Effects of Escherichia coli- and Staphylococcus aureus-induced mastitis in lactating cows

REPRODUCTION

RESEARCH

EffectsofEscherichiacoli-andStaphylococcusaureus-inducedmastitisinlactatingcowsonoocytedevelopmentalcompetence

SAsaf,GLeitner1,OFurman,YLavon,DKalo,DWolfensonandZRoth

DepartmentofAnimalSciences,FacultyofAgriculture,FoodandEnvironment,theHebrewUniversity,Rehovot76100,Israeland1MastitisLaboratory,TheVeterinaryInstitute,BetDagan50250,Israel

CorrespondenceshouldbeaddressedtoZRoth;Email:roth@agri.huji.ac.il

Abstract

Mastitisisassociatedwithdecreasedfertilityindairycows.Inthecurrentstudy,wecreatedanexperimentalmodeltosimulateshort-termmastitisbyasingleintramammaryadministrationofGram-negativeendotoxinofEscherichiacoliorigin(GK),orGram-positivetoxinofStaphylococcusaureusorigin(GC),toexaminetheeffectofmastitisonoocytedevelopmentalcompetence.HealthyHolsteincowsweresynchronized,andfollicular uid(FF)ofcowstreatedwithGCorGKandofuninfectedcows(controls)wasaspiratedfromthepreovulatoryfolliclesbytransvaginalultrasoundprocedure.TheaspiratedFFwasusedasmaturationmediumforinvitro

embryoproduction.ThedistributionofmaturedoocytesintodifferentcorticalgranuleclassesandmeioticstageswasaffectedbyGKadministration(P!0.05)butnotbyGCadministration.Theproportionofoocytesthatcleavedtotwo-andfour-cellstageembryos(44hpostfertilization)waslowerinbothGCandGKgroupsthanincontrols(P!0.05).Blastocystformationrate(7–8dayspostfertilization)waslowerintheGKgroup(P!0.05)andnumericallylowerintheGCgroupcomparedwiththeiruninfectedcounterparts.The

totalcellnumberinblastocystsdidnotdifferamonggroups;however,theapoptoticindexwashigherintheGCgroup(P!0.05),butnotintheGKgroup,relativetocontrols.ExaminingmRNArelativeabundanceinoocytesandearlyembryosrevealedmastitis-inducedalterationsinPTGS2(COX2),POU5F1,andHSF1butnotinSLC2A1(GLUT1)orGDF9.ResultsindicateadifferentialdisruptiveeffectofmastitisinducedbyGKandGConoocytedevelopmentalcompetenceinassociationwithalterationsinmaternalgeneexpression.

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Introduction

Mastitisisoneofthemajordiseasesaffectingdairycattleworldwide.Itcauseslargeeconomiclossestothedairyindustry,duetobothlossofmilkproductionandlowmilkquality.Mastitisalsohasdeleteriouseffectsonreproductiveperformance.Forinstance,thetimefromparturitionto rstinseminationislongerandthenumberofservicesforconceptionislargerinmastiticcows(Schricketal.2001,Maizonetal.2004,Lavonetal.2011a).Otherepidemiologicalstudieshavedemonstratedthatconceptionrates(Loef eretal.1999,Santosetal.2004)andpregnancyrates(Harmanetal.1996)arelowerinmastiticvshealthycows.

Oocytedevelopmentalcompetenceisacquiredinaprogressivemannerthroughoutfolliculardevelopmentandincludesavarietyofmolecularandcellularmodi cationsthatarerequiredfortheoocytetocompletemeiosis,successfulfertilization,maternalzygotetransition,andfurtherpre-andpostimplantationdevelopment(Coticchioetal.2004).Theovarianfollicular uid(FF)inwhichtheoocyteisenclosedanddevelopedisofplasmaorigin(Edwards1974).Thus,itisreasonabletoassumethatelevationofbiomoleculesin

q2014SocietyforReproductionandFertilityISSN1470–1626(paper)1741–7899(online)

