Identification of Extracellular Signal-regulated Kinase 1/2 and p38 MAPK as Regulators of Human ...

13 Pages·2008·0.61 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRY VOL.283,NO.21,pp.14479–14489,May23,2008
©2008byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PrintedintheU.S.A.
Identification of Extracellular Signal-regulated Kinase 1/2 and
p38 MAPK as Regulators of Human Sperm Motility and
Acrosome Reaction and as Predictors of Poor
Spermatozoan Quality*□S
Receivedforpublication,December26,2007,andinrevisedform,March25,2008 Published,JBCPapersinPress,March27,2008,DOI10.1074/jbc.M710492200
TalAlmog‡1,ShlomiLazar‡1,NachumReiss‡,NirEtkovitz§,EyalMilch¶,NirRahamim‡,MashaDobkin-Bekman‡,
RonitRotem‡,MosheKalina†(cid:1),JacobRamon¶,AriehRaziel**,HaimBrietbart§,RonySeger‡‡,andZviNaor‡2
Fromthe‡DepartmentofBiochemistry,GeorgeS.WiseFacultyofLifeSciences,TelAvivUniversity,RamatAviv69978,§TheMina
andEverardGoodmanFacultyofLifeSciences,Bar-IlanUniversity,RamatGan52900,the¶DepartmentofUrology,TheChaim
ShebaMedicalCenteratTelHashomer,TelHashomer,Ramat-Gan52621,the(cid:1)DepartmentofCellBiologyandHistologyand
**MaleInfertilityandInVitroFertilizationUnit,AssafHarofehMedicalCenter,SacklerSchoolofMedicine,TelAvivUniversity,
RamatAviv69978,andthe‡‡DepartmentofBiologicalRegulation,TheWeizmannInstituteofScience,Rehovot76100,Israel
Maturespermatozoaacquireprogressivemotilityonlyafter undergo suppression of progressive motility, become capaci-
ejaculation.Theirjourneyinthefemalereproductivetractalso tated (capable to fertilize), including resumption of another
includes suppression of progressive motility, reactivation, form of motility, hyperactivation, bind the egg, undergo the
capacitation, and hyperactivation of motility (whiplash), the acrosome reaction, and fertilize it (1, 2). Relatively little is
mechanisms of which are obscure. MAPKs are key regulatory knownaboutthemolecularmechanismsthatmediatesperma-
enzymesincellsignaling,participatingindiversecellularfunc- tozoanfunctionsingeneralandthevariousformsofmotilityin
tionssuchasgrowth,differentiation,stress,andapoptosis.Here particular(1,2).
wereportthatERK1/2andp38MAPKareprimarilylocalizedto MAPK3 cascades play a crucial role in metazoan develop-
the tail of mature human spermatozoa. Surprisingly, c-Jun ment, including fate determination, differentiation, prolifera-
N-terminal kinase 1/2, which is thought to be ubiquitously tion, survival, migration, growth, cell cycle progression, and
expressed,couldnotbedetectedinmaturehumanspermato- apoptosis(3–7).MAPKcascadesconsistofseveraltiersofpro-
zoa. ERK1/2 stimulation is downstream to protein kinase C teinkinasesthatactivateeachotherbysequentialphosphoryl-
(PKC)activation,whichisalsopresentinthehumanspermtail ation. Four major MAPK cascades are known in mammals:
(PKC(cid:1)IandPKC(cid:2)).ERK1/2stimulatesandp38inhibitsforward ERK1–2, JNK 1–3, p38 (cid:1)–(cid:2), and ERK5 (big MAPK; BMK)
andhyperactivatedmotility,respectively.BothERK1/2andp38 (3–7).ActivationofMAPKisinitiatedbyactivationofMAP3Ks
MAPKareinvolvedintheacrosomereaction.Usingaproteomic bythesmallG-proteinsoftheRasfamily(e.g.Ras,CDC42,and
approach,weidentifiedARHGAP6,aRhoGAP,asanERKsub- Rac), followed by sequential activation of MAPKKs and
strateinPMA-stimulatedhumanspermatozoa.Inversecorrela- MAPKs. The prototypic ERK cascade is activated by growth
tionwasobtainedbetweentherelativeexpressionlevelofERK1 factors, mitogens, and G-protein-coupled receptors (GPCR)
ortherelativeactivationlevelofp38andspermmotility,for- and consists of Rafs (MAP3K), MEK1/2, ERK1/2, and several
ward progression motility, sperm morphology, and viability. MAPK-activating protein kinases (MAPKAPKs). The stress-
Therefore,increasedexpressionofERK1andactivatedp38can activated protein kinases are now known as JNK and p38
predictpoorhumanspermquality. MAPK.Bothareinitiatedbystressstimuli,GPCRs,inflamma-
tory cytokines, and growth factors. JNK1–3 consists of a
sequentialactivationofRac1/Cdc42,mixedlineagekinasesas
Spermareimmotileinthetestesandtheyacquireprogres- MAP3Ks,MAPKkinases4and7,andJNK1–3.Thep38MAPK
sive motility after ejaculation. They cross the uterine cervix, cascade is composed of sequential activation of MAP3Ks,
MKK3/4/6,p38(cid:1)–(cid:2),andseveralMAPKAPKs(3–7).
*ThisworkwassupportedbytheUnitedNationsDevelopmentProgramme/ Mature spermatozoa are fully differentiated cells that lack
UnitedNationsPopulationFund/WorldHealthOrganization/WorldBank activetranscriptionalmachinery.Hence,itisofgreatinterestto
Special Program of Research Development and Research Training in
HumanReproduction,WorldHealthOrganization,ProjectA15118(2001)
andfromtheMinistryofScienceandTechnologyGrantNOFAR(2003).The 3Theabbreviationsusedare:MAPK,mitogen-activatedproteinkinase;ERK,
costsofpublicationofthisarticleweredefrayedinpartbythepaymentof extracellularsignal-regulatedkinase;PKC,proteinkinaseC;PMA,phorbol
pagecharges.Thisarticlemustthereforebeherebymarked“advertise- 12-myristate13-acetate;OAG,1-oleoyl-2-acetylglycerol;JNK,c-JunN-ter-
ment”inaccordancewith18U.S.C.Section1734solelytoindicatethisfact. minalkinase;GPCR,G-protein-coupledreceptor;MBP,myelinbasicpro-
□S Theon-lineversionofthisarticle(availableathttp://www.jbc.org)contains tein;CASA,computer-aidedspermanalysis;SM,spermmotility;FPM,for-
supplementalvideos1and2. ward progression motility; ROC, receiver operating characteristic; AUC,
†ThispaperisdedicatedtothememoryofProf.MosheKalina,whodiedin area under the curve; BAPTA/AM, 2-bis(2-aminophenoxy)ethane-
2004. N,N,N(cid:1),N(cid:1)-tetraaceticacidtetrakis(acetoxymethylester);PBS,phosphate-
1Bothauthorscontributedequallytothiswork. bufferedsaline;FITC,fluoresceinisothiocyanate;BSA,bovineserumalbu-
2Towhomcorrespondenceshouldbeaddressed.Fax:[phone];E-mail: min;MALDI-TOF,matrix-assistedlaserdesorptionionizationtime-of-flight;
[email protected]. PMA,phorbolmyristoylacetate.
