Sperm Binding to the Zona Pellucida Is Not Sufficient to Induce Acrosome Exocytosis

Home , Acrosome reaction

*Author forcorrespondence (e-mail:[email protected]) Riverside,CA92521,USA. University ofCalifornia, membrane, causingmultiplemembranefenestrationsandrelease of acrosomal membraneofthespermandoverlying plasma zona pellucida.Exocytosis resultsfromfusionbetweentheouter only ifittakes placeinconjunctionwithspermpenetration ofthe cells, theacrosomereactionoccursonceandisbiologicallyrelevant incompletely understood.Unlike recyclable exocytosis insomatic fertilization haslongengagedinvestigative interest,but remains reaction) occursonthesurface ofthezonamatrix. al., 1997)suggeststhatinductionofacrosomeexocytosis (acrosome (Yanagimachi andPhillips,1984;Cherretal.,1986;VandeVoort et presence ofresidualacrosomalshroudsonthezonapellucida to effect fertilization(Austin,1975;Salingetal.,1979).The acrosome-reacted spermfusewiththeplasmamembraneofegg of theegg andtheinneraspectofzonapellucida), andonly are presentintheperivitelline space(betweentheplasmamembrane 1984; Cherretal.,1986).However, onlyacrosome-reactedsperm pellucida, anextracellular matrixthatencirclestheegg (Storey etal., somatic cells)surroundingovulated eggs andbindtothezona cumulus oophorus(ahyaluronicacidmatrixwithinterspersed been reportedthatonlyacrosome-intactmousespermpenetratethe contents duringfertilization(Abou-HailaandTulsiani, 2000).Ithas condensed spermnucleusthatreleasesitspartiallycharacterized acrosome, aGolgi-derived exocytotic vesicle overlying the two competingdemandsaremediatedinpartbytheintegrity ofthe while eventually fusingwiththeplasmamembraneofegg. These travel, spermatozoamustavoid inadvertent fusionwithsomaticcells tracts beforeencounteringeggs intheupperoviduct. Duringthis Mouse spermatozoanavigate boththemaleandfemalereproductive INTRODUCTION Accepted 21November 2006 1 pellucida matrix,Sperm-eggrecognition, Postfertilizationblocktopolyspermy KEY WORDS:Molecularmechanismsofmousefertilization,Spermacrosome exocytosis,Mechanosensorysignaltransduction,Zona hu mouse ZP2butremainsuncleavedafterfertilization.Theobserveddenovobindingof longer bindtoembryos.We assessed exocytosis andpenetratethezonamatrixbeforegametefusion.Followingfertilization,ZP2isproteolyticallycleavedsper At fertilization,spermatozoabindtothezonapellucida(ZP1,ZP2,ZP3)surroundingovulatedmouseeggs,undergoacrosome Boris Baibakov induce acrosomeexocytosis Sperm bindingtothezonapellucidaisnotsufficient to (2007)doi:10.1242/dev.02752 Development 134,933-943 Health, Bethesda,MD20892,USA. zona matrixinitiatesamechanosensorysignaltransductionnecessarytotriggertheacrosomereaction. sufficient toinduceacrosomeexocytosis.Afilterpenetrationassaysuggestsanalternativemechanisminwhichint added beforeorafterfertilization.Thepersistenceofintactacrosomesindicatesthatspermbindingtothezonapellucidais the boundspermremainintactforatleast24hoursinpresenceofuncleavedhumanZP2regardlesswhetherare structure supportiveofspermbinding,independentfertilizationandcorticalgranuleexocytosis.Surprisingly, theacrosome Laboratory ofCellularandDevelopmental Biology, NIDDK,NationalInstitutes of The inductionofspermacrosomeexocytosis inmouse ZP2 rescue micesupportsa‘zonascaffold’ modelofsperm-eggrecognitioninwhichintactZP2dictatesathree-dimensional 1, *, Lyn Gauthier 2 Department ofCellBiologyandNeuroscience, Acr3 1 , PrueTalbot -EGFP spermbindingtowild-typeandhu 2 , Tracy L.Rankin (Rankin etal.,2003). Theseobservations formthebasisof‘zona is observed andthere is noincreasedincidenceofpolyspermy Nevertheless, thenormalpostfertilizationblocktozonapenetration is notcleaved inthechimericmouse-humanzonapellucida. rescue miceandthisobservation correlateswithintacthuZP2,which embryos afterinvitrofertilization ofeggs derived fromhu and arefertile.Unexpectedly, spermcontinuetobind totwo-cell extracellular Ca mediated ligand-receptorinteractions(Ward etal.,1994), endogenous mouseZP2(hu and geneticallyalteredmiceinwhichhumanZP2replaces mouse zonaproteinsare62-71%identical(Hoodbhoy etal., 2005), (Moller andWassarman, 1989).Thehumanhomologstothethree proteolytic cleavage ofZP2hasbeenexperimentally observed Although otherbiochemicalchangeshave beeninferred,onlythe not bindorpenetrate(AustinandBraden,1956;Ducibella,1996). contents, whichmodifythezonamatrixsothatadditionalsperm do Following fertilization,egg corticalgranulesexocytose their ZP1, ZP2andZP3(BleilWassarman, 1980b;Bojaetal.,2003). Saling etal.,1979;Yanagimachi, 1994). effective intriggeringacrosomeexocytosis (Austin,1975; 100% binding orpenetrationofthezonapellucidaduringfertilizationis and Roldan,1995;Satoetal.,2000;SonMeizel,2003),only Meizel, 1985;Roldanetal.,1994;ShiandRoldan,1995;Murase vitro (FlormanandStorey, 1982;BleilandWassarman, 1983; induce theacrosomereactiontwo- tothreefoldover backgroundin or othermechanismsremainsunclear. Althoughmultipleagonists internal inositol1,4,5-triphosphatereceptors(Herricketal.,2005) Whether physiologicmobilizationisinitiatedbyG store thatissufficient totriggerexocytosis (Herricketal.,2005). 1985; KimandGerton,2003).Theacrosomepossessesacalcium accelerated byinteractionswiththezonapellucida(LeeandStorey, beginning withcapacitationofsperm,andisdramatically more recentdatasuggestthattheacrosomereactionisacontinuum, considered abinaryevent (acrosome-intactoracrosome-reacted), acrosomal contents(Barrosetal.,1967).Althoughinitially The mousezonapellucidaiscomposedofthreeglycoproteins, 1 and JurrienDean ZP2 2+ influxes (Jungnickel etal.,2001),activation of Acr3 rescue eggsinwhichhumanZP2replaces -EGFP spermtoembryosderivedfrom ZP2 1 rescue mice)formazonapellucida RESEARCH ARTICLE i not -protein s of m no o the ZP2 933

