protein profile of capacitated versus ejaculated human sperm

Protein Profile of Capacitated versus Ejaculated Human Sperm

Federica Secciani,†,‡ Laura Bianchi,†,§ Leonardo Ermini,‡ Riccardo Cianti,§ Alessandro Armini,§

Giovan Battista La Sala,| Riccardo Focarelli,‡ Luca Bini,*,§ and Floriana Rosati‡

Laboratory of Cell Biology, Department of Evolutionary Biology, Siena University, Siena, Italy, FunctionalProteomics Group, Department of Molecular Biology, Siena University, Siena, Italy, and Unit of Human

Reproduction, Arcispedale S. Maria Nuova, Reggio Emilia, Italy

Received January 13, 2009

Freshly ejaculated sperm acquire the fertilizing potential by a continuing process that occurs duringsperm transport through the female genital tract, and it is physiologically not complete until thespermatozoon reaches the oocyte. The process termed capacitation can be mimicked in vitro by usingappropriate capacitation media. Despite its importance, the molecular mechanisms underlyingcapacitation are poorly understood. This work deals with a proteomic approach to the analysis of proteinprofile variations in human normospermic samples as a consequence of three hours in vitro capacitation.2DE gels were produced per freshly ejaculated sperm and per capacitated sperm and several quantitativeand qualitative significant variations were found. Among the MS obtained identifications, proteins witha significant decrease after capacitation were found to be involved in protein fate, metabolism, andflagellar organization; on the contrary, increasing proteins were found to be related to cellular stress.Interestingly, the detected flagellar organization proteins decreased during capacitation whereas theircorresponding fragments increased. A swim-up selected and three-hour capacitated sperm subpopu-lation has also been resolved by 2DE, and its synthetic gel has been analyzed for the variations observedin the entire sperm population. An immunofluorescence analysis of this sperm typology was carriedout with antiactin and antitubulin antibodies.

Keywords: 2DE • capacitation • human sperm • mass spectrometry

IntroductionSperm represent a unique, differentiated, single-cell popula-

tion destined to travel to, to reach, and to fertilize an oocyte.In mammals, differentiation occurs continuously in the testisthroughout adult life,1 but testicular sperm are not functionalfor fertilization. They progressively acquire this ability byresponding to the changing environment while traveling in themale epididymal ducts and successively in the female genitaltracts. Intracellular and extracellular changes that take placein the epididymis are known as maturation. The intracellularmodifications mainly involve the motile apparatus since it isin this region that sperm acquire the forward motility. Theextracellular changes include loss or modification (i.e., alteredglycosylation) of pre-existing proteins, insertion or adsorptionof epididymally expressed proteins, and changes in lipidcomposition. After ejaculation, mammalian spermatozoa residein the female genital tracts for several hours, first as spermreservoir2 and later on for undergoing all the modificationstermed capacitation. Sperm capacitation includes a cascade ofbiochemical modifications, including PKA-dependent protein

phosphorylation, cholesterol efflux, increase in Ca2+ and, again,changes in motility.3 Since differentiation in the testis includescondensation of their DNA, it was largely accepted thatspermatozoa were translationally silent. By incubating spermin capacitation media containing 35S-methionine and 35S-cystein, it has been recently demonstrated that during spermresidence in the female reproductive tracts nuclear genes areindeed expressed as proteins by mitochondrial-type ribo-somes.4 This is in agreement with previous indications thatmature sperm contain nuclear encoded mRNA5 and are ableto synthesize mitochondrial proteins.6 Spermatozoa then relyon (i) post-translational modifications of pre-existing proteins,(ii) insertion of proteins expressed by the epididymal cells, and(iii) expression of new proteins to regulate cellular processesin view of acquiring their final function. Although work frommany laboratories have successfully analyzed, with differentapproaches, single protein modifications occurring duringmaturation and capacitation, most of the proteins involved inthese processes remain to be characterized.7

As recently reported, proteomics has the potential to trans-form our understanding of the human spermatozoon as aworking machine8-11 and also to acquire new biomarkers ofmale infertility.12 In this regard, here we seek to investigatesperm capacitation by 2DE and mass spectrometry.