This is an open access article under the CC BY license.
MAY23,2008•VOLUME283•NUMBER21 JOURNALOFBIOLOGICALCHEMISTRY 14479
ERKandp38AreRegulatorsofSpermMotility
investigate whether ejaculated human spermatozoa express roomtemperatureusingtheavidin-biotincomplexmethodas
andutilizeMAPKs,whichspecializeingrowth,differentiation, describedpreviously(20).Visualizationwasperformedinsolu-
andstress.Becausespermatozoalackthosefunctions,thestud- tions containing diaminobenzidine (0.25 mg/ml) and 0.01%
iesmightpointtonovelfunctionsmediatedbyMAPKs.During H O inPBS.SpecificityoftheMAPKstainingwasconfirmed
2 2
spermatogenesis,MAPKsmediatecelldivision,differentiation, byomissionoftheprimaryantibodyandbythepreabsorption
survival,adhesion,anddeath(8).MAPKswerereportedtoplay oftheantibodywithanexcessoftherespectiveMAPKpeptide
a role during meiotic progression of mouse spermatocytes (Sigma).Allthereagentsfortheimmunohistochemistrystudies
(9–12)andinadhesionfunctionattheSertoli-germcellinter- wereobtainedfromVectorLaboratories(Burlingame,CA).
face(8).Ontheotherhand,theroleofMAPKinmaturemam- Immunoelectron Microscopy—Human sperm were fixed in
malian spermatozoan motility, capacitation, and acrosome 2%glutaraldehydein0.1Mcacodylatebufferfor60minatroom
reaction is limited and controversial as detailed below (10, temperature(21).Afterwashing,thetissuesamplesweredehy-
13–17).InparticulartheroleofJNKandp38MAPKinejacu- dratedinacetoneandembeddedinaraldite.Thecellswerenot
lated spermatozoa is not known (8). We therefore inquired treatedwithosmiumbecauseoflossofantigenicityinosmium-
whether ejaculated mature human spermatozoa express and treatedcells.Sectionswereplacedonsilvergrids,andimmuno-
utilize the various MAPKs (ERK, JNK, and p38) (6, 7), which goldlabelingwasperformedasdescribedpreviously(21).Sec-
mayorchestratethefine-tuningofspermmotility.Ourresults tionsweretreatedwith0.1%TritonX-100inPBSfor20min,
indicate that ERK1/2 and p38 MAPK, but not JNK1/2, are washedinPBS,andplacedin1%BSAfor1h.Afterdraining,
expressedinthetailofejaculatedhumanspermatozoa.Activa- the sections were incubated with anti-ERK antibody (1:50)
tionofERK1/2isdownstreamtoPKCactivation,andtwoofits (Sigma)for18hat4°C.Afterrinsingin0.05MTris-buffered
isoforms (PKC(cid:3)I and PKC(cid:4)) are also present in the human saline(TBS),pH7.3,thesectionswereincubatedfor1hwith
spermtail(18).Surprisingly,wediscoveredthatERKstimulates 8- or 15-nm gold-conjugated goat anti-rabbit IgG (Biocell,
and p38 MAPK inhibits forward and hyperactivated motility, Cardiff, UK) and diluted 1:10 with TBS, pH 8.4, containing
respectively.WealsofoundthatbothERK1/2andp38MAPK 1% egg albumin. Following the immunostaining, sections
are positively involved in PKC-mediated acrosome reaction. wererinsed,andcontrastwasenhancedwithuranylacetate
Using a proteomic approach, we identified ARHGAP6, a and lead citrate. Sections were then examined with a JEOL
RhoGAP,asanERKsubstrateinPMA-stimulatedhumansper- 100Belectronmicroscope.
matozoa. Finally, we found that inverse correlation exists Indirect Immunofluorescence—Sperm were washed and
betweenERK1andphospho-p38andspermmotility,forward smearedonpolylysineslidesandthenallowedtoair-dry.Cells
progressionmotility,spermmorphology,andviability.Indeed, were washed with PBS, permeabilized with Triton X-100,
increasedexpressionoftotalERK1andactivatedphospho-p38 0.5%bufferedinPBS.Nonspecificbindingwasblockedwith
MAPKcouldpredictpoorhumanspermquality. 3% BSA buffered in PBS. Cells were probed first with mono-
clonalanti-p38(1:100)oranti-ERK1/2(1:100)polyclonalanti-
EXPERIMENTALPROCEDURES
body, washed three times with PBS, and then probed with a
Preparation of Human Spermatozoa—Human semen was secondaryanti-rabbitHilyteFluorTM488-labeledantibodyora
obtained from healthy donors with normal sperm density, secondary anti-mouse Hilyte FluorTM 647-labeled antibody
motility,andmorphologyaccordingtoWorldHealthOrgani- (Anaspec).Slideswereviewedwithalaserscanningmicroscope
zationguidelines(19)orfrommalesattendingeithertheMale (510,Zeiss).
Infertility Unit, Assaf Harofeh Medical Center, Israel, or the ActivationofMAPKCascades—Capacitatedandnoncapaci-
Andrology Laboratory in Assuta Hashalom Medical Center, tatedhumanspermatozoawerepreparedasaboveandstimu-
TelAviv,Israel.Thehumansemenwasliquefiedfor60minat lated with PMA, progesterone, or other drugs as indicated
36°C.SpermwaswashedtwicewithHam’sF-10mediumcon- (Sigma),andcellextractwasusedforWesternblotting.After
tainingbovineserumalbumin(BSA,0.3%)andincubatedwith stimulation, the cells were diluted immediately with excess
themediumfor3hforcapacitation(20).PMAorprogesterone Ham’sF-10medium,precipitatedbycentrifugationat15,000(cid:3)
(Sigma) were added with or without the PKC inhibitor gfor20satroomtemperature,andwashedoncemore,andthe
GF109203x(Calbiochem)ortheMEK1/2inhibitorsPD98059 resultedpelletswerestoredat(cid:4)20°Cuntilused.Thethawed
and U0126, or the p38 inhibitors SB203580 and PD169316 pellets(allthesestepsweremadeat4°C)wereresuspendedina
(Biomol), or the Ca2(cid:2) inhibitors EGTA, nifedipine, and minimalvolumeoflysisbuffer(50(cid:5)lper3(cid:3)107cells)madeof
BAPTA/AM(Sigma)totheabovemediumasdescribedinthe 50 mM Tris-HCl, pH 8.0, 2 mM EGTA, 20 mM NaCl, 1.0 mM
legends. sodium orthovanadate, 25 mM (cid:3)-glycerophosphate, 100 nM
Immunocytochemistry—Humansperm(1.5(cid:3)106)werecol- okadaicacid,0.50%NonidetP-40,1mMbenzamidine,10(cid:5)g/ml
lectedonglassslidesbycytospin(600rpm).Thecellswerefixed aprotinin,10(cid:5)g/mlleupeptin,1mMphenylmethylsulfonylflu-
andpermeabilizedbycoldmethanol(10min),followedbycold oride, and 2 mM dithiothreitol. The suspensions tubes were
acetone (10 min) (20). The cells were treated with the respec- incubated on ice for 10 min and centrifuged (15,000 (cid:3) g, 15
tiveanti-generalMAPK(ERK,JNK,andp38)antibodies(Sigma) min,4°C).Thesupernatants,whichcontainedunboundpro-
orantibodiesagainstmSos,MEK1/2,Raf-1,ortubulin(Santa teins, were collected, and aliquots from each sample (20 (cid:5)g)
Cruz Biotechnology) (1:25) for 18 h at 4°C. Cells were then wereseparatedon10%SDS-PAGEfollowedbyWesternblot-
treated with biotinylated second antibody (1:100) for 30 min. tingwithmousemonoclonalanti-active(doublyphosphoryla-
AfterwashinginPBSthecellswerefurthertreatedfor30minat ted)MAPKs(ERK,JNK,andp38)(Sigma),MPM2(anti-phos-
14480 JOURNALOFBIOLOGICALCHEMISTRY VOLUME283•NUMBER21•MAY23,2008
ERKandp38AreRegulatorsofSpermMotility
conjugated anti-mouse or anti-
rabbit Fab antibodies (Jackson
ImmunoResearch). The blots were
autoradiographed on Kodak X-100
films,andthephosphorylationwas
quantified by densitometry (690
densitometer, Bio-Rad). Each band
from the anti-phospho-MAPK
(ERK, JNK, and p38) was normal-
ized to the corresponding band
from the anti-general MAPK anti-
bodiesblotforevenloading(22).