DEVELOPMENT imaged byconfocal microscopy. Fertilizationwas confirmed bythepresence 1998). Embryoswere thenisolated,fixed, stained withHoechst33342and ovulated eggs obtainedfromgonadotropin-stimulatedmice(Rankinetal., NJ) andincubatedundercapacitatingconditions(1hour, 37°C,5%CO CA) inM16media(SpecialtyMedia,ChemiconInternational,Phillipsburg, Wild-type or In vitro fertilization stained withHoechst33342before imaging. manufacturer’s protocol.Sampleswerefixed in2%paraformaldehydeand (Invitrogen MolecularProbes,Carlsbad,CA)accordingtothe inhibitor (SBTI)(Worthington, Lakewood, NJ)conjugatedwithAlexa 568 samples wereincubatedfor15minutesbeforefixation withsoybean trypsin microscope (Rankinetal.,2003).To detectacrosome-reactedsperm, Confocal laserscanningimageswereobtainedonaZeissLSM510confocal Confocal microscopy and Unless notedotherwise,caudalepididymalspermisolatedfromwild-type Sperm capacitation (Nakanishi etal.,1999),two transgenic mouselines,TgN( Using atransgenesuppliedbyDrMasaruOkabe,OsakaUniversity Establishment oftransgenicmouselines type, hu status andbindingofspermtoeggs andembryosderived fromwild- fluorescent proteinwithintheacrosome,weexamined theacrosome exocytosis (Dean,2004). for spermbinding,independentoffertilizationandcorticalgranule rendering itpermissive (ZP2intact)ornon-permissive (ZP2cleaved) ZP2 regulates thethree-dimensionalstructureofzonamatrix, scaffold’ modelofspermbinding,inwhichthecleavage statusof 934 before useinspermbindingandvitrofertilizationassays. sites ofpEGFP1(Clontech,Palo Alto,CA).Apurified from pXPZP3WT(Millaretal.,1991)andclonedintothe Ace EGFP)1NIH andTgN( treatment withthecalciumionophoreA23187(3 epididymis andincubatedundercapacitatingconditions(seebelow). After material) andwerefertileinvivo. Spermwereisolatedfromthecaudal accumulated EGFPintheiracrosomes(seeFig.S1Athesupplementary persistent spermbindingtohu bound towild-typeandhu model. Unexpectedly, spermacrosomesremainedintactwhen eggs andtwo-cell embryosaspredictedbythe ‘zona scaffold’ approved animalstudyprotocol. Institutes ofHealthunderaDivision ofIntramuralResearch,NIDDK guidelines oftheAnimalCareandUseCommitteeNational these studies.Allexperiments wereconductedincompliancewiththe (to befullydescribedelsewhere). TgN( EGFP inthecytoplasm ofgrowing oocytes andpreimplantationembryos (Rankin etal.,2003)toestablishtwo transgeniclinesthataccumulate fragment was injectedintothemalepronucleus offertilizedFVB/Neggs al., 2003).TgN( granule exocytosis (seeFig.S1Dinthesupplementarymaterial)(Rankinet supplementary material)andeggs fertilizedinvitrounderwentthecortical to ovulated eggs aseffectively aswild-typesperm(seeFig.S1Cinthe Fig. S1Binthesupplementarymaterial).Motile to whichAlexa 568-conjugatedsoybean trypsininhibitor(SBTI)bound(see underwent theacrosomereactionexposing theinneracrosomalmembrane MATERIALS ANDMETHODS penetrate thezonapellucida. the mechanismsbywhichfertilizingspermbind,acrosomereactand than 2and24hours,respectively. Thesedatahave implicationsfor Zp3 Using I- Acr3 Hin -EGFP transgenicmicewereestablishedusing RESEARCH ARTICLE -EGFP micewereplacedunderoil(IrvineScientific, SantaAna, dIII fragmentsofthemouse ZP2 Acr3 Acr3 -EGFP spermthataccumulatesolubleenhancedgreen transgenic andhu Acr3 -EGFP spermwereincubated(1,2, 4or24hours)with -EGFP)1NIH was usedinthesestudies. Acr3 ZP2 EGFP)2NIH, wereestablished.Each rescue eggs orembryosforgreater ZP2 ZP2 Zp3 transgenic andhu Zp3 rescue mice.We observed promoter (1.5kbp)isolated -EGFP)1NIH was usedin ␮ Acr3 mol/l), individual sperm -EGFP spermbound Bgl Bgl Bgl II- ZP2 II- Afl II- Ace II DNA Hin rescue Acr3 I and dIII 2 ) - color mode. respectively, usingAdobePhotoshop(AdobeSystems,SanJose,CA)RGB same blot,digitalimagesweredisplayedingreenorredchannels, Systems, Stamford,CT).To present mouseandhumanZP2resultsonthe acquired byLuminescentImageAnalyzerLAS-3000(FujiFilmMedical immunoblot (Rankinetal.,2003).Chemiluminescencesignalswere or withoutinvivo mating(Rankinetal.,1998)andwerepreparedfor et al.,1999). Tecnai 12transmissionelectronmicroscope(FEI,Hillsborrow, OR)(Rankin Thin sectionswerestainedwithuraniumandleadsaltsexamined ona knife onaRMCMTXultramicrotome(BoecklerInstruments,Tucson, AZ). Supplies, West Chester, PA) andthinsectionswerecutusingadiamond embedded inSpurr’s plastic(SPI-ChemLow Viscosity ‘Spurr’ Kit,SPI (55 were isolatedfromTgN( (Rankin etal.,1998).Asawash control intheseassays,two-cell embryos Millipore, Bedford,MA;12 opening inthefilter support.Polycarbonatefilters (1.2,3,5 incubation (37°C,5%CO was addedontopofthefilter intheupperchamber. Following a30 minute confocal microscopy. analysis ofglass-bead washed spermandconfirmed morphologically by acrosome-intact or-reactedspermon PI/EGFPdotplotwereselectedfrom forward scatterplotswerenotincluded.Regions usedto determine subcellular debris;events below cellularautofluorescenceonEGFPor discrete populationofcellswas selectedthatexcluded clumps aswell Dickinson BD,SanJose,CA).Using forward orsidescatterdensitydata, a analyzed byfluorescenceactivated cellsorting(FACS) (Calibur, Becton nuclei. paraformaldehyde inPBS,andstainedwithpropidiumiodidetovisualize upper andlower chambers,fixed withanequalamountof4% determined. SBTI-positive) andthetotalnumber(Hoechstpositive nuclei)ofspermwere image. Acrosome-intact(EGFP-positive), acrosome-reacted(Alexa 568- collected throughindividual embryosandprojectedontoasingle-plane fertilization withwild-type,hu After washing threetimeswithawideborepipette,embryosfrominvitro Electron microscopy transgenic andhu Eggs ortwo-cell embryoswereisolatedfrom4-week-oldwild-type,hu lmmunoblot analyses bound tothezonapellucida,consecutive 6 of Hoechst-positive spermnucleiwithintheegg cytoplasm. To countsperm M16 media(1hour, 37°C,5%CO and Hancock,1955),450 To enrichforacrosome-intactwild-typeand Filter penetrationassay strain), hu Ovulated eggs andtwo-cell embryoswereisolatedfromwild-type(FVB Sperm bindingassay by confocalmicroscopy (Rankinetal., 1999). EGFP spermbindingtothezonapellucidawas assessedafter1or24hours (2.1 blocked withapieceofplastic.Epididymal orglass-bead-washed sperm of thefilter support,a10mmopeningwas drilledandtheholderoutletwas removed toestablisha10mmopeningintotheupperchamber. Inthecenter (Millipore, Bedford,MA)holderfromwhichthesyringeconnectortipwas on acellometer(Nexcelom Bioscience,Lawrence, MA). media (2ml),spermwerecollectedinasecondfraction(4ml)andcounted the filter holder, 500 in thefilter support,ensuringtheexclusion oftrappedair. Afterassemblyof degassed inPBSundervacuum andplaced,glossysideup,over the opening Aliquots (400 The spermpenetrationassaychamberwas a25mmSwinnex Filter ϫ ϫ 8 mm)packed withthe250-320 10 4 ZP2 sperm, 850 rescue andhu ZP2 ␮ l) ofspermfromtheupperandlower chamberswere ␮ rescue miceafterstimulationwithgonadotropins ␮ l preincubated(1hour, 37°C,5%CO l) wereplacedinthelower chamberthroughthe Zp3 ␮ 2 ), 400 l epididymalspermwas appliedtoacolumn ZP2 ␮ -EGFP)1NIH mice.Wild-type and/or m pores,Whatman,FlorhamPark, NJ)were ZP2 transgenic miceforspermbindingassays 2 ␮ transgenic andhu ). Afterwashing thecolumnwithM16 l was removed sequentiallyfromthe ␮ m glassbeadsandequilibratedwith Acr3 ␮ m opticalsectionswere -EGFP sperm(Bangham Development 134(5) ZP2 rescue eggs were 2 ) M16media ␮ m pores, Acr3 ZP2 -

