Molecular Human Reproduction vol.6 no.2 pp. 163–169, 2000
Expression of cadherin adhesion molecules on human gametes
O.Rufas1,2, B.Fisch2, S.Ziv1 and R.Shalgi1,3
1Department
of Embryology, Sackler School of Medicine and 2Department of Obstetrics & Gynecology, Rabin Medical
Centre, Campus Beilinson, Tel-Aviv University, Israel
3To
whom correspondence should be addressed
The presence of cadherins, Ca2⍣-dependent cell–cell adhesion molecules which may be involved in gamete
interaction, was investigated in human gametes. Expression of cadherin molecules was demonstrated using
an anti-pan-cadherin antibody and specific antibodies against the three classical cadherins: E- (epithelial),
P- (placental) and N- (neural) cadherins. Samples of 48 h old unfertilized oocytes and spermatozoa from
in-vitro fertilizing semen samples were lysed and separated by electrophoresis. Localization of cadherins was
determined on intact, fixed, permeabilized spermatozoa and oocytes by immunocytochemisty assessed by
confocal microscopy. Immunoblotting with the pan-cadherin antibody revealed a single band of ~120 kDa in
spermatozoa (whether ‘fresh’, capacitated, or frozen–thawed) and oocyte extracts. Oocytes presented all
three classical cadherins with the appropriate molecular weights of 120–130 kDa. In sperm lysate we
demonstrated the presence of E-cadherin but not N-cadherin. The anti-P antibody detected a 90 kDa peptide
as the only immunoreactive antigen. Following immunocytochemistry of human oocytes all cadherin molecules
were allocated predominantly to the plasma membrane with only traces in the cytoplasm. In spermatozoa,
several staining patterns were observed with each of the pan-cadherin, N-cadherin and E-cadherin antibodies
mostly confined to different head regions. We conclude that cadherin molecules are present on plasma
membranes of both human spermatozoa and oocytes and may play a role in the intricate recognition process
preceding gamete fusion.
Key words: cadherins/gamete fusion/oocytes/spermatozoa
Introduction
The identity of the molecules that mediate gamete membrane
fusion is as yet unknown but a possible involvement in this
process of cell adhesion molecules (CAM) has been implied.
The role of CAM in embryonic development is well established
and dynamic changes in their expression are involved in the
regulation of morphogenesis (Edelman, 1988). Representatives
of the IgG, selectins, integrin and cadherin superfamilies of
CAM have been demonstrated in oocytes or early preimplantation embryos. N-CAM, a member of the calcium-independent
IgG superfamily, has been detected on unfertilized ovulated
murine oocytes through to the blastocyst stage, although it
appears to function only from the onset of the development
of the nervous system at gastrulation (Kimber et al., 1994).
C-CAM, another member of this family, is expressed by cells
of early blastocysts and is lost during implantation as the
embryo penetrates the lining epithelium of the uterus
(Slavender et al., 1987). L-Selectin, on the other hand, a
member of the selectin superfamily, was detected on oocytes
but not in 8-cell embryos (Campbell, 1995).
The presence of several integrin subunits (α2, α5, α6, αV,
β1, β3), has been demonstrated by a variety of experimental
approaches on hamster, mouse and human oocytes and early
embryos and on human spermatozoa (reviewed by Bronson
and Fusi, 1996). In the mouse, integrin α6β1 appears to play
© European Society of Human Reproduction and Embryology
a key role and antibodies directed at either of its subunits
inhibit gamete binding (Almeida et al., 1995). The β1 integrin
chain has been implicated also in human gamete interaction.
Redistribution of this integrin subunit accompanies maturation
and increase in fusibility of the human oocyte and it labels in
patches at the sites of fused spermatozoa. However, blocking
it with a monoclonal antibody results only in partial inhibition
of gamete fusion, indicating that its function can be bypassed
by other unknown molecules (Ji et al., 1998).
Cadherins are a family of surface glycoprotein CAM, that
bind with high specificity to other cadherins on adjacent cells.
