—Original Article—

Cellular localization, expression and functional implications of the utero-placental endothelin system during maintenance and termination of canine gestationAykut GRAM1), Alois BOOS1) and Mariusz P KOWALEWSKI1)

1)Institute of Veterinary Anatomy, Vetsuisse Faculty, University of Zurich, CH-8057 Zurich, Switzerland

Abstract. Utero-placental(Ut-Pl)angiogenesisandbloodflowarefundamentalforsuccessfuloutcomeofpregnancy.Theyarecontrolledbynumerousvasodilatorandvasoconstrictorsystemssuchasendothelins(EDNs)andthereninangiotensinsystem.Dogspossessaninvasivetypeofplacentation,classifiedasendotheliochorial.DespiteincreasingknowledgeregardingcanineUt-Plfunction,littleinformationexistsonuterineandplacentalvascularactivityduringinitiation,maintenanceandterminationofpregnancyinthisspecies.ThecurrentstudyinvestigatedexpressionofEDNsandtheirreceptors(EDNRAandEDNRB)inthepre-implantationuterusandUt-Plcompartmentsduringgestationandatnormalparturition,aswellasinmid-pregnantdogstreatedwiththeantigestagenaglepristone.TheUt-PlmRNAexpressionofEDN1 and EDNRAwasconstantuntilmid-gestationandincreasedsignificantlyduringprepartumluteolysis.Incontrast,EDN2washighestpre-implantationanddecreasedfollowingplacentation,remaininglowthereafter.ExpressionoftheEDN-activatingenzymeECE1andmRNAofEDNRBincreasedtowardsmid-gestationandwasfurtherelevatedatprepartumluteolysis.AntigestagentreatmentresultedinincreasedlevelsofEDN1 and EDNRA.Atthecellularlevel,theuterineexpressionofEDN1,ECE1andEDNRBwasfoundpredominantlyintheendometrialsurfaceandglandularepithelialcells;uterinesignalsforEDNRAwereweak.InUt-Plalltargetsweremainlylocalizedintheplacenta fetalis,withsyncytiotrophoblaststainingstrongerforECE1andEDNRB.Incontrast,EDNRAstainedstronglyatthebaseoftheplacentallabyrinth.ExpressionandlocalizationofEDNs(EDN1,-2),EDNreceptorsandECE1intheplacenta fetalissuggeststheirinvolvementinthetrophoblastinvasionandproliferation.Key words: Domesticdog,Endothelin(EDN)-system,Placenta,Uterus

(J. Reprod. Dev. 63: 235–245, 2017)

Theendothelins(EDNs)are21aminoacidpeptideswhichareknownfortheirstrongvasoactiveproperties,andtheyaremainly

producedinresponsetohypoxia[1].Theiractiveformsareconvertedfroma212-aminoacidprecursorbythemembrane-boundmetal-loproteinase,endothelin-convertingenzyme-1(ECE1)[2].ThreedifferentisoformsofEDNshavebeenidentifiedsofar,designatedasEDN1,-2and-3[3].Theyaremainlyderivedfromendothelium,andbyactingthroughtheirEDNRAandEDNRBreceptorscaninvokearterialandvenousvasoconstrictionorvasodilatation,respectively.WhereasallEDNsshowsimilaraffinitytoEDNRB,EDN1and2havehigheraffinitytoEDNRAthanEDN3[4,5].AlthoughEDNsaremostlyknownfortheirvasomodulatory

properties,numerousparacrineand/orautocrineeffectshavebeendemonstrated,e.g.,inductionofmitosisinplacentalfibroblastsandvascularsmoothmusclecells[6–8].Inaddition,analysisofpre-implantationlumenflushesanddemonstrationofreceptorexpres-sionbyuterineandconceptuscellsinsheepsuggestthatEDN1mightplayanimportantroleinparacrineregulationofembryo

development,implantationandearlyplacentation[9].Importantly,actingthroughEDNRAandEDNRB,EDN1alsostimulatesin vitro proliferationofhumantrophoblastcellsisolatedfromfirsttrimesterplacenta,whereasitsstimulatorypropertiesontrophoblastinvasionareEDNRB-dependent[10,11].Beinglessabundantduringthefirsttrimester,expressionofEDN1increasesgraduallytowardsterm[7].Itislocalizedintheendotheliumoffetalvessels,syncytiotrophoblastandextravillouscytotrophoblastinthehumantermplacenta[12].TheconcomitantpresenceofECE1inthesecellssupportstheautocrineandparacrinefunctionofEDN1duringplacentation[13].Lessisknownaboutthecellularutero-placentalexpressionandfunctionofEDN2and-3.Otherthanthesedata,knowledgeisverylimitedregardingthe

potentialroleofEDNsandothervasomodulatoryfactorsintheuterusandutero-placental(Ut-Pl)unitsduringimplantationandplacentationinthecaninespecies.However,especiallywhentheinvasive,endotheliochorialtypeofcanineplacentationistakenintoconsideration,adequateproliferationandinvasionoftrophoblastsmustbecriticalforthespecies-specificdecidualization,implantationandplacentationindogs[14–16].Furthermore,inadditiontothephysiologicalimportanceofthe

EDN-systemintrophoblastinvasionandproliferation,itisbelievedtobeinvolvedinthepathophysiologyofpre-eclampsia[17,18].Asapregnancy-specific,multisystemdisorder,pre-eclampsiaischaracterizedbyhighbloodpressurefrequentlyaccompaniedbyseverefunctionaldamagetootherorgansystems,likethekidneysor

