0013-722705$15.000 Endocrinology 146(12)5313–5320 Printed in U.S.A. Copyright 2005 by The

Endocrinology146(12):5313–5320

Copyright©2005byTheEndocrineSociety

doi:10.1210/en.2005-0684

DecidualDifferentiationofStromalCells

PromotesProproteinConvertase5/6ExpressionandLeftyProcessing

MeiyiTang,AnatolyMikhailik,IlsePauli,LindaC.Giudice,AsgerallyT.Fazelabas,SuzanaTulac,DanielD.Carson,DavidG.Kaufman,ClaireBarbier,JohnW.M.Creemers,andSiamakTabibzadeh

DepartmentofObstetricsandGynecology(M.T.,A.M.,S.T.),StonyBrookUniversity,StonyBrook,NewYork11794;

LaboratoryforMolecularCellBiology(I.P.,J.W.M.C.),DepartmentforHumanGenetics,UniversityofLeuvenandFlandersInteruniversityInstituteforBiotechnology,B-3000Leuven,Belgium;DepartmentofGynecologyandObstetrics(L.C.G.,S.T.),StanfordUniversityMedicalCenter,Stanford,California94305;DepartmentofObstetricsandGynecology(A.T.F.),UniversityofIllinoisatChicago,Chicago,Illinois60612;DepartmentofBiologicalSciences(D.D.C.),Universityof

Delaware,Newark,Delaware19707;andDepartmentofLaboratoryPathologyandMedicine(D.G.K.,C.B.),DivisionofReproductiveEndocrinologyandInfertility,UniversityofNorthCarolina,ChapelHill,NorthCarolina27599

Lefty/Ebafpolypeptides,novelmembersoftheTGF-␤super-family,areinvolvedinendometrialdifferentiationandem-bryoimplantation.Recently,weshowedthat,duringundis-turbedestrouscycle,leftyispresentinmouseuterinehornprimarilyinaprecursorform.Here,weshowthatdecidualdifferentiationofendometrialstromaleadstoincreasedlefty(ϳ3.1-to3.6-foldinvivoand5-to8-foldinvitro)andprocessingofitsprecursorprimarilytoitslongform.Thiseventoccursond5ofpregnancy,andisparalleledbyproproteinconver-tase(PC)5/6up-regulation(ϳ6-foldincreaseforPC5Aand3-foldincreaseforPC5B)indecidualizeduterinehorn,inde-pendentofembryoimplantation.Amongtheknownconver-tases,onlyPC5/6Aprocessesleftytoitslongform.Takento-gether,thefindingsshowthatdecidualizeddifferentiationofstroma,whichisaprerequisiteforembryoimplantation,leadstoprocessingofleftybyPC5/6A.(Endocrinology146:5313–5320,2005)

L

EFTY/EBAFPOLYPEPTIDES,novelmembersoftheTGF-␤superfamily,areinvolvedintheformationoftheembryoniclateralpatterningandendometrialdifferentiation(1,2).MembersoftheTGF-␤superfamilyrequireprocessingfortheiractivation,suggestingthatcleavageisanessentialstepforleftyactivation.TransfectionofdifferentcelllineswithhumanleftyAandBformresultedinexpressionofa42-kDaprotein,whichwasproteolyticallyprocessedtoreleasetwopolypep-tidesof34kDa(longform)and28kDa(shortform)(3).Themutationoftheconsensussequencesforproproteinconvertase(PC)cleavageinleftyproteinidentifiedandvalidatedtheleftycleavagesites.ThemutationofthesequenceRGKRtoGGKG(aminoacids74–77)andRHGRtoGHGR(aminoacids132–135)preventedtheproteolyticprocessingofleftyprecursortothe34-and28-kDaforms,respectively(4).InpluripotentP19cells,leftydidnotinduceSmad2orSmad5phosphorylation,Smad2/Smad4heterodimerization,ornucleartranslocationoftheSmad2orSmad4,butactivatedMAPKpathwayinatime-anddose-dependentfashion(4).The28-kDa,butnotthe34-kDa,polypeptideinducedMAPKactivity.Thesedatasup-portedamolecularmodelofprocessingasamechanismforregulationofleftyaction(s).

FirstPublishedOnlineSeptember1,2005

Abbreviations:E2,17-␤Estradiol;FBS,fetalbovineserum;IGFBP-1,IGFbindingprotein1;MPA,medroxy-progesteroneacetate;P,proges-terone;PC,proproteinconvertase.

EndocrinologyispublishedmonthlybyTheEndocrineSociety(http://www.endo-society.org),theforemostprofessionalsocietyservingtheendocrinecommunity.

