2013 Identify Human Sperm Proteins for Zp Binding
Transcript of 2013 Identify Human Sperm Proteins for Zp Binding
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ORIGINAL ARTICLE Reproductive biology
Identification of sperm head proteins
involved in zona pellucida binding
F.M. Petit1, C. Serres2, F. Bourgeon3, C. Pineau3, and J. Auer2,*1AP-HP, Labor atoire de g enet ique moleculaire, Hopital Antoine Becl ere , Clamart 92141, France 2INSERM U1016, Departement de
Genetique et Developpement, Institut Cochin, CNRS UMR8104 and Universite Paris Descartes, Paris 75014, France 3Proteomics Core
Facility Biogenouest, Inserm U1085 IRSET, Campus de Beaulieu, Rennes Cedex 35042, France
*Correspondence address. E-mail: [email protected]
Submitted on September 28, 2012; resubmitted on November 22, 2012; accepted on December 11, 2012
study question: Which human sperm proteins interact with zona pellucida (ZP) glycoproteins, ZPA/2, ZPB/4 and ZPC/3?
summary answer: Co-precipitation experiments with recombinant human ZP (rhZP) coated beads demonstrated interactions withvarious proteins, including glutathione S-transferase M3 (GSTM) with ZPB/4 and voltage-dependent anion channel 2 (VDAC2) with ZPA/2
and ZPC/3.
what is known already: Regarding spermZP binding, several target spot/proteins have been detected in several species, butnot all have been characterized. The limit of these studies was that a mixture of the different ZP glycoproteins was used and did not allow the
identification of the specific ZP glycoprotein (ZPA/2, ZPC/3 or ZPB/4) involved in the interaction with the sperm proteins.
study design, size, duration: To identify the human sperm proteins interacting with the oocyte ZP, we combined twoapproaches: immunoblot of human spermatozoa targeted by antisperm antibodies (ASAs) from infertile men and far western blot of
human sperm proteins overlayd by each of the rhZP proteins.
materials, setting, methods: We used rhZP expressed in Chinese hamster ovary (CHO) cells and ASA eluted from infertilepatients undergoing IVF failure. Sperm proteins separated by two-dimensional (2D) electrophoresis recognized by both sperm-eluted ASAs
from infertile patients and rhZP were identified by mass spectrometry (MALDI-MS/MS). Some of these proteins were further validated by
co-precipitation experiments with rhZP and functional zona binding tests.
main results and the role of chance: We identified proteins that are glycolytic enzymes such as pyruvate kinase 3,enolase 1, glyceraldehyde-3-phosphate dehydrogenase, aldolase A, triosephosphate isomerase, detoxification enzymes such as GSTM or
phospholipid hydroperoxide glutathione peroxidase, ion channels such as VDAC2 and structural proteins such as outer dense fibre 2.
Several of the proteins were localized on the sperm head. However, these proteins have also been described to exert other functions in
the flagellum. Co-precipitation experiments with rhZP-coated beads confirmed the direct interaction of GSTM with ZP4 and of VDAC2
with ZP2 and ZP3.
limitations, reasons for caution: We used recombinant ZP in place of native ZP. Thus, the post-translational modifica-tions of the proteins, such as glycosylations, can be different and can influence their function. However, CHO cell-expressed rhZP are func-
tional, e.g. can bind human spermatozoa and induce the acrosome reaction. Moreover, the identification of relevant proteins was limited by
the need for sufficient amounts of proteins on the preparative 2D-gel to be subsequently analysed in MALDI-TOF MS/MS.
wider implications of the findings: Our results bring new insights on the ability of sperm proteins to exert several func-tions depending on their sub-cellular localization, either the head or flagellum. Their multiple roles suggest that these sperm proteins are
multifaceted or moonlighting proteins.
study funding/competing interest(s): This work was supported by the grant ReproRio (CNRS, INRA, INSERM andCEA) and the Societe dAndrologie de Langue Francaise.
trial registration number: Not applicable.
Key words: spermzona pellucida interaction / antisperm antibodies / far WB / moonlighting / proteome
& The Author 2013. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.
For Permissions, please email: [email protected]
Human Reproduction, Vol.0, No.0 pp. 114, 2013
doi:10.1093/humrep/des452
Hum. Reprod. Advance Access published January 25, 2013
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Introduction
Mammalian fertilization is a complex process involving several mole-
cules distributed on the two gametes, spermatozoon and oocyte.
One of these steps is the binding to, and penetration of, the extracel-
lular coat of the oocytes termed the zona pellucida (ZP) by spermato-
zoa. Immediately after ejaculation, the sperm are not able to recognize
and interact with the ZP. The ability of sperm to bind the ZP is
achieved only after the last maturation event, called capacitation,
which occurs in the female genital tract. This species-specific event sti-
mulates diverse signalling pathways in the spermatozoa leading to the
acrosomal exocytosis. During this acrosome reaction, the sperm
plasma membrane fuses with the outer acrosomal membrane,
causing exposure of the inner acrosome membrane at the surface
and release of the acrosomal content.
A widely used strategy to identify sperm proteins involved in gamete
interaction is based on the inhibition of spermoocyte binding/fusion
by antispermatozoa antibody produced after a mouse/rabbit immun-
ization. This approach requires a time-consuming screening to find in-
hibitory antibodies and then the corresponding antigens. Several
sperm proteins involved in gamete interaction have been identifiedby this procedure, such as acrosin, fertilin, Izumo, SPAM1 and
ADAM (Topfer-Petersen et al., 1990; McLeskey et al., 1998; Nixon
et al., 2007). After validation steps, which often involve the creation
of mouse models, usually loss-of-function mouse models for the
gene of interest, the protein is either confirmed or ruled out for its
role in gamete interaction.
Some groups have studied the immunoproteome of human gametes
revealed by serum or seminal plasma antisperm antibodies (ASAs)
(Shetty et al., 2001, 2008; Bohring and Krause, 2003; Domagala
et al., 2007).Steinet al. (2006) combined a proteomic study with sub-
cellular fractionation in order to identify sperm head proteins that
mediate the spermoocyte interaction. Our group has detected
several proteins targeted by local ASAs (and not systemic ASAs asgenerally used) from patients with autoimmune infertility (Auer
et al., 1997) and identified and characterized a triosephosphate isom-
erase (TPI) involved in spermZP interaction (Auer et al., 2000,
2004).