theplasmauponphysiologicaland/orpathologicalimpairments,suchasmastitis,mightaffectFFcompo-sitionandthefollicle-enclosedoocyte.Insupportofthis,Nakajimaetal.(1997)reportedthepresenceoftumornecrosisfactora(TNFa)andinterleukin6(IL6)inthemilkandserumofcowswithnaturallyoccurringcoliformmastitis.Blumetal.(2000)reportedanincreaseinTNFaandNOx(nitriteandnitrate)inthemilkandplasmauponEscherichiacoli-inducedmastitis.Hisaedaetal.(2001)detectedinterferon-gandTNFainserumandwheysamplesfromnaturallyoccurringcoliformmastitis.Supplementationoflipopolysaccharide(LPS),prostaglandinF2a(PGF2a),nitricoxide(NO)generator,orsodiumnitroprussidetothematurationorculturemediumdeleteriouslyaffectedthedevelopmentalcom-petenceofbovineoocytes(Sotoetal.2003a).ExposingbovineembryostoTNFaincreasedtheproportionofapoptoticblastomeresinthedevelopedblastocyst(Sotoetal.2003b).Inaddition,TNFadecreasesthenumberofcellsintheinnercellmassinmouseembryosandlowersembryonicsurvival(Pampferetal.1994,Wuuetal.1999).Nevertheless,theconcentrationofin ammatorymediatorsintheFFofthepreovulatoryfolliclehasneverbeenexamined.Here,wehypothesizedthat

DOI:10.1530/REP-13-0383

34

SAsafandothers

mastitis-inducedalterationsintheFFcontenthaveadeleteriouseffectonbothmaturationanddevelopmentalcompetenceofthefollicle-enclosedoocyte.

Weestablishedanexperimentalmodelbasedon:i)asingleintramammaryinjectionofGram-negativeendotoxinofE.coliorigin(GK)orGram-positivetoxinofStaphylococcusaureusorigin(GC);ii)invivoaspirationofthepreovulatoryFF;andiii)invitromaturation(IVM)ofbovineoocytesusingtheaspiratedFF(i.e.,physio-logicallyrelevantconditions)asmaturationmedium.Severalanalyseswereperformedtoexaminetheoocytes’competencetoundergonuclearandcytoplasmicmaturation(maturationcompetence),befertilized,cleave,anddeveloptotheblastocyststage(develop-mentalcompetence).Blastocystqualitywasestimatedbytotalcellcountandapoptoticindexinthedevelopingblastocyst.Expressionofstress-relatedgenessuchasPTGS2(COX2)andHSF1(Duboisetal.1998,Santoro2000)andthoseinvolvedinfolliculogenesisandearlyembryonicdevelopmentsuchasGDF9andPOU5F1(Niwaetal.2000,Gui&Joyce2005)andcellmetabolismsuchasSLC2A1(GLUT1)(Leppens-Luisieretal.2001)wasdeterminedbyreal-timePCRinmetaphaseII(MII)stageoocytesandfour-cellstageembryos,beforeactivationoftheembryonicgenome.

Materialsandmethods

Animalsandmilksamples

Thestudywascarriedouton19healthy,cyclicHolsteincowsintheir rstto fthlactation.TheaverageDIMwas125days.Cowsenrolledinthestudywerewithsomaticcellcount(SCC)!150000cell/mlmilkandwithnobacteriological ndingsasdeterminedby3-monthlyroutinemilktestpriortotheexperiment(byFossomatic360instrument,FossElectric,Hillerød,Denmark).Inaddition,SCCwasdetermined(byZ1Coultercounter,CoulterElectronics,Luton,UK)inthreesequentialasepticmilksamplestakenfromeachofthefourmammaryquartersonceaweekbeforetheexperimentwasbegun,asdescribedpreviously(Younisetal.2003,Leitneretal.2006).BacterialexaminationwasperformedaccordingtotheInternationalDairyFederationproceduresusingacceptedstandardsforbacteriologicaltyping(Oliveretal.2004).

Inductionofmastitis

MastitiswasinducedasdescribedpreviouslybyLavonetal.(2011b).TheexperimentwasapprovedbythelocalethicscommitteeoftheHebrewUniversity.Brie y,healthycowswererandomlyseparatedintothreegroupsandreceivedasingleintramammaryinjectionintoonequarterasfollows:i)controlcows(nZ6)received10mlofsterilenonpyrogenicsaline;ii)theGKgroup(nZ6)received10mgLPS(E.coliO55:B5,SigmaChemicalCo.)dissolvedin10mlnonpyrogenicsaline;andiii)theGCgroup(nZ7)received40mgS.aureusFR2449/1extractdissolvedin10mlnonpyrogenicsaline,asdescribedpreviously(Leitneretal.2002,Younisetal.2003).

