2013 identify human sperm proteins for zp binding

8/13/2019 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.

Moonlighting sperm ZP binding proteins 3
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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

4 Petit et al.


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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).



8/14

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).

8 Petit et al.


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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).



10/14

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).

10 Petit et al.


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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



12/14

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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