Inhumans,leftyisexpressedinendometrialstromalcellsthatgraduallyundergothedifferentiatedstateofdecidual-ization(2).Decidualcellsadoptuniquephenotypic,func-tional,biological,secretory,andbiosyntheticfeatures.De-cidualizationispromotedbythecAMPandproteinkinaseApathwaysaswellas,afteraninitialestradiolpriming,byprogesterone(P).Recentstudiesindicatethatthisprocessisassociatedwithexquisitelycontrolledsequentialgenereg-ulationincludingkineticreprogrammingofgenesassociatedwithdecreasedGproteinsignaling,increasedSTATpath-waysignaling,cellproliferation,structuralproteinchanges,cellulardifferentiation,andsecretoryprocesses(5,6).Amongthesechanges,expressionandsecretionofprolactinandIGBP-1havebecomethehallmarksofthisdifferentiatedcellularstate.Decidualizedcellsproduceextracellularmatrixproteinsincludinglaminin,typeIVcollagen,fibronectin,andheparinsulfateproteoglycan.Thekineticreprogramming,asrevealedbymicroarrayanalysisofhumanendometrialstro-malcellsforcedtodifferentiateinvitrobycAMP,includesremarkableelevationintheexpressionoflefty(5,6).Theexpressionofleftyisincreasedfromalowbasallevelby3-foldwithin2h,and123-foldwithin36hofreceivingthedecidualizingstimulus(6).Braretal.validatedthemicroar-raydatawithNorthernblotanalysisandshowedasteadyincreaseofleftyoveraperiodof12dwhenthedecidualcellsarefullydeveloped(5).

Takentogether,thesefindingssuggestthatleftymightbeanintegralcomponentofthedecidualizationprocess.Wewereinterestedinextendingtheseobservationstoamodel

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5314Endocrinology,December2005,146(12):5313–5320Tangetal.•ProcessingofLeftyProteins

systemmoreamenabletoexperimentalmanipulationinvivo.Therefore,thecurrentstudywascarriedouttodefinepre-ciselytheexpressionandprocessingofleftyduringdecidu-alization.Theresultsshowthatinductionofdecidualizationleadstotheconversionofleftyintoitslongform.Thisevent,whichoccursupondecidualizationofstromalcells,ispar-alleledwiththeexpressionofPC5/6,theprimaryenzymeforproteolyticcleavageoftheprecursorformofleftytoitslongform(4).Thesefindingsshowthatleftyexpressionandpro-cessingareintrinsicfeaturesofthedifferentiateddecidual-izedstateofstromalcells.

MaterialsandMethods

Materials

ChemicalswerefromSigma-Aldrich(St.Louis,MO)orFisherSci-entific(Pittsburgh,PA).Theavidin-biotin-peroxidasekitwasfromVec-torLaboratories(Burlingame,CA).Thegoatpolyclonalantibodytoleftypeptide(M-20)mappedtothecarboxylterminusofmouseleftyandrecombinantIGFbindingprotein1(IGFBP-1)werefromSantaCruzBiotechnologyInc.(SantaCruz,CA).TherecombinantleftywasfromR&DSystems(Minneapolis,MN).TheantibodytoactinwasfromAb-cam,Inc.(Cambridge,MA).RabbitantibodytoPC5/6wasfromAlexisBiochemicals(Lau¨felfingen,Switzerland).OutbredCF-1andCD-1micewereobtainedfromCharlesRiverLaboratory(Wilmington,MA).

hornswereremoved.TheapprovaloftheinstitutionalAnimalCareandUseCommitteeofStonyBrookUniversitywasobtainedforcarryingouttheexperimentsondecidualizationofmouseuterinehorns.

Cultureanddecidualizationofhumanendometrialstromalcells

HuFwereisolatedfromdeciduaparietalisdissectedfromthepla-centalmembranesafternormalvaginaldeliveryattermunderanap-provedprotocolbytheUniversityofIllinoisatChicagoCommitteeontheUseofHumanSubjectsinMedicalResearch.Briefly,scrapedcellsweredigestedin0.1%collagenase,0.02%deoxynucleaseincalcium-andmagnesium-freeHanks’balancedsaltsolution.Cellswereplatedinfour100-mmculturedishes(BectonDickinsonandCo.Labware,FranklinLakes,NJ)andplacedintoanincubatorat37Cinpresenceof5%CO2.Thenextday,plateswereextensivelywashedwithPBStoremovenonadherent(mainlydecidual)cells.Atconfluence,cellsweretrypsinizedandusedforexperimentsinpassagenumber3–5.Cellpuritywasassessedbyimmunocytochemistryusingantibodiesagainstcyto-keratin(DakoCorp.,Carpenteria,CA)andvimentin(ZymedLabora-tories,Inc.,SanFrancisco,CA).Thepurityofthefibroblastcellprepa-rationsusedwasmorethan95%.Thesecellsweretreatedwithmediumaloneandwithmediumsupplementedwithhormones[10Ϫ6mmedroxy-progesteroneacetate(MPA)ϩ36nmE2]orwithcAMP(1mm)for14d.ThemediumwascomprisedofDMEMsupplementedwith2%(wt/vol)charcoal-strippedFBSserumandwaschangedevery2d.FBSwasomitted48hbeforetheendoftreatment.Atotalof4ϫ106SHT290cells(immortalizedhumanendometrialstromalcells)wastreatedwithmediumandwithmediumsupplementedwith10Ϫ6mMPAand10Ϫ8mE2,orwithcAMP(1mm)for13d.ThemediumwascomprisedofDMEM,withhighglucose,supplementedwith2%charcoal-strippedFBSandepidermalgrowthfactor(20ng/ml).

