416
Research Article
CD9 controls the formation of clusters that contain
␤1, which are involved
tetraspanins and the integrin ␣6␤
in human and mouse gamete fusion
Ahmed Ziyyat1,*, Eric Rubinstein2,*, Frédérique Monier-Gavelle1, Virginie Barraud1, Olivier Kulski1,
Michel Prenant2, Claude Boucheix2, Morgane Bomsel3 and Jean-Philippe Wolf1,‡
1
Université Paris 13, Laboratoire de Biologie de la Reproduction, UPRES 3410, UFR SMBH, Bobigny, France; AP-HP, Hôpital Jean Verdier,
Service d’Histologie, Embryologie et Cytogénétique, Bondy, France
2
INSERM, U602, Villejuif, France; Université Paris XI, Villejuif, France; Institut André Lwoff, Villejuif, France
3
INSERM, U567, Institut Cochin, Paris, France
*These authors contributed equally to this work
‡
Author for correspondence (e-mail: [email protected])
Journal of Cell Science
Accepted 19 September 2005
Journal of Cell Science 119, 416-424 Published by The Company of Biologists 2006
doi:10.1242/jcs.02730
Summary
The process of gamete fusion has been largely studied in
the mouse and has revealed the crucial role of the
tetraspanin CD9. By contrast, human gamete fusion
␣6
remains largely unknown. We now show that an anti-␣
integrin mAb (GoH3) strongly inhibited human sperm-egg
fusion in human zona-free eggs. Furthermore, a mAb
directed against CD151, a tetraspanin known to associate
␤1, partially inhibited sperm-egg fusion. By
with ␣6␤
contrast, the addition of an anti-CD9 mAb to zona free eggs
␤1, CD151 and CD9
had no effect. The integrin ␣6␤
tetraspanins were evenly distributed on human zona-intact
␤1 and
oocytes. On zona-free eggs, the integrin ␣6␤
tetraspanin CD151 patched and co-localized but the
Key words: Human, Fertilization, Integrins, Tetraspanins, CD9,
CD151
Introduction
Mammalian fertilization depends upon successful binding and
fusion between the spermatozoon and the oocyte plasma
membrane. The fusion process is mediated by a series of
molecular interactions in which sperm fertilin, oolemmal
integrins and members of the tetraspanin family have been
suggested to play a role (Primakoff and Myles, 2002;
Wassarman et al., 2001).
Integrins constitute a family of transmembrane ␣␤ adhesion
proteins involved in cell-cell and cell-extracellular matrix
interactions. The ␤1 subunit can associate with 12 different ␣
subunits (␣1-11 plus ␣v) forming the largest subfamily of
integrins. Integrins are considered as two-way signalling
molecules (Liddington and Ginsberg, 2002). Integrin-mediated
cell adhesion induces regulatory signals, including protein
phosphorylation, that control cell growth, death, migration,
gene induction and differentiation. Reciprocally, the integrin
efficiency in mediating cell adhesion or ligand binding is
modified upon stimulation with a variety of stimuli. This may
be achieved through an increase of integrin affinity for ligand,
but more likely, by an increased avidity which may be
regulated by modifications of integrin clustering at the cell
surface and lateral associations with other transmembrane
proteins (Sanchez-Mateos et al., 1996; Bazzoni and Hemler,
1998; Schwartz, 1992).
Tetraspanins are surface proteins containing four
transmembrane domains and forming complexes with each
other and with various membrane proteins within a network of
molecular interactions called the ‘tetraspanin web’ (Boucheix
and Rubinstein, 2001; Berditchevski, 2001; Hemler, 2003).
They contribute to the formation of cell surface multimolecular
complexes and thereby may participate in the functional
regulation of molecules they associate with. For instance, the
presence of a subset of ␤1 integrins in the tetraspanin web may
explain in part how tetraspanins interfere with cell adhesion
and migration (Boucheix and Rubinstein, 2001; Berditchevski,
2001; Hemler, 2003). Within the tetraspanin web, smaller
primary complexes, containing only one tetraspanin tightly
associated with one partner molecule have been identified,
based on solubility in various detergents and on cross-linking
experiments (Boucheix and Rubinstein, 2001; Hemler, 2003).
Thus, two integrins present on the oocyte, namely the ␣6␤1
and ␣3␤1 integrins, form, on somatic cells, highly
stoichiometric and stable complexes with the tetraspanin
CD151 (Fitter et al., 1999; Serru et al., 1999; Yauch et al.,
1998). A CD151 molecule deleted from its C-ter cytoplasmic
end was shown to act as a dominant-negative inhibitor of
␣6␤1-dependent cord-like structure formation, possibly
through the regulation of ␣6␤1 integrin adhesion
strengthening, showing that CD151 can modulate the function
tetraspanin CD9 remained unchanged. CD9 mAb
␤1 integrin clustering and gamete fusion when
prevented ␣6␤
added prior to, but not after, zona removal. Antibody␤1 yielded patches that
mediated aggregation of integrin ␣6␤
were bigger and more heterogeneous in mouse oocytes
␤1 could be
lacking CD9. Moreover, a strong labelling of ␣6␤
observed at the sperm entry point. Altogether, these data
show that CD9 controls the redistribution of some
␤1 integrin into
membrane proteins including the ␣6␤
clusters that may be necessary for gamete fusion.
Journal of Cell Science
Tetraspanins and integrins in gamete fusion
of this integrin (Lammerding et al., 2003; Zhang et al., 2002).
Biochemical data suggest that within the tetraspanin web,
CD151 links ␣3␤1 and ␣6␤1 integrins to other tetraspanins,
including CD9 (Serru et al., 1999; Yauch et al., 1998; Ito et al.,
2003; Charrin et al., 2003; Berditchevski et al., 2002).