Received:December7,2016Accepted:February10,2017PublishedonlineinJ-STAGE:February20,2017©2017bytheSocietyforReproductionandDevelopmentCorrespondence:AGram(e-mail:aykutgram@gmail.com)This is anopen-access articledistributedunder the termsof theCreativeCommonsAttributionNon-CommercialNoDerivatives(by-nc-nd)License.(CC-BY-NC-ND4.0:https://creativecommons.org/licenses/by-nc-nd/4.0/)

Journal of Reproduction and Development, Vol. 63, No 3, 2017

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liver.Itisthoughttooriginateindysregulatedtrophoblastinvasionandisnowwidelyregardedasasystemicendothelialcelldisease[19–21].Inpatientssufferingfrompre-eclampsia,elevatedlevelsofEDN1werereportedinbloodserum[22].Importantly,applicationofEDNRAreceptorblockerstoapre-eclampticratmodelreducedhypertension[23,24].Nosuchinformationregardingthepotentialroleofvasoactivefactorsinplacentalfunctionisavailableforthecaninespecies,inwhichabnormaltrophoblastinvasion,possiblyconnectedtoaberrantvascularisationandtissueoxygenation,mayresultinsevereconditionslikesubinvolutionofplacentalsites(SIPS)[25,26].Therefore,inordertobetterunderstandtheprocessesofim-

plantationandplacentationinthedogand,thereby,filltheexistingknowledgegap,inthisstudyweinvestigatedtheexpressionandlocalizationpatternsofEDNs(EDN-1,-2,-3)andEDNreceptors(EDNRAandEDNRB),aswellasoftheiractivatorECE1,inthecanineuterusandUt-Plcompartmentsduringpregnancyandatterm.Potentialprogesterone-mediatedeffectswereinvestigatedinanimalsinwhichpretermparturition/abortionwasinducedbyanantigestagen(aglepristone).

Materials and Methods

Tissue collection and preservationUterineandUt-Plsamplesfromhealthy,cross-breedpregnant

bitches(2–8yearsofage)usedinourpreviousstudieswerealsousedhere[27–29].Stagesofpregnancywereclassifiedaspre-implantation(days8–12,n=5),post-implantation(days18–25,n=5),mid-gestation(days35–40,n=5)andprepartumluteolysis(n=3).Inaddition,theprogesterone(P4)receptorblocker(antigestagen)aglepristone(Alizine®,Virbac,BadOldesloe,Germany;10mg/kgbw;2x/24hapart)wasappliedtomid-pregnantdogs(days40–45ofpregnancy;n=10)toinduceabortionsandUt-Plunitswereobtained24h(n=5)and72h(n=5)afterthesecondtreatment.Dogsweremated2daysafterovulation(P4>5ng/mlinperipheral

blood),whichisthetimeneededforoocytematuration.Thedayofmatingwasdefinedasday0ofpregnancyandthepre-implantationstagewasdetectedbyobservationofembryosinuterineflushingsunderastereomicroscope.Pre-partumluteolysiswasdefinedbasedonmeasurementofP4concentrationsinserumevery6hbeginningfromday58ofpregnancy.WhenP4concentrationscontinuedtodeclinebelow3ng/mlin3consecutivemeasurements,ovariohysterectomy(OHE)wasperformed(therespectiveP4concentrationsarepresentedin[30]).TheanimalexperimentsanduseofalltissuesampleswereapprovedbytherespectiveauthoritiesoftheJustus-LiebigUniversity,Giessen(permitno.II25.3-19c20-15cGI18/14andVIG3-19c-20/15cGI18,14),Giessen,GermanyandtheUniversityofAnkara(permitno.Ankara2006/06),Ankara,Turkey.FollowingOHE,alluterinesamples(pre-implantation)andUt-Pl

units(followingimplantation)werecollected,rinsedinphosphate-bufferedsaline(PBS)andclearedofsurroundingconnectivetis-suesasdescribedbefore[29].TheUt-Plunits(full-thickness,allanatomicaltissuelayerscomprisingtheplacentaandtheadjacentuterus)weresampledfromthemiddlepartoftheplacentalgirdleavoidingmarginalhematoma.Fornon-radioactivein situhybridization(ISH)andimmunohistochemistry(IHC)experiments,sampleswere

immediatelyfixedin10%neutralPBS-bufferedformalinfor24hat+4°C,incubatedinPBSforoneweek,dehydratedthroughethanolseriesandembeddedinparaffinwax.InordertoisolateRNA,tissuesampleswereimmersedinRNAlater®(AmbionBiotechnologyGmbH,Wiesbaden,Germany)for24hat+4°Candthenstoredat–80°Cuntilanalysis.

Total RNA extraction, reverse transcription and Real Time (TaqMan) PCRTotalRNAwasextractedfromthepre-implantationuterusand