Humanendometrialsampleswerecollectedfromwomenwithnor-malmenstrualcyclesafterobtaininginformedconsentunderanap-provedprotocolbytheStanfordUniversityCommitteeontheUseofHumanSubjectsinMedicalResearch.Biopsieswereobtainedfromsubjects,26–46yrofage,whohadregularmenstrualcycles(28–35d),weredocumentednottobepregnant,andhadnohistoryofendome-triosis.Endometrialtissuesweresubjectedtocollagenase/hyaluroni-dase(Sigma-Aldrich)digestionfor2hat37C.Afterdigestion,thestromawasdispersed,althoughtheglandularstructuresremainedmostlyintact.Stromalcellswereseparatedfromglandsonasizebasis.Stromalcellswerecentrifuged,andtheresultingpelletwasresuspendedina4:1ratioinDMEM-F12(LifeTechnologies,Inc.)andDMEM/10%FBS.Cellswerepreplatedin10cm2standardcultureplatesfor1hat37Cinthepresenceof9%CO2,andthemediumwasreplacedwithhigh-glucoseDMEM/MCDB-105mediumwith10%charcoal-strippedFBS,insulin(5␮g/ml)(Sigma-Aldrich),gentamycin,penicillin,andstreptomycin.Endometrialstromalcellswerepassagedtwotothreetimesbeforetheexperiments.Thestromalcellsusedwere99%pure,devoidoflymphocytes,endothelialcells,andfibroblasts(5).Culturemediumwaschangedevery2–3d.Forinductionofdecidualization,culturedstromalcellswerepretreatedwithE2(10␮m),and,oncecon-fluent,theyweretreatedinserum-freemediasupplementedwith1␮m8-bromocAMPfor72h.TheconcentrationofIGFBP-1wasdeterminedinconditionedmediaaftercAMPtreatmentusinganIGFBP-1ELISA(DSL,Webster,TX).

Samplepreparationandanalysisofestrouscycleandpregnancy

Inthisreport,theexpressionofmouseleftywasexaminedintheendometriaofmice(nϭ6)duringpregnancy.Eachexperimentwasrepeatedatleasttwicetoconfirmreproducibilityofdata.Day1ofpregnancywasconsideredasthedaywhenthevaginalplugwasde-tected.ApprovaloftheinstitutionalAnimalCareandUseCommitteeoftheUniversityofDelawarewasobtainedforcarryingouttheremovalofuterinehornsduringpregnancy.

Transfection

CHOcellsweremaintainedinDMEM(LifeTechnologies,Inc.,Rock-ville,MD)supplementedwith10%(vol/vol)fetalbovineserum(FBS;LifeTechnologies,Inc.)and1%(wt/vol)antibiotic-antimycoticmixture(LifeTechnologies,Inc.).Fortransfection,cellsweretrypsinizedwith0.05%(wt/vol)trypsin-EDTA(LifeTechnologies,Inc.)andseededintosix-wellplates(Falcon,Franklinlakes,NJ)ataconcentrationof2ϫ105cellsperwell.Cellswereculturedin1.5mloftheculturemedium,inthepresenceofCO2at37Cforabout16h.Uponreaching60%con-fluency,cellswerecotransfectedwithcDNAofleftyAandwithfurin,PACE4,PC5/6A,PC5/6B,orPC7cDNA,usingSuperfectTransfectRe-agent(Qiagen,Valencia,CA)followingthemanufacturer’sprotocol.Inseparateexperiments,293cellsweretransfectedwithanexpressionPC5/6vector.Emptyvectorservedascontrolinthesetransfections.LysatefromthesecellswasusedasacontrolforidentificationofPC5/6onWesternblotting.Intransienttransfections,serum-freemediumwasused.

Inductionofdecidualizationinmouseuterinehorn

Decidualizationwasinducedinovariectomizedmice4–5dafterovariectomy.Micereceivedthreedailyscinjectionsof17-␤estradiol(E2,100ng).After4dofrest,animalsreceivedthreeadditionaldailyscinjectionsofE2(10ng)aswellasscinjectionsofP(1mg).BothE2andPweredeliveredin0.1mlofsesameoil.OnthethirddayoftreatmentwithP,animalsreceivedthedecidualizingsignal,whichwaseithertraumaoroil.Afteranesthesia,anincisionwasmadeintheleftflankandtheleftuterinehornwasexposed.Traumawasproducedbytheinser-tionofablunt25-gaugeneedleseventimesintothehornlumen.Fordecidualizationbyoil,20␮lofsesameoilwasinjectedintotheproximalleftuterinehornatthetubaljunction.AnimalsreceivedthreeadditionaldailyscinjectionsofP(1mg).Seventy-twohourslater,bothuterine

Northernblotanalysis

TotalRNA(20␮g/lane)orpolyARNA(2␮g/lane)wasfractionatedinformaldehyde-agarosegel.Afterdiffusiontransfertonitrocellulosefiltersandcross-linking(Stratalinker;Stratagene,LaJolla,CA),hybrid-izationswereperformedusingdenatured32P-labeledcRNAoflefty,andcDNAprobesformousefur,PC5/6,PACE4,andPC7wereusedasdescribedpreviously(12).Gelswerestainedwithethidiumbromideforrevealingthe18Sand28SribosomalRNAandblotswerestrippedandprobedforactincDNAfornormalization.QuantificationwasperformedusingSigmaGel(Sigma-Aldrich)aswellastheTyphoon9400(GEHealthcare)andImageQuantsoftware(MolecularDynamics).