DEVELOPMENT 2, 4and24hoursafterinvitro fertilizationwithnormalsperm.Scalebar:20 ( contrast (DIC)andconfocalmicroscopy: (1-8)imagesofinvitro fertilizationwith insemination andstainedwithHoechst33342Alexa568-conjugated-soybeantrypsininhibitor(SBTI)before viewingbydiffere ( (>85%) were comparableamonggenotypesasjudgedbydecondensingHoechst-positivespermnucleiintheeggcytoplasm(datanots sperm heads;reacted acrosomes are indicated byred becauseofAlexa568-SBTIbindingtotheinneracrosomal membrane.Fertiliz eggs. Onlythegreen andred channelsofeachimageare displayed.Intactacrosomes are indicatedbygreen (EGFP)ontheanteri eggs; (9-16)sameas1-8,butafterinvitro fertilizationofhu represent linearregression ofaveragesnumberspermbindingtonormal(green), hu 1A, 5-6). or noacrosome-reacted(SBTI-positive) spermwereobserved (Fig. remained acrosome-intact(EGFP-positive) for2-3hours,andfew fragment ofZP2(Fig.1C). Unexpectedly, monoclonal antibodyspecific tothelarger (90 kDa),carboxyl initial cleavage ofmouseZP2,asdetectedbyimmunoblotsusinga 1A,B). Theobserved decrease inspermbindingcorrelatedwith observed over timeandfew boundtotheembryoafter4hours(Fig. decrease inthenumberofspermassociatedwithfertilizedeggs was normal zonaepellucidaeforseveral hours, but aprogressive normal, hu sperm occursshortlyafterbindtothezonapellucida of sperm approachtheegg, andacrosomeexocytosis ofthe‘fertilizing’ capacitating conditions.Duringinvitrofertilization,acohort of epididymal biological samples,8-18eggs each)andinseminatedwith5 from eachofthreegenotypes(threeindependentlyobtained gonadotropins tostimulateovulation, eggs were collected incumulus human ZP2rescuezonae(Rankinetal.,2003).Aftertreatmentwith ZP2 (huZP2),andhuZP2entirelyreplacesendogenousmoZP2in transgenic zonacontainsthesamethreemouseproteins,plushuman into 30kDaand90kDa)following fertilization.Thehuman moZP3), oneofwhich(moZP2)isproteolyticallycleaved (~120kDa ‘normal’) mouseeggs containsthreeglycoproteins(moZP1, moZP2, The zonapellucidasurroundingwild-type(hereafterreferredtoas Persistent spermbindingtohu RESULTS Induction ofspermacrosome exocytosis Fig. 1.Persistentspermbindingtohu C B Postfertilization cleavageofZP2detectedbyimmunoblotusingmonoclonalantibodytomouseZP2,andnormaleggsorembryosis ) Bar graph(meanands.e.m.)ofspermbindingtonormal(A,1-8)hu ) Acrosome-intact sperm(EGFP-positive) continued tobind ZP2 Acr3 transgenic, andhuZP2rescuemice. -EGFP spermthathadbeenincubatedunder ZP2 ZP2 rescue mouseembryosafterfertilization. rescue eggs Acr3 -EGFP sperm ZP2 transgenic eggs;(17-24)sameas1-8,butafterinvitro fertilizationofhu ϫ ZP2 10 5 ZP2 transgenic (A,9-16)andhu and outeracrosomal membranesimmediatelyapposed totheplasma to thezonapellucidawithintactacrosomes containedwithintheinner bound tonormalfertilizedeggs (2hourspost-insemination),adhering were imagedbytransmissionelectron microscopy (Fig.2).Sperm huZP2, moZP3)andhu (moZP1, moZP2,moZP3),hu sperm, embryos(3-5)derived frominvitrofertilizationofnormal To betterdefine theacrosomestatusofEGFP-andSBTI-positive pellucida Electron microscopy ofspermboundtothezona with ZP2-intactzonaepellucidae. exocytosis inducedbytheinitialinteractionsof ‘fertilizing’ sperm may provide mechanisticinsights,but isdistinctfromacrosome Fig. 1B).Thelateacrosomereactionofthese‘non-fertilizing’ sperm EGFP-negative andSBTI-positive by24hours(Fig.1A,9-16and exocytosis beginning 4hoursafterinsemination,withallbecoming transgenic eggs orembryos,however, underwentacrosome not sufficient toinduceacrosomeexocytosis. Spermboundtohu and spermbindingtonormalhu structure towhichspermwillbind fertilization. Thus,thepresenceofintacthumanZP2provides azona intact acrosome(EGFP-positive) forupto24hoursafterinvitro with hu numbers weremodestornon-existent (Fig.1B).Sperm associated females (Fig.1A)andbyregression analysis,changes insperm early embryosderived fromhu ␮ m. More strikingly, spermboundfor Acr3 ZP2 -EGFP sperm(5 rescue-derived embryos(Fig.1A,17-24)maintainedan ZP2 ( A transgenic (blue)orhu ) Embryoswere collected1,2,4and24hoursafter ϫ ZP2 10 ZP2 5 ) andwild-type(referred toas‘normal’) rescue eggs (moZP1,huZP2, moZP3) rescue (A,17-24)embryos.Dottedlines ZP2 ZP2 ZP2 у у RESEARCH ARTICLE transgenic andhu transgenic (moZP1,moZP2, ZP2 24 hoursdespitefertilization, 24 hourstofertilizedeggs and rescue eggs orembryoswas rescue (red) embryos. or surfaceof ntial interference ation rates olated 0,1, ZP2 hown). ZP2 rescue rescue ZP2 935