They comprise the molecular basis for the formation of tissues
and organs in embryonic development and their maintenance
in the adult, by regulating homotypic cellular recognition
(reviewed by Marrs and Nelson, 1996). Cadherins are calcium
dependent and are protected from proteolysis through stabilization by this ion without which the molecule undergoes conformational changes (Takeichi, 1995). There are at present
more than 40 different cadherins known, and among them
three major subfamilies have been identified: E- (epithelial),
N- (neural) and P- (placental) cadherins, that range in molecular
mass between 120 and 140 kDa.
Cell–cell adhesion of the blastomeres in the 8-cell stage
embryo is mediated by E-cadherin (also known as ‘uvomorulin’), which is expressed from unfertilized oocytes throughout
the preimplantation period but presumed to become active
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O.Rufas et al.
only after the third cleavage cycle. Initiation of compaction at
this stage has been shown to rely on redistribution of this
molecule and may be reversed in calcium-free medium (Geiger
and Ayalon, 1992). A null mutation for E-cadherin in mouse
embryos results in the development of abnormal blastocysts
that fail to implant in the uterus (Larue et al., 1994; Riethmacher
et al., 1995). In the mouse, E-cadherin was found to be located
on the oocytes’ plasma membrane by immunocytochemistry
starting only 6 h after fertilization (Clayton et al., 1995).
In the testis, N-cadherin was demonstrated on cells of the
seminiferous epithelium and on spermatogonia and primary
spermatocytes (i.e. diploid cells) but not on spermatids
(Anderson et al., 1994). E-cadherin was demonstrated on
epithelial cells of the human epididymis. A role in intercellular
organization of epithelium was therefore attributed to both
these cadherins in the male genital tract (Anderson et al.,
1994). However, cadherins have not been detected on mature
spermatozoa of any species. Cadherins bind homotypically to
each other, therefore if they participate in sperm–oocyte
interaction it is imperative that they would be presented by
both gametes.
The aim of the present study was to examine the presence
of cadherins on mature human gametes in order to assess their
involvement in gamete recognition and binding that precedes
fertilization. A wide spectrum antibody, recognizing all cadherins, was employed. Cadherins were detected on intact
spermatozoa and oocytes by confocal microscopy and in
homogenized cells by immunoblotting, respectively. Subsequently, specific antibodies raised against E-, N- and
P-cadherins were used.
Materials and methods
Immunoblotting studies
Oocyte preparation
Unfertilized oocytes, 48 h after conventional in-vitro fertilization
(IVF) or intracytoplasmic sperm injection (ICSI) attempts, were
denuded mechanically of adhering cumulus cells and collected in
aliquots of 10–20 oocytes in approximately 20 ml of lysis buffer
containing phosphatase and protease inhibitors (according to
Ben-Yosef et al., 1998, modified after Haffetz and Ziek, 1989).
Samples were dipped into liquid nitrogen for 2 min, thawed at
room temperature to obtain a lysate and refrozen for storage in
liquid nitrogen.
For electrophoretic protein separation, several samples were thawed,
8 µl of Laemmli buffer (Laemmli, 1970) was added to each and
samples were boiled for 5 min. Lysate prepared from a total of
60–90 oocytes was loaded per lane.
Sperm preparation
Spermatozoa left from samples used for insemination were incubated
overnight in culture medium (Sperm preparation medium; Medicult,
Copenhagen, Denmark) and washed by centrifugation (400 g) with
Dulbeco’s phosphate-buffered saline (DPBS). Pellets were resuspended in Tris-buffered saline (TBS: 135 mmol/l NaCl and 10 mmol/l
Tris–HCl buffer, pH 7.4) containing 2% sodium dodecyl sulphate
(SDS), 3% 2-mercaptoethanol followed by addition of 10% glycerol
and 0.001% bromophenol blue. Samples were either boiled for 5 min
or sonicated for 10 s to liquefy the DNA and immediately loaded on
the gel. In addition fresh aliquots, prepared as described but without
164
prior incubation and donor semen samples thawed after liquid nitrogen
cryopreservation, were also examined.