We compared the 2D electrophoretic profile of freshlyejaculated normospermic samples with that of normospermic

* To whom correspondence should be addressed. Tel.: +39 0577 234938.Fax: +39-0577-234903. E-mail: [email protected]. Address: Via Fiorentina, 1 -53100 Siena, Italy.

† These authors equally contributed to the work.‡ Department of Evolutionary Biology, Siena University.§ Department of Molecular Biology, Siena University.| Arcispedale S. Maria Nuova.

10.1021/pr900031r CCC: $40.75 2009 American Chemical Society Journal of Proteome Research 2009, 8, 3377–3389 3377Published on Web 05/04/2009

ones incubated for 3 h in capacitating media. According to thesoftware image analysis, quantitative and qualitative differenceshave been found between the two tested sperm conditions. AsMS protein identification highlighted, proteins that show adecrease after capacitation have functions related to proteinfate, metabolism and flagellar organization. On the other hand,increased proteins were found to be related to cellular stress.In particular, the higher Mr isoforms of flagellar organizationrelated proteins were detected to decrease whereas theircorresponding fragments increased.

Moreover, to evaluate if the variations visualized to occurin a total sperm population after in vitro capacitation were alsopresent in a normospermic sample subpopulation selected forhigh motility, we obtained the 2D protein pattern of a 3 hcapacitated swim-up selected subpopulation. Then we com-pared it to the previously produced 2D electropherograms offreshly ejaculated and 3 h capacitated total sperm population.

A double immunofluorescence analysis with antiactin andantitubulin antibodies was also performed on the swim-up-capacitated sperm.

Materials and Methods

Sperm Preparation and Treatments. Human semen sampleswere collected from 120 patients (mean age 35 years) attendingthe Fertility Unit of the Department of Obstetrics and Gynecol-ogy of the Arcispedale Santa Maria Nuova, Reggio Emilia, Italy.Individual informed consents and Institutional ethical approvalwere secured for the use of human semen samples for thepurposes of this research. All samples used were scored asnormal according to the World Health Organization (WHO)criteria13 with morphology assessed according to Kruger’scriteria.14 Semen parameters are reported in detail in Table 1.

After liquefaction, spermatozoa were washed twice in SidneyIVF sperm buffer (Cook, Sydney, Australia), seminal plasma wasremoved by centrifugation at 500× g for 10 min, and sperma-tozoa were counted. A part of them, considered as freshlyejaculated sample, was immediately kept at -20 °C. Anotherpart was instead incubated in Sidney IVF sperm medium (Cook)for 3 h at 37 °C in 6% CO2 and then kept at -20 °C ascapacitated sample. Successively a high motile sperm sub-population was selected by the swim up procedure, capaci-tated, and separately analyzed. Briefly, sperm were washed inSidney IVF sperm buffer (Cook). Then Sidney IVF spermmedium (Cook) was layered over aliquots of washed sperma-tozoa and, after 30 min at room temperature, the mobilespermatozoa, recovered in the upper layer, were incubated for2 h and 30 min at 37 °C in 6% CO2 in the Sidney IVF spermmedium.

Two-Dimensional Gel Electrophoresis. All sperm sampleswere washed three times in phosphate-buffered saline (PBS:150 mM NaCl, 50 mM KH2PO4, pH 7.4) and then pooledtogether. Two sample pools were obtained per sperm-testedcondition, and three 2D gels were produced for each samplepool. Each freshly ejaculated, capacitated, and swim-up se-lected sample pool was obtained from about 30 donors.Resulting samples were solubilized in a conventional 2D lysisbuffer, consisting of 8 M Urea, 4% w/v CHAPS, and 1% w/vDTE, and protein quantification was determined according tothe Bradford method.15