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SDS-PAGEandWesternblotting

Mediawerecentrifugedat7000ϫgfor3handconcentratedbyabout12-foldusingCentricon10(proteinmolecularsizecut-off:10,000kDa;Amicon,Danvers,MA).TheconcentrationofproteinsinthesesampleswasdeterminedbyBio-RadProteinAssaykit(Bio-RadLaboratories,Hercules,CA).Concentratedconditionedmediaorcelllysates(50␮gproteinperlane)werefractionatedina12%denaturinggeltogetherwithprestainedproteinladder(LifeTechnologies,Inc.)andweresubsequentlyblottedontonitro-cellulosemembraneinaMini-Trans-Blotapparatus(Bio-RadLaboratories).Blotswerestainedwiththegoatpolyclonalantibodytoleftypeptide(M-20)(1–2␮g/ml)orwhereindicatedwithrabbitantibodytoPC5/6.Thesec-ondaryantibodyusedwasdonkeyantigoatIgG-HRPorgoatantirabbit(SantaCruzBiotechnology).Bandsweredetectedbychemiluminescenceasdescribedbythemanufacturer.QuantificationwasperformedusingSigmaGel(Sigma-Aldrich).

Statisticalanalysis

Densityofbandsandratiosofprecursor/longformofleftywerecomparedusingttest.Differenceswereconsideredstatisticallysignif-icantwhenPϽ0.05.

Results

Leftyproteinsinmouseuterinehornduringpregnancy

TransfectionofdifferentcelllineswithhumanleftyAandBformresultedinexpressionofa42-kDaprotein,whichwasproteolyticallyprocessedtoreleasetwopolypeptidesof34kDa(longform)and28kDa(shortform)(4).Toexamineforthepresenceofsimilarprocessedformsinmouseuterinehorn,proteinsfromhornsofpregnantmiceweresubjectedtoWesternblotanalysisforlefty.Ond1ofpregnancy,precursorandlongformofleftywereequallypresent.How-ever,ond3,andparticularlyond5,ofpregnancy,therewasmorelongformofleftycomparedwiththeprecursorform(Fig.1).Ond9,thiswasmorenotableclosetotheimplan-tationsiteratherthanintissuesinterveningtheimplantationsites(Fig.1),suggestingthatprocessingofleftyoccursindecidualizedstromamoreefficientlyaroundtheimplantingembryo.

Leftyinmouseendometriumduringartificialdecidualization

FIG.1.Leftyinmouseuterinehornduringpregnancy.Proteinsex-tractedfrommouseuterinehornsweresubjectedtoWesternblotanal-ysisforleftyondifferentdaysofpregnancy.Ond9,tissueswereremovedaroundtheimplantationsites(9)aswellasfromsitesinterveningtheimplantationsites(9Ј).Blotswerestrippedandreprobedwithantibodytoactintovalidateequalloading.Theupperpanelshowsrepresentativeblotofleftyduringpregnancy.Thelowerpanelshowstheratiosofthemeandensityoftheprecursor/longformsofleftyϮSD.Theratiowassignificantlyincreasedduringpregnancyasstromadecidualizes.PϽ0.05.P,Precursorform;L,longform;S,shortform.

Todeterminewhetherleftyprocessingwasduetothedecidualizationofstromaandwhetheritrequiredpresenceofanembryo,decidualizationwasartificiallyinducedinendometrium.Weintroducedoil,asthedeciduogenicsignal,intotheleftuterinehornsofmiceprimedwithsteroidhor-mones,E2andP.Theleftuterinehorns,removed3dafterreceivingthedecidualizingsignal,wereenlarged,andthedistalpartsofsomerightuterinehornswerealsoenlarged,suggestingtransferofoilfromthelefttotherighthorn(Fig.2).TheintroductionofChicagobluedyehassubstantiatedthistrans-uterinehornflow(7).Forthisreason,theuterinehornsofmiceprimedwithhormonesthatdidnotreceivethedecidualizingsignal,ratherthantherighthornofanimalsthatreceivedthedeciduogenicstimulus,wereusedascon-trols.Traumawasusedasaseconddeciduogenicsignalintheleftuterinehorn.Thelefthornsremoved3dlaterwerealsoenlarged(Fig.2).Thewetweightoftheuterinehornsinjectedwithoilwassignificantlyhigherthantheweightsofhornsthatweretraumatized(datanotshown).Thesectionsofuterinehornsexaminedafterhematoxylinandeosinstain-ingshowedthetypicalmorphologyofadecidualizedstroma

(datanotshown).WesternblotanalysisforIGFBP-1wascarriedouttoconfirmthedecidualizationofstromaintheuterinehorns.IGFBP-1wasfoundinbothuterinehorns,butunlessbothhornswereenlargedtothesameextent,moreIGFBP-1immunoreactivitywaspresentintheleftcomparedwiththerighthorns(Fig.2).

Northernblotanalysisshowedboth2.4-and1.5-kbbandsofleftymRNAinmouseuterinehornsofbothnondecidu-alizedcontrolanddecidualizedhorns.Ascomparedwithcontroluterinehorns,leftywasexpressedatasignificantlyhigherlevel(ϳ3.1-to3.6-fold)inthedecidualizedhornsirrespectiveofthetypeofthedecidualizingsignal(Fig.3A).AlthoughbothprecursorandlongformsofleftywerepresentinthenondecidualizeduterinehornsprimedwithhormonesE2andP,thelongformwasthepredominantforminthedecidualizedhornsirrespectiveofthedeciduogenicsignal(Fig.3B).