Immunoblotting
Proteins were separated on 10% SDS–polyacrylamide 1.5 mm mini
gels (40 mA constant current) for ~1 h alongside marker proteins of
known molecular weight (SDS MW prestained standards, Sigma Co.,
St Louis, USA).
Western blots were prepared by electrically transferring the separated proteins to nitrocellulose paper (NC, Hybond ECL; Amersham,
Buckinghamshire, UK) by wet transfer (40 mA current for 18 h).
The nitrocellulose membrane was incubated overnight in DPBS
containing 0.05% polyoxyethylene sorbitan monolaurete (Tween-20)
and 3% skimmed milk powder at room temperature for blocking nonspecific binding of antibodies and rinsed with DPBS containing
0.05% Tween-20.
Blots were probed by overnight incubation with one of the following
anti-cadherin primary monoclonal antibodies (1:250 to 1:1000
concentrations, room temperature): pan-cadherin (C1821, Sigma);
E-cadherin (C208220, Transduction Laboratories, Lexington, KY,
USA); N-cadherin (N-19, Santa Cruz Biotechnology Inc., Santa Cruz,
CA, USA); P-cadherin (C24120, Transduction Laboratories). The
blots were exposed to appropriate horseradish peroxidase (HRP)conjugated anti-IgG secondary antibodies (Sigma, diluted to 1:5000
to 1:10 000, 2 h incubation at room temperature). Bands were
visualized on X-ray films following enhanced chemiluminescence
(ECL; Amersham) detection, according to the manufacturer’s
instructions.
Immunocytochemistry
Immunostaining of oocytes
Forty-eight hour old unfertilized oocytes were fixed by a 10 min
incubation in a freshly prepared solution of 0.01% gluteraldehyde
and 3% paraformaldehyde in IVF culture medium (IVF medium;
Medicult). Zonae pellucidae (ZP) were removed by a brief exposure
to acid Tyrode’s solution (pH 2, Sigma) and the plasma membrane
perforated with 0.05% nonidet (NP-40, Sigma). The same perforating
protocol has been employed by us for introduction of antibodies to
cytoplasmic proteins (Raz et al., 1998; Talmor et al., 1998) ensuring
that it allows penetration of antibodies to fixed oocytes.
The expression of cadherins on the cell surface and cytoplasm of
oocytes was probed with the following antibodies: Pan cadherin
(C3678, polyclonal, raised in rabbit; Sigma). E-Cadherin (N-20,
polyclonal, raised in goat; Santa Cruz Biotech.), N-cadherin (N-19,
polyclonal, raised in goat, Santa Cruz Biotech); P-cadherin (C24120,
monoclonal, Transduction Laboratories). Oocytes were incubated for
2 h with the various antibodies diluted in culture medium containing
0.005% NP-40 and 3% fetal calf serum (the former to maintain
membrane pores and the latter to mask non-specific binding sites).
Oocytes were rinsed by several transfers to fresh droplets of the
above medium and introduced to medium containing appropriate
secondary antibodies (raised in goat or rabbit) bound to either
fluorescein isothiocyanate (FITC) or Cy fluorescent markers (Jackson
Laboratories, West Grove, PA, USA) for 30 min incubation in
darkness. Secondary antibody only staining was used as control.
Immunostaining of spermatozoa
Sperm samples left over from specimens used for insemination were
incubated overnight in IVF culture medium (Sperm Preparation
Medium, Medicult) washed by centrifugation (400 g) with DPBS.
Cells were perforated by slowly dripping pellets along the side of a
test tube immersed in ice. Samples were warmed to room temperature
and the procedure was then repeated. Anti-cadherin antibodies were
Expression of cadherins on human gametes
Figure 1. Detection of cadherins in human oocytes by Western
immunoblotting. A lysate of 66 oocytes was separated by
electrophoresis and probed with 1:1000 anti-pan cadherin antibody.
Figure 2. Detection of cadherins in human spermatozoa by Western
immunoblotting. An extract from ~5⫻106 spermatozoa was
subjected to electrophoresis. Proteins were probed with 1:1000 antipan cadherin antibody.
added to reach a final concentration of 1:100 or 1:200 for 2 h.