The potential role of the integrin ␣6␤1 and the tetraspanin
CD9 in mouse fertilization has been extensively studied. The
role of the integrin ␣6␤1 is still controversial. Based on
antibody-inhibition assays, it had been suggested that the
integrin ␣6␤1 could be a receptor for sperm binding to the egg
(Almeida et al., 1995), but this conclusion was not reached in
a later study using a different method of removing the zona
pellucida surrounding the egg (Evans, 1999). Recently, the
analysis of oocytes from mice lacking the integrin ␣6 or ␤1
genes showed that these integrins were not essential for spermoocyte binding and fusion in the mouse (Miller et al., 2000).
These data could not, however, completely exclude a role for
the integrin ␣6␤1. This integrin has been suggested to mediate
the binding of fertilin ␤ (ADAM 2) and cyritestin (ADAM 3),
two members of the ADAM family (A Disintegrin And
Metalloprotease) expressed on sperm (Chen et al., 1999; Chen
and Sampson, 1999; Takahashi et al., 2001; Tomczuk et al.,
2003), but others have concluded that fertilin ␤ interacts with
an alternate, but as yet unidentified, ␤1 integrin on the egg
(Evans et al., 1997; Zhu and Evans, 2002).
The crucial role of CD9 in sperm-egg fusion was
demonstrated by the inability of CD9 knock-out mouse oocyte
to fuse with sperm (Le Naour et al., 2000; Miyado et al., 2000;
Kaji et al., 2000) and by the ability of anti-CD9 antibodies to
inhibit fusion (Chen et al., 1999; Le Naour et al., 2000; Miller
et al., 2000). How CD9 functions in sperm-egg fusion is largely
unknown. In a recent study, recombinant proteins including the
large extracellular loop of CD9 were shown to partially inhibit
fusion when added to the oocyte prior to insemination, but not
when preincubated with sperm. This indicated that CD9 was
probably not a sperm receptor and drew the attention to
molecules that associate with CD9 at the oocyte surface and
thus to the tetraspanin web (Zhu et al., 2002).
In humans, the fertilization mechanism is rarely studied,
mainly because oocytes are rare and precious. While the
expression of tetraspanins on human oocytes has not been
reported, they express several integrins, including the ␣6 and
␤1 subunit (Fusi et al., 1992; Ji et al., 1998; Campbell et al.,
1995b). We have previously suggested the implication of ␤1
integrin in the fusion process, as fused spermatozoa colocalize with ␤1 integrin oolemmal patches and as a functionblocking anti-␤1 integrin mAb significantly inhibited spermoocyte fusion (Ji et al., 1998). In this study, we now
demonstrate that CD9 controls the formation of integrin
␣6␤1-containing clusters on the oocyte plasma membrane,
both in human and mouse. Moreover, we show a role for the
␣6␤1 integrin and the tetraspanin CD151 in human spermoocyte fusion.
Results
In human zona-pellucida-free oocytes, the integrin ␣6␤1
and the tetraspanins CD151 and CD81 are present in
the same patches
We first investigated the presence of the tetraspanins CD9,
CD81 and CD151 and of ␣6 and ␤1 integrin subunits on the
oolemma (the oocyte plasma membrane) of human oocytes. All
417
Fig. 1. Distribution of integrins and tetraspanins on human eggs.
Intact (A-E) or zona pellucida free (F-O) human eggs were labelled
with anti-␣6 integrin (A,F,I,L), anti-␤1 integrin (B,G), anti-CD151
(C,J), anti-CD81 (D,M) and anti-CD9 (E,O), mAbs. A merged
image (H) of ␣6 (F) and ␤1 (G) shows that the subunits co-localize
on the plasma membrane. Merged images (K and N) of ␣6 (I and
L) with CD151 (J) or CD81 (M), respectively, show that the
integrin ␣6␤1 co-localizes with these two tetraspanins on the
oolemma. All these molecules are evenly distributed on ZP-intact
eggs. On ZP free eggs, ␤1 integrin subunit, CD81 and CD151
tetraspanins co-localize with ␣6 integrin subunit. CD9 remained
evenly distributed. Bar, 50 ␮m.
five molecules were detected on human unfertilized zona
pellucida (ZP) intact oocytes (Fig. 1A-E).
Owing to French bioethical laws, in vitro human gamete
fusion assays can only be performed using ZP-free oocytes.
The ZP is a thick extracellular coat surrounding the egg that is
composed of three glycoproteins (ZP1, 2, 3). It selects the
fertilizing spermatozoon, triggers its acrosomal reaction and
prevents the fusion with any extra spermatozoon (blockade to
polyspermy) (Wasserman, 2001). As chemical and enzymatic
␣6␤1 integrin and CD151 control human gamete fusion
In vitro fertilization of ZP-free human eggs was performed to
test for a role of the ␣6␤1 integrin and tetraspanins in the
fusion process in human. As shown in Fig. 2A, in control
experiments, the average number of sperms fusing with an
oocyte was about 25 after 18 hours incubation. Except for ZPremoval, these experiments were done under the standard
routine conditions used for in vitro fertilization of human
intact oocytes before transfer to the uterus for clinical
purposes. Because only one sperm fuses with zona-intact
eggs under these conditions, these data further show the lack
of blockade to polyspermy at the level of the plasma
membrane in human. The same conclusion was reached using
the SUZI technique, in which the number of sperms injected
directly into the perivitelline space (the space between the ZP
and the plasma membrane) correlated linearly to the number
of fused sperm (Wolf et al., 1997; Tesarik and Kopecny,
1989). This is in contrast to what was reported in mice
(Horvath et al., 1993).