Ut-PlcompartmentsusingTRIzol®reagent(Invitrogen,Carlsbad,CA,USA)followingthemanufacturer’sprotocol.ThequalityandquantityofisolatedRNAweremeasuredusingaNanoDrop2000C®spectrophotometer(ThermoFisherScientificAG,Reinach,CH).Inthenextstep,DNase-treatmentwasperformedwithRQ1RNase-freeDNase(Promega,Dübendorf,CH,Switzerland),followedbycDNAsynthesisbyreversetranscription(RT)withrandomhexamersusedasstartersandotherRTreagentspurchasedfromAppliedBiosystems,FosterCity,CA,USA,followingthemanufacturer’sinstructionsandinaccordancewithourpreviouslypublishedprotocol[31,32].Semi-quantitativeRealTime(TaqMan)PCRforallEDN-systemmemberswasperformedonanABIPRISM7500SequenceDetectionSystem(AppliedBiosystems)asdescribedpreviously[32].Allreactionswereruninduplicateandcontaining200nMTaqManProbe,300nMofeachprimer,12.5μlFastStartUniversalProbeMaster(ROX)®(RocheDiagnostics,Mannheim,Germany)and5μlcDNAcorrespondingto100ngtotalRNAforeachtarget.Asacontrol,experimentswererunintheabsenceoftheenzymeduringreversetranscriptionsteptocheckforanypotentialgenomicDNAcontamination(theso-calledminus-RTcontrol).Additionally,waterwasusedinsteadofcDNA.Theamplificationconditionswereasfollows:denaturationat95°Cfor10min,followedby40cycles:95°Cfor15secand60°Cfor60sec.SelectedPCRproductsfromalltargetsweresentforsequencing(Microsynth,Balgach,CH,Switzerland).ResultswerequantifiedwiththecomparativeCT(ΔΔCT)methodusingthreereferencegenes(GAPDH,18SrRNA and CYCLOPHILIN A)asnormalizers.Thesamplewiththelowestconcentrationofthetargetgenewasusedasthecalibrator.ThereactionefficiencywascalculatedwiththeCTslopemethodandwassetupforapproximately100%.ThefollowingprimersandTaqManProbeswereused:GAPDHforward:5’-GCTGCCAAATATGACGACATCA-3’,reverse:5’-GTAGCCCAGGATGCCTTTGAG-3’,TaqManprobe:5’-TCCCTCCGATGCCTGCTTCACTACCTT-3’(GenBank:AB028142,ampliconlength:75bp);18SrRNAforward:5’-GTCGCTCGCTCCTCTCCTACT-3’,reverse:5’-GGCTGACCGGGTTGGTTT-3’,TaqManprobe:5’-ACATGCCGACGGGCGCTGAC-3’(GenBank:FJ797658,ampliconlength:125bp);EDNRAforward:5’-GGCCCCAACGCACTGATA-3’,reverse:5’-CCCGCCAGAAGCTTAAACAC-3’,TaqManprobe:5’-CCAGCCTTGCCCTTGGAGACCTTATC-3’(GenBank:NM_001031632.1,ampliconlength:92bp);EDNRBforward:5’-CATCATCGGGAACTCCACACT-3’,reverse:5’-CAGAGCCAGGCTGGCTATCA-3’,TaqManprobe:5’-CAAGAACAAGTGCATGCGAAACGGC-3’(GenBank:NM_001010943.2,ampliconlength:91bp).ThefollowingcommerciallyavailableTaqManGeneExpressionAssaysfromAppliedBiosystemswereused:EDN1(prod.

CANINEPLACENTALEDN-SYSTEM 237

no.Cf02622421_m1),EDN2(prod.no.Cf02622240-m1),EDN3 (prod.no.Cf02622419-g1),ECE1(prod.no.Cf02627515-m1)andCYCLOPHILIN A(prod.no.Cf03986523-gH).

ImmunohistochemistryOurstandardimmunoperoxidaseIHCassaywasperformed[31,33]

inordertoidentifythelocalizationofEDN1,ECE1andthetwoEDNreceptors(EDNRAandEDNRB)intheuterusandUt-Plcompart-ments.Briefly:tissuesweresectionedat2–3μmthickness,mountedontoSuperFrostmicroscopeslides(Menzel-Glaeser,Braunschweig,Germany),deparaffinizedwithxyleneandrehydratedinanethanolseries.Thiswasfollowedbyantigenretrievalinamicrowaveovenat560Wfor15minin10mMcitratebufferpH6.0.Then,sectionswerecooleddownandtreatedwith0.3%hydrogenperoxideinmethanolfor30min,blockedwith10%normalserumfromthesamespeciesinwhichthesecondaryantibodywasproduced,andoverlaidwithprimaryantibodiesovernightat4°C.Thesewere:affinitypurifiedgoatpolyclonalanti-EDNRA(sc-21194;dilution1:50),affinitypurifiedgoatpolyclonalanti-EDNRB(sc-21196;dilution1:200),affinitypurifiedgoatpolyclonalanti-ECE1(sc-27558;dilution1:300),allfromSantaCruzBiotechnology,CA,USA;andmousemonoclonalanti-EDN1(E166;dilution1:300)purchasedfromSigma-Aldrich,St.Louis,MO,USA.Nocommercialanti-EDN2canine-specificantibodywasavailableforthepresentstudy.Inaddition,monoclonalmouseanti-VIMENTINIgG2a(M7020;Clone3B4),dilution1:100(DakoSchweizAG,Basel,Switzerland)andaffinity-purifiedpolyclonalrabbitanti-CYTOKERATIN,widespectrumscreening,dilution1:300(DakoSchweizAG)antibodieswereused.Thespecificityofallantibodieswascheckedbyreplacingtheprimaryantibodywithnon-immuneIgGsofthesamespeciesinsteadoftheprimaryantibody(servingastheso-calledisotypecontrol),appliedatthesameproteinconcentration.Inthenextstep,sectionswerewashedwithIHCbuffer/0.3%TritonXpH7.2–7.4(0.8mMNa2HPO4,1.47mMKH2PO4,2.68mMKCl,137mMNaCl),andincubatedwiththefollowingbiotin-labeledsecondaryantibodiesat1:100dilution:horseanti-goatIgGBA-9500,horseanti-mouseIgGBA2000andgoatanti-rabbitIgGBA1000(allpurchasedfromVectorLaboratories,Burlingame,CA,USA).Theintensityofsignalswasenhancedbyapplyingthestreptavidin-peroxidaseVectastainABCkit(VectorLaboratories)for30min.ImmunereactionswerevisualizedwiththeLiquidDAB+substratekit(DakoSchweizAG).Finally,slideswerecounter-stainedinhematoxylin,dehydratedinagradedethanolseriesandxylene,andmountedinHistokit®(Assistant,Osterode,Germany).