Another method, also called blot-overlay or far western blot (WB),
involves the separation of sperm proteins on one- or two-dimensional
(2D) electrophoresis gels, transfer to polyvinylidene difluoride (PVDF)
membranes and overlay of the sperm proteins by solubilized ZP gly-
coproteins. Several target spot/proteins have been detected with
this strategy, but not all have been characterized [Shabanowitz and
ORand, 1988 (human); Tanii et al ., 2001 (mouse); Manaskova-
Postlerova et al., 2011(boar)]. The limit of these studies was that a
mixture of the different ZP glycoproteins was used and did not
allow the identification of the specific ZP glycoprotein (ZPA/2,
ZPC/3 or ZPB/4) involved in the interaction with the sperm proteins.
In humans, the difficulty in obtaining ZP material in adequate
quantity and quality made the above approach less realistic but, in
the last 10 years, such a problem has been overcome by the use of
human recombinant ZP obtained from diverse cells (Harris et al.,
1999; Martic et al., 2004; Chakravarty et al., 2005; Marin-Briggiler
et al., 2008;Chirinos et al., 2011). Notably, this approach allows the
interactions with the different glycoproteins (ZPA/2, ZPC/3 or
ZPB/4) to be distinguished.
Here, we made use of the relatively recently developed proteomic
tools and of recombinant human ZP (rhZP) glycoproteins and
studied the human sperm receptors for ZP2, ZP3 and ZP4 by
direct interaction between rhZP2, rhZP3 or rhZP4 glycoproteins
and solubilized sperm membrane proteins using the far WB tech-
nique. We compared the results obtained by this approach with
those obtained when sperm proteins separated by 2D electrophoresis
were recognized by sperm-eluted ASA from infertile patients. The pro-
teins recognized by both ZP glycoproteins and ASA were then identi-
fied by mass spectrometry. Some of these proteins were further
validated by co-precipitation experiments and functional zona binding
tests.
With this study, we identified a set of sperm proteins involved in
spermZP interaction. Several of them are involved in functions
other than ZP interaction, which highlights the moonlighting functions
of these sperm proteins.
Materials and Methods
rhZP glycoproteins and specific antibodies
rhZP produced in Chinese hamster ovary (CHO) cells and their specific
anti-sera obtained by rabbit immunization were gifts of Harris et al.
(1999). These CHO cells-expressed rhZPs are secreted in the medium
and are highly purified.
Sperm membrane fraction
Sperm samples with normal semen parameters (WHO, 2010) were
obtained from fertile donors. The motile spermatozoa were selected on
Percoll gradients as previously described (McClure et al., 1989). After
overnight capacitation in B2 medium (CCD, Paris, France), spermatozoa
were washed in 0.05 mmol/l Tris buffer (TB). A pool of capacitated
spermatozoa from several donors was constituted for co-precipitation,
WB or far WB assays, while individual samples were used for functional
tests.
For co-precipitation experiments, 1 108 washed spermatozoa were
solubilized with 1% NP-40 or 0.1% Triton X-100 detergent in 100m l
of TB supplemented with a protease inhibitors cocktail (Sigma-Aldrich,
St Quentin Fallavier, France) for 1 h at 48C. The supernatant was stored
at 2808C until use.
For electrophoretic separation, spermatozoa were solubilized either in
Laemmli-reducing sample buffer [SDS-polyacrylamide gel electrophoresis
(PAGE)] at 958C or in 9 mol/l urea, 2% Triton X-100, 60 mmol/l dithio-
threitol, 2% immobilized pH gradient (IPG) buffer (2D) and a protease
inhibitors cocktail at 48C. Supernatants were stored at 2808C until use.
Antisperm antibodies
Sperm samples with high levels of ASA as detected by Immunobeads test
(IBT, Sigma-Aldrich) were obtained from infertile men undergoing several
unsuccessfulin vitrofertilization attempts. Sperm samples from three fertile
men with no ASA detectable by IBT were used as negative controls.
To obtain ASAs, sperm samples were centrifuged at 600g for 10 min
and then sperm pellets were washed twice with phosphate-buffered
saline (PBS). The washed pellets containing 5 107 to 1 108 motile
spermatozoa were resuspended in 1 2 ml of 100 mmol/l glycine-HCl
buffer at a pH of 2.8 under gentle rotation for 15 min at room tempera-
ture (RT) and centrifuged for 5 min at 12 000g. The supernatants were
neutralized with 3 mol/l Tris, dialysed against PBS overnight at +48C
and filtered through 0.2 mm sterile Acrodisc filters (Gelman Sciences,
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France). The titre, immunoglobulin (Ig) class and localization of ASAs were
determined by an indirect Immunobeads test (Clarke et al., 1984).
All experiments with human samples were conducted in accordance
with ethical guidelines and were approved by the ethics evaluation
committee (comitede qualification institutionnelle) of the Institut National
de la Sante et de la Recherche Medicale, INSERM (authorization no.
01-013).
Electrophoresis separationSperm membrane proteins were further fractionated using 2D gel electro-
phoresis. Briefly isoelectrofocalization was performed on linear immobi-
lized pH 3 10 gradient gel strips of 13 cm, using the Multiphor II
system (GE Healthcare, Saclay, France). Strips were cup-loaded at the
anodic end with 70 mg of sperm proteins (2530 106 spz) for analytical
gels and isoelectric focalization was performed at 208C for a total of
18.5 kVh. The second dimension was performed in SDS-PAGE on 12%
polyacrylamide gels.
For preparative gels, the 24 cm 3 10 linear IPG strips were cup-loaded
with 90 mg of sperm proteins and focalization was conducted in IPGphor
(GE Healthcare) for a total of 71.4 kVh. The second dimension was per-
formed on 12.5% polyacrylamide gels using the DALTSix system (GE
Healthcare). At each step, the protein concentration was determinedusing the bicinchoninic acid protein assay (Sigma-Aldrich).
Following migration, 2D gels were silver stained, as previously described
(Shevchenko et al., 1996) with minor modifications (Com et al., 2003).
Gels were scanned with an ImageScanner (GE Healthcare) and then
stored at 48C in 1% acetic acid until spot excision. Finally, a pick list was
generated using ImageMaster 2D Elite software (GE Healthcare).
SDS-PAGE was also performed after co-precipitation of the sperm pro-
teins with rhZP. The protein complexes were separated using a mini
PROTEAN II Cell apparatus (Bio-Rad, Marne la Coquette, France) on
12% polyacrylamide gels.
Overlay assay (or far WB)
Sperm proteins separated in 1D or 2D electrophoresis were electrotrans-
ferred onto PVDF membranes (Hybond-P, GE Healthcare) in renaturing
conditions (Dunn, 1986). Prior to incubation, an rhZP solution was pre-
absorbed with a blank piece of nitrocellulose (Shabanowitz and ORand,
1988). To avoid any non-specific binding, anti-rhZP sera were pre-
absorbed on human spermatozoa.
