecs to sialylated ligands. Siglec-9 binds to sLex and other sialylated glycans (Angata and Varki, 2000). It is primarily associated with
monocytes, granulocytes and CD16-, CD56 cells, but is also weakly
expressed on half of all B cells and NK cells (Angata and Varki,
2000). Minor subsets of CD8+ T cells and CD4+ T cells are also
weakly positive for siglec-9. The ability of siglec-9 interactions to
inhibit immune responses has been confirmed. Certain strains of
Group B streptococci have been shown to induce a tolerizing effect
on neutrophils via the interaction of their sialylated capsular polysaccharide with siglec-9 presented on these leukocytes (Carlin et al.,
2009). The hypothesis that carbohydrate sequences presented on
the mammalian ZP could be employed as ligands for both gamete
binding and immune protection of the egg or the human embryo
before it hatches out of the blastocyst was presented in previous
reviews (Clark et al., 1997; Clark et al., 2001; Clark and Dell,
2006). Immune modulatory factors such as PP14 and CA125
become elevated during the temporal window of implantation, and
could block immune responses directed against the embryo after it
exits the blastocyst (Dalton et al., 1995).
There are good reasons to believe that the same type of overlap
exists in the mouse as well. The complex type N-glycans that act as
ligands for galectins have previously been implicated in murine
gamete binding (Clark and Dell, 2006; Clark, 2010). Galectin-3 is
the specific immune recognition molecule that promotes the suppression of specific T cell-mediated responses (Demetriou et al., 2001).
574
Siglecs could also play a role in mediating immune protection of the
mouse egg and embryo, as murine ZP glycans are also sialylated
(Noguchi and Nakano, 1993; Easton et al., 2000; Dell et al., 2003).
However, sLex sequences are not expressed on the mouse ZP,
which could explain why human sperm do not bind to mouse eggs
(Easton et al., 2000).
In 1984, a mouse monoclonal antibody (CSLEX1) was developed
that was highly reactive with human tumor cells derived from the pancreas, stomach, colon, lung and bladder, but not with normal tissues.
CSLEX was bound to 68% of all human tumors tested (Fukushima
et al., 1984). This antibody specifically reacted with sLex and sLex –
Lex sequences that are expressed on the human ZP, but not with
sLea sequences (Fukushima et al., 1984; Pang et al., 2011) (Table II).
The sLex sequences on human tumor cells have been proposed to
act as ligands for selectins, promoting their binding to the vascular
endothelium and metastasis (Irimura et al., 1993; St. Hill, 2012).
However, they could also act as protective signals that engage
Siglec-9 and perhaps other immunomodulatory lectins to attenuate
potential immune responses. The proposal that aggressive human
tumor cells would be selected by the immune system to express
the same immunomodulatory glycans associated with human
gametes was previously presented (Clark et al., 1997).
The lectin-like EBP that mediates sperm –egg recognition in either
the human or the mouse has not been unequivocally identified. It is
unlikely that the EBPs are either selectins or siglecs, but these adhesion
molecules cannot be ruled out completely without more definitive investigation. The protein or proteins that subsequently trigger acrosomal exocytosis in both the human and mouse are also unidentified at
this time. These are important topics for future study for both clinical
application and basic scientific understanding.
Summary
For many years now, scientists and clinicians have observed human
sperm binding to eggs during IVF, often wondering about how this
interaction occurs. This cell adhesion event seemed to be beyond molecular definition, due to the lack of access to substantial numbers of
human eggs. However, ultrasensitive biophysical tools have finally
revealed some of the details about this interaction, making the
human egg more accessible for study. The concept was initially presented years ago that human sperm –egg binding involved the specific
recognition of a selectin ligand. The recent demonstration that the universal selectin ligand sLex is profusely expressed on human ZP glycans,
and that, in a soluble form, it also inhibits human sperm– ZP binding
provides powerful supporting evidence for this hypothesis.
However, the validity of this paradigm continues to be challenged by
advocates of the supramolecular complex model. As discussed here,
the sLex sequence could also act as a marker for the immune recognition of the egg and the histoincompatible human embryo before it
hatches out of the blastocyst. The neoexpression of the sLex
sequence on aggressive tumor cells could also be linked to this
same protective effect. Although a complete molecular understanding
of human gamete binding is not yet available, substantial progress has
been made since Rock and Menkin first observed human sperm –egg
binding .70 years ago. Other biological roles of the human ZP will be
defined in the future.
Clark
Acknowledgements
The author thanks Lynn Stevenson for her editorial assistance in preparing the manuscript. The author also thanks Drs Randy Prather and
Danny Schust for reviewing the manuscript and making useful suggestions for change.
Funding
Studies focused on human sperm–egg interactions carried out by the
author and coworkers have been supported by the Life Sciences
Mission Enhancement Reproductive Biology Program funded by the
State of Missouri, a Research Board Grant (CB000500) supported by
the University of Missouri System, and a grant from the Jeffress Memorial Trust of Virginia. Support from the Breeden-Adams Foundation has
also been obtained to investigate potential linkages to tumor evasion.
Conflict of interest
None declared.
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