Expressionofconvertasesinmouseuterinehornduringartificialdecidualization

BecausemembersofthePCfamilycleavedifferentTGF-␤proteins,thedatasuggestedthatprocessingofleftymightbeduetoexpressionofaconvertaseinthedecidualizedstroma.Forthisreason,Northernblotanalysisfortherelevantcon-

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5316Endocrinology,December2005,146(12):5313–5320Tangetal.•ProcessingofLeftyProteins

FIG.2.Decidualizationofmouseuterinehornbyoilandtrauma.UterinehornsofmicewereprimedwithPandE2asdescribedinthetext.Thentheleftuterinehornofeachmousewasdecidualizedbyintraluminalinjectionofoilorbytrauma.Threedayslater,hornswereremovedandphotographed.Lefthornsweregreatlyenlarged.ProteinsextractedfromuterinehornsweresubjectedtoWesternblotanalysisforIGFBP-1.IGFBP-1increasesinthedecidualizedhorns.Blotswerestrippedandreprobedwithantibodyactintovalidateequalloading.Dataarerepresentativeofexperimentsin16mice.

vertasesincludingFurin,PC5/6,PC7,andPACE4,werecar-riedoutontheRNAisolatedfromthedecidualizedandcontrolnondecidualizedmouseuterinehorns.Amongtheseconvertases,onlytheexpressionofPC5/6wassignificantly

increasedinthedecidualizedhorns(Fig.4A).Bothdecid-uogenicsignalsincreasedtheexpressionofPC5/6andboththeA(shortform)andB(longform)isoformsweresignif-icantlyincreased(ϳ6-foldincreaseforPC5Aand3-foldin-creaseforPC5B).Westernblotanalysiswasperformedonthetissuelysatesofthedecidualizedandnondecidualizeduter-inehorntovalidateincreasedPC5/6.Thelysateof293cellstransfectedwithPC5/6servedasapositivecontrolfortheidentificationofPC5/6,whereasthelysatefrom293cellstransfectedwithemptyvectorservedasanegativecontrol.AsshowninFig.4B,PC5/6wasincreasedinthedecidual-izeduterinehorn,whereasitwaslowtoundetectableincontrolhormone-primednondecidualizeduterinehorn.

ProcessingofleftybyPC5/6A

FIG.3.A,Leftyexpressionindecidualizedmouseuterinehorn.PolyARNA(2␮g)fromeachcontrolmouseuterinehornprimedwithhormonealoneandthosedecidualizedwithoilortraumaweresub-jectedtoNorthernblotanalysisforleftyusinga32P-labeledlefty-1cRNAprobe.Theblotwaswashedandreprobedwith32P-labeledactinprobe.Theupperpanelshowsarepresentativeblotofmultipleex-periments(nϭ6)incontrolhorn(C)andhornsdecidualizedwithtrauma(T)andoil(O).ThelowerpanelshowsthemeanODofa2.5-kbbandnormalizedwiththedensityofactinbandsϮSD.Thedensityofleftybandswasincreasedindecidualizedhorns.PϽ0.05.B,Pro-cessingofleftyindecidualizedmouseuterinehorn.Proteinextracts(250␮g)fromeachcontrolmouseuterinehornprimedwithhormonealoneandthosedecidualizedwithoilortraumaweresubjectedtoWesternblotanalysisforlefty.Toshowequalloading,theblotwasstrippedandreprobedforactin.Theupperpanelshowsarepresen-tativeblotofmultipleexperiments(nϭ6)incontrolhorn(C)andhornsdecidualizedwithtrauma(T)andoil(O).ThelowerpanelshowstheratiosofnormalizedmeanODofprecursor/longformofleftyϮSD.PϽ0.05.Theratiowassignificantlyincreasedupondecidualizationofhorn.P,Precursorform;L,longform;S,shortform.

ThesefindingssuggestedthatleftywasbeingprocessedinthedecidualizedstromabyPC5/6.TodirectlyshowthatPC5/6wasresponsibleforleftyprocessing,CHOcellsweretrans-fectedsimultaneouslywithleftycDNAandcDNAsofvariousPCsincludingFurin,PACE4,PC5/6A,PC5/6B,andPC7.FromtheseconvertasesonlythePC5/6wasabletoconvertleftytoitslongform.PC5/6AwasmoreefficientthanPC5/6Bintheprocessingofleftytoitslongform(Fig.5).