As seen in Fig. 2A, pre-incubation of eggs with the anti-␣6
blocking mAb GoH3 inhibited fusion in a dose-dependent
fashion. GoH3 at a concentration of 10 or 50 ␮g/ml inhibited
sperm-egg fusion by 71% and 96%, respectively (P<0.001). By
contrast, an anti-␣3␤1 blocking mAb (P1B5) (range 10 to 200
␮g/ml) had no effect on sperm-egg fusion (not shown). The
60
A
17
50
40
5
30
*
20
8
*
10
*
*
5
7
8
50
100
200
0
0
1
10
GoH3 (␮g/ml)
Number of sperm fused by oocyte
ZP removal methods significantly alter oolemmal proteins and
may reduce fertilization (Santella et al., 1992; Kinn and Allen,
1981; Kellom et al., 1992), a mechanical technique for ZP
removal was performed using a pair of microdissection scissors
under a binocular microscope (Ji et al., 1998). This technique
is particularly gentle as no recovery time is necessary before
insemination (Ji et al., 1998). However, it resulted in a
modification of the ␣6 and ␤1 integrin subunits distribution as
well as that of CD81 and CD151, that all formed patches (Fig.
1F,G,J,M). By contrast, CD9 remained evenly distributed (Fig.
1O) showing that the patching of surface molecules induced by
ZP removal was restricted to a subset of surface molecules.
These patches were observed whether labelling was done at
4°C or at room temperature, and were similar whether fixation
was performed after or before labelling, indicating that
patching was not induced by mAb after ZP removal.
Merging of ␣6 and ␤1 subunit staining (Fig. 1H) showed
a co-localization of both integrin subunits in patches,
suggesting the existence of oolemma ␣6␤1 integrin
heterodimers. All the ␣6 patches contained the ␤1 integrin
subunit. However, some ␤1 integrin patches were devoid of
␣6, probably because it is associated with other ␣ integrin
chains. Interestingly, as shown in Fig. 1K, all ␣6 patches
contained CD151, indicating their association on oocyte
surface, as previously shown in other cell types (Fitter et al.,
1999; Serru et al., 1999; Yauch et al., 1998). Some CD151
patches did not contain ␣6 integrin. In these patches, CD151
may interact with ␣3␤1 integrin, another known CD151
partner (Serru et al., 1999) expressed on the human oocytes
(Capmany et al., 1998; Campbell et al., 1995b). As shown in
Fig. 1N, ␣6 patches are also co-localized with tetraspanin
CD81 patches. In contrast, CD9 was not concentrated in ␣6
patches, which is consistent with CD9 being only indirectly
associated with the ␣6␤1 integrin (Serru et al., 1999; Charrin
et al., 2003; Berditchevski et al., 2002).
Number of sperm fused by oocyte
Journal of Cell Science 119 (3)
35
B
16
30
8
25
*
*
20
8
9
100
200
15
10
5
0
0
10
Anti-CD151 (␮g/ml)
Number of sperm fused by oocyte
Journal of Cell Science
418
45
C
20
6
40
35
7
30
5
25
20
15
10
5
0
Ctrl
SYB-1
50␮g/ml
SYB-1
100␮g/ml
ALB-6
50␮g/ml
Fig. 2. Effects of an anti-␣6, anti-CD151 or anti-CD9 mAb on
human sperm-egg fusion. Zona pellucida-free human eggs were
incubated with the blocking anti-␣6 mAb GoH3 (A) the anti-CD151
mAb 11B1G4 (B) or the anti-CD9 mAbs SYB-1 and ALB-6 (C)
before and during insemination with human sperm. Lines represent
the mean ± s.d. from five different experiments. The number of eggs
used in each condition is given above. *Significantly different from
control (P<0.001).
anti-CD151 mAb also inhibited fusion by up to 50% (P<0.001)
(Fig. 2B). This suggests that CD151 participates with the ␣6␤1
integrin in the formation of a functional complex involved in
human sperm-egg fusion. Surprisingly, although anti-mouse
CD9 mAb inhibits fusion of ZP-free mouse oocytes with sperm
(Chen et al., 1999), two anti-human CD9 mAb did not affect
the fusion of sperm with human ZP-free oocytes (Fig. 2C).
Tetraspanins and integrins in gamete fusion
Anti-human CD81 mAb (Z81; range 10 to 200 ␮g/ml) had no
effect either on gamete fusion (data not shown).
continuously present during ZP removal and the fusion assay.
As shown in Fig. 3A, the presence of anti-CD9 mAb
throughout the procedure inhibited ␣6 integrin patch formation
upon ZP removal in a dose-dependent manner, and importantly,
as seen in Fig. 3B, decreased fusion by 78% at 50 ␮g/ml [the
number of fused sperm were 4±2 (mean ± s.d.) in treated
oocytes versus 18±8 in control eggs; P<0.001]. The same
experiment performed with the anti-human CD81 mAb (Z81,
100 ␮g/ml) did not inhibit sperm-egg fusion (data not shown).
The CD9 mAb did not affect patching of CD151 and CD81
tetraspanins (Fig. 3C) and the CD81 mAb did not affect
patching of the ␣6 integrin subunit (Fig. 3D).
CD9 controls antibody-induced ␣6␤1 integrin
redistribution on the mouse oocyte surface
The above data showed that in human oocytes, CD9 controls
the formation of ␣6␤1 integrin-containing patches. To further
provide evidence that CD9 controls the lateral mobility of the
integrin ␣6␤1, integrins were aggregated at the surface of wildtype and CD9–/– ZP intact eggs using a combination of antiintegrin ␣6 and goat anti-mouse antibodies. The distribution of
the ␣6␤1 integrin was then studied by confocal microscopy.