DEVELOPMENT IAM, inneracrosomal membrane.Scalebar:1 rescue embryos(12hourspost-insemination)withanintactacrosome. insemination) havingundergoneacrosomal exocytosis;and( with anintactacrosome; ( (referred toas‘normal’)mouseembryos(2hourspost-insemination) three-dimensional matrixarchitecturethatispermissive for sperm been ascribedtotheabilityofuncleaved humanZP2 topreserve a persistence ofspermonthesurface ofhu Normally, spermdonotbindtotwo-cell embryos, andthe cell embryos Quantification ofdenovo spermbindingtotwo- early embryoscontaininghumanZP2inthezonapellucida. and bothacrosome-intactacrosome-reactedspermcanbind to provide realisticproxiesoftheultrastructuremouseacrosome, controls. Thus,confocalimagesofEGFP-andSBTI-positive sperm and boundbyintactplasmamembrane(Fig.2C),muchlike normal insemination) weremostlyacrosome-intact(42of51sperm,82%), sperm thatboundtohu sperm observed byconfocalmicroscopy (Fig.1A,16).Bycontrast, fenestrations andconsistentwiththesimilarlyboundSBTI-positive 2B), presumablyleadingtolossofEGFPthroughtheresulting plasma membraneandouteracrosomalmembraneshadfused(Fig. insemination), but forthemostpart(31of34sperm, 91%)their EGFP spermboundto:( sperm bindingtomouseembryos. also boundtohu membrane overlying thespermhead(15of15sperm,Fig.2A).Sperm Fig. 2.Transmission electron photomicrographs of 936 RESEARCH ARTICLE ZP2 A ) thezonapellucidasurrounding wild-type B ) hu ZP2 transgenic embryos(12hourspost- ZP2 rescue embryos(12hourspost- transgenic embryos(12hourspost- After invitro fertilization, ZP2 ␮ m. rescue embryoshas Acr3 C -EGFP ) hu Acr3 ZP2 - Additional rescue micewereflushedfromoviducts afterinvivo mating. samples, teneggs each)was similar (100±10,112±8,103±5, hu The numberofspermbinding tonormal,hu and two-cellembryos Reversibility ofspermbinding toovulatedeggs hours withoutinducingtheacrosome reaction. hu embryos andcomparablenumbersboundtoderived from adherent sperm(Fig.3A,1,4,7),noboundtonormaltwo-cell EGFP two-cell embryosaswash controls. samples, tenembryoseach)inaspermbindingassayusing fertilized two-cell embryos(threeindependentlyobtainedbiological additional 24hours,almostallofthespermboundtohu positive) (Fig.3A,4-9).Iftheembryoswereincubatedforan evidence ofhaving undergone theacrosomereaction(SBTI- remained acrosome-intact(EGFP-positive), withfew showing derived fromboththehu respectively). Virtually allofthespermassociatedwithembryos cell embryosderived fromnormal,hu binding (Rankinetal.,2003).To furtherexamine thismodel,two- mouse ZP2issufficient tosupportmousespermbindingfor (Rankin etal.,2003).Thus,uncleaved humanZP2intheabsenceof differences inthetwo homologsprevent cleavage ofhumanZP2 within the‘humanized’ zonaarchitectureandsuggeststhatintrinsic mouse, but nothuman,ZP2inhu by theegg following fertilization.Theability oftheproteasetocleave 1-4). Normally, ZP2iscleaved byacorticalgranuleproteasereleased antibodies crossreactedwithZP2fromtheotherspecies(Fig.4,lanes mice (Fig.4,lanes3-6).Neitherthemouse-norhuman-specific from normal,hu of mouseandhumanZP2eggs andtwo-cell embryosderived To investigate themechanismofthisdichotomy, thecleavage status proteins Cleavage statusofmouseandhumanZP2 are presentinthezonamatrix. ZP2, but doesoccur, albeitdelayed,ifbothmouseandhumanZP2 acrosome reactionwhenhumanZP2replacesendogenousmouse (up to24hours)thezonapellucidaisnotsufficient toinducethe the acrosomereaction(Fig.3B,1-3).Thus,persistentspermbinding sperm boundtothehu zonae remainedacrosome-intact(Fig.3B,4-6),whereasallofthe embryos derived fromhu contrast, humanZP2remaineduncleaved inbotheggs andtwo-cell and hu was intactinnormalandhu carboxyl terminusofmouse(green)orhumanZP2(red), immunoblots (Fig.4)usingmonoclonalantibodiesspecific tothe hu (Moller andWassarman, 1989;Bauskinetal.,1999). ~35 kDa,65kDaforhuman)thatremainlinked bydisulfide bands fertilization resultingintwo fragments(~30kDa,90kDaformouse; modifications. EachZP2proteinisnormallycleaved following 110 kDa,respectively) duetodifferences inpost-translational identical, but theirmolecularmassesdiffer (120-140kDaand90- (599 aminoacids)andhumanZP2(602are62% examined (Fig.4).Theprimarystructuresofsecreted mouse ZP2 ZP2 After washing withawide-borepipettetoremove loosely Eggs andtwo-cell embryos(10-15)wereisolatedfromnormal, ZP2 ZP2 ZP2 transgenic andhu transgenic andhu rescue eggs (threeindependently obtainedbiological Acr3 transgenic two-cell embryos(Fig.4,lanes1,2,5,6).By -EGFP sperm(5 ZP2 transgenic andhu ZP2 ZP2 ZP2 ZP2 ZP2 transgenic zonaepellucidaeunderwent ZP2 transgenic andhu transgenic eggs andcleaved innormal transgenic andhu ZP2 ϫ rescue mice.Asdeterminedby rescue eggs (49±8and73±15, 10 transgenic miceindicatesaccess 5 ) wereaddedtotheseinvitro ZP2 ZP2 rescue femalemicewas transgenic andhu Development 134(5) ZP2 ZP2 ZP2 transgenic and rescue female rescue eggs ZP2 rescue Zp3 у ZP2 24 -