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Intramammaryinjectionswereperformedfollowingcarefulcleaningoftheteat,usingasterilesyringemountedwithasterileadapterviatheteatcanalintothegland,asdescribedpreviously(Lavonetal.2008).Clinicalsymptomssuchasswelling,rigidity,andhighsensitivityoftheudderwerecheckedfrequentlythroughoutthedayofadministration.Bodytemperatureswererecordedhourlyfor12h,andSCCwasdetermined24hfollowingtoxininjection.

Synchronizationandfollicularaspiration

CowsweresynchronizedbytheOvsynchprotocol(Fig.1).GNRHanalog(200mgGonadorelin,Gonabreed,ParnellLaboratories,Alexandria,NSW,Australia)wasinjected(i.m.,dayK8)followedbyinjectionofPGF2aanalog(500mgCloprostenol,Estroplan,ParnellLaboratories;dayK2)andasecondGNRHadministration(day0).Ultrasonographicscanning(7.5MHzlinearprobewithSSDAloka900,Tokyo,Japan)wasperformedthroughdays0–6ofthecycletocon rmovulationandcorpusluteumformationandtocharacterizethepatternofgrowthofthe rst-wavedominantfollicle.Ondays6and7ofthesynchronizedcycle,cowsreceivedaninjectionofPGF2atoinduceregressionofthecorpusluteumanddevelopmentofpreovulatoryfollicle.

FFofthedevelopingpreovulatoryfolliclewasaspirated42hafterthesecondinjectionofPGF2a(Fig.1A).Brie y,cowsweresedatedwithxylazinehydrochloride(Sedaxylan;EurovetAnimalHealthBV,Bladel,Holland),andcaudalepiduralanesthesiawasinducedwithlidocaine.Aspirationwasperformedwithan

A

G–, G+

–8

–2

6 and 78

Cycle day

B

COCs collection

Day 0

IVM in FF

IVFIVC

22 h

18 h

42–44 h PFDays 7–8Nuclear and cytoplasmic

Cleavage (%)

Blastocyst (%)

maturation

Figure1Theexperimentalmodelcomprisedtwostages.Intheinvivostage(A),cowsweresynchronizedusingtheOVSYNCprotocol.GNRHanalogwasinjected(dayK8)followedbyinjectionofPGF2aanalog(dayK2)andasecondGNRHadministration(day0).Ultrasonographicscanningwasperformedthroughdays0–6ofthecycle.Ondays6and7,cowsreceivedaninjectionofPGF2atoinduceluteolysisand

developmentofapreovulatoryfollicle.Mastitiswasinduced36hlater,byinjectionofE.coliLPS(GK),S.aureusextract(GC),orsterilesaline(control).Follicular uid(FF)wasaspiratedfromthepreovulatoryfollicleonday8ofthecycle(6haftermastitisinduction).Inthesecondstage(B),cumulus–oocytecomplexes(COCs)wereaspiratedandinvitromatured(IVM)in50mldropletsofoocytematurationmedium(OMM)or

previouslyaspiratedFF.Subgroupsofmaturedoocyteswerecollectedforexaminationofnuclearandcytoplasmicmaturation,whereasthe

remainingoocyteswereIVFfor18h.Afterfertilization,putativezygotesweredenudedofcumuluscellsandinvitrocultured(IVC)for7–8days.

ultrasoundscanner(PieMedical,Maastricht,TheNetherlands)equippedwitha7.5MHzvaginaltransducerand19Gneedleconnectedtoasterilesyringe.Foreachexperimentalgroup,theaspiratedFFswerepooledandkeptatK208C.

Hormoneconcentrations

EstradiolconcentrationsintheFFweredeterminedbysolid-phaseRIAkit(DiagnosticProductsCorp.,LosAngeles,CA,USA)asdescribedpreviously(Lavonetal.2011b).Theassaysensitivitywas8pg/ml,andtheintra-assaycoef cientofvariationwas3%.ProgesteroneconcentrationintheFFwasanalyzedwiththesolid-phaseRIAkitagainstastandardcurvepreparedfromovari-ectomizedcowplasma(Lavonetal.2011b).Theminimumdetectedamountwas0.2ng/mlandtheintra-andinter-assaycoef cientsofvariationwere8.6and9.9%respectively.