2DE was performed using Immobiline polyacrylamide sys-tem as previously described.16,17 The IEF was carried out onpreformed immobilized nonlinear pH gradient, from pH 3 to10, of 18 cm length (GE Healthcare, Uppsala, Sweden) andachieved using an Ettan IPGphor IEF system (GE Healthcare).Analytical-run IPG strips were rehydrated, at 16 °C, with 60 µgof protein in 350 µL of lysis buffer and 0.2% v/v carrierampholyte, for 1 h at 0 V and for 8 h at 30 V. The strips werethen focused according to the following electrical conditions,at 16 °C: 200 V for 1 h, from 300 to 3500 V in 30 min, 3500 Vfor 3 h, from 3500 to 8000 V in 30 min, and 8000 V until a totalof 80 000 Vh was reached.

MS-preparative IPG strips were instead rehydrated with 350µL of lysis buffer and 2% v/v carrier ampholite for 12 h at roomtemperature. Sample load, 800 µg per strip, was successivelyperformed by cup loading in the IPGphor Cup Loading StripHolders (GE Healthcare). To obtain a successful focusing onbasic pH values, sample was applied at the cathodic end ofthe strip. IEF was then achieved according to the followingvoltage steps, at 16 °C: 30 V for 30 min, 200 V for 2 h, 500 V for2 h, from 500 to 3500 V in 30 min, 3500 V for 5 h, from 3500 to5000 V in 4 h, from 5000 to 8000 V in 30 min, 8000 V until atotal of 95000 Vh was reached.

After focusing, analytical and preparative gel strip equilibra-tion occurred in 6 M urea, 2% w/v SDS, 2% w/v DTE, 30% v/vglycerol, and 0.05 M Tris-HCl pH 6.8 for 12 min; and for afurther 5 min in 6 M urea, 2% w/v SDS, 2.5% w/v iodoaceta-mide, 30% v/v glycerol, 0.05 M Tris-HCl pH 6.8 and a trace ofbromophenol blue. The gel strips were then placed on 18 cm× 20 cm × 1.5 mm 9-16% polyacrilamide linear gradient gelsfor the protein separation in the second dimension. The SDS-PAGE was carried out at 40 mA per gel constant current, at 10°C, until the dye front reached the bottom of the gel.

Analytical gels were stained with ammoniacal silver nitrateas previously described,18 while the MS-preparative gels werestained according to a silver staining protocol compatible withMS.19 Stained gels were digitalized using a Molecular Dynamics300S laser densitometer (4000 × 5000 pixels, 12 bits per pixel;Molecular Dynamics, Sunnyale, CA), and image analysis wascarried out with MELANIE 4 computer system (GeneBio,Geneva, Switzerland).

Statistical analyses of protein variations were performed with1-way ANOVA, at 95% level of significance (p < 0.05), andTukey’s posthoc multiple comparison test using the ExelTemplate inerSTAT-a 2.0.

The reported pI and Mr (Da) values were experimentallydetermined by comigration with human serum as an internalstandard.20,21

Protein Identification by Mass Spectrometry. Protein iden-tification was carried out by PMF on an Ettan MALDI-TOFPro mass spectrometer (GE Healthcare) as previouslydescribed.22,23 Electrophoretic spots, visualized by MS-

Table 1. Semen Analysis: All Samples Were Scored AsNormal According to the World Health Organization (WHO)Criteria with Morphology Assessed According to Kruger’sCriteria

WHO parameters specimen parameters

volume >2.0 mL 3.23 ( 1.34 mLconcentration >20 × 106/mL 72.06 ( 25.44 × 106/mLmotility >50% 52.17 ( 14.11%morphologya >5% with normal

morphology8.47 ( 3.12%

WBCb <1 × 106/mL <1 × 106/mL

a According to Kruger’s criteria. b White blood cells.

research articles Secciani et al.