DEVELOPMENT hu embryos were acrosome-reacted (97%),andthose bindingtohu challenged with rinsed byserialpassagethroughthreedropsofmedia(30 with Hoechstdye(Fig.5,1,2,5,6,9,10).Thefertilizedeggs were respectively) ina1hourbindingassaywhichspermwerelabeled Induction ofspermacrosome exocytosis Fig. 3.Denovo Zp3 (1-3, 49±8s.e.m.perembryo)andhu respectively. ( (1,4,7) andconfocalmicroscopy toobserveEGFP(2,5,8)orSBTIbinding(3,6,9),reflecting acrosome-intact andacrosome-reacte previously observed (Fig.lA),thespermbound tohu rescue embryos(48±9,56±7,respectively) (Fig. 5,13,14,17,18).As numbers ofsperminitiallyboundtohu bound tonormalembryos(datanotshown) andcomparable from eachgenotype.Asexpected fromprevious results,nosperm derived byinvitrofertilizationwithnormal spermandeggs isolated independently obtainedbiologicalsamples,tenembryoseach) reversible 1hourafterinsemination. and hu of spermboundtothezonapellucidanormal,hu 4,8,12). Thus,undertheseassayconditions,afairly constantnumber bound spermhadbeenreplacedwith (92±12, 115±17,104±22,respectively), 84-88%oftheinitially total spermboundnormal,hu controls (Fig.5,3,4,7,8,11,12).Althoughcomparablenumbersof washing with0.2mmpipettesusing rescue zonaepellucidaewereacrosome-intact. zonae pellucidaewereacrosome-reacted,andthoseboundtohu numbers ofspermremainedbound tohu controls) afteranadditional hour ofincubation.Comparable were addedandembryoswashed (using egg, hu the zonapellucidaisreversible bothforacrosome-intact(normal (>90%) wereEGFP-positive (Fig.5,15,16,19,20).Thus,bindingto rescue embryos(59±10,68±16, respectively), andineachcasemost embryos) andacrosome-reacted (hu ZP2 A similarassaywas performedwithtwo-cell embryos (three -EGFP two-cellmouseembryoswere usedascontrols (insets1,4,7).Embryoswere stainedwithAlexa568-SBTIandfixedbefore im transgenic (4-6)andhu ZP2 ZP2 rescue eggs, andthebindingwas almostcompletely B ) Aliquotsofembryosfrom Awere incubatedforanadditional23hours,andsimilarnumbersofspermboundtohu transgenic egg, hu Acr3 Acr3 -EGFP spermbindingtotwo-cellembryos. -EGFP sperm(1hourincubation)followed by ZP2 ZP2 rescue (7-9)micewere incubatedwith5 ZP2 transgenic andhu ZP2 ZP2 Zp3 rescue egg, hu rescue (4-6,73±15s.e.m.perembryo)embryos.However, spermbindingtohu Acr3 -EGFP two-cell embryosas transgenic embryos) sperm. ZP2 ZP2 -EGFP sperm(Fig.5, transgenic andhu transgenic andhu Zp3 Acr3 ZP2 -EGFP two-cell ZP2 ZP2 -EGFP sperm ZP2 rescue eggs transgenic transgenic ␮ rescue ZP2 l) and ZP2 ZP2 ZP2 -rescue-derived embryosremained acrosome-intact (94%).Scalebars:30 ( A ) Two-cell embryosderivedfrom wild-type(referred toas‘normal’)(1-3), ϫ mice. AfterSDS-PAGE rununderreducing conditions(5% exocytosis byasignaltransductionpathway predicatedonazona embryos appearsinconsistentwiththeinductionofacrosomal for 2-3hourswithnormalor The abilityof Mechanical inductionoftheacrosome reaction on theleft. antibody andchemiluminescence. Molecularmasses(kDa)are indicated green) orhuman(fauxcolored red) ZP2andvisualizedwithasecondary antibodies specifictothecarboxyl fragmentofmouse(fauxcolored mercaptoethanol), immunoblots were incubatedwithmonoclonal hu (lanes 2,4,6)from wild-type (referred toas‘normal’)(lanes1and2), fertilization. Fig. 4.Mouse,butnothuman,ZP2iscleavedfollowing 10 ZP2 5 motile, capacitated rescue (lanes3and4) hu Immunoblot ofeggs(lanes1,3,5)andtwo-cellembryos Acr3 -EGFP spermtobindandremainacrosome-intact Acr3 -EGFP spermfor1hourandwashed; у 24 hourswithhuZP2rescueeggs or ZP2 RESEARCH ARTICLE transgenic (lanes5-6)female ZP2 -transgenic-derived d sperm, ZP2 ␤ aging byDIC transgenic - ␮ 937 m.

DEVELOPMENT decreased, thenumber ofsperminthelower chambergradually acrosome-reacted, respectively (Fig.6B).Asthefilter poresize alone andconfirmed morphologicallyasacrosome-intactand were observed according tothepresenceofEGFPandDNA orDNA (nuclear stain)andanalyzedby FACS. Two populationsofsperm the filters wereobtained, fixed, treatedwithpropidiumiodide After incubation(30minutes), aliquots ofspermfromeachside incubation afterwashing. (4,8,12,16,20) reflect thosethatwere notdisplacedbyEGFP-sperm.Insets(3,7,11,15,19), were modifiedinAdobePhotoshoptoremove nuclearstainingfrom EGFP-positivesperm;thus,Hoechst-positive,EGFP-negativespe and confocalmicroscopy toobserveSBTIandEGFP(4,8,12,16,20),reflecting acrosome-reacted andacrosome-intact sperm,respect hour. Afterwashingtoremove non-adherent sperm,eggs andembryoswere stainedwithAlexa568-SBTIbefore imagingbyDIC(3,7, inert filters withporesizesrangingfrom1.2to12 penetration assayinwhichtheprogressofspermwas impeded by independently obtainedbiologicalsamples)wereusedinafilter- acrosome-intact. Theseglass-bead-treatedsperm(three and Hancock,1955),resultedinapopulationthatwas 90-95% acrosome-reacted, non-viablespermpreferentiallybind(Bangham intact. However, passagethroughacolumnofglassbeads,towhich conditions, ~70%ofepididymal ligands (Fig.6).Normally, after1hourofincubationincapacitating with inertpolycarbonatefilters unadornedwithzona pellucida This possibilityhasbeeninvestigated usingafilter penetrationassay presence ofonlyacrosome-reactedspermintheperivitelline space. signal transduction,leadingtoexocytosis, andaccountforthe penetration intothezonamatrixmighttriggeramechanosensory ligand bindingtoaspermsurface receptor. However, initial Fig. 5.Reversiblespermbindingtowild-type,hu 938 (1,2), hu confocal microscopy (2,6,10,14,18).Afterabriefrinse,5 with normalspermfor1hour(1,5,9)or24hours(13,17)andstainedAlexa568-SBTIHoechstbefore imagingbyDIC(1,5 RESEARCH ARTICLE ZP2 transgenic (5,6)andhu Acr3 ZP2 -EGFP spermwereacrosome- rescue (9,10)eggsorhu ZP2 ␮ ϫ m (Fig.6A). transgenic andhu 10 5 motile, capacitated ZP2 transgenic (13,14)andhu increased from1.5to1.9 (13-15%) asporesizesdecreased from12 acrosome-reacted sperminthe lower chambersremainedconstant (Fig. 7B).However, nonewas observed andthepercentof chambers ofthesmaller, comparedwiththe larger, pore-sizefilters a decreaseinthepercentofacrosome-reactedspermlower the upperchamberreflected‘preferentialpassage’,thereshould be sperm head(Fig.2).Ifthepresenceofacrosome-reacted in exocytosis doeslittletodecreasethemaximalwidth ofthemouse interpretation oftheseexperimental results,even thoughacrosome reacted spermthroughthepolycarbonatefilters mightaffect 93±5.8% with1.2 sperm increaseddramaticallyto85±5.3%infilters with 3 size (Fig.6C,Fig.7A),but theproportionofacrosome-reacted chamber afterpassagethroughfilters decreasedwith smallerpore Fig. 7B).Asexpected, thenumberofspermrecovered intheupper exocytosis remainedcomparable,rangingbetween13-15%(Fig.6C, minutes inthe filter penetrationassay. Inthreeindependently with glassbeads,theotherwas not,andeachwas observed after30 same epididymalspermsample were compared.Onewas pretreated ZP2 However, ‘preferentialpassage’ ofspontaneouslyacrosome- In addition,two capacitated Acr3 rescue eggsandembryos. -EGFP spermwere addedandincubatedforanadditional1 Zp3 EGFP control two-cellmouseembryosfrom each ZP2 ␮ m pores(Fig.6C,Fig.7B). rescue (17,18)two-cellembryoswere incubated ϫ 10 4 (Fig. 7A),but spontaneousacrosome Acr3 Wild-type (referredWild-type toas‘normal’) -EGFP spermaliquotsfromthe ␮ m to3 Development 134(5) ␮ ,9,13,17) and ively. Images rm m (Fig.7B). 11,15,19) ␮ m and