In situ hybridizationNon-radioactiveISHwasperformedonparaffin-embeddedtissue

slidesinordertolocalizeEDN1,-2,ECE1,EDNRA and EDNRB mRNAbasedonourpreviouslypublishedprotocol[33,34].Briefly,RT-PCRswereperformedforcomplementaryRNA(cRNA)probesynthesisusingthefollowingcanine-specificprimers:

EDN1forward:5`-CCACAGGAAGAGATGCCAGT-3`;EDN1reverse:5`-GATGGCGTCCAACCTTCTTA-3`(amplicon

length205bp);EDN2forward:5`-ACATCATCTGGGTGAACACT-3`;EDN2reverse:5`-CCTAGGAAAGCGGATCTT-3`(amplicon

length281bp);

ECE1forward:5`-CCCCTGATGGAGCTAATTGA-3`;ECE1reverse:5`-GCCCAGTTGGACCATGTAGT-3`(amplicon

length276bp);EDNRAforward:5`-AGGGTGAACAGCACAAAACC-3`;EDNRAreverse:5`-CTTAAGTGAAGCGGGAACCA-3`

(ampliconlength280bp)EDNRBforward:5`-ACGTTACCTGCGAATCTGCT-3`;EDNRBreverse:5`-GCTTCAAAATCCTGCTGAGG-3`

(ampliconlength293bp).FollowingPCR,ampliconswereseparatedonethidiumbromide-

stained2%agarosegel,cutfromthegel,purifiedusingtheQiaexIIgelextractionsystem(QiagenGmbH,Hilden,Germany)andligatedintothepGEM-Tvector(Promega,Duebendorf,CH,Switzerland).TheincorporationofPCRproductsintothepGEM-Tvectorwas

verifiedbycontroldigestionwithNcoIandNotIrestrictionenzymes(NewEnglandBiolabs,Frankfurt,Germany).Plasmidscarryingtheampliconsweresentforsequencing(Microsynth).Then,selectedcloneswerelinearizedwithNcoI(antisense,positiveprobe)andNotI(senseprobe,negativecontrol)restrictionenzymesandlabeledusingtheDIG-RNAlabellingkitaccordingtothemanufacturer’sprotocols(RocheDiagnostics).Tissuesections(2–3μm)werecut,mountedonSuperFrostPlus

microscopeslides(Menzel-Glaeser),deparaffinizedinxylene,rehydratedinanethanolseriesanddigestedwithproteinaseK(70μg/ml,Boehringer,Mannheim,Germany),followedbyhybridiza-tionovernightat37°CusingcRNAprobesasdescribedpreviously[34].Then,followingincubationoftheprobedslideswithalkalinephosphatase-conjugated,sheepanti-DIGFabFragments(RocheDiagnostics,dilution1:5000),signalsweredetectedwiththesubstrate5-bromo-4-chloro-3-indolylphosphateandnitrobluetetrazolium(NBT/BCIP;RocheDiagnostics).

StatisticsTheeffectsoftheobservationgroupontargetgeneexpressionwere

assessedbyapplyingaparametricone-wayANOVA.IncaseofP<0.05,eithertheTukey-Kramermultiplecomparisonspost-testwasperformedinordertodetecttheeffectofpregnancystageonexpressionofEDNsystemmembersatdifferenttimepointsduringpregnancy,orDunnett’smultiplecomparisontestinexperimentsinvestigat-ingtheeffectsofaglepristonetreatmentontargetgeneexpression;resultsshowthen-foldchangeingeneexpressioncomparedwithitsexpressionatmid-gestation(non-treatedcontrol).ThestatisticalsoftwareprogramGraphPad3.06(GraphPadSoftware)wasusedforalltests.Numericaldataarepresentedasthemean±S.D.

Results

Expression profile of the EDN system in canine uterus and Ut-Pl compartments throughout gestation and at normal prepartum luteolysis.Atime-dependentexpressionprofileofEDN1,-2,ECE1 and

bothEDN receptors (EDNRA and EDNRB)wasdetectableinallsamples(Fig.1A,C,EandFig.2A,C).Specifically,EDN1mRNAwasexpressedconstantlyfrompre-implantationuntilmid-gestation,andincreasedstrongly(P<0.001)duringprepartumluteolysiscomparedwithpreviousgestationalstages(Fig.1A).Theopposite

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Fig. 1. ExpressionofEDN1,-2 and ECE1-mRNAasdeterminedbyRealTime(TaqMan)PCR(mean±SD)inthecaninepre-implantationuterusandutero-placental(Ut-Pl)compartmentsduringpregnancy(A,C,E)andataglepristone-inducedparturition/abortioninmid-pregnantdogs(B,D,F).(B,DandF;comparedwiththemid-pregnancygroupasanon-treatedcontrol).BarswithdifferentlettersdifferatP<0.001in(A),P<0.05in(B)oratP<0.01in(D).

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expressionpatternwasobservedforEDN2mRNA:itwashighestatthepre-implantationstageofpregnancyandwassignificantlysuppressed(P<0.001)followingimplantationandplacentation,remaininglowuntilprepartumluteolysis(Fig.1C).TheexpressionofEDN3wasgenerallylowandevenfrequentlybelowthedetectionlimitinbothuterineandUt-Plsamplesthroughoutpregnancy,normalandinducedparturition,precludingquantificationofitsexpression(datanotshown).TheexpressionofmRNAencodingforECE1, responsibleforconvertingandtherebyactivatingEDNs,showedagradualincreasefollowingplacentationtowardsmid-gestation(P<0.05)(Fig.1E).Itreachedthehighestlevelsatprepartumluteolysiscomparedwithpre-implantationandpost-implantationstages(P<0.001andP<0.05,respectively),resemblingtheexpressionpatternobservedforEDN1.