Recombinant Expression of
pERK—To obtain doubly phos-
phorylated ERK, activated human
GST-ERK2wasco-expressedwith
constitutively active MEK1 in
BL21 bacteria, as described (23).
The bacteria were grown in 2YT
mediumat30°Ctoanabsorbance
of 0.6, and then 1 mM isopropyl
1-thio-(cid:3)-D-galactopyranoside was
added for an additional 4 h. Pro-
FIGURE1.A,localizationofMAPKinejaculatedhumanspermatozoa.Humanspermatozoawereimmuno- teins were purified over glutathi-
stainedwithantibodiestoERK(panelsaandb),Raf-1(panelc),MEK1(paneld),p38(panelseandf),JNK(not one-Sepharose 4B (Amersham
shown),andtubulin(panelg).ForERKandp38,butnotJNK,immunostainingcanbeobservedinthesperm
Biosciences) or nickel-NTA-agar-
neckandalongtheentiretail.Preabsorptionoftheantibodieswiththerelevantpeptideantigenresultedin
disappearanceofthestaining(panelsbandf).ThestainingofmSos(notshown),Raf-1(panelc),andMEK1 ose (Qiagen) according to the
(paneld)issimilartothatofERK,withheavystainingoftheneckandalongthetail.Notethelackoftubulin manufacturer’sinstructions.
immunostainingintheneck(panelg).Cellsshownarerepresentativeofatleastfourindependentexperiments.
B,localizationofERKinhumanspermatozoabyimmunogoldlabeling.Ananti-ERKpolyclonalantibodyand CellCulture,Transfection,andIm-
goatanti-rabbitgold-labeledparticles(8and15nm)wereusedonaraldite-embeddedthinsectionsofglutar- munoprecipitation—Human embry-
aldehyde-fixed(unosmicated)humanspermatozoa.Panela,localizationofERKincross-sectionoftheprincipal
onickidney293cells(HEK293)were
piece.Goldparticlesaredistributedintheouterdensefibers.Panelb,lackofstainingwiththepeptide-preab-
sorbedantibody.Cellsshownarerepresentativeofthreeindependentexperiments. growninDulbecco’smodifiedEagle’s
mediumcontaining10%heat-inacti-
vated fetal bovine serum in 95% air,
5%CO at37°C.Cellswereplatedin
2
100-mmplates24hpriortotransfec-
tionandweretransfectedwith4(cid:5)gof
plasmidDNAARHGAP6-GFP,using
the calcium phosphate method, and
grown as above. 16 h post-transfec-
tion, the medium was replaced, and
48 h later the cells were lysed. Lysis
buffer contained 100 mM NaCl, 20
mMTris-HCl,pH7.4,0.5mMEDTA,
15% glycerol, 0.2% Triton X-100, 1
mM phenylmethylsulfonyl fluoride,
andproteaseinhibitorsmixture.The
FIGURE2.FluorescencemicroscopyforexpressionofERK1/2andp38inhumanspermatozoa.Precapaci- lysate was subjected to immunopre-
tatedspermatozoawerereactedwithanti-ERK(A)andanti-p38(E)antibodies.Phase-contrastimagesare cipitationwithananti-greenfluores-
shownforERK1/2andp38immunolabelinginBandF,respectively,andnegativecontrolswithsecondary
centproteinantibody(RocheApplied
antibodiesareshowninCandG.Phase-contrastimagesareshownforcontrolswithsecondaryantibodiesinD
andH.LikethediaminobenzidinestainingofFig.1,ERK1/2wasdistributedalongthetail,whereasp38seems Science) conjugated to protein A/G
tolocalizealsoinpost-acrosomalregions,themid-piece,andthetail(C).Scalebarsindicate10(cid:5)m. PLUS-agarose (Santa Cruz Biotech-
nology)bygentleshakingfor3–4hin
pho-Ser/Thr-Pro, Upstate Biotechnology, Inc.), or anti- 4°C,andwashedthreetimeswithlysisbuffer.Sampleswerefrozen
ARHGAP6 mouse monoclonal antibody (Abnova). Total in(cid:4)20°Candwerelatersubjectedtoinvitrophosphorylation.
MAPKsweredetectedwithpolyclonalantibodiesforthevari- In Vitro Phosphorylation—Immunoprecipitated ARHGAP6
ousgeneralMAPKs(Sigma)asacontrol.Theblotsweredevel- attachedtobeads(15(cid:5)l)(0.5(cid:5)gperreaction)wasmixedwith
oped with alkaline phosphatase- or horseradish peroxidase- immunoprecipitatedERKattachedtobeads(15(cid:5)l).Thebuffer
MAY23,2008•VOLUME283•NUMBER21 JOURNALOFBIOLOGICALCHEMISTRY 14481
ERKandp38AreRegulatorsofSpermMotility
reactionmix(3(cid:3))(75mM(cid:3)-glycerophosphate,1.5mMdithio-
threitol,3.8mMEGTA,0.15mMorthovanadate,30mMMgCl ,
2
30 (cid:5)M calmidozolium, 0.3 mM ATP), containing 100 (cid:5)M
[(cid:6)-32P]ATP(4000cpm/pmol),wasaddedtothereactionina
final volume of 30 (cid:5)l and incubated for 20 min at 30°C. The
reactionwasterminatedbyadding10(cid:5)lof4(cid:3)Samplebuffer,
andthephosphorylatedproteinswereresolvedonSDS-PAGE.
PhosphorylationofMBP(8(cid:5)gperreaction)byERKwascarried
outasapositivecontrol.