After incubation in blocking buffer [1% gelatine in TB salin (TBS)] blots
were incubated with rhZP (0.75 mg for 1 106 of spz) in TBS modified
(TBSm) with 0.05% Tween20, 1 mmol/l Ca2+ and 1 mmol/l Mg2+. Glyco-
proteins that interacted with sperm proteins were detected with specific
anti-rhZP sera (1:4000 in TBSm). After incubation with peroxidase-
conjugated anti-rabbit IgG, the binding was detected with the enhanced
chemiluminescence (ECL)+ detection western blotting system (GE
Healthcare).
Immunodetection with ASA
Sperm proteins were electrotransferred as described above. The mem-
branes were saturated for 1 h at RT in PBS with 5% low-fat milk
powder, and then incubated for 1 h at 378C and overnight at 48C with
ASAs obtained from spermatozoa of infertile men complemented with
0.1% Tween20 (PBST) and 1% low-fat milk powder. After washing, the
blots were incubated for 1 h at RT with affinity-purified goat anti-human
Ig antibodies conjugated to peroxidase (Biosys, Compiegne, France)
diluted to 1:8000 in PBST with 1% gelatine. The blots were washed
twice in PBST and once in PBS. Bound peroxidase was detected by an
ECL+ western blotting system (GE Healthcare).
After the far WB assay as well as after immunodetection, 2D-PVDF
membranes were silver stained (Kovariket al., 1987) for the precise local-
ization of the reactive spots.
In-gel trypsin digestion
Protein spots were excised from 2D gels, and further processed for mass
spectrometry thanks to an EttanTM Spot Handling Workstation (GE
Healthcare). Briefly, before drying, the gel plugs were washed three
times in MilliQ water, once in 50% ethanol/50 mmol/l ammonium bi-
carbonate and once in 75% acetonitrile. Dried plugs were then incubated
for 60 min in 20 mmol/l NH4HCO3 supplemented with 8.3mg/ml se-
quencing grade modified porcine trypsin (Promega, Charbonnieres-
les-Bains, France). Digested peptides were extracted in two successive
steps by incubation of gel plugs in 70% acetonitrile and 0.1% trifluoroacetic
acid. Digested peptides were then dissolved in 0.6 mg/ml a-cyano-
4-hydroxycinnamic acid in 55% ethanol/27% acetone/0.1% trifluoroacetic
acid, and further spotted onto a MTP AnchorchipTM MALDI target (384
Scout MTP 600 mm Anchorchip; Bruker Daltonik, GmbH, Bremen,
Germany).
Mass spectrometry analysis
Protein identification by mass spectrometry was performed using aMALDI-TOF/TOF mass spectrometer (Ultraflex; Bruker Daltonik). Peak
lists were generated from MALDI-MS spectra using the FlexAnalysis soft-
ware (version 3.0; Bruker Daltonik). Following internal calibration with
trypsin autodigestion peptides, the monoisotopic masses of tryptic pep-
tides were used to query the NCBInr sequence database (version
20092604, 6833826 sequences), using Mascot server version 2.2 (www.
matrixscience.com). Search conditions used were an initial open mass
window of 70 ppm for an internal calibration and one missed cleavage
allowed. Carbamidomethylation of cysteines was set as fixed modifications
whereas methionine oxidation was set as variable modifications. Peptide
identifications were scored using the probability-based Mowse score
(the protein score is 2108log (P) where P is the probability that the
observed match is a random event). In the present experimental condi-
tions, a score .78 corresponded to a significant identification (P, 0.05).
Immunofluorescence staining
For immunostaining on live spermatozoa, a capacitated sperm suspension
(5 106 cells/ml) was incubated for 30 min in PBS-5% bovine serum
albumin (BSA) to block non-specific staining sites, then with primary
antibodies at 1:50 for 1 h at RT (anti-GST, Uptima, Interchim, France;
anti-ALDOA, Abnova, Interchim, France) or overnight at 48C [anti-voltage-
dependent anion channel 2 (VDAC2), Proteintech, Manchster, UK]. After
washing, spermatozoa were incubated with the corresponding fluorescein
isothiocyanate (FITC)-conjugated secondary antibodies or biotinylated sec-
ondary antibodies and FITC-conjugated streptavidine (for VDAC2).
An immunostaining procedure was also conducted on fixed cells. For
this, capacitated spermatozoa were incubated for 1 h in 1% paraformalde-
hyde (PFA) in PBS. To neutralize free reactive aldehyde groups, the cells
were then incubated for 30 min in 200 mmol/l glycine in PBS. After
washing, spermatozoa were resuspended at a density of 5 106 cells/
ml in PBS-1% BSA and smeared on slides and air dried. They were first
incubated for 30 min in PBS-5% BSA to block non-specific staining sites
and then with primary antibodies at a 1:50 for 1 h at RT. The staining
with corresponding fluorescent secondary antibodies or biotinylated sec-
ondary antibodies and FITC-conjugated streptavidine (for VDAC2) was
done at RT.
An addition of 0.005% saponin to PBS/BSA before and during the
staining procedure was needed to facilitate the access of anti-ALDOA
and anti-VDAC2 to their sperm targets.
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Rabbit or mouse IgG in place of primary antibodies was included as
negative controls.
After immunostaining procedures, stained spermatozoa were mounted
in glycerol-PBS (Citifluor, London, UK) for observation. The fluorescence
was examined, using an epifluorescence microscope (Nikon E600, Cham-
pigny sur Marne, France) at 630 and 1000 magnifications.
Co-precipitationTo obtain beads coated with rhZP, Carboxyl-Adembeads (Ademtech,France) were firstly activated according to the manufacturers instructions.
Then, 6075 mg of rhZP2, rhZP4 and rhZP3 proteins were incubated
with 900 mg of magnetic activated beads for 2 h at 408C under agitation.
After saturation of the binding sites by 40 mmol/l ethanolamine and three
washing steps, 100 ml of sperm membrane protein extract (corresponding
to 1 108 cells) was added to the rhZP-coated beads in the presence of a
protease inhibitor cocktail. After 2 h of incubation at RT under agitation,
the beads were washed three times in 500 m l of lysis buffer, then resus-
pended in Laemmli reducing buffer and boiled at 958C. Supernatants
were then subjected to electrophoresis and WB analysis.
Functional testsZona binding test
To ensure that the antibody effect on a binding test is not related to their
effect on motility, we analysed sperm motility after incubation with anti-
bodies using a Computer-Assisted Sperm movement Analyser (CASA,
Hamilton Thorn, USA).