DEVELOPMENT through a3 status ofepididymal(left)andglass-bead-treated (right)spermwasassayedbyFACS before (lowerchamber)andafter(upperch were isolatedandcapacitatedfor1houranaliquotwaspassedoveraglassbeadcolumntoremove acrosome-reacted sperm.T individual filters(pore size,1.2-12 was confirmedbyconfocalmicroscopy (right).( stained withpropidium iodideanddividedintoacrosome-intact and-reacted populationsbyFACS (left).Theacrosome statusof chamber) penetrationofindividualpolycarbonatefilters(pore size,1.2-12 placed inthelowerchamberandincubatedfor30minutes.Thespermacrosome statuswasassayedbefore (lowerchamber)andafte compared withtheglass-bead-treated, increased numberofspermwould beanticipatedintheuntreated, ‘preferential passage’ ofspontaneouslyacrosome-reactedsperm,an acrosome-reacted spermintheupperchambermerelyreflected acrosome-reacted intheupperchamber(Fig.6D).Ifpresenceof reacted, whereasmorethan90%ofspermfromeachsamplewere the glass-bead-treatedsperminlower chamberwereacrosome- obtained biologicalsamples,21-31%oftheuntreatedand5-7% Induction ofspermacrosome exocytosis Fig. 6.Passagethrough inertpolycarbonatefiltersinducesthespermacrosome reaction. the single‘fertilizing’ sperm rapidlyundergoes acrosomal membrane overlying theiranteriorhead.Currentdatasuggestthat cohort ofcapacitatedspermbinds tothezonamatrixviaplasma induction oftheacrosomereaction. Duringinvitrofertilization,a permissive for spermbindingtoovulated eggs andsubsequent Mouse fertilizationrequires a zonapellucidathatisinitially DISCUSSION reaction duringnormalfertilization(Fig.8). forces mayhave physiologicalimportforinductionoftheacrosome acrosome reactionandraisethepossibilitythatsimilarmechanical of motilespermthroughtheinertmatrixissufficient toinducethe samples. Taken together, theseobservations suggest thatthepassage despite fourfoldmoreuntreatedthantreatedsperminthestarting respectively) betweentheglass-bead-treated anduntreatedsamples, acrosome-reacted spermwas similar(318.3±202.6,141.7±81.0, However, nosuchincreasewas observed, andthenumberof ␮ m pore-size polycarbonatefilter. ␮ m), were fixed,stainedwithpropidium iodideandscored byFACS foracrosome status.( Acr3 -EGFP spermsamples. C ) Acr3 -EGFP sperm,isolatedbefore (lowerchamber)andafter(upperpassagethrough ␮ m); twoexamplefiltersare imagedontheright.( embryo (Wassarman etal.,2005;Shur etal.,2006).The‘zona toaccountfortheabsenceof spermbindingtotheearly granules, removed byaglycosidase releasedpostfertilizationbyegg cortical postulates thatspermbindto acarbohydrateligand,whichis dichotomy, two modelsareconsidered.The‘glycanrelease’ model embryos, ispermissive forspermbinding.To accountforthis The zonapellucidasurrounding ovulated eggs, but nottwo-cell Sperm-egg recognition sperm following fertilization. the processbywhichzonamatrixbecomesimpenetrable by mechanism bywhichthespermacrosomereactionisinduced; and the molecularbasisofspermbindingtozonapellucida; the non-permissive forspermbinding.Majorunresolved issuesinclude: more gradualenzymaticcleavage ofZP2rendersthezonamatrix bound spermareunabletopenetratethematrix.Within hours,the zona pellucidaisalteredinayet-to-be-determinedmanner, andthese their acrosomesintact.However, shortlyfollowing fertilizationthe continues toassociatewiththesurface ofthezonapellucida with equatorial region. Therestofthecohort(‘non-fertilizing’ sperm) ‘fertilizing’ sperm fusestotheegg viathemembraneofposterior penetration ofthezonamatrix.Onceinperivitelline space,the into scything movements ofthedenseheadthatfacilitates complete zona interaction.Theforward motilityofthespermthentranslates exocytosis andtheresultantacrosomalshroudstabilizessperm- ( A ) Capacitated Acr3 RESEARCH ARTICLE -EGFP sperm(2.1 D B ) Fixedspermwere ) Epididymalsperm amber) passage each population he acrosome r (upper ϫ 10 4 ) were 939

DEVELOPMENT dwell timeofacrosome-intact spermonthesurface onthezona and again,spermremainacrosome-intact (Fig.1A,17-24).Thelong EGFP spermbindingpersistsfor atleast24hoursafterinsemination, hu for several hours (Fig.1A,1-8).Ineggs andembryosderived from bound tothesurface ofthe zonapellucidaremainacrosome-intact with partialproteolyticcleavage ofmoZP2.Thosesperminitially in vitrofertilization,but bindingisabsentafter4hours andcorrelates Acr3 Acrosome reaction egg recognition. matrix shouldprovide insightsintothemolecularbasisofsperm- relationship withinthesupramoleculararchitectureofzona definition ofindividual zonapellucidaproteinsandtheir cognate bindingsite,andfurtherhighresolutionstructural the zonapellucidaprobablyplaysacrucialroleinforming the 1999; Rankinetal.,2003).Thus,thethree-dimensionalstructure of as aspermadhesionmolecule(Rankinetal.,1998;Rankin al., replaced withhumanhomologsdonotsupportasinglezonaprotein genetic studiesinwhichindividual zonaproteinsareabsentor Boja etal.,2003;Lowe andMarth,2003;Shietal.,2004), al., 1995;LuandShur, 1997;Elliesetal.,1998;Easton2000; carbohydrate ligand(Nagdasetal.,1994;Liu1995;Thall increasingly severe limitsontherangeofpossibilitiesforasingle or both.However, biochemicalandgeneticresultshave placed contacts betweenspermandegg arebasedonprotein,carbohydrate al., 2003). postfertilization spermbindingtohu model byconfirming earlierobservations ofpersistent, with the‘glycanrelease’ modelandsupportsthe‘zonascaffold’ hu of denovo zona matrixbecauseofintactZP2.Theexperimental observation the glycanligand,whereassecondpredictsspermbindingtoa predicts nospermbindingbecauseofpostfertilizationrelease cortical granuleexocytosis (Jungnickel etal.,2003).Thefirst from hu concerning denovo spermbindingtotwo-cell embryosderived Dean, 2004).Thesetwo modelsmake disparatepredictions permissive bythepostfertilizationcleavage ofZP2(Hoodbhoy and is initiallypermissive forspermbindingandisrenderednon- scaffold’ modelhypothesizesathree-dimensionalzonamatrixthat 940 .Nme fSperm of Number A. ZP2 Of note,the‘zonascaffold’ modeldoesnotdistinguishwhether ZP2

-EGFP spermbindreversibly tonormaleggs andembryosafter Total sperm 12,000 17,000 22,000 1,100 1,650 2,200 rescue miceinwhichhuZP2isnot cleaved, reversible transgenic andhu RESEARCH ARTICLE 550 ZP2 0 Acr3 itrPr ie(m) ( Size Pore Filter rescue miceinwhichZP2remainsintactdespite 12 -EGFP spermbindingtoembryosderived from 5 Chamber Upper 31.2 Chamber Lower ZP2 µ rescue mice(Fig.3)isnotconsistent .Arsm ttso Sperm of Status Acrosome B.