As a control, wild-type eggs that were fixed before labelling
Journal of Cell Science
The tetraspanin CD9 controls ␣6 integrin patch
formation and human gamete fusion
The lack of effect of the anti-CD9 mAb on human ZP-free
oocytes was in apparent contrast to the mouse system in which
CD9 mAb efficiently inhibits the fusion of ZP-free oocytes
with sperm (Chen et al., 1999; Le Naour et al., 2000; Miller et
al., 2000). We therefore hypothesized that in human oocytes,
the formation of ␣6 integrin patches, induced by mechanical
ZP removal, put the oocyte at a stage in which CD9 is no longer
required for fusion. To challenge this hypothesis, oocytes were
preincubated with anti-CD9 mAb, and the mAb was kept
419
Fig. 3. Effects of anti-CD9 mAb added before zona removal. (A) ␣6
integrin subunit distribution of a control zona pellucida-free egg (a)
or of eggs pretreated before zona pellucida removal with the mAb
SYB-1 directed against CD9 (b,c). At 50 ␮g/ml, the patches were
smaller (b) whereas at 100 ␮g/ml (c) the ␣6 distribution was similar
to that of ZP intact oocytes (d). Bar, 50 ␮m. (B) Effect of mAb
against CD9 on gamete fusion. Eggs pretreated with 100 ␮g/ml of
SYB-1 prior to zona pellucida removal were tested in the presence of
50 ␮g/ml of SYB-1 for 18 hours. *The number of fused spermatozoa
was significantly reduced (P<0.001). Lines represent the mean ± s.d.
from three different experiments. The number of eggs used in each
condition is given above. (C) CD151 (e) and CD81(f) tetraspanins
distribution on eggs pretreated before zona pellucida removal with
the mAb SYB-1 (100 ␮g/ml). (D) ␣6 integrin subunit distribution on
eggs pretreated before zona pellucida removal with the mAb antiCD81, Z81 (100 ␮g/ml).
Fig. 4. Antibody-induced redistribution of ␣6␤1 integrin in wild-type
(WT) and CD9–/– oocytes. ␣6␤1 integrins were aggregated at the
surface of WT and CD9–/– ZP intact eggs using a combination of
anti-integrin ␣6 and goat anti-mouse antibodies and its distribution
was studied by confocal microscopy as described in the Materials
and Methods. As a control, wild-type eggs that were fixed before
labelling were analysed. Note that the ␣6␤1 integrin patches were
bigger and more heterogeneous in CD9-null oocytes than in WT
oocytes. Bar, 40 ␮m.
420
Journal of Cell Science 119 (3)
Journal of Cell Science
were analysed. A finely punctate distribution of this integrin in
the microvillar region was observed both in control and CD9–/–
oocytes (Fig. 4A). This is similar to the previously reported
repartition of the integrin ␣6␤1 on ZP-free eggs (Tarone et al.,
1993; Takahashi et al., 2000). In wild-type oocytes, antibodymediated aggregation of ␣6 integrins resulted in the formation
of small, regularly arranged patches. By contrast, the patches
were bigger and more dispersed in CD9-null oocytes (Fig. 4A).
There were ~40% less patches in CD9–/– oocytes after
antibody-mediated aggregation of ␣6␤1 integrins (Fig. 4B).
Interestingly, aggregation of the integrin also resulted in a
higher level of internalization in CD9–/– oocytes (data not
shown). These results indicate that CD9 restricts the mobility
of the integrin on the cell surface.
Redistribution of CD9 and ␣6␤1 integrin during
fertilization
The dual inhibition by anti-CD9 mAb of both patch formation
and human gamete fusion when added before ZP removal
strongly suggested that these integrin ␣6␤1-containing patches
were involved in fusion. Thus we examined the distribution of
CD9 and integrin ␣6␤1 during fertilization. The distributions
of CD9 and ␣6␤1 integrin were subjected to dramatic changes.
Four major trends were observed. (1) A labelling of CD9 and
␣6␤1 was observed at the site of interaction of the sperm and
oocyte (Fig. 5D,K). (2) A large area surrounding the sperm
entry point was devoid of CD9 and of integrin ␣6␤1. This area
became larger as meiosis 2 division proceeded (Fig. 5F,H). No
Fig. 5. Dynamics of CD9 and ␣6␤1 integrin during fertilization.
Intact mouse oocytes were left uninseminated (A,B) or were
inseminated (C-M). After incubation, the eggs were fixed, labelled
with DAPI, stained with an anti-␣6 integrin mAb (green) and a CD9
mAb (red), and analysed by confocal microscopy as described in the
Materials and Methods. Composite images were generated by
superimposition of the green and red signals, with areas of overlap
appearing yellow. On the left (A,C,E,G,I) are shown
superimpositions of a transmission image with the DAPI staining
(blue) to identify the stage of the oocytes. (A,B) Non-inseminated
metaphase II oocyte. The integrin ␣6␤1 and CD9 have a finely
punctuate distribution and strongly overlap. (C,D) An inseminated
metaphase II oocyte with a sperm attached to the membrane (arrow).
The distribution of both molecules is very similar to non-inseminated
oocytes. Note the strong labelling where the sperm is attached, seen
at a higher magnification in K where the DAPI staining is also
shown. (E,F) After fusion has occurred, the integrin ␣6␤1 and CD9
are excluded from the region (see the area delimited by short lines)
surrounding the sperm entry point (arrow) and start to gather into
small clusters. Note in E the sperm DNA starting to decondense
(arrow) and the anaphase of the oocyte (arrowheads). The region
surrounding the oocyte DNA (*) is poor in microvilli (the
amicrovillar region) and poorly expresses CD9 and the integrin
␣6␤1. (G,H) An egg at the pronuclei stage. The pronuclei are
indicated by arrowheads in G. The disappearance of both molecules
from the region surrounding the sperm entry point (delimited by
short lines) has continued and the molecules concentrate into
heterogeneous patches. Note the concentration of these molecules in
the meiotic cleavage furrow. The polar body is indicated (PB).