We used zona intact unfertilized oocytes recovered after IVF failure.
Spermatozoa capacitated overnight (5 105) were added to 500 ml of
B2 medium containing zona intact unfertilized oocytes and incubated for
3 h (when primary binding was examined) or 18 h (when zona penetration
was examined) at 378C in a 5% CO2/95% air atmosphere in the presence
of antibodies. After 3 h of incubation, the oocytes were washed in B2
medium by three to four successive aspirations through a glass pipette
(inner diameter of 250 mm) to remove loosely bound spermatozoa. A
first aliquot of washed oocytes was then deposited in a glass depression
slide in order to count the spermatozoa bound to each oocyte. The
remaining washed oocytes were repeatedly aspirated through a pipette
having an inner diameter slightly smaller than the oocytes (125 mm) to
detach spermatozoa tightly bound to the ZP. Then, the acrosomal
status of the latter spermatozoa was determined.
To determine the effect of antibodies on the zona penetration, the
oocytes were incubated with spermatozoa for 18 h in the presence of anti-
bodies. After removing adhering spermatozoa from the oocytes, their
acrosome reaction rate was determined as described below and
oocytes were placed on a slide in a 20m l drop of 0.2% BSA-PBS to
examine the number of spermatozoa that remained firmly bound to or
embedded within the ZP or present in the perivitelline space.
Acrosome reaction measurement
The acrosomal status of spermatozoa was examined using the modified
method ofCrosset al. (1986). The suspension of spermatozoa was depos-
ited on slides, air dried and fixed in ethanol for 30 min at 4 8C. Cells were
then stained by tetramethyl-rhodamine isothicyanate (TRITC) or FITC-
Pisum sativum agglutinin (PSA) (25 mg/ml in PBS) for 15 min. After
washing in distilled water, the slide was mounted with Citifluor and 200
spermatozoa were examined using an epifluorescence microscope. Sperm-
atozoa displaying a fluorescent equatorial segment or with no staining of the
head were recorded as acrosome-reacted. As only motile sperm were able
to bind to the ZP, no viability staining was performed.
Statistical tests
Studentsttest and x2 test were employed as statistical tests and P, 0.05
was considered as significant.
Results
Detection of sperm proteins interacting with
ZP glycoproteins by the far WB method
The sperm proteins targeted by ZP glycoproteins were detected in
a far WB assay. This analysis revealed that each ZP glycoprotein
interacts with several sperm proteins. About 15 spots or groups of
spots were targeted with rhZP2. The molecular weights of these
sperm proteins ranged from 18 to 90 kDa. The majority of them
were basic with an isoelectric point (pI) between 5.0 and 8.5
(Fig. 1A). Among the basic proteins recognized by rhZP4, three
groups of spots had a high intensity. Their molecular weights ranged
from 15 to 90 kDa and their pI ranged from 5.5 to 8.0 (Fig. 1B).
The signals of the acidic spots with rhZP4 were less intense. Spots tar-
geted with rhZP3 were more numerous, and their pI ranged from 5.2
to 8.2. Their molecular weights ranged from 15 to 75 kDa (Fig. 1C).Control blots which were incubated without rhZP or with the rhZP
but without anti-rhZP antibody did not show any spots. For further
analysis, we focused on the proteins that were recognized by rhZP
in at least two far WB experiments out of the three performed.
Detection of sperm antigens targeted by
ASA eluted from sperm of infertile patients
In this study aimed at identifying sperm proteins involved in ZP recog-
nition/binding, we used ASAs directly eluted from spermatozoa pro-
vided by infertile patients who underwent IVF failures. In the majority
of the cases, a defect of binding and/or penetration of the ZP had
been noticed.All the samples of ASAs isolated from spermatozoa of infertile men
contained IgA and IgG antibodies. The percentages of the sperm
binding immunobeads were 4087% for IgA and 1095% for IgG
and at least 71% of spermatozoa bound to beads in each sample.
We analysed the sperm antigens recognized by ASA isolated from
the spermatozoa of six individual infertile men and from the pool of
five patients constituted according to their class of ASA (predominant-
ly IgA or IgG). As reported in our earlier study ( Aueret al., 1997), we
found heterogeneity in the response to ASA targets among individual
ASA samples (Fig.2), as well as among Ig classes. Only a small propor-
tion of sperm proteins resolved in the 2D gels appeared to be targets
of ASAs. Thus, 530 protein spots were recognized by ASAs with a
group of basic proteins of 50 kDa, an area of proteins at around60 kDa with a rather neutral pI, and an area of more acidic proteins
(pI , 5) of 80 kDa. We also noted that the pool of eluted ASA con-
taining IgG antibodies recognized more antigens than those containing
predominantly IgA antibodies. No spots appeared in WBs targeted
with ASA samples from fertile men.
Identification of sperm proteins targeted byboth eluted ASA and ZP glycoproteins
We limited our identification work to the spots targeted by both
eluted ASAs and rhZP. For a precise localization of the targeted
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spots on 2D gels, exposed film and corresponding silver-stained blots
were scanned and overlapped. The resulting spots were then posi-
tioned on a scanned preparative gel (Fig. 3) and analysed with the
ImageMaster 2D Platinum software able to take into account the dif-
ferences between the gels. Only spots that were well visible on the
preparative silver-stained gel were excised, digested and analysed by
mass spectrometry. Among the spots selected for mass spectrometry
analysis, 14 were identified with a high score and high coverage
(between 22 and 75%) and these corresponded to 9 different proteins
(Table I). Five glycolytic enzymes represented the most important
group [pyruvate kinase 3 (PK3), enolase 1 (ENO1), glyceraldehyde-3-
phosphate dehydrogenase (GAPDH), aldolase A (ALDOA) and TPI];
two proteins were known to be involved in detoxification processes,
glutathione S-transferase M3 (GSTM) and phospholipid hydroperoxide
glutathione peroxidase (PHGPx); one protein was an ionic channel,
VDAC2 and another protein, outer dense fibre 2 (ODF2), was a cyto-
skeletal protein.
Co-precipitation of sperm proteins with
recombinant ZP glycoproteins
To further assess the ability of sperm proteins to interact specifically
with rhZP, we carried out co-precipitation experiments using sperm
extract incubated with rhZP-coated magnetic beads. We focusedthis analysis on ALDOA, TPI, GSTM and VDAC2, four of the sperm
proteins shown to interact with one or two ZP glycoproteins in the
far WB/ASA assay and for which an antibody was commercially avail-
able. After co-incubation of sperm extract with rhZP-coated beads,
we precipitated TPI, GSTM and VDAC2 with rhZP-coated beads.