Chemicalsandmediaforinvitroprocedures

Allchemicals,unlessotherwisestated,werepurchasedfromSigma.Follicle-stimulatinghormone(FSH)isolatedfromovinepituitaryextract(Ovagen)wasfromICPBio(Auckland,NewZealand).Double-distilledwater(DDW)wasfromMerck.Dulbecco’sPBS,FCS,andRQ1RNase–freeDNaseIwerefromPromega.Diethylpyrocarbonate(DEPC)-treatedwaterwasfromBiologicalIndustries(BeitHaemek,Israel).Paraformal-dehyde(16%)wasfromElectronMicroscopySciences(Hat eld,PA,USA).SuperscriptIIreversetranscriptase,DynabeadsmRNADIRECTKit,non-essentialaminoacids,andessentialaminoacidswerefromInvitrogen.DyNAmoColorFlashSYBRGreenqPCRKitwasfromFinnzymes(Espoo,Finland).InsitucelldeathdetectionkitwasfromRoche.FluoromountwasfromDiagnosticBiosystems(Pleasanton,CA,USA).TheculturemediaHEPES–Tyrode’slactate(TL),SP-TL,andIVF-TLwerepreparedinourlaboratory:HEPES–TLwassupplementedwith0.3%(w/v)BSA,0.2mMsodiumpyruvate,and0.75mg/mlgentamicin(HEPES–TALP);SP–TLwassupple-mentedwith0.6%BSA,1mMsodiumpyruvate,and0.2mg/mlgentamicin(SP–TALP);IVF–TLwassupplementedwith0.6%(w/v)essentialfattyacid–freeBSA,0.2mMsodiumpyruvate,0.05mg/mlgentamicin,and0.01mg/mlheparin(IVF–TALP)asdescribedbyParrishetal.(1986).Oocytematurationmedium(OMM)wasmadeupofTCM-199andEarle’ssaltssupple-mentedwith10%(v/v)heat-inactivatedFCS,0.2mMsodiumpyruvate,50mg/mlgentamicin,2.2g/lsodiumbicarbonate,2mg/ml17b-estradiol,and1.32mg/mlFSH.Potassiumsimplexoptimizedmedium(KSOM)contained95mMNaCl,2.5mMKCl,0.35mMKH2PO4,0.2mMMgSO4.7H2O,0.8%(v/v)sodiumlactate,0.2mMsodiumpyruvate,0.2mMD(C)-glucose,25mMNaHCO3,0.01mMphenolred,1mML-glutamine,.and0.01mMEDTAsupplementedwith1.7mMCaCl22H2O,0.1mg/mlpolyvinylalcohol,10ml/mlessentialaminoacidsand5ml/mlnon-essentialaminoacids,100U/mlpenicillin-G,and0.1mg/mlstreptomycin.

Invitroproductionofembryos

Invitroproduction(IVP)ofembryoswasperformedasdescribedpreviouslybyGendelman&Roth(2012a,2012b).

Mastitisimpairsoocytecompetence

35

Brie y,Holsteincowovarieswereobtainedfromalocalabattoirandtransportedtothelaboratorywithin60–90mininphysiologicalsalinesolutionat378Cwith50mg/mlpenicillin–streptomycin.Ovarieswereplacedoveratrans-illuminator(Arav2001)andcumulus–oocytecomplexes(COCs)wereaspiratedfrom3to8mmfollicles.GroupsoftenCOCsweretransferredto50mldropletsofOMMorFFoverlaidwithmineraloilandincubatedinhumidi edairwith5%CO2for22hat38.58C.Groupsof30maturedoocytesweretransferredtofour-wellplatescontaining,perwell,600mlIVF–TALPand25mlPHE(0.5mMpenicillamine,0.25mMhypotaurine,and25mMepinephrinein0.9%NaCl)andIVFwithPercoll-puri edspermatozoa(w1!106)fromthesamebull(i.d.Pazil3421‘Sion’Hafetz-Haim,Israel)for18hat38.58C,5%CO2.Afterfertilization,putativezygotesweredenudedofcumuluscellsbygentlevortexinginHEPES–TALPcontaining1000U/mlhyaluronidaseandrandomlyplacedingroupsoftenin25mldropletsofKSOM.Allembryodropletswereoverlaidwithmineraloilandculturedfor8days(38.58C,5%CO2,and5%O2).TheexperimentsincludedsixIVPrunswith57–68oocytesperexperimentalgroupperrun.