AssessmentofSpermMotility—Progressiveflagellarmotility
was determined manually (20) or by using Computer-aided
Sperm Analysis (CASA) (Sperm Analysis System version
12-IVOS,HamiltonThorneBiosciences,Beverly,MA),which
wasalsousedtomeasurehyperactivation(24).Hyperactivation
parameters were as follows: curvilinear velocity (cid:5)100 (cid:5)m/s,
linearity(cid:6)60%,amplitudeoflateralhead(cid:5)5(cid:5)m.
AssessmentofSpermAcrosomeReaction—Thepercentageof
acrosome-reacted sperm was determined microscopically on
air-driedspermsmearsusingFITC-conjugatedPisumsativum
agglutinin, which is a fluorescent lectin capable of binding to
theacrosomalcontent.Washedcells(107cells/ml)werecapac-
itatedfor3hat37°Cand0.5%CO inF-10mediumsupple-
2
mentedwithBSA3mg/ml(20).Theinhibitorswereaddedfor
the last 10 min of incubation, and then PMA (100 nM) was
addedforanotherhour.Attheendofincubationanaliquotof
thespermwasspreadonmicroscopeslidesandallowedtoair-
dry. The sperm were then permeabilized by methanol for 15
minatroomtemperature,washedoncewith25mMTris-buff-
ered saline, pH 7.6, for 5 min and twice with H O at 5-min
2
intervals,air-dried,andthenincubatedwithFITC-P.sativum
agglutinin(60(cid:5)g/ml)for1h,washedtwicewithH Oat5-min
2
intervals,andmountedwithFluoroGuardAntifade(Bio-Rad).
For each experiment, at least 150 cells per slide in duplicates FIGURE3.ActivationofMAPKinhumanspermatozoa.Capacitatedhuman
spermatozoaweretreatedwithorwithoutPMA(50nM),progesterone(5
wereevaluated.Cellswithgreenstainingovertheacrosomalcap (cid:5)g/ml),orvanadate(0.2mM)for15min,andmousepituitarygonadotrope
were considered acrosome-intact; those with equatorial green L(cid:3)T2cellsweretreatedwithPMA(50nM,5min)asapositivecontrol.Cell
lysatesweresubsequentlyprepared.MAPK(ERK,p38,andJNK)activitywas
staining or no staining were considered acrosome-reacted. The
determinedbyWesternblottingwithtype-specificantibodiesforphospho-
analysisofmultiplegroupswasperformedbyone-wayanalysisof MAPK(activeform)andanti-ERKantibodies.Eachbandfromtheanti-phos-
varianceusingSPSSsoftwarewithp(cid:6)0.05consideredsignificant. pho-MAPKwasnormalizedtothecorrespondingbandfromtheanti-ERKblot
forevenloading.Arepresentativeblotisshown,andsimilarresultswere
Correlation between MAPK Levels and Sperm Quality
observedintwootherexperiments.
Parameters—Human spermatozoa from 47 males attending
the Male Infertility Unit were analyzed for MAPK levels and tected least significant difference tests, and statistical signifi-
activity as above and correlated with percent sperm motility cancewasacceptedwhenp(cid:6)0.05.Otherstatisticaltestsare
(SM), forward progression motility (FPM), morphology, and detailedinthelegends.
viability (19). The correlations were obtained using Pearson
RESULTS
correlation coefficients. Logarithmic transformation (when
needed)wasusedtoobtainnormaldistribution.Aimingtodis- ERK1/2(anditscascademembersSos/Raf-1/MEK1)andp38
criminatenormalandabnormalmotilityandFPMsamples,sin- MAPK,butnotJNK,wereidentifiedinthetailofmatureejac-
glevariableanalysiswasusedutilizingStudent’sttest.Stepwise ulatedhumanspermatozoabyimmunocytochemistry(Fig.1A).
logistic regression was used for multivariate analysis. p value TheERKcascade(Sos/Raf-1/MEK1/2/ERK1/2)andp38were
(cid:6)0.05 was considered statistically significant. To define opti- localizedintheneckanddistributedalongthemid-,principal,
malcutpointsforMAPKlevels,receiveroperatingcharacter- andendpiecesofthetail.Tubulinintheaxonemewasevenly
istic(ROC)analysis(25)wasuseddiscriminatingnormalversus distributed along the tail. Electron microscopy revealed (Fig.
abnormalmotilityandFPM.Wecalculatedtheareaunderthe 1B)thatERK1/2waslocalizedtotheouterdensefibers.Confo-
curve(AUC),whichrepresentedtheaccuracyofthetest,anda cal microscopy revealed that ERK1/2 is distributed mainly to
pvalue(cid:6)0.05wasconsideredpredictive. theentiremid-piece(Fig.2),whereasp38isprimarilylocalized
DataAnalysis—Resultsfromtwoorthreeexperimentswere totheuppermid-piece(Fig.2).
expressed as mean (cid:7) S.D. Data were subjected to statistical WethenidentifiedthepresenceofactiveERK1/2andp38in
analysis with one-way analysis of variance and Fisher’s pro- humanejaculatedspermatozoabyWesternblottingandcom-
14482 JOURNALOFBIOLOGICALCHEMISTRY VOLUME283•NUMBER21•MAY23,2008
ERKandp38AreRegulatorsofSpermMotility
FIGURE4.ActivationofERK1/2byPMAinhumanspermatozoa.Human
spermwereincubatedincapacitationmedium.Attheendofeachpreincu-
bationtime,cellsweretreatedwithPMA(100nM)for15min.Celllysateswere
subsequentlyprepared.ERK1/2activity(upperandlowergraphs,respectively)
wasdeterminedasinFig.3.Arepresentativeblotisshown,andbarsare
mean(cid:7)S.D.fromthreeexperiments.*,p(cid:6)0.05;**,p(cid:6)0.01.
pareditwiththeknownPMA-stimulatedMAPKinthemouse
pituitarygonadotropeL(cid:3)T2cells(22)(Fig.3).Humanejaculate
spermatozoa express mainly ERK2, which was stimulated by
PMAorbythegeneralactivatorvanadateperoxide(Na VO ).
3 4
PMA serves as an analog of diacylglycerol, the physiological
activatorofPKC,andbothstimulantsrevealedtheactivationof
ERK1. The sperm-ligand progesterone, reported to activate
humanspermERK(26),hadnoeffect.p38MAPKinitsphos-
phorylated form was very low in control L(cid:3)T2 cells but was
presentinspermatozoa.Wecouldnotdetectfurtheractivation
ofphospho-p38byPMA,vanadate,norprogesteroneversusa
positiveeffectofPMAinL(cid:3)T2cells(Fig.3).Thedatasuggest
thatejaculatespermp38MAPKisfullyphosphorylated,hence
activated.p38MAPKisencodedbyfourgenesthatyieldnine
isoforms(5),andwecoulddetectmainlyp38(cid:1)andoccasionally
p38(cid:3).JNK1/2ispresentinL(cid:3)T2cellsandisactivatedbyPMA
but is undetectable in PMA-, vanadate-, or progesterone- FIGURE5.A,PMA-inducedERKactivationinhumanspermatozoaissensitive
treatedspermatozoa. toMEK1/2inhibitorsandinsensitivetop38inhibitor(leftpanel)andtothe
pan-PKCinhibitorGF109203X(rightpanel).Capacitatedhumanspermwere
Wethenincubatedspermatozoafor2.5hinacapacitation preincubatedwiththespecificMEK1/2inhibitorsPD98059(50(cid:5)M)andU0126
medium(Fig.4)toreachcapacitationinvitro(20).Exposureto (50(cid:5)M),thep38inhibitorSB203580(50(cid:5)M)andthePKCinhibitorGF109203X
PMA(15min)throughouttheentirecapacitationprocessfur- (3(cid:5)M)for15minandPMA(100nM)wasaddedforanother15min.Celllysates
weresubsequentlypreparedandERK2activitywasdeterminedasinFig.3.