3378 Journal of Proteome Research • Vol. 8, No. 7, 2009

compatible silver staining protocol, were manually excised,destained and acetonitrile (ACN) dehydrated. Successively,they were rehydrated in trypsin solution, and in-gel proteindigestion was performed by an overnight incubation at 37°C. From each excised spot, 0.75 µL of recovered digestedpeptides were prepared for MALDI-TOF MS by spotting themonto the MALDI target, allowed to dry and then mixed to0.75 µL of matrix solution (saturated solution of R-cyano-4-hydroxycinnamic acid in 50% v/v ACN and 0.5% v/vtrifluoroacetic acid). After the application of the matrix tothe dried sample and its own drying, tryptic peptide masses

were acquired. PMF searching was carried out in the Swiss-Prot/TrEMBL database using MASCOT (Matrix Science Ltd.,London, UK, http://www.matrixscience.com) online availablesoftware. The taxonomy was limited to Homo sapiens, a masstolerance of 100 ppm was allowed, and the number ofaccepted missed cleavage sites was set to one. Alkylation ofcysteine by carbamidomethylation was assumed as a fixedmodification, while oxidation of methionine was consideredas a possible modification. The experimental mass valueswere monoisotopic. No restrictions on protein molecularweight and pI values were applied. The criteria used to

Figure 1. Silver stained electropherograms of freshly ejaculated (A), capacitated (B), and swim-up selected capacitated (C) humansperm. The quantitative differences are indicated by a letter/number (An or Bn). Numbers from A1 to A40 visualize spots more abundantin freshly ejaculated sperm and from B1 to B38 those more abundant in capacitated sperm. Qualitative differences are marked by squaresand indicated by a letter/greek letter: AR for that only present in ejaculated sperm and from BR to Bδ for those only present in capacitatedsperm.

Protein Profile of Capacitated versus Ejaculated Human Sperm research articles

Journal of Proteome Research • Vol. 8, No. 7, 2009 3379

accept identifications included the extent of sequence cover-age, number of matched peptides, and probabilistic scoresorted by the software, as detailed in Tables 2 and 3.

Tryptic digests that did not produce MALDI-TOF unambigu-ous identifications were subsequently acidified with 2 µL of 1%TFA solution and then subjected to ESI-IT MS/MS peptide-sequencing on a LCQ DECA IonTrap mass spectrometer(Thermo Fisher, San Jose, CA). Using the Zip-Tip pipette tipsfor sample preparation (Millipore, Billerica, MA), previouslyequilibrated in 50% ACN solution and abundantly washed in0.5% TFA, acidified samples were enriched. Tryptic digestionelution from the Zip-Tip matrix was achieved with a 70%methanol and 0.5% formic solution, and 3 µL of such concen-trated sample solutions were then loaded in the nanosprayneedle. The collision energy was set based on the mass of theprecursor ions, that are doubly charged, and spectra wereacquired using Excalibur software (Thermo Fisher). MS/MSdatabase searching was performed by TurboSEQUEST (ThermoFisher) and Mascot MS/MS ion search software (http://www-.matrixscience.com) in the Swiss-Prot/TrEMBL or NCBInrdatabases. The following criteria were applied: MS massaccuracy ( 1.2 Da, MS/MS mass accuracy ( 0.6 Da, peptideprecursor charge 2+, monoisotopic experimental mass values,trypsin digestion with one allowed missed cleavage, fixedcarbamidomethylation of cysteine, and variable oxidation ofmethionine.

Fluorescence Microscopy. Aliquots of ejaculated and swim-up selected capacitated sperm, as previously described (2 ×106) from different ejaculates, were smeared onto ethanol-cleaned glass slides and allowed to attach at room temperature.Specimens were fixed with methanol and permeabilized withacetone. After extensive washings in PBS, the smears wereblocked for 30 min with 2% BSA in PBS and then wereincubated 1 h at room temperature with the first primaryantibody, the antibeta tubulin Mab (1:100; Sigma-Aldrich, SaintLouis, MO), and followed by Alexa Fluor 488 conjugatedantimouse IgG (1:200; Invitrogen, Carlsbad, CA) for 1 h at roomtemperature.