Nuclearandcorticalgranulestaining

Attheendofmaturation,oocytesweredenudedofcumuluscellsandplacedinpermeabilizationsolutioncontainingPBSwith1mg/mlpolyvinylpyrrolidone(PVP)and0.1%(v/v)TritonX-100for5minat398Cintheoven.Thenoocyteswere xedin4%(v/v)paraformaldehydeinPBSfor15minatroomtemperatureandstoredinPBSwith1mg/mlPVP(PBS–PVP)at48C.OocyteswerewashedthreetimesinPBS–PVPandplacedinblockingsolutioncontainingPBSwith1mg/ml0.1%(v/v)TritonX-100,2%(v/v)normalgoatserum,0.1Mglycine,1%(w/v)powderedskimmilk,and0.5%(w/v)BSA(pH7.4)for1hat398Cintheoven.

Corticalgranuleswerestainedwith100mg/mlFITC–peanutagglutinin(PNA)inPBS–PVPfor30minat398Cintheoven.Nuclearstainingwasperformedwith10mg/ml40,6-diamidino-2-phenylindoledihydrochloride(DAPI)inPBS–PVPfor15minatroomtemperature.StainedoocyteswerewashedandplacedindropsofFluoromountandexaminedunderinverted uorescencemicroscope(Nikon,Tokyo,Japan)usingNisElementsSoftware(Nikon).

Theoocyteswereclassi edintothreetypesaccordingtotheobserveddistributionalpatternofthecorticalgranulesasde nedbyIzadyaretal.(1998):classI,largeaggregatesofcorticalgranulesdistributedovertheentirecytoplasm;classII,corticalgranuleslocalizedinthecorticalcytoplasmanddistributedasindividualparticlesaswellassmallaggregates;andclassIII,corticalgranulesmoreorlessevenlydispersedinthecorticalcytoplasmaligningtheoolemma(Fig.2A,B,andC).Thesameoocyteswerealsoclassi edintofourmeioticnuclearstagesasdescribedpreviouslybydelosReyesetal.(2005):germinalvesicle(GV),germinalvesiclebreakdown(GVBD),metaphaseI(MI),andMII(Fig.2D,E,F,andG).Theexaminationincluded50oocytespergroupfrom vedifferentIVMruns.

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SAsafandothers

Figure2Cellularfeaturesinmaturedoocytesandblastocysts.CorticalgranuleswerestainedwithFITC–PNA(A,B,andC).Oocyteswereclassi edintoclassI(A),classII(B),andclassIII(C).NucleiofthesameoocyteswerestainedwithDAPI(D,E,F,andG)andclassi edintofourmeioticstages:germinalvesicle(D),germinalvesiclebreakdown(E),metaphaseI(F),andmetaphaseII(G).Representativeimagesof8-dayblastocyststainedwithHoechst33342(H,bluenuclei),stainingpositiveinTUNELassay(I,greennuclei),andmergedpictures(J).

DetectionofDNAfragmentationbyTUNELassay

TUNELassay(Roche)wasusedtodetectDNAfragmentationaspreviouslyperformedinourlaboratory(Kalo&Roth2011).Brie y,8-dayembryoswerewashedthreetimesinPBS–PVP, xedin4%paraformaldehydeinPBSfor15minatroomtemperature,andstoredinPBS–PVPat48C.Onassay,sampleswereplacedinpermeabilizationsolutioncontainingPBSwith1mg/mlPVP,0.3%TritonX-100,and0.1%(w/v)sodiumcitratefor20minatroomtemperatureinanhumidi edbox.Forpositiveandnegativecontrols,sampleswereincubatedin50mldropsof50U/mlRNase–freeDNaseat378Cfor1hinthedark.AfterRNase–freeDNasetreatment,sampleswereincubatedin50mldropletsofTUNELreactionmixture(containingFITC-conjugateddUTPandTdT)for1hat378Cinthedark.Thenegativecontrolwasincubatedunderthesameconditions,butwithoutTdT.Finally,sampleswerestainedwith1mg/mlHoechst33342inPBS–PVPfor15minatroomtemperature,washedthreetimesinPBS–PVP,belingwasexaminedunderaninverted uorescencemicroscopeusingNisElementsSoftware(Fig.2H,I,andJ).TheapoptoticcellratioforeachblastocystwasdeterminedbycalculatingthenumberofTUNEL-positive