Samples were washed twice by centrifugation and incubated for
30 min with the appropriate fluorescein isothiocyanate (FITC) or
Cy-conjugated anti-IgG secondary antibody (Jackson Laboratories).
Excess antibody was removed by three successive wash and centrifugation cycles. Secondary antibody only staining was used as
control.
Gametes were mounted on microslides for confocal microscopic
analysis of the localization and fluorescent staining intensity of
cellular compartments (Zeiss confocal laser scanning microscope,
CLSM 410, equipped with 25 mW Krypton–Argon laser and 10 mW
HeNe laser).
Results
Immunoblot analysis
Spermatozoa and oocytes were collected and lysed, and proteins
of whole cell lysates were separated by electrophoresis and
transferred to nitrocellulose paper as described in Materials
and methods. In the first stage, anti-pan-cadherin, a general
antibody raised against an intracellular domain that is common
to most known cadherins, was employed to allow a broad
detection of different cadherins. Each experiment was repeated
at least three times with comparable results.
Pan-cadherin detection
Oocytes. Immunoblots of oocytes revealed a major specific
protein band of 120 kDa when incubated with the monoclonal
anti-pan-cadherin antibody diluted to 1:1000 followed by
exposure to an anti-mouse IgG HRP-conjugated antibody at a
concentration of 1:5000. In addition, a lower molecular weight
polypeptide band of ~40 kDa was observed, probably representing a degradation fragment (Figure 1). These bands were
apparent when a lysate of 艌60 oocytes was mounted on the gel.
Spermatozoa. Immunoblotting with the pan-cadherin antibody
(diluted to 1:1000) revealed a single band of 120 kDa in
sperm extracts of 2–10⫻106 spermatozoa (Figure 2). Only
normozoospermic fertilizing samples were used and similar
results were obtained whether fresh, capacitated or frozen–
Figure 3. Identification of classical cadherin molecules: epithelial
(E), neural (N) and placental (P) cadherins in human oocytes by
Western immunoblotting. E: immunoblot of a lysate of 96 oocytes
probed with an anti-E-cadherin antibody (1:500). N: immunoblot of
a lysate of 66 oocytes probed with an anti-N-cadherin antibody
(1:500). P: immunoblot of a lysate of 96 oocytes probed with an
anti-P-cadherin antibody (1:500).
thawed samples were solubilized. However, the intensity of
the immunoreactive band varied between samples of different
patients, containing comparable numbers of cells on the
same gel.
Identification of specific cadherin types
Immunoblotting was used to reveal the identity of the cadherin
molecules initially detected with the anti-pan cadherin antibody.
Expression of E-, N- and P-cadherins was studied using
commercial specific antibodies as detailed in Materials and
methods.
Oocytes. Oocytes presented all three cadherins with the appropriate molecular weights as shown in Figure 3. A single band,
calculated at 120 kDa, was visualized using a monoclonal
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O.Rufas et al.
Figure 4. Identification of classical cadherin molecules: epithelial
(E) and placental (P) cadherins in human spermatozoa. Lysates
from ~5⫻106 spermatozoa were subjected to electrophoresis.
Immunoblots were probed with anti-E-cadherin antibody (1:1000),
and anti-P-cadherin antibody (1:250). N-Cadherin could not be
demonstrated.
anti-E-cadherin antibody (diluted to 1:500 followed by 1:5000
HRP-conjugated anti-mouse IgG). Figure 3E shows the result
of an experiment on a Western blot using a lysate of 96
oocytes. A weak, albeit distinct, band resulted when an
extraction of 59 oocytes was separated on the gel (results
not shown).
The antigen detected by the anti-N-cadherin polyclonal
antibody (diluted to 1:500 followed by 1:5000 HRP-conjugated
anti-goat IgG secondary antibody) was estimated to have a
molecular weight of 127 kDa in a lysate of 66 human oocytes
(Figure 3N). This result conforms to the size of N-cadherin
in human ovary and granulosa cells cited in the literature
(MacCalman et al., 1995).
Immunoblotting with the monoclonal anti-P-cadherin antibody (diluted to 1:500, followed by 1:5000 anti-mouse IgG
HRP-conjugated secondary antibody) exhibited a single band
of 120 kDa (Figure 3P).