The second primary antibody, the antiactin PAb (1:100;Sigma-Aldrich), was applied for 1 h at room temperature andfollowed for 1 h at the same conditions by the appropriate AlexaFluor 555-conjugated goat antirabbit antibody (1:200; Invitro-gen, Carlsbad, CA). Slides were then washed, mounted in 20mmol/L Tris-HCl pH 8.0, 80% v/v glycerol, and 4% w/vN-propylgallate as antifade and observed with a Leitz Diaplanfluorescence microscope (Leica, Heidelberg, Germany).

Results

Analysis of 2DE Images of Ejaculated and CapacitatedNormospermic Samples. To investigate the modifications ofthe human sperm protein profile related to the capacitationprocess, ejaculated and 3 h capacitated human normospermicsamples, from 120 healthy donors, were resolved using 2DE.For this analysis we did not use any selective separationprocedure for spermatozoa, such as Percoll, so the resulting2D gels would be representative of the average spermatozoonpresent in the normospermic sperm sample before and aftercapacitation.

To validate the resulting data, technical and biologicalreplicates were produced.23 Two sample pools were preparedper sperm condition, with equal contribution from each sampleforming the pool, and three 2D gels were obtained per eachsample pool. Six gels were thus produced per freshly ejaculated

sperm and six gels per capacitated sperm, and then a syntheticgel was elaborated by MELANIE 4 software for each spermtypology.

Synthetic gels were qualitatively and quantitatively analyzedand, to avoid the overestimation of qualitative variations, adetailed gel by gel matching was performed. Actually, eachsynthetic image represents all spots constantly present in allgels examined from the same sperm typology. Typical unca-pacitated and capacitated human sperm electropherograms areshown in Figure 1A and B. According to the silver stainingsensitivity and to the 18 cm, 3-10 nonlinear IPG strip resolutionlimit, 78 quantitative and 5 qualitative variations were foundbetween the two sperm samples. We considered significant allthe quantitative differences with a variation of at least two timesin spot relative volume, %V (Vol ) integration of OD over thespot area; % Vol ) Vol of single spot divided by the total spotVol), and with a statistical probability less than 0.05 (p < 0.05),as reported in Materials and Methods. The quantitative varia-tions, numbered from A1 to A40 and from B1 to B38, have beenpointed out in Figure 1A and B. Among them 40 (from A1 toA40) corresponded to more abundant spots in ejaculated thanin capacitated sperm, and 38 (from B1 to B38) corresponded tothose more abundant in capacitated versus ejaculated sperm.Among the qualitative variations, one spot was exclusivelydetected in ejaculated sperm (AR) and four spots exclusively incapacitated sperm (from BR to Bδ).

The general trend emerging from the visualized quantitativedifferences (Figure 1A and B) was that capacitation caused a%V value decrease of peptides present at the higher Mr gel-area and the counterbalancing increase of those at the lowerMr area.

MS Protein Identification. Twenty-five protein spots of themore abundant proteins in ejaculated sperm and 23 of thosemore abundant in capacitated sperm were successfully identi-fied by MALDI-TOF MS and ESI-IT MS/MS. Sixty-eight furtherprotein spots were identified whose %V variation betweencapacitated and uncapacitated samples did not fit with theabove-described parameters. Proteins with significant %V valuevariation are listed in Table 2, and proteins with no significantvariation, used to build a reference human sperm map, areshown in Table 3.

The identified proteins were classified according to theirbiological activities in five functional classes. In Table 2 proteinsare specified by letter/number (An or Bn), as used to point outthe corresponding spots in Figure 1A and B, and by anabbreviation of their protein name, as also done for proteinslisted in Table 3, obtained by SwissProt/TrEMBL database.Tables 2 and 3 abbreviations have been used to indicate theprotein spot identity in the reference sperm map (Figure 2).

According to the sequence coverage and to the discrepancyexisting between theoretical and experimental pI and Mr values,some protein spots are supposed to be protein fragments, asindicated below the experimental pI/Mr values in Tables 2 and 3.

Table 2 also reports mean and SD of %V values fromejaculated, capacitated and swim-up selected and capacitatedsperm gels, in order to visualize the different protein profile ofsperm before and after the capacitation process. Swim-up-subpopulation spots that significantly differ from ejaculated orcapacitated sperm identified proteins are pointed out in Table2 by footnotes according to their statistical significance (f p <0.05, g p < 0.01).