The characteristic 36 kDa band of sperm TPI (Auer et al., 2004)
was detected in 1% NP40 extract and found to interact with rhZP3
and rhZP4 to a larger extent than with rhZP2 (data not shown).
GSTM was detected as a 26 kDa band in 0.1% TX100 as well as in
1% NP40 sperm extract and preferentially co-precipitated with
rhZP4 when compared with rhZP2 and rhZP3 (Fig. 4A). VDAC2
which runs as a 3334 kDa protein in 1% NP40 sperm extract was
precipitated with rhZP2 or rhZP3 but not with rhZP4-coated beads(Fig. 5A). Similar results were obtained for these three proteins in
two independent experiments. For ALDOA, a 44 kDa band (consist-
ent with its theoretical molecular weight) was detected in 1% NP40
sperm extracts but not after precipitation with rhZP2, rhZP4 or
rhZP3 (Fig. 6A).
Localization of sperm proteins interacting
with ZP
In human spermatozoa, double staining of capacitated spermatozoa
with anti-GSTM and PSA lectin revealed GSTM immunoreactive
sites in the head region overlying the acrosome of intact (PSA positive)
spermatozoa (Fig.4B a and a). The GSTM staining was not observed
on acrosome-reacted (PSA negative) spermatozoa. No GSTM staining
was observed in the control incubation with rabbit IgG instead of
anti-GSTM antibody (Fig. 4B b), nor in sperm treated with 0.05%
Triton X100.
On PFA-fixed sperm cells, VDAC2 was detected in the region over-
lying the acrosome of intact cells and at the flagellum level (Fig. 5B a and
c). We failed to detect any staining with anti-VDAC2 antibody on live
spermatozoa. However, anti-VDAC2 immunodetection performed
on spermatozoa kept in suspension and treated with saponin worked.
Under such conditions, VDAC2 staining was observed in the post-
Figure 1 Sperm proteins targeted by rhZP in far western blots (WB)
assays. 2D WB of human sperm proteins was probed with solubilized
rhZP2 (A), rhZP4 (B) and rhZP3 (C) and revealed with corresponding
anti-rhZP2, anti-rhZP4 or anti-rhZP3 antibodies as described in the
Materials and Methods section. Molecular identities of some proteins
identified by mass spectrometry are indicated on the blots. The spots
with low intensity areshown on theright of the blots with increasedcon-
trast. The pI gradient (310) used for IEF is reported at the top of each
blot and the molecular mass (Mr) standards in kDa used for the second
dimension are indicated on the left of each blot.
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equatorial band and at the base of the head of acrosome intact sperm-
atozoa (Fig.6B a and c). Anti-ALDOA antibody also stained the flagel-
lum. Of note, ethanol or 0.05% Triton X100 treatments eliminated
the sperm head staining but preserved the immunoreactivity of the fla-
gellar principal piece (Fig. 6B d). Control staining using mouse IgG in
place of anti-ALDOA is shown in Fig. 6B b.
............................................................................................................................................................................................
Table I ZP and ASA binding human sperm proteins identified by Mass fingerprinting.
Function Protein
name
Species Accession
numberaMr
expbpI
exp
Matched
peptides/
totalc
%
coverage
Mascot
score
Target
Glycolytic
enzyme
PK3 isoform 2 Homo
sapiens
NP_872270 66 7.3 10/24 24 102 ZP2-ZP3-ZP4 ASA
ENO1 Homosapiens
AAH04458 47 6.8 22/51 53 200 ZP2-ZP3-ZP4 ASA
GAPDH Homo
sapiens
NP_055179 47 7.4 5/9 19 133 ZP2-ZP3-ZP4 ASA
ALDOA Homo
sapiens
NP_000025 42 8.6 14/29 59 189 ZP4 ASA
TPI 1 Homo
sapiens
AAH17917 33 5.2 10/25 51 142 ZP3-ZP4 ASA
Detoxification GSTM Homo
sapiens
NP_000840 27 4.9 7/20 38 83 ZP3-ZP4 ASA
PHGPx 4 Homo
sapiens
P36969 17 8.5 10/35 41 74 ZP2-ZP3-ZP4 ASA
Ion transport VDAC Homo
sapiens
AAB59457 34 6.7 10/28 47 126 ZP2-ZP3 ASA
Cytoskeleton ODF of sperm tails Homo
sapiens
NP_702915 47 5.5 16/42 22 100 ZP3-ZP4 ASA
PI, Isoelectric point.aNational Center for Biotechnology Information (NCBI) sequence identification number.bExperimental relative mass in kDa.cThe number of matched peptides versus the total number of peptides.
Figure 4 Validation of GSTM as spermZP binding protein. (A) Sperm proteins extracted in 1% NP40 were precipitated using magnetic beads
coated with rhZP glycoproteins. The WB of sperm proteins co-precipitated with rhZP2 (A), rhZP4 (B) or rhZP3 (C) and of sperm extract (E)were revealed with an anti-GSTM antibody. (B) Localization of GSTM on human spermatozoa. Double staining of capacitated spermatozoa using
an anti-GSTM and a secondary antibody conjugated to FITC (a) followed by FITC- Pisum sativum agglutinin (PSA) lectin conjugated to tetramethyl-
rhodamine isothicyanate (TRITC) to reveal the acrosomal content (a). GSTM staining is visible in the acrosomal region of intact spermatozoa
(PSA positive). The arrows indicate two acrosome-reacted spermatozoa (PSA negative) without GST labelling. Rabbit IgG was used in place of
anti GSTM antibody in the control staining experiment (b).
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Functional testsWe next tested the role of ALDOA and VDAC2 in sperm binding to
zona intact oocytes. We thus performed gamete interaction tests in
the presence of anti-ALDOA or anti-VDAC2 antibody (Tables IIV,
respectively). In four independent experiments, the number of
sperm bound to the ZP was significantly decreased in the presence
of anti-ALDOA when compared with incubation with control IgG
(Table II). During the binding tests, we verified that anti-ALDOA did
not immobilize the spermatozoa. In one experiment measuring
sperm motility by CASA, a slight decrease in the percentage of
motile spermatozoa was noted with anti-ALDOA (62%) when
compared with IgG (81%). We examined the acrosomal status of
those spermatozoa bound to the ZP after 3 h of interaction, in the
presence of anti-ALDOA or control IgG. In three separate experi-
ments, no difference in the rate of acrosome reaction induced by
ZP was observed between the two groups (data not shown).