therstimulatedERK1/2levelsincomparisonwithcontrols(*, Barsaremean(cid:7)S.D.fromthreeexperiments.Meansdesignatedbydifferent
p(cid:6)0.05;**,p(cid:6)0.01).Thus,ERKactivationbyPMAcanbe lettersaresignificantlydifferent(p(cid:6)0.05).B,PMA-inducedERKactivationis
Ca2(cid:2)-independent. Capacitated human spermatozoa were preincubated
detectedinbothnoncapacitatedandcapacitatedspermatozoa. withorwithoutvariousCa2(cid:2)blockers:nifedipine(L-typevoltage-dependent
ERK2activationbyPMAwassensitivetotheMEK1/2inhibi- Ca2(cid:2) channel blocker, 1 (cid:5)M), EGTA (a general Ca2(cid:2) chelator, 5 mM) and
torsPD98059andU0126(Fig.5A,leftpanel),confirmingthe BAPTA/AM(intracellularCa2(cid:2)chelator,50(cid:5)M)for30min.PMA(100nM)was
thenaddedfor15minandERK2activitywasdeterminedasinFig.3.Arepre-
functioning of a canonical MEK1/2-ERK1/2 cascade (6, 7) in sentativeblotisshownandbarsaremean(cid:7)S.D.fromthreeexperiments.
spermatozoa and the use of the two drugs in the studies Meansdesignatedbydifferentlettersaresignificantlydifferent(p(cid:6)0.05).
describedbelow.Theselectivep38inhibitorSB203580hadno
effectonPMAtoERKsignaling,rulingoutaninhibitorycross- dependent(Fig.5A,rightpanel).ERKactivationbyPMAwas
talkbetweenp38MAPKandERK(27).Theselectivepan-PKC insensitivetoEGTA(extracellularCa2(cid:2)chelator),tonifedipine
inhibitor,GF109203X,completelyabolishedthePMA-induced (voltage-dependentCa2(cid:2)channelblocker),ortoBAPTA/AM
activationofERK1/2,indicatingthatthePMAeffectwasPKC- (intracellular Ca2(cid:2) chelator) (Fig. 5B), unlike L(cid:3)T2 cells, in
MAY23,2008•VOLUME283•NUMBER21 JOURNALOFBIOLOGICALCHEMISTRY 14483
ERKandp38AreRegulatorsofSpermMotility
MEK1/2selectiveinhibitors,U0126
and PD98059, reduced basal and
abolished the PMA response for
bothforward(Fig.6C)andhyperac-
tivated motility (Fig. 6D). In con-
trast, as in the manual examina-
tions, the p38 inhibitors SB203580
and PD169316 enhanced forward
(Fig.6C)andhyperactivatedmotil-
ity (Fig. 6D) and had no additive
effectonthePMAresponse(Fig.6,
CandD).Thus,p38andERK1/2are
involved in sperm motility in an
opposing fashion; p38 decreases
forward and hyperactivated motil-
ity,whereasERK1/2increasesbasal
and mediates PKC-activated for-
ward and hyperactivated motility.
Indeed,thecell-permeablediacylg-
lycerol analog, 1-oleoyl-2-acetylg-
lycerol (OAG), also stimulates
ERK1/2(Fig.7A)andenhancesfor-
ward (Fig. 7B) and hyperactivated
motility (Fig. 7C) (see also supple-
mentalvideo2).Thesedataprovide
further support for the role of
FIGURE6.EffectofPMAandMAPKinhibitorsonhumanspermatozoanforwardandhyperactivated
ERK1/2 in human spermatozoan
motility.A,normalhumanspermatozoa((cid:5)20(cid:3)106/ml;(cid:5)50%motility)wereincubated(5(cid:3)107/ml)withor
withouttheselectiveMEK1/2inhibitorPD98059(50(cid:5)M)forthetimeindicated,andpercentmotilespermato- motility.
zoawasdeterminedin10-(cid:5)laliquotsbyfollowingprogressivemotility,underamicroscope.Similarly,abnor- The role of ERK and p38 in the
malspermsamples((cid:6)20(cid:3)106/ml;(cid:6)30%motility)wereincubatedwithorwithoutthep38inhibitorSB203580
(50(cid:5)M),andmotilitywasdeterminedasabove.Resultsaremeans(cid:7)S.E.fromthreeexperiments.B,abnormal acrosome reaction that spermato-
spermsamples((cid:6)20(cid:3)106/ml;(cid:6)30%motility)wereincubatedwithorwithoutthep38inhibitorsSB203580 zoamustundergoinordertofertil-
andPD169316(50(cid:5)M)for10min,andPMA(100nM)wasaddedasindicatedforanother15min.Percent izetheegg(30)wasthenexamined
forwardmotilitywasdeterminedasabove.Resultsaremean(cid:7)S.D.fromthreeexperiments.CandD,human
spermatozoa(50–60%motility)werepreincubatedwithorwithouttheselectiveMEKinhibitorsPD98059and (Fig. 8). Human spermatozoa were
U0126orthep38inhibitorsSB203580andPD169316(50(cid:5)Meach)for15min,andPMA(100nM)wasadded incubated with PMA, a known
whereindicatedforanother15min.Forwardmotility(C)andhyperactivationofmotility(hyperactivated/total
motilecells)(D)weremeasuredusingCASA.Resultsaremean(cid:7)S.D.fromthreetosixexperiments.Means inducer of the acrosome reaction
designatedbydifferentlettersaresignificantlydifferent.*,p(cid:6)0.05;**,p(cid:6)0.01. (31–33),andtheMEK1/2inhibitors
PD98059 and U0126 or the p38
whichactivationofERKbyPMAwasmarkedlyreducedbythe inhibitors SB203580 and PD169316. Stimulation of the acro-
aboveCa2(cid:2)inhibitors(Fig.5B). somereactionbyPMA(20)wasnearlyabolishedbytheMAPK
Incubation of spermatozoa with normal motility ((cid:5)50% inhibitors. Further support comes from the observation that
motility) (19) with the selective MEK1/2 inhibitor PD98059 OAG,whichactivatesERK(Fig.7A),isalsocapableofactivat-
reducedflagellarmotility(Fig.6A)incomparisonwithcontrol ingtheacrosomereaction(18).Hence,despitetheiropposing
(*,p(cid:6)0.05;**,p(cid:6)0.01).Incontrast,incubationofspermato- role in motility, ERK1/2 and p38 MAPK are positively impli-
zoawithlowmotility((cid:8)30%motility)(19)withthep38inhib- catedintheacrosomereaction.