RegardingtheEDNreceptors,likeEDN1,EDNRAmRNAwasconstantlyexpressedduringpregnancyandwasstronglyupregulatedatprepartumluteolysis,comparedwithallothergestationaltimepoints(P<0.05)(Fig.2A).AsimilarexpressionpatternwasobservedforEDNRB,theexpressionofwhichwas,however,stronglyelevatedatmid-gestation(P<0.01andP<0.001comparedwithpre-implantationandpost-implantationstages,respectively)(Fig.2C).Therewasafurther,significantstrongincreaseofEDNRBtowardsprepartumluteolysis(P<0.01)(Fig.2C).

Effects of antigestagen treatment on expression of the EDN system in canine Ut-Pl units during mid-gestationInordertoexaminetheeffectsoftheantigestagenaglepristonetreat-

mentontargetgeneexpressionduringmid-gestation,corresponding

Fig. 2. ExpressionofEDNRA and EDNRB-mRNAasdeterminedbyRealTime(TaqMan)PCR(mean±SD)incaninepre-implantationuterusandutero-placental(Ut-Pl)compartmentsduringpregnancy(A,C)andataglepristone-inducedparturition/abortioninmid-pregnantdogs(B,D).(BandD;comparedwiththemid-pregnancygroupasanon-treatedcontrol).BarswithdifferentlettersdifferatP<0.05in(A,B)oratP<0.001in(D).

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samplesfrommid-pregnantdogswereusedasnon-treatedcontrols.TheexpressionlevelsofEDN1 and EDNRAincreasedsignificantly(P<0.05andP<0.05,respectively)24hafterthesecondtreatment.TheirmRNAlevelswere,however,diminished48hlater,i.e., at the72htimepoint,anddidnotdiffersignificantlyfromcontrols(Fig.1BandFig.2B).WhereasEDN2decreasedgraduallybutsignificantly(P<0.01)towards72hafterthesecondapplicationofaglepristone,theexpressionofEDNRBwassignificantlysuppressedat72hcomparedwiththecontrolandthe24htimepoint(P<0.01)(Fig.1DandFig.2D).TheexpressionofECE1remainedunaffectedbytreatmentwithantigestagen(P>0.05)(Fig.1F).

Localization of EDN system in the canine uterus and placentaLocalizationofEDN1,ECE1andEDNreceptors(EDNRAand

EDNRB)wasdeterminedincaninepre-implantationuterusandUt-PlcompartmentsattheproteinandtranscriptlevelsbyIHCandISH.Duetothelackofcanine-specificand/orcross-reactingantibody,expressionofEDN2waslocalizedonlyatthemRNAlevel.Duringpre-implantation,strongpositivesignalsforEDN1wereobservedintheendometrialsurfaceepithelium,superficialanddeeputerineglandsandmyometrium(Fig.3A,B).Relativelyweakerstainingwasobservedintheconnectivetissuesofstromalcompartments.AsimilarlocalizationpatternofEDN1intheuterinepartoftheUt-Plcompartmentswasalsoobservedfollowingimplantation,however,withsignalsappearingstrongeratprepartumluteolysis(Fig.3F,G),compared,e.g.,withmid-gestation(Fig.3C,D).TheplacentallocalizationofEDN1wasobservedinfetaltrophoblastcells,predominantlyincytotrophoblast,throughouttheplacentationperiod,withsignalsnoticeablystrongeratprepartumluteolysis(Fig.3E,H).AsforECE1,duringpre-implantationitsuterineendometrial

signalswerelocalizedtotheepithelialcellsoftheendometrialsurface,superficialanddeeputerineglands,aswellastothemyometriumandmediaofthevessels(Fig.4A,B).Theunevendistributionpatternofsignalsinthesurfaceepitheliumcanbemostprobablyattributedtotherelativelylowproteinexpressionlevelinthiscompartmentortothesensitivitylimitsoftheantibody.Followingimplantationuntilprepartumluteolysis,nooronlyweaksignalsweredetectedintheuterus(Fig.4C,D,H,I).InordertofacilitatecharacterizationofthelocalizationpatternofECE1withinthespecificcellularcomponentsoftheplacenta(i.e.,inendothelialcells,fetaltrophoblastandmaternaldecidualcells),differentialstainingwasperformedonconsecutivesectionsusinganti-VIMENTINoranti-(pan)CYTOKERATINanti-bodies(Fig.4EandF,respectively).Whereasduringearlierstagesofgestation(post-implantationandmid-gestation)bothcyto-andsyncytiotrophoblaststainedintenselyforECE1,duringpre-partumluteolysisthesignalsweremainlylocalizedinthecytotrophoblast(Fig.4G,J).Muchweakerstainingwasobservedinmaternalstroma-deriveddecidualcells(Fig.4G,J).TheuterineexpressionofEDNRAreceptorwasgenerallyweak

throughoutpregnancy,withsomesignalslocalizedtouterineepithelialcompartments(Fig.5A,B,C,D).Thisstronglycontrastedwithitsplacentalexpressionduringprepartumluteolysis.Followingrelativelylowexpressionduringearlierstagesofpregnancy,strongEDNRAsignalsweredetectedprepartuminthefetalcytotrophoblast,especiallyatthebaseoftheplacentallabyrinth(Fig.5E,F).RegardingEDNRB,itsuterineexpressionandcellularlocalization

patternsweresimilartothoseobservedforEDN1andECE1.Thus,signalsweredetectedinluminalepithelialcells,superficialanddeeputerineglandsandmyometrium(Fig.6A,B).However,afterplacentation(Fig.6C,D)andduringprepartumluteolysis,noorrelativelyweaksignalswereobservedintheuterinepartofUt-Plunits.Intheplacentallabyrinth,strongEDNRBsignalsweredetectedpredominantlyinsyncytiotrophoblastcellsfrompost-implantation