Incubation with anti-VDAC2 antibody during the in vitro gamete
interaction led to a reduction by half of the number of spermatozoa
bound to or penetrating the ZP (TablesIII and IV). Exposure to anti-
VDAC2 during the binding tests did not significantly alter the sperm
motility. Indeed, the percentage of motile spermatozoa measured by
CASA after 3 h of contact with anti-VDAC2 was 60.0+6.24%
Figure 5 Validation of VDAC2 as sperm ZP binding protein. (A) Sperm proteins extracted in 1% NP40 were co-precipitated using magnetic beads
coated with rhZP glycoproteins. The WB of sperm extract (E) and sperm proteins co-precipitated with rhZP2 (A), rhZP4 (B) or rhZP3 (C) were
revealed with anti-VDAC2 antibody. (B) VDAC2 localization on PFA-fixed (ad) or live (e and f) human spermatozoa permeabilized by 0.005%
saponin. Spermatozoa were PFA-fixed before (a and b) or after (c and d) spreading on slides. Spermatozoa were labelled with anti-VDAC2 and
FITC secondary staining (a, c and e) and TRITC-PSA lectin (a ). The arrow indicates the VDAC2 staining over the acrosomal area of intact sperm-
atozoa (PSA positive). Rabbit IgG was used in place of anti-VDAC2 in the control staining experiments (b, d and f).
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compared with 70.0+ 8.54% in the presence of IgG control (P
0.09;n 3). The induction of the acrosome reaction in the spermato-
zoa bound to ZP was unaffected by the presence of anti-VDAC2
(TableV).
Discussion
New strategy for identification of ZP
receptor proteins
In the present study, we aimed to identify human sperm proteins that
interact with ZP glycoproteins using a double approach: a far WB
Figure 6 Aldolase localization on human spermatozoa. (A) Sperm proteins extracted in 1% NP40 were co-precipitated using magnetic beads
coated with rhZP glycoproteins. The WB of sperm proteins co-precipitated with rhZP2 (A), rhZP4 (B) or rhZP3 (C) and of sperm extract (E)
were revealed with anti-ALDOA antibody. (B) Live spermatozoa were permeabilized by 0.005% saponin (ac) or ethanol (d) treatment before immu-
nostaining. They were stained with anti-ALDOA and FITC-conjugated secondary antibody (a and d) or double stained with anti-ALDOA and FITCsecondary staining followed by TRITC-PSA to label the acrosomal content (c). Mouse IgG replaced anti-ALDOA in the control experiment (b).
........................................................................................
Table II Sperm binding to zona-intact unfertilized
human oocytes in the presence of anti-ALDOA
antibody.
Experiment Control Anti-ALDOA
1 98.20+24.54 (5)a 44.50+21.38 (4)*
2 62.71+13.49 (7) 20.62+3.82 (13)***
3 17.71+1.78 (7) 9.50+4.04 (8)*
4 97.00+15.31 (8) 48.17+12.47 (6)**
Results are expressed as the number of spermatozoa per oocyte (mean + SEM).aThe number of oocytes is given in parentheses.
Significantly different from control oocytes (Studentsttest, *P, 0.05, **P, 0.02
and ***P, 0.0001).
........................................................................................
Table III Sperm binding onto ZP of intact unfertilized
human oocytes in the presence of anti-VDCA2
antibody.
Experiment Control Anti-VDAC2
1 30.00+6.73 (8)a 16.43+4.84 (7)
2 72.71+12.04 (7) 40.50+10.99 (8)
3 32.14+4.06 (14) 13.00+4.86 (9)*
Results are expressed as the number of spermatozoa per oocyte (mean +SEM).aThe number of oocytes is given in parentheses.
*Significantly different from control oocytes (Students ttest,P, 0.01).
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assay testing for direct interaction between sperm proteins and ZP gly-
coproteins and an indirect approach using ASAs directly eluted from
spermatozoa of infertile patients with failure of conventional IVF.
With this strategy, we increased the probability of identifying sperm
proteins actually involved in gamete interaction.
These two approaches have already been independently employed
to identify sperm proteins presumed to play a role in gamete inter-
action. Indeed, several studies have been carried out to identify
sperm antigens that react with ASAs, which are responsible for
2 15% of IVF failure. They therefore constitute an appropriate
tool for such a research. In the most recent studies using 2D gel elec-
trophoresis coupled to MALDI-TOF-MS/MS, diverse proteins such as
HSP70, Caspase3, LDHC4, VDAC2, ODF1 and GSTMu5 have
been identified as the targets of serum or seminal plasma ASAs
(Shettyet al., 1999; Bohring et al., 2001; Bohring and Krause, 2003;
Chiu et al., 2004; Paradowska et al., 2006; Domagala et al., 2007).
Although the humoral response has been described to interfere
with fertility, local ASAs would be a better tool for identifying sperm
membrane antigens actually involved in IVF failure. For this reason, we
rather used ASAs bound to and eluted from spermatozoa of infertile
men to identify relevant sperm-specific antigens. We confirmed thepreviously observed heterogeneity of the immune response of each
infertile individual in intensity and in the number and range of protein
spots that were revealed. Despite this heterogeneity, we noted that
some antigens were common to different individuals distributed over
three to four areas of proteins recognized by a majority of samples.
Only a few studies report the use of a far WB assay, a method
routinely used to identify interacting proteins (Edmondson and Roth,
2001; Machida and Mayer, 2009), with the aim of characterizing
human sperm receptors for the ZP. Using radio-labelled solubilized
ZP, ORand et al . (1985) and Shabanowitz and ORand (1988)
detected several sperm receptors at 16, 18, 19, 35 and 60 kDa
without further characterization. More recently in the porcine
species, multiple sperm plasma membrane proteins interacting with
native ZP fragments were identified, including spermadhesin
AQN-3, SED-1 (also known as P47), fertilin-beta and peroxiredoxin
(van Gestel et al., 2007). In humans, fucosyltransferase has been pro-
posed to be a sperm receptor for the intact and solubilized ZP ( Chiu
et al., 2007).
In the present study, we adapted the far WB technique to optimize
the protein protein or protein carbohydrate interaction between
sperm proteins and human rhZP. Protein conformation is known to
play a role in protein protein interaction. Since sperm proteins
were denatured for electrophoresis separation, we used renaturing
transfer conditions, which allow the proteins to refold partially into
their native structure.