itor SB203580, significantly stimulated flagellar motility (Fig. WethentookaproteomicapproachtoidentifyERKsubstrates
6A). Similarly, we could significantly increase sperm forward inhumanspermatozoa.ControlandPMA-treatedcellsweresub-
motility, even for spermatozoa with very low motility ((cid:6)30% jectedtogelelectrophoresisfollowedbyWesternblottingusing
motility) by incubation with PMA, or the p38 inhibitor phospho-specific anti-ERK (MAPK) substrates antibodies
SB203580,oranotherselectivep38inhibitorPD169316for15 (MPM2)(Fig.9A).WesearchedforbandsthatshowedPMA-in-
min,withnoadditiveeffectwithPMA(Fig.6B).Similarresults ducedphosphorylation.Amajorband((cid:8)80kDa)wasexcisedand
werefoundwithspermofnormalmotility.Wealsoexamined subjected to MALDI-TOF mass spectrometry, and the primary
forwardandhyperactivatedmotilityusingCASA(Fig.6,Cand identifiedproteinwasARHGAP6,aRhoGAP(Fig.9B).Onceidenti-
D,respectively).Studieshaveindicatedthatthedirectquanti- fied,wecouldnowverifytheresultsbyimmunoblottingspermato-
tativeassessmentofspermmotilitybyCASAaccuratelyreflects zoanlysatewithanti-ARHGAP6(Fig.9C).Indeed,ARHGAP6canbe
thefertilizingabilityofhumanspermatozoainvitroinparticu- phosphorylated in vitro by ERK2, myelin basic protein (MBP), a
larwheretheconventionalsemenanalysisisoflimiteddiagnos- knownERKsubstrateservingasapositivecontrol(Fig.9D).
ticvalue(28,29).PMAincreasedforward(Fig.6C)andhyper- TofindoutwhetherMAPKcanbelinkedtohumansperm
activatedmotility(Fig.6D)(seealsosupplementalvideo1).The quality,wecorrelatedMAPKwithSM,FPM,morphology,and
14484 JOURNALOFBIOLOGICALCHEMISTRY VOLUME283•NUMBER21•MAY23,2008
ERKandp38AreRegulatorsofSpermMotility
FIGURE8.RoleofMAPKinacrosomereaction.Humanspermwerepreincu-
batedincapacitationmediumfor3h.ThespecificMEK1/2inhibitorsPD98059
(PD)andU0126,orthep38inhibitorsSB203580(SB)andPD169316(50(cid:5)M,
each)wereaddedforthelast10minofthepreincubation,andPMA(100nM)
wasaddedforanotherhour.Thepercentageofacrosome-reactedcellswere
determined using FITC-conjugated P. sativum agglutinin (PSA-FITC). The
basalacrosomereactionwas21%.Thedatarepresentthemean(cid:7)S.D.of
duplicatesfrom6to8experimentsaftersubtractingthebasalpercentage
fromtheinducedacrosomereaction.Meansdesignatedbydifferentletters
aresignificantlydifferent(p(cid:6)0.05).
FIGURE7.EffectofthediacylglycerolanalogOAGonERK1/2activation DISCUSSION
(A)andforward(B)andhyperactivated(C)motilityinejaculatedhuman
WefirstidentifiedtheSos/Raf-1/MEK1/ERK1/2pathwayand
spermatozoa. A, capacitated human spermatozoa were stimulated with
increasingconcentrationsofOAGorwithPMA(100nM)for15min.After p38MAPKinmatureejaculatedhumanspermatozoabyimmu-
treatment,celllysateswereanalyzedforERKactivitybyWesternblotusingan nocytochemistry,immunofluorescencemicroscopy,andWestern
antibodyforphospho-ERK.TotalERKwasdetectedwithpolyclonalantibod-
blotting.Surprisingly,wecouldnotdetecttheotherstress-acti-
iesasacontrolforsampleloading.Arepresentativeblotisshownandsimilar
resultswereobservedintwootherexperiments.BandC,capacitatedhuman vatedMAPK,namelyJNKbytheabovemethods.Wetherefore
spermatozoa((cid:8)40%motility)wereincubatedwithOAG(60(cid:5)g/ml)for15
postulatedthatJNKmaybeinvolvedinspermatogenesisbutnotin
min.Forwardmotility(B)andhyperactivationofmotility(hyperactivated/
totalmotilecells)(C)weremeasuredusingCASA.Resultsaremean(cid:7)S.D. maturespermatozoa.Indeed,wefoundthatJNKexpressionistran-
fromthreeexperiments.Meansdesignatedbydifferentlettersaresignifi- sientasitappearsinhamsterroundspermatids,decliningthereafter.4
cantlydifferent(p(cid:6)0.05).
Nevertheless,thelackofexpressionofJNKinejaculatedspermatozoa
isinterestingbecauseJNKisthoughttobeubiquitouslyexpressed
viability(19).AmongERK1,ERK2,andp38MAPK(phospho- (34)andJNK1/2/3knock-outmicearefertile(35).
rylated and nonphosphorylated forms), only total ERK1 and WefoundERKandp38MAPKintheneckofmaturehuman
phosphorylatedp38gavehighlysignificantinversecorrelation spermatozoa,andlikePKC(cid:3)IandPKC(cid:4)(20),theyweredistrib-
toallthespermqualityparameters(Table1).Univariateanal- utedalongthemid-,principal,andendpiecesofthetail.Mature
ysisrevealedthatERK1andphospho-p38arestatisticallysig- humanspermexpressmainlyERK2underbasalconditions,and
nificant discriminators for abnormal sperm motility ((cid:6)50%) activation by PMA revealed the presence of ERK1. The p38
(19) and abnormal forward progression motility ((cid:6)3) (19) MAPK is apparently fully activated, because PMA, vanadate,
andprogesteronehadnoeffectonp38MAPKphosphorylation.
(Table 2). Multivariate analysis (logistic regression), which
p38MAPKisencodedbyfourgenesthatyieldnineisoforms
includedtheageofthedonor,enzymelevels,andspermquality
(5),andwecoulddetectmainlyp38(cid:1).
parameters,revealedphospho-p38tobeasingleandindepend-
ent discriminator for abnormal sperm motility (p (cid:9) 0.001). AsPMAservesasananalogofdiacylglycerol,thephysiolog-
icalactivatorofPKC,wereasonedthatdiacylglycerolmaybea
Similarly,ERK1isasinglediscriminatorforforwardprogres-
sion motility (p (cid:9) 0.003). Using ROC analysis, a cut point of spermactivator,andthereforeweevaluateditsabilitytostim-
ulateERK(36).Indeedweshowherethatacell-permeableana-
1.40 for log phospho-p38 (at 25.12% compared with control)
coulddistinguishnormal((cid:7)50%)fromabnormalmotilitywith logofdiacylglycerol,namelyOAG,iscapableofactivatingERK
andalsomimicsotherfunctionsofPMAonhumanspermato-
80% sensitivity and 81.5% specificity; and abnormal forward
progression motility ((cid:6)3) with 84.2% sensitivity and 82.1%
specificity;AUCis0.880(Table3andFig10). 4R.Golan,L.Shochat,T.Almog,andZ.Naor,manuscriptinpreparation.