Protein spots that result to mainly decrease during in vitrocapacitation were those related to the protein fate, metabolism



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and flagellar organization, whereas spots increasing were thoserelated to cellular stress. Interestingly, the %V decrease ofproteins related to flagellar organization in capacitated spermhas been shown to be counterbalanced by an increase ofprotein fragments from the same proteins.

Swim-Up Selected and Capacitated Sperm Subpopulation.Since the obtained differences were representative of theaverage spermatozoon present in the normospermic samples,we also evaluated if the same variations characterized a spermsubpopulation selected for high motility, that is generally usedfor artificial fertilization. Six 2DE gels, three per sperm pool,of a swim-up selected and 3 h capacitated sperm subpopulationwere produced. The corresponding synthetic gel was elaboratedby MELANIE 4 software according to criteria used to obtainthe other two synthetic gels. A typical electropherogram ofswim-up selected and capacitated human sperm is shown inFigure 1C. In the swim-up subpopulation, 55% of spotscorresponding to quantitative differences more abundant inejaculated than in capacitated sperm show %V values closerto those of ejaculated than to those of capacitated sperm. Suchspots are pointed out in Figure 1C by letter/number, An, as inFigure 1A and B. On the other hand, the 32% of swim-up spotscorresponding to quantitative differences more abundant incapacitated than in ejaculated sperm has %V values closer tothose of capacitated than to those of ejaculated sperm. InFigure 1C, these spots are visualized by letter/number, Bn, as inFigure 1A and B.

A peculiar picture emerges by comparing the three spermtypology. The swim-up selected capacitated sperm subpopu-lation differs from the entire capacitated population for nothaving a significant quantitative decrease in proteins relatedto protein fate and metabolism, and for having about half ofthe %V value decrease of proteins related to flagellar organiza-tion and motility, and half of the %V value increase of thecorresponding fragmented products. Figure 3 (A, B) shows byhistograms the %V variations occurring among the quantitativedifferences detected between ejaculated and capacitated totalsperm population, and the corresponding spots detected in theswim-up subpopulation.

Immunofluorescence Experiments. Since variations mainlyconcerned flagellar proteins, we also performed an immunofl-uorescence analysis with antitubulin and antiactin antibodieson freshly ejaculated and swim-up capacitated sperm. Resultswere in line with those obtained by the proteomic approach.As shown in Figure 4, a very bright signal for tubulin character-ized the stiff flagellum of the great part of freshly ejaculatedsperm (Figure 4A) whereas the flagella of at least 30% of swim-up selected capacitated sperm appeared undulated and morefaintly fluorescent (Figure 4B). A bright signal for actin wasdetected in the head and along the first part of the tail ofejaculated sperm with the bright signal for tubulin (Figure 4A1),whereas in swim-up selected capacitated sperm this signalremained in the acrosomal portion of the sperm head with thedecreased signal for tubulin, and almost completely disap-peared from their tails (Figure 4B1).

Discussion

Freshly ejaculated sperm are unable to fertilize an oocyte.They acquire the fertilizing potential by a continuous processthat occurs during sperm transport throughout the femalegenital tract, and it is physiologically not complete until thespermatozoon reaches the oocyte. The process termed “ca-pacitation” can be mimicked in vitro by using appropriateT

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capacitation media. However, since mammalian sperm areheterogeneous and among a sperm population only a very fewwill become fertilizing sperm, changes of in vitro capacitatedsperm generally reflect the average population modificationsirrespective of those directly related to fertility.

We applied a proteomic approach to examine whether andhow the global protein profile of human sperm changes as aconsequence of in vitro capacitation. The present studydemonstrated that about 4% of the detected silver-stained spotsfrom freshly ejaculated normospermic samples quantitativelychanged after 3 h of incubation in capacitation media. Interest-ingly, the protein spots quantitatively decreasing in capacitatedversus ejaculated sperm were localized in the upper part ofthe sperm electropherograms whereas those increasing werelocalized in the lower part of the electropherograms. Thispeculiar feature was in part explained by the detection of a setof proteins observed as fragments in capacitated sperm andas full length proteins in ejaculated sperm.