Under such conditions, we observed different target proteins dis-
tributed between 18 and 100 kDa with a pI ranging from 5 to 9 de-
pending on the recombinant ZP used. This relatively large pattern in
molecular weight corresponds quite well to the proteins already iden-
tified as ZP binding proteins in humans by ORand et al. (1985) andShabanowitz and ORand (1988). Several spots of the same protein
were also revealed, undoubtedly corresponding to post-translational
modifications (PTMs), such as phosphorylation and/or glycosylation.
These two PTMs have been described to play an essential role in dif-
ferent steps of fertilization (i.e. capacitation, binding) via modification
of protein function and/or of the antigenicity of spermatozoa .
From this point of view, it is noticeable that in our far WB assays we
used rhZP(s) expressed in CHO cells in place of native solubilized ZP.
These recombinant proteins expressed in the CHO cell undergo
PTMs that can be different from the native protein produced in
human oocytes. This may affect the protein function since the sugar
moieties of ZP glycoproteins play a critical role in the gamete inter-
action. However, it has been reported that CHO-expressed rhZPCcan bind human spermatozoa and induce the acrosome reaction
(Bray et al., 2002; Marin-Briggiler et al., 2008). So we believe that
certain sugars (if not the totality) of these CHO-expressed rhZP are
similar to those of the native ZP. Other rhZP(s) able to interact
with spermatozoa have been produced in Escherichia coliand baculo-
virus expression systems (Chakravartyet al., 2005). It remains to be
seen up to which point the results of the studies can be influenced
by the system of expression of proteins.
Five of the sperm proteins identified by our double approach belong
to the category of glycolytic enzymes involved in energy production
. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . ..
.............................................................................................................................................................................................
Table V Induction of acrosome reaction by ZP in the presence of anti-VDCA2 antibody.
Experiment 3 h 18 h
Control Anti-VDAC2 Control Anti-VDAC2
1 78.00 53.00* 91.00 86.00
2 71.50 72.80 70.22 62.60
3 73.47 67.33 58.67 62.04
Results are expressed as percentage of acrosome-reacted spermatozoa.
*Significantly different from control oocytes (x2 test,P, 0.02).
........................................................................................
Table IV Sperm penetration of ZP of intact
unfertilized human oocytes in the presence ofanti-VDCA2 antibody.
Experiment Control Anti-VDAC2
1 10.30+2.47 (10)a 5.33+1.84 (9)
2 35.18+10.03 (11) 27.18+5.13 (11)
3 6.17+2.19 (12) 1.79+0.87 (14)*
Results are expressed as the number of spermatozoa per oocyte (mean +SEM).aThe number of oocytes is given in parentheses.
*Significantly different from control oocytes (Students ttest,P, 0.01).
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(PK3, GAPDH, ENO1, ALDOA, TPI). The VDAC2 can also be
included in this group. Indeed, VDAC2 transports adenine nucleo-
tides, Ca2+ and other metabolites at the level of the outer mitochon-
drial membrane and therefore participates in energy production.
Other proteins identified in the present study include proteins
involved in detoxification processes (GSTM3 and PHGPx) and a struc-
tural protein belonging to the flagellar cytoskeleton (ODF2). At first
sight, these results may seem surprising, as these proteins are primarily
described to be located in the sperm tail. However, the literature
reports, and our immunofluorescence experiments revealed that
they are also localized on the sperm head (on the plasma membrane
or acrosomal membrane, insofar as soft permeabilization is sometimes
necessary to visualize them). Additionally, co-precipitation experi-
ments confirmed that some of them actually interact with ZP proteins.
This is the case for GSTM with rhZP4, VDAC2 with rhZP2 and rhZP3,
and TPI with rhZP3 and rhZP4. We thus demonstrate here that pro-
teins with several functions according to their localization exist in the
spermatozoa. The phenomenon by which a protein can perform more
than one function has already been described and the protein is then
termed moonlighting (Sirover, 1999; Kim and Dang, 2005; Sriram
et al., 2005). Moreover, it can be noted that several of these proteinshave germ cell-specific isoforms, differing from isoforms expressed in
somatic cells (GAPDH, ENO1, ALDOA, TPI, etc.) (Edwards and
Grootegoed, 1983;Welchet al., 1992;Russell and Kim, 1996;Vemu-
ganti et al., 2007). This could also explain why the function of these
proteins may be different in the gametes compared with somatic cells.
Glycolytic enzymes identified as ZP binding
proteins
With regard to the group of glycolytic enzymes identified in this work,
it is worth noting that the majority have a double distribution, mito-
chondrial and extra-mitochondrial, in sperm cells. Recent studies
reported the presence of PK3, GAPDH or TPI in the acrosome, inaddition to the expected flagellar localization. In the present work,
we found ALDOA in the flagellum and in the head of human sperm-
atozoa, precisely at the equatorial band level of intact cells. The fact
that the detection of ALDOA required permeabilization by saponin
suggests that ALDOA is located at the internal side of the plasma
membrane or at the acrosomal membrane. The observation of
ALDOA in both the head and the flagellum of human spermatozoa
is an original result. The loss of the ALDOA staining in the sperm
head and at the midpiece following ethanol treatment despite its pres-
ervation in the principal piece seems to indicate that ALDOA is not
similarly organized in the head and in the principal piece where it
is anchored to the fibrous sheath.
Our group has already shown that TPI which is targeted by sperm
autoantibodies is localized at the acrosomal level in human spermato-
zoa (Aueret al., 2004). Two other enzymes of the glycolytic pathway,
hexokinase 1 and 6-phosphofructokinase (PFK), are also present in the
acrosomal area of boar sperm (Kamp et al., 2007), increasing to six
the number of glycolytic enzymes localized in the sperm head.
Up to now, the function of these glycolytic enzymes in the human
sperm head had not been demonstrated. Feiden et al . (2008)
described GAPDH on the acrosome of boar spermatozoa and con-
cluded that GAPDH is implicated in local ATP supply which drives
ion pumps involved in the initiation of the acrosome reaction. But it
remains to be addressed whether these glycolytic enzymes truly
exert their catalytic activity when localized in the sperm head.
Almost of all these glycolytic proteins are described as multifunctional
with non-enzymatic moonlighting properties (Kim and Dang, 2005;
Sriramet al., 2005) and may therefore have a different role according
to their localization. Here, we propose a new role for these sperm
glycolytic enzymes: they would act as potential sites of recognition
and/or binding for ZP glycoproteins when located in the sperm
head. We showed that ALDOA interacts with a ZP glycoprotein
(ZP4) directly, in a far WB assay, and indirectly, in the functional ZP
binding test. Its localization on the equatorial segment and at the
basis of the head of acrosome-intact spermatozoa is also in agreement
with its interaction with ZP4, a glycoprotein involved in the first steps
of the gamete interaction, before the acrosome reaction.