(I,J) In some case the sperm heads fused before the sperm tail had
entirely crossed the ZP. A high local concentration of integrin ␣6␤1
was observed at the sperm entry site (arrow). L and M show a higher
magnification of the region of the sperm entry site. Arrowheads in I
indicate pronuclei. Note again the high concentration of these
molecules in the meiotic cleavage furrow. Bar 40 ␮m.
particular staining of the sperm entry point could be observed
except when the flagellum remained partially outside (Fig.
5J,M). (3) These molecules gathered into clusters that were
heterogeneous in size and shape (Fig. 5F,H,J). These patches
occurred rapidly as they were observed in eggs before
completion of sperm nucleus decondensation and entry into
telophase (Fig. 5F). (4) The ␣6␤1 integrin and to a lesser extent
CD9, accumulated in the mitotic cleavage furrow (Fig. 5J).
Since this reorganization was not observed in CD9–/– oocytes
(which do not fuse), we suggest that it was consecutive to
fusion (data not shown).
Discussion
CD9 is strongly expressed on the oocyte surface where it is
essential for sperm-egg fusion (Le Naour et al., 2000; Miyado
et al., 2000; Kaji et al., 2000; Chen et al., 1999; Miller et al.,
2000). So far, the mechanisms by which it controls this process
are unknown. An isolated large extracellular loop of CD9 was
recently shown to inhibit fusion when preincubated with eggs,
but not with sperm (Zhu et al., 2002). This indicated that CD9
was not a receptor for a sperm molecule. Because of the
Journal of Cell Science
Tetraspanins and integrins in gamete fusion
exceptional ability of CD9 to associate with other
transmembrane proteins including the ␣6␤1 integrin
(Boucheix and Rubinstein, 2001), these data suggested that
CD9 could in some way modulate the function of a sperm
receptor on the oocyte surface.
Our data show that the fusion of human gametes is more
dependent on ␣6␤1 integrin than mouse gamete fusion. Indeed,
the mAb GoH3 strongly inhibited the fusion (up to 96%
inhibition), and this was achieved using concentrations that
were similar to the concentrations necessary to block the
binding of somatic cells to laminin, a major ␣6␤1 integrin
ligand (Sonnenberg et al., 1990). Consistent with this role of
␣6␤1 integrin, we also found that a mAb directed to CD151,
a tetraspanin that intimately associates with ␣6␤1 (Serru et al.,
1999), could partially inhibit the fusion. This is the first
demonstration of a role for this tetraspanin in this process. This
result contrasts with the finding that CD151 knockout mice are
fertile (Wright et al., 2004). However, mice lacking CD151
appear to be grossly normal and healthy while human patients
with mutations in this molecule have severe disorders of skin
and kidney (Karamatic Crew et al., 2004). This discrepancy
was suggested to be linked by the expression in the mouse, but
not in humans, of a molecule closely related to CD151 that
may compensate for CD151 deficiency in knockout animals.
The strong inhibitory effect of the anti-␣6 integrin mAb on
human sperm-egg fusion contrasts with the controversial role
of this integrin in the mouse. The notion that ␣6␤1 integrin in
mouse could play a role in gamete fusion came from a study
showing that the anti-␣6 integrin-blocking mAb GoH3
diminished in a dose-dependent fashion sperm binding to
mouse eggs whose ZP had been previously removed by
protease digestion. In this study, fusion was only inhibited at
400 ␮g/ml, concentration that almost completely blocked
sperm binding to eggs (Almeida et al., 1995). More recently,
these data were challenged by the finding that GoH3 did not
inhibit binding of sperm to mouse oocytes after ZP removal
using an acidic treatment (Evans et al., 1997), and that it did
not reduce the fertilization rate of ZP-intact mouse oocytes at
500 ␮g/ml (Miller et al., 2000). Moreover, oocytes lacking the
␣6 integrin subunit could be fertilized (Miller et al., 2000).
Thus the integrin ␣6␤1 does not play an essential role in
sperm-egg fusion in the mouse, although its participation
cannot be excluded. This apparent discrepancy may be due to
the different techniques used to remove the ZP. Protease
treatment may modify some proteins resulting in a loss of
function of the main receptor, making the egg susceptible to
GoH3 inhibition (Miller et al., 2000). This is the reason why
we used a totally chemical- and enzyme-free mechanical
technique to remove the ZP in human eggs in which ␣6␤1
appears crucial for fusion. This technique of zona removal
appears less deleterious since in this condition the oocyte is
immediately competent for fertilization and does not need
further incubation time to recover its ability to be fertilized.
Use of ZP-free eggs for sperm-fusion assays is common
because its removes any possible influence of the ZP and
cumulus cells. Moreover, for ethical reasons, removal of ZP is
mandatory to study the process of fusion in human.
Further data will be necessary to determine the precise role
of the ␣6␤1 expressed by human oocytes in gamete fusion. By
analogy with the mouse system, it is possible that in human,
the integrin ␣6␤1 functions as a sperm receptor through its
421
ability to bind sperm ADAM proteins. Indeed in the mouse,
based on the capacity of specific mAb to inhibit the binding of
fertilin ␤ recombinant proteins to the oocyte, and the direct
binding to this integrin of fertilin ␤ peptides, the integrin ␣6␤1
has been suggested to be a receptor for fertilin ␤ (ADAM2)
(Chen and Sampson, 1999; Chen et al., 1999; Takahashi et al.,
2001), but these data have not been confirmed by others (Evans
et al., 1997; Zhu and Evans, 2002). More recently, this ␣6␤1
integrin has been shown to mediate the binding of cyritestin
(ADAM3), another member of the ADAM family expressed on
sperm (Takahashi et al., 2001). The role of fertilin ␤ in the
fusion process is highlighted by the fact that sperm lacking this
protein have a 50% decrease ability to fuse with the oocyte
(Cho et al., 1998). In human, a peptide containing the integrin
recognition sequence of human fertilin ␤ (Phe-Glu-Glu) was
shown to also inhibit sperm adhesion to oocytes and their
penetration (Bronson et al., 1999).