MAY23,2008•VOLUME283•NUMBER21 JOURNALOFBIOLOGICALCHEMISTRY 14485
ERKandp38AreRegulatorsofSpermMotility
zoasuchasincreasedmotility,hyperactivationofmotility,and its insensitivity to both extracellular and intracellular Ca2(cid:2)
inductionofacrosomereaction. removal.Inmostcells,activationofERKbyGPCRsandviaPKC
ActivationofERKdownstreamtoPKCasrevealedbyPMA is Ca2(cid:2)-dependent (37, 38), whereas we present evidence
stimulationinhumanspermatozoadiffersfromsomaticcellsin here that activation of spermatozoan ERK by PMA is
Ca2(cid:2)-independent.
Mammalian spermatozoa ex-
press the G-protein G , phospho-
q
lipase C(cid:1), and inositol 1,4,5-
trisphosphate receptors (39, 40).
WefoundPKC(cid:4)inthetail(20),and
PKC(cid:4)is implicated in ERK activa-
tion in general (41). Hence, a
G /phospholipase C(cid:1)/diacylglyc-
q
erol/PKC(cid:4)/ERK signaling cascade
seems to operate in the human
sperm,apparentlyinparalleltothe
progesterone or bicarbonate/
cAMP/Ca2(cid:2)system(42).
We also looked for the role of
ERKandp38MAPKinspermfunc-
tions. The role of ERK in sperm
motilityiscontroversial.Apositive
role for ERK was suggested in fowl
sperm (43). ERK was shown to be
gradually activated throughout the
transition of mouse spermatozoa
FIGURE9.ARHGAP6,aRhoGAP,isanERKsubstrateinhumanspermatozoa.Humanspermwerewashed fromthecaput(immotile)tothevas
andstimulatedwith100nMPMA,andcellswerelysedasmentionedunder“ExperimentalProcedures.”A,part deferens (fully motile) (10). A con-
ofthelysatewasresolvedon10%SDS-PAGEandWestern-blottedwithMPM2,aphospho-specificanti-MAPK
tradictory report concluded that
substrateantibody,whereastheotherpartwasresolvedinthesamemanner,followedbygelstaining.
CON,control.B,majorPMA-stimulatedband((cid:8)80kDa)wassubjectedtoMALDI-TOFmassspectrometry. ERK inhibits motility of human
Peptidecoveragemapofthespecificidentifiedprotein,ARHGAP6,aRhoGAPisshown.C,ARHGAP6is spermatozoa (16). We report here
presentinhumanspermatozoaasrevealedbyimmunoblotsofthespermlysatewithspecificanti-ARH-
GAP6antibodies.D,HEK293cellsweretransfectedwithpEGFPN1(Clontech)fusedtoARHGAP6bythe that PMA increased forward and
calciumphosphatemethod.Cellswerelysed,andARHGAP6wasimmunoprecipitatedwithananti-green hyperactivated motility in an ERK-
fluorescentproteinantibody.Theprecipitatewassubjectedtoaninvitrophosphorylationassaywith
dependent manner. Interestingly,
activatedERKasmentionedunder“ExperimentalProcedures.”MBPphosphorylationisalsoshownasa
positivecontrol. wefoundthatERKandp38MAPK
TABLE1
CorrelationbetweenMAPKlevelsandspermqualityparameters
Humansemensamplesfrom47malesattendingtheMaleInfertilityUnitwereanalyzedforSM,FPMgrade,morphology,andviability.MAPKlevelsweredeterminedas
describedunder“ExperimentalProcedures.”ThecorrelationbetweenMAPKlevelsandspermqualityparameterswascarriedoutafterlogarithmictransformationby
Pearsoncorrelations.rindicatesPearsoncorrelationcoefficient.
Forwardprogression
Viability Morphology motility Motilesperm
pvalue r pvalue r pvalue r pvalue r
% %
ERK1 0.000002 (cid:4)0.630 0.000001 (cid:4)0.676 0.0001 (cid:4)0.605 0.0001 (cid:4)0.553
ERK2 0.063 (cid:4)0.274 0.106 (cid:4)0.239 0.006 (cid:4)0.398 0.026 (cid:4)0.324
P38 0.014 (cid:4)0.355 0.528 (cid:4)0.094 0.003 (cid:4)0.431 0.008 (cid:4)0.383
Phos-ERK1 0.151 (cid:4)0.213 0.135 (cid:4)0.221 0.186 (cid:4)0.196 0.17 (cid:4)0.214
Phos-ERK2 0.211 (cid:4)0.186 0.375 (cid:4)0.133 0.072 (cid:4)0.265 0.253 (cid:4)0.170
Phos-p38 0.0005 (cid:4)0.493 0.004 (cid:4)0.409 0.0003 (cid:4)0.568 0.0002 (cid:4)0.582
TABLE2
CorrelationbetweenMAPKlevelsandspermqualityparameters
UnivariateanalysisofSM(abnormal(cid:6)50%)andFPM(abnormalgrade(cid:6)3)versuslogarithmictransformationofMAPKlevelswereobtainedusingStudent’sttest.NS
indicatesnotsignificant.
Forwardprogressionmotility(0–4) %Motilesperm
<3(n(cid:3)19) >3(n(cid:3)28) <50%(n(cid:3)20) >50%(n(cid:3)27)
pvalue pvalue
S.D. Mean S.D. Mean S.D. Mean S.D. Mean
ERK1 0.000007 0.853 1.151 0.366 0.093 0.001 0.920 0.951 0.512 0.208
ERK2 0.015 0.221 2.064 0.157 1.913 NS 0.243 2.022 0.153 1.934
Phos-p38 0.00003 0.518 1.631 0.504 0.960 0.000004 0.428 1.658 0.517 0.915
14486 JOURNALOFBIOLOGICALCHEMISTRY VOLUME283•NUMBER21•MAY23,2008
ERKandp38AreRegulatorsofSpermMotility
TABLE3
ROCanalysisforoptimalcutpointsofERK1andphospho-p38levelsdiscriminatingnormalversusabnormalFPMandmotility
Reducedmotility(n(cid:3)20) Abnormalforwardprogressionmotility
Areaunderthecurve Logcutoff Sensitivity Specificity Areaunderthecurve Logcutoff Sensitivity Specificity
% % % %
ERK1 0.727 1.56 45 100 0.821 0.55a 68.4 92.9
Phospho-p38 0.880 1.40 80 81.5 0.862 1.39 84.2 82.1
aThisvalueisanaverageofanintervalofvaluesrangingfrom0to1.1,inwhichtheaccuracyofmeasurementisconstant.