Spermatozoa. Immunoblots of spermatozoa exhibited the
expected 120 kDa protein and a distinguished smaller protein of
~40 kDa, when incubated with an anti-E-cadherin monoclonal
antibody (diluted to 1:1000) (Figure 4E). The anti-P monoclonal antibody detected an 88 kDa peptide as the major
immunoreactive antigen (Primary antibody 1: 250, secondary
antibody 1:5000) (Figure 4P). The presence of N-cadherin
could not be demonstrated under the present experimental
settings in sperm lysates.
Detection of cadherins on intact gametes by immunocytochemistry
Immunocytochemical analysis was performed, as detailed
above, to identify and localize cadherin molecules in intact
oocytes and spermatozoa. To examine the possibility of
involvement of cadherins in gamete recognition and interaction
166
leading to fusion, their presence on the plasma membrane of
both gametes was investigated. A polyclonal anti-pan cadherin
antibody was employed for broad-spectrum cadherin detection
as described. Preliminary experiments with the monoclonal
antibody used in the immunoblotting experiments proved that
it was inadequate for intact cell probing. Immunoreactivity of
cellular compartments was visualized by confocal microscopy
and controls of secondary antibody staining indicated background fluorescence.
Pan-cadherin detection
Oocytes. Fixed, permeabilized oocytes stained intensely and
uniformly on their plasma membrane when incubated with the
polyclonal anti-pan cadherin antibody (1:100 dilution, 2 h,
room temperature) followed by a Cy-3 conjugated goat antirabbit IgG secondary antibody (1:200, 30 min, room temperature). Very faint staining was observed within the cytoplasm.
A representative result of 10 oocytes examined on two experimental days is presented (Figure 5). No significant differences
were observed in the intensity or distribution between oocytes
not fertilized by conventional IVF or not exhibiting pronuclear
formation following a sperm injection by ICSI (not shown).
Since the ZP was dissolved by acid, reactivity of adhering
spermatozoa could not be studied.
Spermatozoa. Spermatozoa were immobilized and permeabilized by cold shock treatment as detailed above. Following
immunocytochemistry with the anti-pan cadherin antibody
(1:200 dilution, 2 h, room temperature) and a Cy-3-conjugated
goat anti-rabbit IgG secondary antibody (1:1000, 30 min, room
temperature) several patterns were observed. Staining was
mainly confined to the head region but varied from entire
head to postacrosomal restricted fluorescence (representative
examples are shown in Figure 6). Staining was observed in
40 to 70% of the cells on five different experiments indicating
both inter- and intra-sample heterogeneity.
Identification of specific classical cadherins
Oocytes. Indirect immunofluorescence on oocytes demonstrated expression of E- and N-cadherins (probed in 19 and
16 oocytes in three experiments respectively). Fluorescence
was allocated predominantly to the plasma membrane with
traces of stain within the cytoplasm (Figure 7). A dilution of
1:50 was used for the primary antibodies. The polyclonal
E-cadherin and N-cadherin were followed by 1:200 FITCconjugated rabbit anti-goat IgG.
Spermatozoa. Only weak immunoreactiviy was noted when
spermatozoa were probed for N- and E-cadherins (not shown).
Very faint staining of the post-acrosomal zone of the head was
demonstrated by 20–50% of the cells examined in seven
different semen samples. No cells stained with the monoclonal
anti-P-cadherin monoclonal antibody. As E- and P-cadherins
were shown to be present by immunoblotting in cell lysates,
the results obtained by confocal microscopy may be attributed
to a low concentration of these molecules on membranes of
individual spermatozoa.
Discussion
In contrast to the significant progress which has been made in
identifying the molecules that mediate the first steps of sperm
Expression of cadherins on human gametes
Figure 5. Localization of cadherins in human oocytes by confocal microscopy. A representative result of 10 oocytes examined on two
experiments is presented. For broad-spectrum detection of cadherins oocytes were incubated with a polyclonal anti-pan-cadherin antibody
(1:100). The plasma membrane exhibited intense uniform staining whereas the cytoplasm presented faint fluorescence. (A) Light image;
(B) fluorescent image; (C) control (incubation with secondary antibody only) fluorescent image.