Processingofleftyinendometrialstromalcellsdecidualizedinvitro

Todirectlyshowthatleftywasprocessedindecidualizedcellsirrespectiveofotherinvivosignals,decidualizationwasinducedinHuFscells.ThesecellswerederivedfromhumanplacentaandhavebeenshowntodecidualizeinvitrobothwithcAMPandwithE2/Ptreatment(8).BothcAMPandE2/PledtothedecidualizationofthesecellsasconfirmedbyreleaseofIGFBP-1intotheculturemedium(Fig.6A).Bothdecidualizingsignalsledtotheaccumulationofthelongformofleftyinthecelllysate.However,thelongformofleftywaspresentintheculturemediumofcellsdecidualizedwithcAMPbutnotwithE2/P(Fig.6A).Tofurthervalidatethesefindings,SHT290cells,whichwerederivedfromhuman

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FIG.4.A,Expressionofconvertasesindecidualizedmouseuterinehorn.A,TotalRNA(30␮g)frommouseuterinehornsoffourcontrol(C)miceprimedwithhormonealone(pooled)andthosedecidualizedwithtrauma(T)andoil(O)weresubjectedtoNorthernblotanalysisforPC5/6,PC7,PACE4,andFurinusing32P-labeledcDNAprobes.Theblotwaswashedandreprobedwith32P-labeledactinprobe.Theupperpanelshowsleftyincontrolhorn(C)andhornsdecidualizedwithtrauma(T)andoil(O).Thelowerpanelshowsactinbands.AlthoughthedensityofPC5/6bandswasincreasedsignificantlyindecidualizedhorns,theden-sityofotherbandsdecreasedordidnotchangesignificantly.PϽ0.05.B,PC5/6indecidualizeduterinehorn.Thetissuelysatesofcontrolnondecidualized(C)anduterinehornsdecidualizedwithtrauma(T)andoil(O)weresubjectedtoWesternblotanalysis.TheblotswereprobedwitharabbitpolyclonalantibodytoPC5/6.Thelysatesof293cellstransfectedwithemptyvectorandPC5/6expressionvectorserved,respectively,asnegativeandpositivecontrolforidentificationofPC5/6.Blotwasstripped,washed,andsubjectedtoWesternblotanalysisforactinfordeterminationofequalloading.

FIG.6.A,ProcessingofleftyindecidualizedHuFcells.HuFscellsweretreatedwithmediumaloneandwithmediumsupplementedwitheitherhormones(10Ϫ6MMPAϩ36nME2)for14d,orwithcAMPfor48hafter12dinmediumwithhormones.MediumwascomprisedofDMEMsupplementedwith2%charcoal-strippedFBSandwaschangedevery2d.FBSwasomitted48hbeforetheendoftreatment.CelllysatesandculturemediawerethensubjectedtoWesternblot-tingusingrecombinantIGFBP-1(rIGFBP-1)andleftyasapositivecontrol.BlotswereprobedforIGFBP-1andforlefty.P,precursor;L,longprocessedformoflefty;S,shortprocessedformoflefty.B,Pro-cessingleftyindecidualizedSHT290cells.SHT290cells(immortal-izedhumanendometrialstromalcells)weretreatedwithmediumandwithmediumsupplementedwith10Ϫ6MMPAϩ10Ϫ8ME2orwithcAMP(1mM)for13d.MediumwascomprisedofDMEM,withhighglucose,supplementedwith2%charcoal-strippedFBSand20ng/mlepidermalgrowthfactor.CelllysateswerethensubjectedtoWesternblottingusingcelllysateofleftyϩcells(GPEϩ86cellstransducedwithleftyvector)asapositivecontrol.Blotwasprobedforlefty.P,Pre-cursorform;L,longform;S,shortform.

endometrialstromalcellsandimmortalizedbytransfectionwithtelomerasewereused.SHT290stromalcellswereverysimilartotheparentalstrainfromwhichtheywerederivedaccordingtocriteriaofproliferation,karyotype,cellularlo-calizationofcytoskeletalmarkersandnuclearstaining,andbasalgeneexpressionbasedonmicroarrayanalysis(9).ThesecellswereshowntodecidualizewithE2/Ptreatment

FIG.5.Processingofleftybyconvertases.CHOcellsweretransfectedwithanexpressionplasmidencodingleftyandwithemptyvector(control)orwiththesamevectorwithmousecDNAofFur,PACE4,PC5/6A,PC5/6B,andPC7.ProteinsextractedfromtransfectedcellsweresubjectedtoWesternblotanalysisforlefty.PC5/6andnototherconvertasesprocessleftytoitslongform.P,Precursorform;L,longform;S,shortform.

(9).ThesecellsweredecidualizedinvitrowithE2/PandwithcAMP.Untreatedcellsservedascontrols.Inthesecells,onlyE2/PandnotcAMPledtotheprocessingoflefty(Fig.6B).However,neithertreatmentledtothereleaseofleftytotheculturemedium.Thissuggestedthatcultureconditionortransfectionwithtelomerasemighthavehamperedtheabil-ityofthesecellstorespondtocAMP.

ToshowthatleftywasalsoprocessedinresponsetocAMP,andwasreleasedintoculturemedium,primarycul-turesofhumanendometrialstromalcellsderivedfromfivedifferentsubjectsweredecidualizedwithcAMP.Northernblotanalysisofthesecellsshowedsignificantincrease(5-to8-fold)inleftyexpressionwithintheshortdurationoftreat-ment(Fig.7A).ThequantityoftheIGFBP-1wasdeterminedintheculturemediaofthesecellsbyELISA.IGFBP-1waspresentintheculturemediaofdecidualizedcellsandnottheculturemediaofuntreatedcells(Fig.7B).Westernblotanal-ysisofculturemediaofnondecidualizedcellsshowedtheprecursorformoflefty(Fig.7B).Ontheotherhand,decidu-alizationofstromalcellsledtoaccumulationofbothlongandshortformsofleftyintheculturemedia(Fig.7B).