ZP2 Acrosome reacted (%) 100 25 75 50 0

rescue eggs (Rankinet 38% Controls

Epi di dym al Tre (28 ated %) (6% ) oe Chamber Lower pe Chamber Upper

12 14%

itrPr ie(m) ( Size Pore Filter 10%

Acr3 15% 5 11% -

13% 3 reacted iftheporesizeswere differing glycanisoforms. multiple spermsurface receptorsarerequiredtoaccommodate all glycanligandsataparticularattachmentsiteareutilizedor 2006) furthercomplicatesthesemodelsbysuggestingthateithernot residues) inthezonapellucida(Eastonetal.,2000;Chalabi heterogeneity ofO-glycansidechains(1-7monosaccharide vivo fertilityintransgenicmice(Liuetal.,1995).Theobserved and mutationofthesitestoprecludeglycosylationdoesnotaffect in by microscalemassspectrometryofnative ZP3(Bojaetal.,2003) glycans implicatedaszonaligands(Chenetal.,1998)isnotdetected 1997). Moreover, occupancy ofputative attachmentssitesbyO- (Thall etal.,1995;LuandShur, 1997;Asanoetal.,Liu ablation ofleadingcandidatesdoesnotprevent invivo fertility or thespermsurface receptoraswelltheobservation thatgenetic eroded bytheinabilitytodefine definitively eitherthezonaligand Confidence intheroleofO-glycansacrosomeexocytosis hasbeen surprisingly sparse(Nagdasetal.,1994;Boja2003). (Rankin etal.,1999),andO-linked glycosylationofZP2andZP3is Leyton andSaling,1989).However, ZP1isnotrequiredforfertility inducers oftheacrosomereaction(BleilandWassarman, 1983; signal transductionpathway. receptor toinduceacrosomalexocytosis viaaclassicligand-receptor is seeminglyinconsistentwithazonaligandinteractingsperm pellucida ofnormal(2-3hours)andhuZP2rescue( sperm intheupper chamber(Fig.7B)asthepore sizedecreased(2 exocytosis. The dramatic thresholdincreaseinacrosome-reacted shear forcetoelicitamechanosensory signalandacrosome contact betweenmotilesperm and filter would generatesufficient (Wyrobek etal.,1976;Cummins andWoodall, 1985). Atthatpoint, approaches thesizeofacrosome-intact sperm(~3.5 would belittle ornophysicalconstraintuntiltheporesize(3 in virtuallyallsperm.This‘induction’ modelpredictsthatthere zona pellucidamatrixmechanically‘induces’ acrosomeexocytosis data isthatpassagethroughfilter porescomparable insizetothe if poresizeswere1.2 zona ligand,93±5.8%ofthespermunderwentacrosomeexocytosis by passagethroughinertpolycarbonatefilters. Inthe absence ofany Acr3 explored asapossiblealternative. Inthefilter penetrationassay, exocytosis, initialpenetrationintothezonapellucida matrixwas pellucidae isseeminglynotsufficient toinduceacrosome

µ 85% Because spermbindingtonormalorhu O-glycan ligandsinthezonapellucidahave been implicatedas

1.2 14% -EGFP spermwereinducedtoundergo theacrosomereaction 93% determined byFACS andconfirmedmorphologically. sperm inthelower(blue)andupper(red) chamberswas penetration assay, thepercent ofacrosome-reacted (controls) byFACS. Followingthe30minutesfilter (treated) passagethrough theglassbeadcolumn sperm wasdeterminedbefore (epididymal)andafter penetration assay. Fig. 7.Quantificationofpolycarbonatefilter ␮ passage through filterswithpore sizes1.2,3,5and12 141.7±46.8, 1356.7±165.9,and2165.0±188.0,after recovered from theupperchamberwas123.3±38.1, by FACS andhandcounting.Thenumberofsperm s.e.m.) inthelowerandupperchamberswasdetermined polycarbonate filters.Theaveragenumberofsperm(± EGFP spermwere assayedforpenetrationthrough m, respectively. ( ␮ m, whereasonly10-11%wereacrosome- у B 5 ) Theacrosome statusof ␮ ( A m. Asimpleexplanation ofthese ) Approximately 2.1 Development 134(5) ZP2 у 24 hours)mice rescue zonae ϫ Acr3 10 4 ϫ Acr3 -EGFP 8 ␮ ␮ - m) m)

DEVELOPMENT leads toincreased intracellularCa forward motilityofthespermtransducesamechanosensorysignalthat inhibiting furtherprogression ofthesperm.However, the continued size ofthematrixinterstices)immobilizesspermplasmamembrane, approach andbindtothezonapellucida.Thisbinding(orlimiting mechanosensory signalthatleadstoincreased intracellularCa the filter. Thecontinuedforward motilityofthespermtransducesa size ofthepore, thespermisslowedorstoppedasitseekstopenetrate the inertpolycarbonatefilterlackingzonaligands.Dependingon can bedirect,as instretch-activated channels,orindirect, whereby forces imposedonthelipidbilayer toregulate ionfluxes. Thiseffect transducing asignal,mechanically gatedchannelscanrespondto (Kung, 2005).Unlike thelockandkey bindingofligandtoreceptor molecular detailshave beenmoreextensively described inbacteria signaling iswidespreadamong plantsandanimals,although induction oftheacrosomereaction (Fig.8).Mechanosensory enter intotheperivitellinespace. surface ofthezonapellucidamatrix,andonlyacrosome-reacted sperm reaction. Theresidual acrosomal shroud isleftbehindboundtothe side ofthefilter. ( smaller pores, mostlyacrosome-reacted sperm are observedonthefar induction oftheacrosome reaction. Afterpassagethrough 3 ␮ Induction ofspermacrosome exocytosis reaction. Fig. 8.Modelofmechanosensoryinductionspermacrosome mobilization ofacrosomalCa thrusting ofthetailtransducesamechanosensorysignalleading to stops theforward progressionofmotilespermandthe vigorous zona pellucida(orthelimitingsizeofmatrixinterstices)slows or exocytosis. reacted) isconsistentwiththe‘induction’ modelofacrosome m) from5 If physiologicallyrelevant, theseresults suggestthatbindingtothe .Zn Pellucida Zona B. .PlcroaeFilter Polycarbonate A. Perivitelline ( A Inside