The essential role of CD9 in sperm-egg fusion in the mouse
has now been largely documented using knockout mice (Le
Naour et al., 2000; Miyado et al., 2000; Kaji et al., 2000) and
mAbs that have been shown to block sperm-egg fusion (Chen
et al., 1999; Le Naour et al., 2000; Miller et al., 2000).
Surprisingly, anti-human CD9 mAb failed to inhibit the fusion
of human ZP-free oocytes with sperm, when added at the time
of insemination, under conditions in which anti-mouse CD9
mAb inhibit the fusion in mouse. However, anti-CD9 mAb
strongly inhibited the fusion when added to human intact
oocytes and kept continuously present during ZP removal and
fusion, indicating that mechanical ZP removal put the oocyte
at a stage where CD9 is no longer required for fusion. A major
effect of ZP removal in human was to induce the formation of
patches containing the integrin ␣6␤1 and CD151. Actually,
CD9 mAb added before ZP removal inhibited the formation of
these patches along with fusion, showing that these patches
play a key role in human gamete fusion and that the role of
CD9 in sperm-egg fusion (in human) is to control the formation
of these patches. Study of CD9–/– mouse oocytes provided
additional evidence for a role of CD9 in the control of
␣6␤1 integrin lateral mobility. Indeed, antibody-mediated
aggregation of ␣6␤1 integrin yielded patches that were more
dispersed and larger in the absence of CD9. We hypothesize
that CD9 is necessary for the maintenance of a tetraspanin web
to which the integrin ␣6␤1 is linked, thus controlling its lateral
mobility. Surprisingly, the tetraspanin CD151 still formed
patches in the presence of the CD9 mAb. This suggests that
this treatment may dissociate the interaction of the integrin
␣6␤1 with CD151. Alternatively, as a fraction of CD151
formed patches not containing this integrin (Fig. 1), it is
possible that only the patches containing CD151 but not the
␣6␤1 integrin are still observed after CD9 mAb treatment.
The fact that a CD9 mAb inhibits the formation of the patches
formed after ZP removal of human oocytes strongly suggests
that it uncovers a physiological mechanism of patch formation.
The perivitelline space of unfertilized oocytes (the space
between the oolemna and ZP) contains a hyaluronan-containing
matrix, well developed in humans but scant in rodents including
the mouse (Talbot and Dandekar, 2003). The hyaluronan-rich
matrix attaches to the plasma membrane of the oocytes. CD44,
a major hyaluronan receptor is actually present on human
oocytes (Campbell et al., 1995a) and has been shown to associate
with tetraspanins (Jones et al., 1996). Moreover, during
Journal of Cell Science
422
Journal of Cell Science 119 (3)
fertilization, acrosome-reacted sperm carry substantial amount
of hyaluronidase localized at the inner acrosomal membrane that
may facilitate fusion (Cowan et al., 1991). The hyaluronan-rich
matrix attached to the membrane may have a stabilizing effect
that is abolished upon ZP removal, which removes proteins and
vesicles present in the perivitelline space. This role is further
supported by the fact that adding exogenous hyaluronan
decreases polyspermy during conventional porcine IVF (Suzuki
et al., 2000). Therefore, although this remains speculative, it is
possible that hyaluronan participates in maintaining unclustered
the tetraspanin/integrin ␣6␤1-containing membrane structures.
The fact that hyaluronan-containing matrix is well developed in
human and less in mice could explain the differences observed
in integrin relocation upon ZP removal between these two
species (Talbot and Dandekar, 2003).
In addition to the dual inhibition by CD9 mAb of fusion and
patch formation, several lines of evidence suggest that ␣6␤1
integrin-containing patches play a role in sperm binding and
fusion. Concerning humans, we have previously shown that
fused spermatozoa co-localized with ␤1 integrin patches (Ji et
al., 1998). In the mouse (using ZP-free eggs), clustering of
integrins at sperm contact and fusion occurred concomitantly,
and the percentage of eggs in which the integrin ␣6 and ␤1
subunits appeared clustered at a sperm-binding site was closely
correlated to the percentage of eggs with fused sperm. This led
Takahashi et al. to suggest that sperm-egg fusion occurs at sites
where the ␣6␤1 integrin is clustered (Takahashi et al., 2000).
In our study, we observed, before fusion, a strong labelling of
CD9 and to a lower extent of integrin ␣6␤1 at the site of
interaction of the sperm with the oocyte. After fusion occurred,
we detected a local high concentration of the integrin ␣6␤1
(and to a lower extent CD9) when the flagella had remained
partially outside the ZP. At latter time, the sperm entry point
could not be detected based on CD9 and integrin labelling,
suggesting that these molecules diffuse rapidly in the plasma
membrane after fusion. This result is consistent with the study
of Takahashi et al. who observed that the ␣6␤1 integrin
disappeared from the site of sperm penetration (Takahashi et
al., 2000). These data further suggest a role for integrin ␣6␤1
patches during sperm-egg fusion.
The non-essential role of the integrin ␣6␤1 in the mouse
(Miller et al., 2000) seems to conflict with a model in which
␣6␤1 integrin-containing clusters play a major role in fusion.