Figure 6. Localization of cadherins in human spermatozoa by
confocal microscopy. Cells were perforated as described in the text
and incubated with anti-pan-cadherin polyclonal antibody. Paired
light images (A, B) and fluorescent images (A⬘, B⬘) of two
representative results are shown.
interaction with the oocyte’s ZP, very little concrete information
exists on the participants in the subsequent binding and fusion
of the plasma membranes. It remains to be elucidated whether
binding and fusion are two separate events carried out by
different molecules or by a dual function receptor. Several
sperm surface proteins have been proposed as candidates for
these roles in different species, among them a family of
proteins containing a disintegrin and a metalloprotease domain
(ADAM) (Wolfsberg et al., 1995). The first and the most
extensively characterized member of this family is ‘fertilin’
(previously named PH30) identified on guinea-pig spermatozoa
(Primakoff et al., 1987). The detection of a disintegrin domain
on its β chain led to the hypothesis that fertilin binds to an
integrin receptor on the oocyte plasma membrane and thus
implicated adhesion molecules in gamete interaction. Integrins
were later shown to be present on the plasma membranes of
hamster, mouse and human oocytes (Almeida et al., 1995;
Evans et al., 1995; Bronson and Fusi, 1996) and the integrin
α6β1 has been proposed as the mouse egg receptor for
spermatozoa (Almeida et al., 1995).
Cell–cell contact may involve the interplay of more than
one set of molecules. This is even expected in the case of the
most intricate binding and fusion of gametes. Increasing the
number of participating molecules may refine specificity by
complicating the recognition code or, on the other hand, offer
alternative pathways to avoid failure. If gamete interaction
involves mechanisms common to somatic cell adhesion and
signalling, a logical partner for integrins would be the closely
related cadherin family of Ca2⫹-dependent adhesion molecules.
Cadherins form a large family of glycoproteins encoded by
a multigene family present from Drosophila and Xenopus to
mammals (Suzuki, 1996) and are expressed in a cell-typespecific manner (Geiger and Ayalon, 1992). They participate
mainly in homophilic interactions via junction complexes and
are responsible for tissue formation, structure regulation, cell
polarity, cell sorting and cell migration (Takeichi, 1991, 1995).
Cadherin expression correlates with the cell’s adhesiveness
(Takeichi, 1988) and variations in its presentation on the cell
membrane have been associated with tumour development
(Behrens et al., 1989). Cadherins act as key regulators of
embryonic development, appearing or being redistributed at
specific stages and acting as signalling molecules in the
coordination of morphogenetic processes (Takeichi, 1995). The
first function assigned to this family was the induction of
compaction in the murine morula (Hyafil, 1980, 1981).
As cadherins interact with cadherins on adjacent membranes,
a possible role for them in sperm–oocyte recognition would
entail their presence on both cells. The present study demonstrated the expression of cadherin molecules on both human
gametes. Immunobloting with the pan-cadherin antibody
revealed the presence of cadherins in sperm and oocyte extracts.
They were localized to the oocyte plasma membrane and the
sperm head in the intact cells by immunocytochemistry.
Oocytes presented all three classical cadherins probed (E-, Pand N-), while in sperm lysates E-cadherin was detected at
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O.Rufas et al.
Figure 7. Immunolocalization of N- and E-cadherins on human oocytes by confocal microscopy. Paired light (top, A–C) and fluorescent
(bottom, A⬘–C⬘) images of: (A) oocytes probed with N-cadherin-specific antibody (1:50); representative result of 19 oocytes from three
experiments, showing intense fluorescence of the plasma membrane; (B) oocytes probed with E-cadherin-specific antibody (1:50).
Representative result of 16 oocytes from three experiments, showing a weak but distinctive fluorescence of the plasma membrane;
(C) control, secondary antibody only.
the appropriate molecular weight and the specific anti-Pcadherin antibody recognized a 90 kDa fragment. It may also
be speculated that the immunoreactive band demonstrated by
the broad-spectrum anti-pan-cadherin represents other known
or novel cadherins with the typical cadherin molecular weight.