Three of the identified fragmented proteins are involved inflagellar organization: tubulin beta-2C chain (TBB2C: B11), theouter dense fiber protein 1 (ODFP1: B34), and the A-kinaseanchor protein 4 (AKAP4: B23); other two protein fragmentswere identified as metabolic enzymes: the ATP synthasesubunit alpha (ATPA: B14) and the L-asparaginase (ASGL1: B32);and, finally, further four spots are related to protein fate: theheat shock-related 70 kDa protein 2 (HSP72: A35,36, B12,16).

TBB2C, which is specifically expressed in the testis, is oneof the major constituents of the flagellum microtubular struc-ture. A large TBB2C N-terminal fragment, B11, was found to beprevalent in capacitated sperm. In this context, it could be ofinterest that tubulin beta-2C chain may bind cations such ascalcium at its highly acidic C-terminal region.25 The ODFP1 is

one of the main components of the outer dense fiber (ODF) ofthe sperm tail whose transcription is restricted to testis tissueand more specifically to round spermatids.26 ODF are filamen-tous structures located on the outside of the axoneme in themidpiece and principal piece of the mammalian sperm tail andare described to help maintain the passive elastic structuresand the elastic recoil of the sperm tail. For this protein, aC-fragment, B34, was found to be more abundant in thecapacitated sperm map. Finally, AKAP4 is the most abundantfibrous sheath protein27 and a major structural component.28

Its synthesis and incorporation into the nascent fibrous sheathoccurs late in spermatid development.29 According to theclassical view, the function of the fibrous sheath is to modulatethe plane of the flagellar beat by imposing a restraint to slidingof axonemal doublets and to flagellar bending. However, areasonable scaffold function may also be assigned to AKAP4for components involved in signal transduction. In particular,it has been suggested to anchor, in a restricted region, cAMP-dependent protein kinases involved in this process.30 Thefinding that AKAP4 knockout mice completely lack spermforward motility28 clearly evidenced the key role of AKAP4 insperm motility. A small N-fragment, B23, of AKAP4 was detectedas more abundant in the total capacitated sperm populationthan in the freshly ejaculated one.

The amount of other cytoskeleton proteins, such as tubulinalpha-3C/D chain (TBA3C: A17-19) and actin-related protein T2(ACTT2: A32,33), also decreases in capacitated sperm, but nofragments or modified forms of these proteins were identifiedamong the detected differences occurring between capacitatedand ejaculated spermatozoa.

In vitro capacitation also resulted in a reduction of proteinsrelated to metabolism and in particular to energy production.

Figure 2. Silver stained map of capacitated human sperm. Circles show all the identified protein spots by MALDI-TOF MS and ESI-ITMS/MS. Identified quantitative variations are indicated with letters/numbers (An or Bn) as used in Figure 1A and B.



Such reduction might be, directly or indirectly, related to theflagellar disorganization. As a matter of fact, enzymes of theglycolytic pathway have been recently proposed to be orderlybound to the fibrous sheath of the sperm tail.31

Proteins that, instead, increased as a consequence of in vitrocapacitation were seminal proteins such as clusterin (CLUS:B2,4-7,9) and prolactin-inducible protein (PIP: B22,24,25,27,28). Clus-terin is a disulfide-linked heterodimeric protein present in allhuman fluid, including seminal plasma, and in the humansperm plasma membrane. It has been associated with apop-tosis, and clusterin positive sperm have been negatively cor-related with motility.32 The physiological function of PIPremains unknown, but its ability to bind IgG-Fc has suggestedthat PIP may have an immunomodulatory role.33

Overall, our results indicate that in an entire normospermicpopulation many of the capacitating sperm undergo modifica-

tions targeted to stop them. Interestingly, proteins regulatingsperm motility were described to be differently expressed inasthenozoospermic patients compared with normozoospermicdonors.34