Fig. 3. Immunohistochemical(IHC)localizationofEDN1inthecanineuterusandutero-placental(Ut-Pl)compartmentsatselectedtimepointsduringpregnancy;atthepre-implantationstage(AandB),intheUt-Plunitsduringmid-gestation(C,DandE),andintheUt-Plcompartmentsatprepartumluteolysis(F,GandH).(A,B)Atpre-implantation,EDN1islocalizedtotheendometrialluminal(surface)epithelialcells(solidarrowsinA),glandularepithelialcells of the superficial (open arrows in A) and deep uterineglands(openarrowsinB)andmyocytes(solidarrowheadsinB).Duringmid-gestation,withintheUt-PlunitsendometrialEDN1expressionappearsweakerandisdetectedinthesuperficialglands(theso-calledglandularchambers)anddeeputerineglands(openarrows inC andD, respectively).During prepartum luteolysis,endometrialEDN1expressionisdetectedinthesuperficialglands(the so-called glandular chambers), deep uterine glands (openarrows in F and G, respectively) and endothelial cells (solidarrowheadsinG).Intheplacentallabyrinthduringmid-gestation,signals are localized in fetal trophoblast cells (cytotrophoblast)(open arrows in E). At prepartum luteolysis, fetal trophoblast(cytotrophoblast)stainsstrongly(openarrowsinH).Nooronlyweaksignalsweredetectedinmaternalstroma-deriveddecidualcells(solidarrowsinEandH).Thereisnobackgroundstainingintheisotypecontrol(insertedinH).

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untilmid-gestation(Fig.6E).Interestingly,however,thesignalswerespreadovertheentiretrophoblast(i.e.,syncytio-andcytotrophoblast)atthetimeofprepartumluteolysis(Fig.6F).Anti-VIMENTINstainingwasusedonconsecutivesectionsforeasierdifferentiationofcells(Fig.6G).SimilarlocalizationpatternsofEDN1,ECE1,EDNRAandEDNRB

wereobservedinplacentallabyrinthbynon-radioactiveISH(Fig.7).Duetothelackofcommerciallyavailablecanine-specificantibody,

localizationofEDN2wasdeterminedatthetranscriptlevel.ThelocalizationpatternofEDN2mRNAgreatlyresembledthatofEDN1.Intheuterus,itwaslocalizedpredominantlyinepithelialcompart-ments(Fig.8A,B).Itwasclearlydetectableduringpre-implantation,whereasduringprepartumluteolysisitwasbarelydetectable(Fig.8C,

Fig. 4. Immunohistochemical(IHC)localizationofECE1inthecanineuterus and utero-placental (Ut-Pl) compartments at selectedtime points during pregnancy and at prepartum luteolysis; atthepre-implantation stage (AandB), in theUt-Pl units duringmid-gestation (C,D andG), and in theUt-Pl compartments atprepartumluteolysis(H,I,J).(A,B)Atpre-implantation,signalsarepredominantlylocalizedtotheendometrialluminal(surface)epithelial cells (solid arrows inA), glandular epithelial cells ofthesuperficialanddeeputerineglands(openarrowsinAandB),inmedia of the vessels (open arrowheads in B) andmyocytes(solidarrowheadsinB).Duringmid-gestationandatprepartumluteolysis, within the Ut-Pl units, endometrial expression ofECE1 appears weaker and is detected in tunica media of thevessels(openarrowheadsinCandD),thesuperficialglands(so-calledglandularchambers)anddeeputerineglands(openarrowsinC,H,andD,I,respectively).Intheplacentallabyrinthduringmid-gestation,signalsarelocalizedinfetaltrophoblastcells(i.e.,syncytio- and cytotrophoblast) (open arrows in G). In order todistinguishbetweenfetalandmaternalcelltypeswithinthecanineplacenta, i.e., endothelial, trophoblast and decidual cells, (pan)CYTOKERATIN(widespectrum)andVIMENTINstainingwasapplied to consecutive sections following thoseused forECE1.Trophoblast cells stain positively for CYTOKERATIN (openarrowsinF),whereasendothelialcells(openarrowheadinE)anddecidualcells(solidarrowinE)stainpositivelyforVIMENTIN.At prepartum luteolysis, fetal cytotrophoblasts stain strongly(openarrowsinJ)forECE1.Nooronlyweaksignalscanbeseeninmaternaldecidualcells(solidarrowsinGandJ).Thereisnobackgroundstainingintheisotypecontrol(insertedinJ).

Fig. 5. Immunohistochemical(IHC)localizationofEDNRAinthecanineuterineandutero-placental(Ut-Pl)compartmentsatselectedtimepoints during pregnancy; at the pre-implantation stage (A andB), and in theUt-Pl compartments at prepartum luteolysis (C,D,EandF).(A,B)Atpre-implantation,thesignalsforEDNRAin endometrial surface epithelial cells (solid arrows in A),superficialanddeeputerineglandsareweak(openarrows inAandB,respectively).Duringprepartumluteolysis,withintheUt-Plunits,onlyweakendometrialEDNRAexpressionisobservedin thesuperficialglands(theso-calledglandularchambers)anddeeputerineglands(openarrowsinCandD,respectively).Intheplacental labyrinthduringprepartum luteolysis, cytotrophoblastcells at the base of the placental labyrinth stain strongly (openarrowsinEandF).Thereisnobackgroundstainingintheisotypecontrol(insertedinF).