Co-expression of different cadherins in the same cell as
demonstrated here is common to many tissues (kidney: Okada
et al., 1988; skin: Hirai et al., 1989; Geiger and Ayalon, 1992)
and it is important to note that the requirement for exclusive
homophilic interaction is not absolute. It was shown that when
cells expressing either N-cadherin or E-cadherin are mixed,
there is a preference (higher affinity) for homotypic contacts
(i.e. of similar cells), yet weaker but significant heterotypic
junctions were also found (Volk et al., 1987). A similar process
may occur in gamete recognition enabling the union of distinct
cells through distinct cadherin molecules.
Gamete fusion elicits oocyte activation via triggering of
calcium oscillations (Swann and Ozil, 1994) and a putative
receptor on the plasma membrane is required to initiate a
signalling cascade. Cadherins are involved in diverse signal
transduction pathways through several protein tyrosine kinases
associated with adhesion sites, affecting the intracellular association with the cytoskeleton (reviewed by Geiger and Ayalon,
1992). If cadherins are involved in gamete membrane interaction, modulation of their adhesive capacity may also provide
a means to block polyspermy at the level of the plasma
membrane.
168
The oocyte contains proteins that function later in embryonic
development and the presence of E-cadherin in oocytes has
been interpreted in this context. Moreover, it was reported that
cadherin molecules were allocated to the plasma membrane
only 6 h after fertilization (Clayton et al., 1995). We have
shown that at least E- and N-cadherins (and perhaps other
cadherin molecules recognized by the anti-pan cadherin antibody) are present on the oolema of unfertilized human oocytes,
and, as previously demonstrated, these aged oocytes are capable
of fusing with spermatozoa (Rufas et al., 1994). Similar results
were obtained in our laboratory when mature rat oocytes were
analysed (S.Ziv et al., unpublished data). Sperm maturation
involves the casting away of most of the cytoplasm, and
capacitation comprises rearrangement of essential surface proteins. The expression of cadherins on the spermatozoon head
is therefore likely to be related to its functional state. The
presence of cadherins on spermatozoa has not been previously
reported in the literature.
The mere presence of cadherins on the cell membrane does
not necessarily imply their involvement in a physiological
process. Uvomorulin synthesized in 2-cell embryos was shown
to cause cell compaction only at the 8-cell stage (Vestweber,
1987). Activation can be exerted through biochemical or
conformational changes of the binding domain or may depend
on expression of a critical density of the protein. The oocyte
plasma membrane attains its adhesive capacity following
maturation in the follicle prior to ovulation. A steroid hormone
Expression of cadherins on human gametes
involved in this process, 17β-oestradiol, has been implicated
as a major regulator of N-cadherin levels in the ovary and a
mediator of granulosa cell interaction affecting both adhesion
and differentiation (MacCalman et al., 1995).
In mammalian spermatozoa, fusogenic transformation
accompanies the acrosome reaction, which physiologically
occurs after the encounter with the ZP. Molecules responsible
for binding and fusion with the oolema may become activated
or exposed at this stage. The intra-sample heterogeneity
observed in staining of spermatozoa with the pan-cadherin
antibody may reflect differences in capacitation or acrosome
reaction of individual cells. It could, however, result from
insufficient permeabilization by the cold shock treatment
that was used to introduce the antibody that recognizes an
intracellular domain of the molecules. As E- and P-cadherins
were demonstrated in cell lysates by Western blot analyses,
the results obtained by confocal microscopy may be attributed
to a low concentration of these molecules or a relative low
affinity of the specific antibodies employed.
Investigation of the relevance of the study of adhesion
molecules to the clinical search for mechanisms involved in
unexplained infertility is appealing, but characterization of the
cadherin profile in specific subgroups of infertile patients is
not yet feasible. Further refinement of the procedures may
enable quantification of cadherin presentation, and determination of its correlation with fertilization potential.
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Received on July 16, 1999; accepted on November 17, 1999
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