Discussion

Wepreviouslyshowedthatleftywasexpressedinhumanendometriumatahighlevelimmediatelybeforeandduringmenstrualbleeding(2).Insituhybridizationshowedthatlefty

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5318Endocrinology,December2005,146(12):5313–5320Tangetal.•ProcessingofLeftyProteins

FIG.7.A,Expressionofleftyindecidualizedhumanendometrialstromalcells.Primaryculturesofhumanendometrialstromalcellsfromfivedifferentsubjectsweretreatedwithserum-freemediumaloneormediumsupplementedwithcAMP(1mM)for72h.TotalRNA(50␮g/lane)extractedfromthesecellswassubjectedtoNorthernblotanalysisforleftyusinga32P-labeledlefty-1cRNAprobe.Becausereprobingoftheblotforactinshowedanapparentdecreaseinactinindecidualizedcells,equalloadingwasverifiedbytheintensityofethidiumbromide-stained18Sand28SribosomalRNA.Theupperpanelshowstheblotinthreerepresentativesamples.ThelowerpanelshowsthemeanODofeachbandnormalizedwiththedensityof18SbandsϮSD.Thedensityofbandswassignificantlyincreasedindecidualizedcells.PϽ0.05.B,Processingleftyindecidualizedhumanendometrialstromalcells.Primaryculturesofhumanendometrialstromalcellsfromfivedifferentsubjectsweretreatedwithserum-freemediumaloneandmediumsupplementedwithcAMP(1mM)for72h.Culturemediawereconcentratedandanequalamountofprotein(250␮g/lane)wassubjectedtoWesternblottingforlefty.TheamountofIGFBP-1intheculturemediawasquantifiedwithELISAandindicatedbeloweachlane.Theupperpanelshowstherepresentativeblotinculturestreatedwithout(Ϫ)andwith(ϩ)cAMP.ThelowerpanelshowstheratiosofmeanODofprecursor/longformofleftyϮSD.Theratiowassignificantlyincreasedupondecidualizationofcells.PϽ0.05.P,Precursorform;L,longform;S,shortform.

wasexpressedincellsthatwereundergoingdecidualization,arequisiteforsuccessfulimplantation(2).Inhumanendo-metrium,decidualizationoccursintheabsenceoftheem-bryo.However,inmice,decidualizationoccursonlyinthepresenceofanembryoorbyapplicationofadecidualizingsignaltoaproperlyprimedendometrium.Duringpreg-nancy,bythetimetheembryoarrivesintothemouseuterinecavityond4andbyd5,thereisevidenceofdecidualizationofstroma.Here,weshowthatthisdecidualizationisasso-ciatedwithanincreaseinthelongformofleftyinmouseendometrium.However,presenceoftheembryoisnotre-quiredforthisevent,becauseleftyisefficientlyprocessedtoitslongformwhenuterinehornisartificiallyforcedtode-cidualizewithoilortrauma.Althoughbothprecursorandlongformsofleftyareincreasedwithhormonalprimingalone,decidualizationappearstobeimportantfortheeffi-cientconversionofleftytoitslongform.

Decidualizationofmouseuterinehornaswellasdecidu-alizationofvarioushumanendometrialstromalcellsleadstoincreasedleftyexpression.ThesefindingsareconsistentwiththemicroarraydataandNorthernblotanalysis,whichshowasteadylevelofincreaseofleftyasthedecidualcellsdif-ferentiate(5,6).Invitro,cAMPismoreefficientthanE2ϩPintheinductionofdecidualizationinleftyexpressioninprimaryculturesofhumanendometrialstromalcells.Treat-mentofendometrialstromalcellswithcAMPinducesde-cidualizationofcells,IGFBP-1releaseintothecultureme-dium,andleftyprocessingwithin72h;longertreatmentwithE2/P(12d)arerequiredfordecidualizationandleftyprocessing.

Theprocessingofleftyproteinanditssecretionappeartobedependentonthedecidualizingsignalandcelltype.InSHT290cells,E2/PandnotcAMPledtotheprocessingoflefty,andinHuFscells,leftywasreleasedintotheculturemediumonlyaftertreatmentwithcAMPandnotafterE2/P-induceddecidualization.Similarly,Menoetal.re-portedthattheprocessingoflefty-1wasdependentonthecellsthatweretransfectedtoexpresstheprotein(1).Whentransfectedwithlefty-1cDNA,the293Tcellsproducedtheleftyprecursorbutfailedtoreleaseitintotheculturemedium(10).Ontheotherhand,theBALB/3T3cellsproduceda32-kDaproteinandreleaseditalongwitha25-to27-kDaproteinintotheculturemedium(10).Thesefindingsshowthattheprocessingandreleaseofleftyproteinshavespecialrequirementsandnotallcellshavetherequiredmachinerytocarryoutbothofthesefunctions.