GRAMet al.242

D).InUt-Plunits,theplacentallocalizationofEDN2wastargetedtothefetaltrophoblastcells(Fig.8E).

Fig. 6. Immunohistochemical(IHC)localizationofEDNRBinthecanineuterineandutero-placental(Ut-Pl)compartmentsatselectedtimepointsduringpregnancy;atthepre-implantationstage(AandB),intheUt-Plunitsduringmid-gestation(C,DandE),andintheUt-Pl compartments at prepartum luteolysis (F). (A,B)At pre-implantation,stainingofEDNRBispredominantlylocalizedtotheendometrialluminal(surface)epithelialcells(solidarrowsinA),glandularepithelialcellsofthesuperficialanddeeputerineglands(openarrowsinAandB)andmyocytes(solidarrowheadsinB).Duringmid-gestation,within theUt-Plunits,weakendometrialEDNRBexpressionisdetectedinthesuperficialglands(theso-calledglandularchambers)anddeeputerineglands(openarrowsinCandD,respectively).Intheplacentallabyrinthduringmid-gestation,strongsignalsarelocalizedinfetalsyncytiotrophoblastcells (open arrows in E). At prepartum luteolysis, only fetaltrophoblast (i.e., syncytio- and cytotrophoblast) stains strongly(openarrowsinF).No,orsporadicallyonlyweaksignals,canbeidentifiedinmaternaldecidualcells(solidarrowsinEandF).Inordertodistinguishbetweenfetalandmaternalcelltypeswithinthe canine placenta, i.e., endothelial, trophoblast and decidualcells, VIMENTIN staining was performed on consecutivesections following those used for EDNRB. Endothelial cells(openarrows inG)anddecidualcells (solidarrows inG)stainpositivelyforVIMENTIN.Thereisnobackgroundstainingintheisotypecontrol(insertedinF).

Fig. 7. LocalizationofEDN1,ECE1,EDNRA and EDNRBmRNAinthecanineutero-placental (Ut/Pl) unitsduringprepartum luteolysisas determined by in situ Hybridization (ISH). All factors arelocalized in trophoblast cells (open arrows;A–D). There is nobackgroundstaininginthenegativecontrols(inserttoA–D).

Fig. 8. Localization of EDN2-mRNA as determined by in situ Hybridization(ISH)inthecanineuterusatthepre-implantationstage(AandB)andintheutero-placental(Ut-Pl)compartmentsduring prepartum luteolysis (C, D and E). (A, B) At pre-implantation, EDN2-mRNA is localized to the endometrialluminal (surface) epithelial cells (solid arrows inA), glandularepithelial cells of the superficial (open arrows inA) and deeputerineglands(openarrowsinB).Duringprepartumluteolysis,onlyweakornosignalsarevisibleforEDN2expressionin thesuperficial glands (the so-called glandular chambers) and deeputerine glands (open arrows in C and D, respectively). In theplacentallabyrinthduringprepartumluteolysis,fetaltrophoblastcellsstainstrongly(openarrowsinE).There isnobackgroundstaininginthenegativecontrol(insertedinE).

CANINEPLACENTALEDN-SYSTEM 243

Discussion

Unequivocally,angiogenesisandvasculogenesisarecriticalstepsresponsibleforsuccessfuldecidualization,implantationandplacentation.Whiletheyhavebeenintensivelyinvestigatedinothermammalianspecies,sofar,littleisknownabouttheseprocessesinthecaninespecies.Therefore,inthecurrentstudy,wefeltpromptedtoinvestigatetheexpressionandlocalizationpatternsofthemembersoftheEDNfamily,i.e.,EDN1,-2,-3,aswellasoftheEDN-activatingenzymeECE1andthetwoEDNreceptors,EDNAandEDNB,inthecanineuterusandUt-Plcompartmentsduringselectedtimepointsofgestation(pre-implantation,post-implantation,mid-gestation),andduringnormalparturition(prepartumluteolysis)andantigestagen-inducedabortion.Inagreementwithpreviouslypublisheddatafromotherspecies,

e.g.,humans[35,36]orsheep[37],therewasclearlydetectableproteinexpressionofEDN1and-2,theconvertingenzyme(ECE1)andoneofthereceptors,EDNRB,particularlyintheepithelialuterinecompartmentsduringthepre-implantationstageofpregnancy.Interestingly,EDN2seemstobethemajorisoformrepresentedduringtheearlystagesofpregnancyinthedog.Thisfindingcorrespondspositivelywithobservationsmadeinmice,inwhichEDN2washighlyrepresentedinsomeorgansliketheuterus,ovaryorintestine[38].Itappearsplausiblethatalsointhedog,priortoimplantation,EDN2,

whichiscolocalizedwithEDNRB,mightbeinvolvedinfacilitat-ingthereleaseofNO[39].This,inturn,wouldimplytheindirectinvolvementofEDN2inregulationofuterineproliferation,vascularpermeabilizationandedemathatareimportantforuterineremodeling,embryoattachmentandimplantation.ItisalsointerestingthatEDN2wasabsentfromtheovariesofknockoutmicelackingexpressionoftheP4receptor(PGR),indicatingafunctionalrelationshipbetweenexpressionandfunctionofthesetwoentities[40].Takingintoac-countthehighuterinecontentofthispeptidepriortoimplantationanditssignificantlydecreasedexpressioninUT-Plcompartmentsofmid-pregnantdogsinresponsetoaglepristone-treatment,suchrelationshipmightalsoapplyinthedoguterus.Furthermore,theexpressionandcolocalizationofEDN-family

members,i.e.,thereceptors,theirligandsandtheEDN-activatingECE1,withintheplacentafetalis(trophoblast)oftheUt/Plcompart-ments,suggestsapossiblefunctionalroleofthissystemintrophoblastproliferationandinvasion.Ifproved,thiswouldresemblethesituationdescribedpreviouslyforhumantrophoblasts,whoseproliferativeandinvasivepropertiesaremediatedbytheEDNsreceptor[10,11].EDNRBisknownasapotentvasodilator[41],whereasEDNRA

exhibitsstrongvasoconstrictorproperties[42].Thus,thegradualincreaseofEDNRBexpressiontowardsmid-gestation,parallelingtheprogressionofcaninegestation,anditsabundantpresenceinthefetaltrophoblast,impliesthepossibleinvolvementofEDNRBin