It is known that sperm receptors for ZP act as lectin-like proteins
that specifically recognize sugar chains on ZP (Wassarman, 1995).
We propose that glycolytic enzymes, which have oligosaccharides as
substrates of their catalytic activity in the flagellum, bind to the sugar
moieties of ZP glycoproteins without exerting their catalytic activity,
when located on the sperm head. Recently, the concept of multi-
recognition molecules assembled in a functional complex located inlipid rafts of the sperm membrane has emerged (Nixon et al., 2009)
and these would interact with the carbohydrate segment of ZP glyco-
proteins. Multimolecular complexes involving glycolytic enzymes have
already been described (Campanella et al., 2005). In spermatozoa,
three testis-specific isoenzymes, HK1-S, muscle-type PFK and
GSTM5 form a molecular complex associated with the mouse
fibrous sheath (Nakamura et al., 2010). The existence of similar com-
plexes with glycolytic enzymes remains to be demonstrated in the
human sperm head.
Detoxification enzyme GSTM and ZP
binding
We have identified GSTM as a ligand for human ZP4 and ZP3. This
result confirms the receptor activity of GSTM, which has already
been reported in goat spermatozoa (Gopalakrishnanet al., 1998). In
the present study, GSTM was found at the surface of the human
sperm head in a region overlying the acrosome of intact spermatozoa
and was not maintained after the acrosome reaction. This suggests
that the GSTM-ZP4/ZP3 interaction occurs in the first steps of
gamete recognition. In the goat, GST was characterized as a protein
of the sperm surface and was shown to bind specifically to ZP
during the first phase of spermoocyte interactions (Hemachand
et al., 2002). Using the far WB method, the authors identified GST
as a ligand for a ZP3-like protein. Our study agrees and reinforces
the idea that GSTM is a sperm molecule for ZP3/ZP4 recognitionduring initial binding. This sperm-specific role of GST adds the detoxi-
fication function of GSTM which, by eliminating reactive oxygen
species via glutathione, prevents lipid membrane peroxidation, a
process highly damaging to sperm membrane integrity (Hemachand
and Shaha, 2003). Thus, in spermatozoa, GSTM would also have a
double role of cell protection and oocyte recognition.
VDAC2 as a site of ZP binding
Interestingly, we found by the two approaches (ASA WB and rhZP far
WB) that VDAC2 interacts with ZP2 and ZP3 glycoproteins. The facts
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that the presence of anti-VDAC2 antibodies decreases its ability to
bind to ZP and that VDAC2 protein is localized on the sperm head
corroborate the role of VDAC2 as a sperm ZP ligand. More precisely,
VDAC2 was found on the acrosome of fixed human sperm, in addition
to its localization on the flagellum, as reported in bovine spermatozoa
(Hinschet al., 2001,2004;Triphanet al., 2008). On live permeabilized
sperm cells, we observed VDAC2 staining at the base of the head
(calyx), similarly to that found by Liu et al. (2009). As VDAC2 was
only detected after a permeabilization treatment by saponin during
the immunostaining procedure, we deduced that the epitopes recog-
nized by commercial antibody were on the internal side of the plasma
membrane or on the outer acrosomal membrane. Thus, in our condi-
tions, we could not differentiate between the plasma membrane and
the outer acrosomal membrane for the localization of VDAC2, as
Triphanet al. (2008) found in their study.
According to the literature, VDAC2 is a voltage-dependent, and not
an ion-specific, channel commonly located on the outer mitochondrial
membrane (topical review: Shoshan-Barmatz and Israelson, 2005).
However, it also exhibits other extra-mitochondrial localizations as in
the plasma membrane of lymphocytes (De Pinto et al., 2010) or the
endoplasmic/sarcoplasmic reticulum membrane of skeletal muscle(Shoshan-Barmatz and Israelson, 2005). Recently, it has been reported
that VDAC2 could play a role in the rapid transfer of the calcium
released from the endoplasmic reticulum through a ryanodine receptor
(RyR) and IP3 receptor (IP3R) to the outer mitochondrial membrane
(Csordas and Hajnoczky, 2009). The presence of the same receptors
RyR and IP3R in the acrosomal membrane suggests that VDAC2
could be localized at the level of this membrane in sperm cells where
it could be involved in ATP and calcium transport between the acro-
some and the cytoplasm (Triphanet al., 2008). A scaffolding function
of VDAC2 has been evoked byDe Pintoet al. (2010) suggesting that
VDAC2 has the potential to assemble with other proteins to form a
complex. Indeed, the association of VDAC2 with cytosolic enzymes in-
cluding HK-1, ALDOA and GAPDH has been reported in the skeletalmuscle (Shoshan-Barmatz and Israelson, 2005) and, in this complex,
HK1 could modulate VDAC channel activity (Azoulay-Zohar et al.,
2004). With this in view, we can hypothesize, in the context of the
sperm head, that the glycolytic enzymes localized on the acrosome
could contribute to VDAC biological function. Whether the interaction
ZP-VDAC2, reported in this study, is able to modulate the permeability
of VDAC2 and, as a consequence,regulate thecalcium signal remains to
be demonstrated.
Conclusion
Our work led to the identification of human sperm proteins that arerecognized both by ASA eluted from the spermatozoa of patients who
have had IVF failures and by rhZP glycoproteins. This double approach
allows us to reliably assign to these proteins a role in gamete inter-
action and specifically in binding/recognition of the ZP. These proteins
were already known for exerting functions at the flagellar level such as
glycolysis (ALDOA, TPI, ENO1) or ion transport (VDAC2). Here, we
propose an additional function for these proteins (zona binding) when
they are located in the sperm head. Their multiple roles depending on
their sub-cellular localization suggest that these sperm proteins are
multifaceted or moonlighting proteins.
Acknowledgements
The authors thank Drs Marta De Almeida and Luc Camoin for their
advice and scientific expertise and Drs Julie Coquet and Sandrine
Barbaux for their critical reading of the manuscript. The authors
also thank Prof. Pierre Jouannet (Service de Biologie de la Reproduc-
tion, Hopital Cochin, Paris) for his kind collaboration notably in pro-
viding human gametes of infertile patients and donors.
Authors roles
F.M.P., C.S. and J.A. contributed to the study design, execution, ana-
lysis of results and drafting of the manuscript. F.B. and C.P. contributed
to the proteome analysis and a critical reading of the manuscript.
Funding
This work was supported by the grant ReproRio (CNRS, INRA,
INSERM and CEA) and the Societe dAndrologie de Langue Francaise.
Conflict of interest
None declared.
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