However, because other molecules (integrins and other types)
associate with tetraspanins within the tetraspanin web
(Boucheix and Rubinstein, 2001; Berditchevski, 2001; Hemler,
2003), it is possible that the actual sperm receptor(s) in the
mouse oocyte, that remain(s) to be identified, also cluster(s) in
a CD9-dependent mechanism. The recent demonstration that
none of the integrins known to be present on the mouse egg
are essential for sperm-egg binding and fusion suggests that the
actual sperm receptor in mice is not an integrin (He et al.,
2003). We propose that the ␣6␤1-containing patches constitute
a marker for the presence of multimolecular complexes
assembling in large clusters necessary for fusion. Inside these
clusters, the avidity for the ligand(s) may be increased. In this
regard, it has recently been demonstrated that an anti-CD9
mAb inhibited the binding to eggs of fertilin ␤ disintegrin
domains immobilized onto beads, but not the binding of the
soluble protein (Zhu and Evans, 2002). In contrast to soluble
proteins, beads are subjected to strong detaching shear forces
during washing steps, and thus their binding to oocytes
depends not only on the affinity of the receptor to fertilin, but
importantly on avidity. Our results suggest that this effect of
the CD9 mAb on the binding of fertilin ␤-coated beads reflects
a decreased avidity of the receptor linked to an inefficient
clustering. This hypothesis is strengthened by the recent
finding that the tetraspanins CD151 and CD81 regulate
adhesion strengthening of the integrins ␣6␤1 and ␣4␤1,
respectively (Lammerding et al., 2003; Feigelson et al., 2003).
We have also shown that fertilization induces a major
redistribution of CD9 and of the ␣6␤1 integrin. In particular,
a large area surrounding the sperm lacked these two molecules
after fusion. This is probably a consequence of the formation
of a fertilization cone, that does not discernibly bind conA, in
relation with the absence of microvilli in this region (Davies
and Gardner, 2002; Maro et al., 1984). The intense colocalization of CD9 and integrins into heterogeneous patches,
together with the control of integrin ␣6␤1 diffusion by CD9,
suggest that CD9 may play a role in this reorganization.
However, CD9 does not play an essential post-fusion role, as
intracytoplasmic sperm injection into CD9–/– eggs results in
normal stage 2 embryo formation and implantation efficiency.
Moreover, we have observed that the ␣6␤1 integrin strongly
accumulates in the mitotic cleavage furrow, raising the
possibility that it may play a role in cytokinesis. In this regard,
integrins are linked to the actin cytoskeleton, which
reorganizes at the growing end of the cleavage furrow of
Xenopus egg during cytokinesis (Noguchi and Mabuchi, 2001).
Thus, the integrin ␣6␤1 may also play a role in cytokinesis.
In general, mAb directed to various tetraspanins trigger the
same functional effects, and this is thought to be related to their
presence in the tetraspanin web (Boucheix and Rubinstein,
2001). In this study, we found that the three tetraspanins
examined behave differently with respect to sperm-egg fusion
in human. Thus, no effect of CD81 on fusion could be
observed, although in previous studies anti-mouse CD81 mAb
was shown to partially inhibit sperm binding to the oocyte and
fusion (Takahashi et al., 2001), and CD81 was shown to
compensate for the lack of CD9 when exogenously expressed
in the oocyte (Kaji et al., 2002). These discrepancies may relate
to differences in the human and mouse processes. Two other
tetraspanins were shown to be involved at different stages, with
CD9 controlling the formation of clusters that contain the
tetraspanin CD151, the ␣6␤1 integrin and possibly a sperm
receptor at the surface of human oocyte. We have demonstrated
for the first time that CD151 plays a role in sperm-fusion, since
mAb directed to CD151 could partially inhibit this process.
CD151 plays its role downstream of CD9 since this inhibition
was achieved after the formation of patches induced by ZP
removal. This is consistent with the tight association of CD151
with the integrin ␣6␤1 (Serru et al., 1999) and with a role of
this integrin in the fusion process in human. This is the first
demonstration that two tetraspanins are involved sequentially
in a physiological process and this shows the dynamic
behaviour and plasticity of the tetraspanin web.
Materials and Methods
Antibodies
The function blocking mAb against human ␤1 integrin (DE9), and against ␣6
integrin (GoH3) were purchased from Chemicon International (London, UK). The
mouse anti-human CD151 mAb TS151, the two mouse anti-human CD9 antibodies
Tetraspanins and integrins in gamete fusion
(ALB-6, SYB-1) and the rat anti-mouse CD9 antibody 4.1F12, coupled or not with
Alexa Fluor 488, were described previously (Charrin et al., 2001; Le Naour et al.,
2000; Rubinstein et al., 1996). 11B1G4 (anti-CD151) was provided by L. K.
Ashman (Fitter et al., 1999), Z81 (CD81) by F. Lanza (Azorsa et al., 1999) and 1D6
(CD81) by S. Levy (Higginbottom et al., 2000). Fluorescein isothiocyanateconjugated donkey anti-mouse immunoglobulin and rhodamine-conjugated donkey
anti-rat immunoglobulin (Jackson Laboratories) were used for second antibodies in
immunocytochemistry experiments.
Preparation of human gametes and fusion assays
Oocytes preparation
Immature human oocytes and unfertilized human oocytes were donated by patients
undergoing intracytoplasmic sperm injection and in vitro fertilization respectively,
with the approval of local Ethical Committee. Exceptionally, fresh cohorts of mature
oocytes were used when no sperm were available. Similar results were obtained
with these fresh oocytes and unfertilized or in vitro matured oocytes for
immunostaining and functional test of fusion. Unfertilized normal metaphase II
(MII) stage human oocytes were collected 48 hours after insemination. In vitro
matured metaphase II were obtained by incubating germinal vesicle or metaphase
I stage oocytes under a 5% CO2 atmosphere at 37°C for 24 hours. ZP was removed
with a chemical- and enzymatic-free technique, using a pair of microdissection
scissors under a binocular microscope, in order to preserve the integrity of the
surface proteins (Kellom et al., 1992).
Journal of Cell Science
Sperm preparation
Semen from donors of proven fertility were collected after 3 days of abstinence.
Sperm samples were kept at 37°C until liquefaction was completed. A two-step
90/45% Puresperm® gradient (Pharmacia), was used to select motile spermatozoa.