Fig. 9. SchematicrepresentationofplacentalEDNsystemdistributionwithincanineutero-placentalcompartments(placenta endotheliochorialis).Post-implantationandatmid-gestationsimilarlocalizationpatternsareobservedforEDNRB,ECE1andEDN1.WhereasECE1islocalizedboth,insyncytiotrophoblastandcytotrophoblastcells,EDN1targetsonlytocytotrophoblastcells,andEDNRBstainspositivelyinsyncytiotrophoblast.Duringprepartumluteolysis,bothtypesoffetaltrophoblastcellsstainpositivelyforEDN2andEDNRB.EDN1,ECE1andEDNRAarelocalizedonlyincytotrophoblastcells.Asdeterminedbyin situhybridization(ISH),localizationpatternsofEDN1,ECE1,EDNRA and EDNRBmRNAweresimilarwithexpressionprofilesoftherespectiveproteins(investigatedbyimmunohistochemistry).Duetothelackofcommerciallyavailablecanine-specificantibody,localizationofEDN2wasdeterminedatthetranscriptlevelbyISH.

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modulatingvascularpermeabilityandtherebyfacilitatingbloodflowinfeto-maternalunits.Itisnoteworthythatitsplacentalexpressionshownhereiniscolocalizedwithcellularlocalizationofanotherpotentvascularpermeabilityfactor,whichisVEGFA[43].Duringprepartumluteolysis,placentalexpressionoftheEDN

receptors,aswellasECE1andEDN1,beingpredominantlyassociatedwithtrophoblastcells,stronglyresemblestheplacentallocalizationofprostaglandin(PG)familymembersregulatingtheavailabilityofprepartumprostaglandinsinthedog,e.g.,cyclooxygenase(COX2/PTGS2),PTGFS/AKR1C3orPTGES.Thefunctionalinvolve-mentofEDNsinPGsynthesishasbeenshownpreviously[27–29],implyingthatEDNsplayaroleintheregulationofutero-placentalhemodynamicsaffectingthephysiologicalmechanismsofparturition.Whetherthiscouldalsoapplyinthecaninespeciesremainstobeelucidated.Nevertheless,theupregulationofEDN1andEDNRAexpressionnotonlyduringnormalprepartumluteolysisbutalsoduringantigestagen-inducedparturition/abortion,whichinbothsituationsisassociatedwithincreasedPGoutput,impliesatemporalassociationbetweenPGsandEDNsduringprepartumluteolysisinthedog.ItisnoteworthythatthespatialexpressionofECE1andEDNRB

changedduringgestation.WhereasECE1wasinitiallyexpressedbothinsyncytio-andcytotrophoblast,itsexpressionprepartumwaspredominantlyrestrictedtothecytotrophoblastandwascolocalizedwiththeincreasedavailabilityofEDN1andEDNRAinthesamecells.Incontrast,theexpressionofEDNRB,whichwasinitiallylocalizedmostlyinthesyncytiotrophoblast,spreadovertheentiretrophoblastatthetimeofitsincreasedexpressionduringprepartumluteolysis.Evenifthisimpliesspatio-temporaldifferencesinthebiologicaleffectsevokedbyEDNsduringthemaintenanceandterminationofcaninegestation,thefunctionalmeaningoftheseobservationremainstobefurtherinvestigated.SchematicrepresentationofplacentalEDNsystemdistributionwithincanineutero-placentalcompartmentsisshowninFig.9.Thetime-dependentuterineandplacentalexpressionoftheEDN

system,andinparticularitsstrongabundanceinfetaltrophoblastcellsthroughoutpregnancy,impliesitsinvolvementinthefunctionalproliferativeandinvasivepropertiesofthesecells.Thefunctionalapproachpresentedhere,consistingofantigestagen-treatedanimals,leadingtotheupregulationoffetalplacentalEDN1anditsvasocon-strictorreceptorEDNRA,resemblingtherebythesituationobservedatnormalparturition,suggeststhattheyareinvolvedininitiatingthesignalingcascadeofPGsynthesisleadingtotheinductionofparturitioninthedog.

Acknowledgements

AuthorsaregratefultoProfDrSelimAslan,NearEastUniversi-ty,Cyprus(formerlyoftheUniversityofAnkara,Turkey),andProfDrBerndHoffmann,Justus-LiebigUniversityGiessen,Germany,andtheirteamsforprovisionofthetissuematerial,andtoDrBarryBavisterforcarefuleditingofthemanuscript.Thetechnicalexper-tiseandcontributionsofElisabethHöggeraregreatlyappreciated.PartofthelaboratoryworkwasperformedusingthelogisticsattheCenter forClinicalStudies,VetsuisseFaculty,UniversityofZu-rich.ThisresearchwassupportedbytheSwissNationalScienceFoundation (SNSF); researchgrantnumber31003A_160251.The

authorsdeclarethattheyhavenoconflictofinterests.Allauthorsreadandapprovedthefinalversionofthemanuscript.

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