N N

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c ) Capacitated,motileandacrosome-intact spermapproach

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and inductionoftheacrosome stores (Herricketal.,2005)and

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␮ c c Shroud r Nucleus r Nucl o o s s o o m (85%acrosome- m m eus e e idn rConstrain or Binding Signal Signal hsclConstraint Physical Mechanosensory Mechanosensory owr Motility Forward Motility Forward nue AR Induced Binding Sperm-Zona Key Transduction Transduction ␮ 2+ m or and t complete cleavage takes >4hours(Fig. 1C)andhu two fragmentsthatremaindisulfide-bonded (Bleiletal.,1981), fusion. Althoughthisblockhasbeenascribedtocleavage ofZP2into 2000), indicatesthattheblocktopenetrationoccursaftergamete deficient insperm-egg fusion(LeNaouretal., 2000;Miyadoetal., supernumerary spermwithintheperivitelline spaceof penetration israpidaswell.Theobserved accumulation of space (Sato,1979)indicatesthatthepostfertilizationblocktozona the observation offew, ifany, supernumeraryspermintheperivitelline to polyspermyattheplasmamembraneofegg (Wolf, 1978)and Bedford, 2004).Aftergametefusion,thereisaprompt,effective block membrane oftheegg occurswithinminutes(Tollner etal.,2003; acrosome reaction,zonapenetrationandfusionwiththeplasma is rapid,andthebindingtozonamatrix,inductionofsperm egg. Theinteractionofthe‘fertilizing’ spermwiththezonapellucida penetrating thematrixandfusingwithplasmamembraneof The ‘fertilizing’ spermsucceedsbybindingtothezonapellucida, Block tozonapenetration sperm playaroleintheacrosomereactionremainstobedetermined. other TRPCmechanosensorysignaltransducerspresentinmouse the humanhomologisapseudogene(Wes etal.,1995).Whetherthe TRPC2 remainfertile(Stowers etal.,2002;Leypold etal.,2002)and exocytosis (Arnoultetal.,1999;Jungnickel etal.,2001),micelacking been incorporatedintoaligand-receptormodelofacrosome (Jungnickel etal.,2001;Trevino etal.,2001).AlthoughTRPC2has (Neill etal.,2004)andTRPCl,2,35arepresentinmousesperm of theacrosomereaction 2005). Seaurchinpolycystin-2 hasbeenimplicatedintheinduction of thetransientreceptorpotential(TRP)family (BarrittandRychkov, (Orr etal.,2006),includingpolycystins (Delmas,2004)andmembers force. Multiplemechanotransducershave beendescribedinanimals cytoskeletal orextracellular matricesaugmentminorperturbationsin matrix. chemoattraction orchemotaxis andtheinabilitytopenetrate the surface ofthezonapellucida mayreflectaconflictbetween Thus, theperceived, reversible bindingof‘non-fertilizing’ spermon because oftherapid,effective postfertilizationblocktopenetration. chemoattractant, but be unabletoprogressthroughthezonamatrix sperm would remain under theinfluenceofegg’s bind tothezonapellucida.Inthisformulation,‘non-fertilizing’ mechanosensory signaltransductionwithoutrequiringthatsperm ‘fertilizing’ spermcouldinduceacrosomeexocytosis by Giojalas, 2006),thentheinitialpenetrationofzonamatrixby the (Cohen-Dayag etal.,1995;Spehr2003;Eisenbach and sperm areattractedtotheegg bychemoattractantguidancesystems the ‘fertilizing’ spermthroughthezonapellucida.Alternatively, if glycoprotein(s) seeminglywould impedetheforward progressionof invoking repeatedcycles ofattachmentandrelease,bindingtozona Wassarman, 1980a;Bleil andWassarman, 1986).However, without (Hartmann etal.,1972;HartmannandHutchison,1974;Bleil and ‘secondary’) spermbindingtothesurface ofthezona matrix has beendescribedas‘loose’ andthen‘tight’ (or‘primary’ and sperm onthezonapellucida(Fig.5).Initialsperm-egg recognition accumulation ofreversibly adherent,‘non-fertilizing’ rapidly modifythezonapellucidatoprevent spermpenetration. stored materials,presumablysmallandhighlydiffusible, fromtheegg cleavage ofZP2,anditseemslikely thatpostfertilizationreleaseof penetration appearstobeindependentoftherelatively late-occurring zona penetration(Rankinetal.,2003).Thus,theblockto which ZP2isnotcleaved have aneffective postfertilizationblockto The rapidblocktozonapenetrationmayaccountforthe Strongylocentrotus purpuratus RESEARCH ARTICLE ZP2 Cd9 rescue micein Acr3 -null eggs, -EGFP sperm 941

DEVELOPMENT Boja, E.S.,Hoodbhoy, T., Fales,H.M.andDean,J. Bleil, J.D.,Beall,C.F. andWassarman, P. M. Chalabi, S.,Panico, M.,Sutton-Smith,Haslam,S. Patankar, M.S., Bedford, J.M. Arnoult, C., Kazam, I. G., Visconti, P. C.,Kazam,I.G.,Visconti, Arnoult, M.andFlorman, E.,Kopf,G.S.,Villaz, Abou-Haila, A.andTulsiani, D.R. References http://dev.biologists.org/cgi/content/full/134/5/933/DC1 Supplementary materialforthisarticleisavailableat Supplementary material Health, NlDDK. supported bytheIntramuralResearch Program ofthe NationalInstitutesof the laboratory andcolleaguesinthefieldaswellinitialcharacterizationof We appreciate themanythoughtfuldiscussionswithmembersof will requirefurtherinvestigations. sperm toadhereembryos.Thevalidity ofthese disparate models postfertilization lossoftheglycanaccountingforinability binding aswelltheinductionofacrosomeexocytosis, withthe release’ models,inwhichZP3glycansactasligandsforsperm induction ofacrosomeexocytosis. Thismodeldiffers from‘glycan zona matrixaseliciting‘mechanosensory’ signalssufficient for model hasbeenextended toimplicateinitialpenetrationintothe transduction inacrosomeexocytosis. Thus,the‘zonascaffold’ with widelyembracedinvolvement ofligand-receptorsignal normal andhu sperm toremainacrosome-intactdespitehours-longadherence fertilization andcorticalgranuleexocytosis. Theobserved abilityof permissive (ZP2cleaved) forspermadherence,independentof dimensional zonamatrixwillbepermissive (ZP2intact)ornon- in whichthecleavage statusofZP2determineswhetherthethree- These datasupporta‘zonascaffold’ modelofsperm-egg recognition Conclusions 942 Bauskin, A.R.,Franken,D.Eberspaecher, U.andDonner, P. Bleil, J.D.andWassarman, P. M. Bleil, J.D.andWassarman, P. M. Bleil, J.D.andWassarman, P. M. Bleil, J.D.andWassarman, P. M. Asano, M.,Furukawa,K.,Kido,Matsumoto,S.,Umesaki,Y., Kochibe,N. Barritt, G.andRychkov, G. Bangham, A.D.andHancock,J.L. Barros, C.,Bedford, J.M.,Franklin,L.E.andAustin, C.R. 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