The sperm were then kept under capacitating conditions for 2 hours before use.
Sperm-egg fusion assay
ZP-free mature eggs were inseminated with 4000 capacitated motile human
spermatozoa in 20 ␮l Ferticult Medium (FertiPro, Belgium) for 18 hours under
paraffin oil. These are the standard conditions (except that ZP is not removed) for
in vitro fertilization of human oocytes before implantation in women. In some
experiments, the eggs were preincubated with different concentrations of mAb for
30 minutes before insemination. For analysis, oocytes were washed and loaded with
DNA-specific fluorochrome Hoechst 33342 (Sigma) at 5 ␮g/ml for 20 minutes.
After washing they were fixed in 4% paraformaldehyde in 1% PBS-BSA for 30
minutes at room temperature. Fusion was considered to have occurred when the
nucleus of the spermatozoa stained with Hoechst and was decondensed. The
samples were analysed using a Zeiss Axiophot microscope equipped with a camera
and connected to an Imaging System Package (Applied Imaging, Newcastle-uponTyne, UK).
Preparation of mouse gametes
Preparation of oocytes
CD9–/– and inbred wild-type female mice (6- to 12-weeks old) were obtained as
previously described (Le Naour et al., 2000). They were superovulated by
intraperitoneal injections of 5 IU PMSG (Folligon, Intervet, France) and 5 IU hCG
(Chorulon, Intervet, France) 48 hours apart. Fifteen to 16 hours after hCG injection,
the animals were sacrificed by cervical dislocation. Cumulus-oocyte complexes
were collected by tearing the oviductal ampulla, then inseminated in Whittingham
medium with 3% BSA and 106 spermatozoa/ml.
Sperm preparation
Spermatozoa were collected from CD9+/+ male mice after squeezing two caudae
epididymis in a 500 ␮l drop of Whittingham’s medium supplemented with 30 mg/ml
BSA under mineral oil (Whittingham, 1971). Spermatozoa were incubated at 37°C
in 5% CO2 for 90 minutes for capacitation.
In vitro fertilization
Cumuli were inseminated with 106/ml capacitated spermatozoa for 3 hours. There
were then washed and loaded with the DNA-specific fluorochrome DAPI at 1.5
␮g/ml for 15 minutes. After washing they were fixed in 4% paraformaldehyde in
1% PBS-BSA for 30 minutes at room temperature. Fertilization was considered to
have occurred when the oocyte nucleus and decondensed sperm head showed
fluorescence under UV light and the second polar body had been extruded.
To analyse different fertilization steps, insemination was sequentially interrupted
(at 30 minutes, 1 hour, 2 hours and 3 hours) by dropping them in 4%
paraformaldehyde.
Fluorescent staining of eggs
Human eggs
Single or double staining of human eggs was performed by incubating intact or ZPfree eggs with primary antibodies for 1 hour at the following concentrations: anti␣6 (20 ␮g/ml) and/or anti-␤1 (10 ␮g/ml) and/or anti-CD151 (10 ␮g/ml) or antiCD9 antibodies (10 ␮g/ml). These incubations were followed by extensive washing
with Ferticult® IVF culture medium prior to a staining procedure with a mixture of
423
FITC-conjugated donkey anti-mouse IgG (10 ␮g/ml) and/or a rhodamineconjugated donkey anti-rat IgG (10 ␮g/ml) antibody. Eggs were then fixed with 4%
paraformaldehyde solution for 30 minutes. Oocytes were mounted in Immumount
antifade solution and analysed under a fluorescence microscope. The primary
antibodies were omitted for control staining. To verify that clustering was not
mediated by the antibodies, labellings were also performed after paraformaldehyde
fixation and identical results were obtained.
Mouse eggs
Fertilized oocytes were separated from unfertilized oocytes. Both were incubated
with 20 ␮g/ml rat mAb GoH3 for 1 hour. Eggs were then treated with a donkey
anti-rat IgG (10 ␮g/ml) antibody for 1 hour. For additional labelling of CD9, the
eggs were treated with rat IgG and then with an Alexa Fluor 488-coupled CD9 mAb.
The eggs were deposited in a small drop of medium on a Labteck coverslip and
covered with mineral oil. Confocal analysis of ␣6 integrin and CD9 tetraspanin
expression was performed with a TCS SP2 confocal microscope (Leica, Wetzlar,
Germany), using a 63⫻ objective. Images of about 100 sections, representing nearly
half of the eggs were collected, and non-specific labelling in the ZP was removed
in each using the program Paintshop Pro. The maximum projection function of the
LCS software (Leica) was then used to superimpose the different sections.
Antibody-mediated aggregation of ␣6␤1 integrin
Wild-type and CD9–/– intact eggs were successively incubated with anti-␣6 GoH3
(20 ␮g/ml) and, after washing, with Alexa Fluor 488-labelled goat anti-rat
antibodies (10 ␮g/ml) for 45 minutes at 37°C under 5% CO2. They were then fixed
with 4% paraformaldehyde and analysed by confocal microscopy. There were two
control groups of oocytes: one group was fixed prior to any antibody incubation and
the other before the secondary antibody. The distribution of the integrin ␣6␤1 was
identical in both groups. The number of patches were quantified using the ‘particle
analysis’ menu of Image J.
We thank P. Fontanges (Hôpital Tenon, Paris, France), S. Chambris
(Université Paris 13, Bobigny, France) for their technical assistance,
L. Ashman, F. Lanza and S. Levy for the gift of monoclonal
antibodies. We are grateful to A. Hazout, P. Cohen-Bacrie and A.-M.
Junca from Eylau laboratories for providing us with human material.
This work was supported by grants from Inserm, the Association pour
la Recherche sur le Cancer, NRB-Vaincre le Cancer, and the Institut
de Cancérologie et Immunogénétique.
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