(44).Itisthereforereasonabletoassumethatproteinphospho-
rylation is involved in flagellar motility. We therefore took a
proteomicapproachtoinitiatetheidentificationofERKsub-
strates in human spermatozoa. Using phospho-specific anti-
MAPKsubstratesantibodies(MPM2)andMALDI-TOFmass
spectrometry,weidentifiedARHGAP6,aRhoGAP,asanERK
substrate in PMA-stimulated spermatozoa. We further con-
firmed the identification by showing the presence of ARH-
GAP6 in human spermatozoa and the phosphorylation of
ARHGAP6byERK2invitro.AstudyofARHGAP6functional
analysisfoundthatARHGAP6co-localizeswithactinfilaments
throughitsN-terminaldomainandrecruitsF-actinintogrow-
ingprocesses(45).ARHGAP6hastwoindependentfunctions
as follows: a specific GAP for RhoA, and a promoter of actin
remodeling(45).AsRhoAisimplicatedincellmotility(55,56)
and actin remodeling is implicated in hyperactivated motility
andtheacrosomereaction(46,47),phosphorylationofsperm
ARHGAP6byERKmaybeinvolvedinspermmotility,capaci-
tation,andacrosomereactionviaRhoA.Otherpotentialsub-
stratesarethedyneins,thespermmotormoleculesoftheaxon-
eme(48),knowntobephosphorylatedduringactivation(49).
ERKandp38mayhaveopposingeffectsonthephosphorylation
ofaxonemalproteinssuchasthedyneins.Hence,ERKandp38
maybeinvolvedinspermchemotaxis(2),asamolecularmech-
anism navigating the sperm to the egg. Further studies are
requiredtoexaminethisproposal.
Capacitationisassociatedwithreorganizationoftheplasma
membraneasaresultofcholesterolefflux,calciuminflux,tyro-
sine phosphorylation (50), and an increase in cAMP (51).
Capacitation culminates in the process known as acrosome
reaction, which is an exocytotic secretion of proteolytic
enzymesthatfacilitatesthepenetrationbythespermatozoaof
FIGURE10.ROCanalysisfornormalversusabnormalmotilityusingphos-
the oocyte zona pellucida and reorganizes the sperm head in
pho-p38levels.HumansemensampleswereanalyzedasinTable1.Loga-
rithmictransformationwasusedtonormalizethedistributionofenzymelev- preparation for the sperm-oocyte fusion (30). Interestingly,
els. Using ROC analysis, a cut point of 1.40 for log-phospho-p38 could
although we found here an opposing role for ERK and p38
distinguishnormal((cid:7)50%)fromabnormalmotilitywith80%sensitivityand
81.5%specificity;AUCis0.880. MAPKinmotility,botharepositivelyimplicatedintheacro-
somereaction.
regulatespermatozoanforwardandhyperactivatedmotilityin Sperm quality is determined by measuring the volume,
anopposingfashion.ERKwasfoundtostimulate,whereasp38 spermcount,SM,FPM,morphology,andviabilityaccordingto
MAPK inhibits forward and hyperactivated motility. Indeed, theWorldHealthOrganizationguidelines(19).Wetherefore
wecoulddoubletheforwardandthehyperactivatedmotility, analyzedwhetherERK1/2andp38MAPK(boththephospho-
evenwhenitwasverylow(20–30%motility;normalisregarded rylatedandthenonphosphorylatedforms)canbecorrelatedto
as(cid:5)50%)(19),byincubatingthespermwiththep38inhibitors the sperm quality parameters as above. Interestingly, total
SB203580andPD169316orPMA.Henceforwardprogressive ERK1andphospho-p38levelswereinverselycorrelatedtothe
motility,suppressionofmotility,andhyperactivationofmotil- sperm quality parameters. Univariate analysis revealed that
ity(1)maybemaintainedbytheERK/p38ratio. ERK1 and phospho-p38 could discriminate abnormal SM
Sperm flagellar motility requires active sliding of microtu- ((cid:6)50%)andabnormalforwardprogressionmotility(FPM(cid:6)3)
bulesbytheATPaseactivityonthedyneinarmsoftheouter (19).Logisticregression(multivariateanalysis)foundERK1and
doublet microtubules, but the sliding mechanism is not clear phospho-p38tobesingleandindependentdiscriminatorsfor
MAY23,2008•VOLUME283•NUMBER21 JOURNALOFBIOLOGICALCHEMISTRY 14487
ERKandp38AreRegulatorsofSpermMotility
various forms of sperm motility (Fig. 11). Our data might be
applied to unravel possible unbalanced expression of ERK1/2
andp38inthevariousformsofmaleinfertility(e.g.oligotera-
toasthenozoospermia).MAPKactivatorsandinhibitorsmaybe
appliedasawaytoimprovespermqualityofinfertilemenor
when cryopreserved spermatozoa are used during assisted
reproductive techniques. ERK1 and phospho-p38 MAPK can
becontemplatedasameansforspermatozoandiagnostics.Our
studiesmightalsopavethewayforthepotentialuseofMAPK
activators,inhibitors,substrates,orsperm-specificMAPK-in-
teracting proteins as potential tools for the development of
nonhormonal contraceptives and immunocontraceptives for
menandwomen.
Acknowledgments—WethankIrenaGlassandYelizabetaMakovsky
fromtheAndrologyLaboratory,AssutaHashalom,Tel-Aviv,Israel,
FIGURE11.ProposedmodelfortheroleofERK1/2andp38inhuman and Sarita Kaufman and Ana Umansky from the male infertility
spermatozoanmotilityandacrosomereaction.ERK1/2andp38MAPK,but unit,AssafHarofehMedicalCenter,Israel,forthespermsamples.We
notJNK,arefoundinthetailofmaturehumanspermatozoa.ERK1/2stimula-
tionisdownstreamtoPKC(mostlikelyPKC(cid:4)),whichisalsopresentinthe thank Drs. Siddharth K. Prakash and Ignatia B. van den Veyver,
humanspermtail(20).ERKstimulatesandp38inhibitsforwardandhyperac- DepartmentofObstetricsandGynecology,BaylorCollegeofMedi-
tivatedmotility,respectively.Still,bothERKandp38MAPKarepositively cine,Houston,TX,fortheARHGAP6-GFPconstruct.Wealsothank
involvedintheacrosomereaction.ERK1/2mayexertitsfunctionsbyphos-
Dr.GiladGiborforassistancewiththeinvitrokinaseassayandProf.
phorylatingARHGAP6.
ProbalGhoshforcriticalreviewofthismanuscript.Wealsothank
IlanaGelernterforhelpwiththestatisticalanalysis.
abnormal FPM and SM, respectively. ROC analysis revealed
that phospho-p38 and ERK1 could diagnose abnormal FPM
andreducedspermmotilitywithhighspecificityandsensitiv-
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major mediator of ERK1/2 activation in the sperm because E., Hollweck, R., Ring, J., and Kohn, F. M. (2005) Hum. Reprod. 20,
MEK1knock-outmiceareembryonicallylethal(55),whereas 456–461
MEK2knock-outmicearefertile(56). 17. Chen,W.Y.,Ni,Y.,Pan,Y.M.,Shi,Q.X.,Yuan,Y.Y.,Chen,A.J.,Mao,
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field. To understand the underlying mechanisms involved in CambridgeUniversityPress,Cambridge
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spermdysfunction,oneshouldfirstdecipherthenormalbio-
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chemical mechanisms of its functions. Our findings reported
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hereshedfurtherlightonthisbasicaspectofhumanfertility.
chem.43,439–445
We have provided a potential molecular mechanism for the 22. Bonfil,D.,Chuderland,D.,Kraus,S.,Shahbazian,D.,Friedberg,I.,Seger,
14488 JOURNALOFBIOLOGICALCHEMISTRY VOLUME283•NUMBER21•MAY23,2008