MembersoftheTGF-␤familyaresynthesizedasprecursorproteins,whichareproteolyticallyprocessedtoreleasethebioactivepolypeptides(11).ProteinsoftheTGF-␤super-familyarecleavedbymembersofthePCfamilyofendo-

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Tangetal.•ProcessingofLeftyProteinsEndocrinology,December2005,146(12):5313–53205319

proteases(12–15).Thusfar,inmammals,sevenmembersofthisfamilyofproteinshavebeenidentified.Theseincludefurin,PC1/3,PC2,PC4,PC5/6AandB,PACE4,andPC7(16,17).TheseCa2ϩ-dependentserineproteasescleaveproteinsmostlyatsubstratesontheC-terminalsideofarginine-X-X-arginine(R-X-X-R)orlysine-X-X-arginine(K-X-X-R)pairsofbasicaminoacids(18–20).Furinwasthefirstconvertasetobeextensivelycharacterized,includingintwoknockoutmousemodels(21,22),andhasbeenshowninvitrotoberequiredfortheproperprocessingofseveralTGF-␤pro-proteins.Furin-deficientLoVocellsfailtocleaveTGF-␤1,whereascellstransfectedwithFurinregaintheabilitytoproperlyprocessTGF-␤1(13).Thus,specificconvertasesap-peartobeimportantintheprocessingofmembersoftheTGF-␤family.Leftyhastwocleavagesites,RGKRandRHGR.BecausethemutationofthesequenceRGKRtoGGKG(aminoacids74–77)andRHGRtoGHGR(aminoacids132–135)preventedtheproteolyticprocessingofleftyprecursor,thesesequencesarebonafidesitesfortheactionofconvertases(4).

DespitetheclearimplicationofthePCintheprocessingofTGF-␤1,littleisknownabouttheprocessingofleftybytheseproteasesduringdecidualization.Inthisstudy,weshowtheimportanceofthePCfamilyofendoproteasesinleftyprocessing.NeitherFurinnorPACE4orPC7wereabletoprocessleftytoitslongform.OnlyPC5/6wascapableofthisprocessingandtheAisoformwasthemostefficient.Asimilarobservationwasmadeforpro-lactase-phlorizin-hy-drolase,whichiscleavedbyPC5/6AbutnotPC5/6B(23).Also,theproinsulin-likegrowthfactor-IAisprocessedlessefficientlybyPC5/6AthanbyPC5/6B(24).ThePC5/6AandBsharethesamecatalyticdomain(23).Therefore,itisnotclearwhytheyseemtodiffersodrasticallyintheiractivityofprocessingleftyprotein.Thereasonforthisdiscrepancyislikelyduetothedifferentcompartmentalizationoftheisoforms.PC5/6AandPC5/6Baresortedtodifferentcom-partments(25).PC5/6Ahasbeenshowntobelocalizedintheextracellularmatrix(26)andinthelargedense-corevesicleintheneuroendocrinecells,whereasPC5/6Bisconcentratedinthetrans-Golgi(27).Furthermore,PC5/6Aisasolubleprotein,whereasPC5/6B,duetoitsC-terminalmembraneanchor,ismembrane-bound(28–30).

Interestingly,bothPC5/6AandBareincreasedduringdecidualizationinmouseuterinehorns.However,morePC5/6AthantheBisoformispresentindecidua,suggestingamoreprominentroleofPC5/6Aintheprocessingoflefty.ThesefindingsareconsistentwithpreviousreportsofstrongPC5/6expressionduringpregnancyindifferentiatedde-cidua(31)andinhumanendometrialstromalcellsdecidu-alizedinvitrobyE2/Ptreatment(32).Wongetal.(33)showedPC5/6AexpressioninmouseuterinedeciduafromE5.5throughtoE8.5andinartificiallydecidualizedstroma.PC5/6appearstoberequiredfordecidualizationandforembryoimplantation(32,34).Okadaetal.(32)haverecentlyshownthatantisensemorpholinotoPC5/6decreasesdecidu-alizationofhumanendometrialstromalcellsincultureasevidencedbyreducedprolactinproduction.Intraluminalad-ministrationofantisensemorpholinotoPC5/6inhibitsim-plantation(34).TherequirementofPC5/6doesnotappeartobelimitedtomouse,becausePC5/6isgreatlyup-regulated

bothinrhesusmonkeyandhumansduringthephaseofendometrialreceptivityandatimplantation(34).Thein-creaseintheprocessedformofleftyinuterinehornstreatedwithhormonesoccurredintheabsenceofdecidualizationorPC5expression(Fig.2B).ThissuggeststhatPC5-indepen-dentpathwaysmightexistforleftyprocessingorthatleftyaccumulatesincellsduetodecreaseddegradationandorsecretion.

Takentogether,thefindingsshowthatleftyisaprimarytargetforPC5/6actionandthatup-regulationofPC5/6alongwithprocessingofleftyareessentialfeaturesofthedecidualizationprocess.

Acknowledgments

ReceivedJune7,2005.AcceptedAugust23,2005.

Addressallcorrespondenceandrequestsforreprintsto:S.Tabibza-deh,M.D.,FrontiersinBioscience,POBox160,Searingtown,NewYork11507.E-mail:tabibzadeh@bioscience.org.

ThisworkwassupportedbygrantsfromtheNationalInstituteofChildHealthandHumanDevelopment/NationalInstitutesofHealth:1U01HD43165-01(toS.T.),U01HD29963(toD.C.),andHD42298(toL.C.G.),andRO1HD42280(toA.T.F.)aspartoftheNationalCooperativeProgramonTrophoblast-MaternalTissueInteractions.J.C.isholderofascholarshipfromthe“FondsvoorWetenschappelijkOnderzoekVlaanderen.”

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