We also analyzed if similar variations occur in a swim-upselected and capacitated sperm population. Although to lessan extent with respect to the entire capacitated sperm popula-tion, a decrease of proteins related to flagellar organization anda corresponding increase of their fragments also take place inthese spermatozoa selected for high motility. Immunofluores-cence analysis of swim-up capacitated sperm with antitubulinconfirmed that part of the capacitated versus ejaculated spermundergoes modifications of proteins such as tubulin, which isone of the most important flagellum protein, and actin, thathas been suggested as motility regulator.35 At least 30% of themshowed an antitubulin signal in the flagellum minor than that

Figure 3. Histograms highlight spot volume variations (mean ( SD) of quantitative differences occurring between ejaculated (E) andcapacitated (C) sperm and the corresponding %V × 10-4 values of the swim-up spots (Su). Quantitative differences more abundant inejaculated than in capacitated sperm, An(1-40), are in the upper level and those more abundant in capacitated than in ejaculated sperm,Bn(1-38), are in the lower level.



detected in ejaculated sperm, and an antiactin faint signalremaining only in the acrosomal region whereas stronglypresent in the head and in the first part of the tail of ejaculatedsperm, as previously reported.36 In general, the high motile-selected sperm subpopulation did not show quantitative varia-tions of clusterin and proteins related to metabolism(A8,15,16,25,28,30). It is possible that these variations are subsequentto the fragmentation process and that this process occurs insome of the selected high motile sperm later on with respectto that occurring in the average of the entire sperm population.

These results suggest that capacitation includes a processtriggering precise events of flagellar protein fragmentation thatleads many sperm to stop and it is tempting to speculate thatthis is an apoptotic-like process. Indeed cytoskeletal proteinsare described as targets of caspases37 and it is known that HIV-Tat protein induces apoptosis by targeting the microtubulenetwork.38 Furthermore, sperm fractions with low motility areknown to exhibit more active caspase-positive cells than thehigh motile ones.39 Apoptosis in human sperm is controversial.Although all apoptosis markers (active caspase, annexin Vbinding and DNA fragmentation) have been demonstrated inejaculated sperm39-41 and indicated as higher in infertilepatients than in donors as well as greater in the low versus thehigh motility fractions,39-42 statistical significance was oftenindicated as marginal42-44 and the capacity of non apoptoticselected sperm to improve fertilization is controversial.41,45 Acentral question in this subject is what the role of apoptosis isfor sperm cells. Paasch et al.46 recently suggested that sperm,being terminally differentiated, may exhibit a limited lifetime,even if slightly different, or a programmed cell death fate whenleaving testicles. Moreover, the apoptotic sperm cells could bethen eliminated by macrophages during their journey without

releasing proinflammatory signals. Since phagocytosis does notseem to interfere with fecundity47 and the process occurs inthe uterus several hours after insemination,48 it has beenproposed that its role is to selectively remove, in a silent andclean way, spermatozoa that are no longer functional.49

Concluding Remarks

The capacity of sperm to fertilize is acquired only after aperiod of residence in the female reproductive tract and, amongthe million spermatozoa from a single ejaculate, only a veryfew end up the race toward the oocyte. During travel, thegreatest part of them are arrested by mechanisms largelyunknown.

Capacitation can be accomplished in vitro by providingculture conditions similar to those occurring in vivo. Thepresent proteomic study indicates that motility apparatusalterations, probably mediated by apoptotic-like mechanisms,take place in a high percentage of in vitro capacitating sperm.This information may provide the basis for developing newparameters and criteria in the evaluation of the capacitationprocess.

Moreover, the annotated sperm map, that will be soonavailable in the next update of the SIENA-2DPAGE (http://www.bomol.unisi/2d/2d.html), may be a useful tool in thestudy of human sperm.

Acknowledgment. We thank Mrs Ellen Beranek forthe mother tongue revision of the manuscript.

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PR900031R



protein profile of capacitated versus ejaculated human sperm

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