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blastomeresoutofthetotalnumberofblastomeres.Theexaminationincluded7-dayblastocysts,20perexperimentalgroup,fromthreedifferentruns.

Genequanti cation

Oocyteswerecollectedafter22hofIVManddenudedofcumuluscellsasdescribedabove.Theexaminationincluded100oocytes( vesamplesof20oocyteseach)takenfrom vedifferentIVPruns.Inaddition,four-cellstageembryoswerecollectedat42–44hpostfertilization.Theexaminationincluded50embryos( vesamplesoftenembryoseach)takenfrom vedifferentIVPruns.AllcollectedsampleswerewashedinPBS,snapfrozeninliquidnitrogen,andstoredatK808CuntilRNAextraction.

Poly(A)RNAwasisolatedusingDynabeadsmRNADIRECTKitaccordingtothemanufacturer’sinstructions(Invitrogen)asdescribedpreviouslybyGendelman&Roth(2012a,2012b).Inbrief,oocytesandembryoswerelysedbyadding100mllysis-bindingbuffertoeachsample.Prewashedoligo(dT)25Dynabeads(20ml)wereaddedtoeachtubeandmixedfor5minatroomtemperaturetoallowbindingofpoly(A)tothebeads.ThesampleswereputintoamagneticseparatortoremovethelysisbufferwhileretainingtheDynabeads.TheDynabeadswerewashedtwicewith100mlwashingbufferA(100mMTris–HCl,pH7.5,500mMLiCl,10mMEDTA,pH8,and5mMdithiothreitol),twicewith100mlwashingbufferB(10mMTris–HCl,pH7.5,0.15MLiCl,and1mMEDTA),andoncewith100ml10mMTris–HCl.AfterremovalofHCl,8mlsterileDEPCwaterwasaddedandthesampleswereimmediatelysubjectedtoRT.RTwasperformedinatotalvolumeof20ml.The rststepwasincubationat708Cwith8mlRNAsample,1mloligo(dT)12–18(500mg/ml),1mlRNAseout,1mldNTPs(10mMeach),and1ml(50ng)randomprimer,followedby50minincubationat428Cand5minat708CwithRTmixcontaining4ml5!reversetranscriptasebuffer,200USuper-scriptIIreversetranscriptase,2ml0.1Mdithiothreitol,andDEPCwater.ThesamplesweretransferredtoK208Cuntiluse.QuantitativeRT-PCRwascarriedoutwithprimersforPTGS2(alsoknownasCOX2),HSF1,GDF9,POU5F1,andSLC2A1(alsoknownasGLUT1),usingYWHAZasareferencegene(Gendelman&Roth2012a,2012b).TheprimerswerederivedfrombovinesequencesfoundinGenBankanddesignedusingPrimerExpressSoftware(LifeTechnologies,Carlsbad,CA,USA;Table1).Brie y,real-timePCRwasconductedonanMx3000pcycler(Stratagene,LaJolla,CA,USA)usingSYBRGreenina nalvolumeof20mlcontainingultrapurewater,500nMofeachprimer,and3mldilutedcDNA.Thereactionef ciencyrangedbetween90and110%withR2O0.995.Theampli cationprogramincludedpreincubationat958Cfor7mintoactivatetaqpolymerase,followedby40ampli cationcyclesofdenaturationat958Cfor10sandannealing–elongationat608Cfor15s.Allsampleswereruninduplicatein96-wellplates.Ameltingcurveanalysiswasperformedattheendoftheampli cationtocon rmsingle-genespeci city.Fluorescencewasrecordedtodeterminethethresholdcycleduringthelog-linearphaseofthereactionatwhich uorescencerisesabovebackground.Geneexpressionwasquanti edandanalyzedbyMxPROQPCRSoftware

for

Table1Primersusedinthisstudyforreal-timePCR.AccessionGene

Primersequence

numberSize(bp)YWHAZForward:GCATCCCACA-NM_174814

124

GACTATTTCC

Reverse:GCAAAGACAAT-GACAGACCA

GDF9Forward:

NM_174681202

TGGTCCTTGCTGAAG-CATCTAGA

Reverse:ACAGTGTTGTA-GAGGTGGCTTCT

POU5F1Forward:ATATACC-AY490804201

CAGGCCGATGTGGReverse:TGCA-CAAGGGTCTCTGCCTT

SLC2A1Forward:CAGCCA-M60448167

GAGTCTCCTTCACCReverse:CATAGC-CACCTCTTGGGGTA

HSF1Forward:AAAGATTCGC-NM

151

CAGGACAGTG

001076809Reverse:CTGGAT-GAGCTTGTTGACGA

PTGS2Forward:GAAATGATC-AF031698161

TACCCGCCTCA

Reverse:TCTGGAA-NM174445

CAACTGCTCATCG

Mx3000pandMx3005pQPCRver.3,andtheDDCtmethodwasusedtocalculatetherelativeexpressionofeachgene.

Experimentaldesign

Theexperimentalmodelincludedtwostages.Inthe rst(Fig.1A),mastitiswasinducedinvivousingdosesofGKorGCtoxinsasdescribedaboveandaspiratedpreovulatoryFF.Brie y,lactatingHolsteincowsweresynchronized,andondays6and7ofthecycle,PGF2awasinjectedtoinducedluteolysisanddevelopmentofapreovulatoryfollicle.Mastitiswasinduced36hpost-PGF2ainjectionandthepreovulatoryfollicleswereaspirated6hlater.Foreachexperimentalgroup,theaspiratedFFswerepooledandkeptatK208C.

Inthesecondstage(Fig.1B),oocytesaspiratedfromHolsteincowovariesobtainedfromalocalabattoirwerematuredinvitrointheundilutedFFaspiratedinthe rststage.Therationaleforusingthismodelwastomatureoocytesinaphysiologicallyrelevantfollicularenvironment.Attheendofmaturation,fromeachexperimentalgroup,subgroupsofoocyteswereexaminedforcorticalgranuledistribution(FITC–PNA)andmeioticstatus(DAPI).Inaddition,maturedoocyteswerefertilized,culturedfor8days,andtheproportionofoocytesthatcleavedanddevelopedtoblastocystswasrecorded44hand7and8dayspostfertilizationrespectively.Blastocystqualitywasestimatedbytotalcellcountandapoptoticindex(TUNELassay).Geneexpressionwasexaminedinbothmaturedoocytesandfour-cellstageembryos(real-timePCR).

Statisticalanalysis

Differencesbetweentreatmentsweresubjectedtoone-wayANOVA(JMP-7;SASInstitute,Cary,NC,USA)followedby

Mastitisimpairsoocytecompetence

37

Tukey–Kramertest.Toexaminethemodel’sreliability,oocytematurationinastandardOMMwascomparedwiththatinundilutedFFaspiratedfromcontroluninfectedcows.ToexaminetheeffectofmaturationinFFaspiratedfrommastiticcows,GKandGCgroupswerecomparedwithcontrols.Thevariableswereasfollows:SCCinmilksamples,estradiolandprogesteroneconcentrationsintheFF,proportionofoocytesthatcleavedtothetwo-andfour-cellstagesanddevelopedtoblastocysts(datawerearcsine-transformedbeforeanalysis),relativemRNAabundanceinmaturedoocytesandfour-cellstageembryos,totalcellcount,andproportionofTUNEL-positiveblastomeresin8-dayblastocysts.DataarepresentedasmeanGS.E.M.andP!0.05wasconsideredsigni cant.Analysisoflikelihoodratiowasperformedtoexaminedifferencesinoocytedistributiontocorticalgranuleclasses(I–III)andnuclearmeioticstages(GV,GVBD,MI,andMII).P!0.05wasconsideredsigni cant.

Results

Mastitiseffectsonbodytemperature,milkyield,andSCC

InductionofGKmastitisincreasedbodytemperatureby28C(P!0.05),whichlastedfor4habovethebodytemperatureofthecontrolgroup.InthecowstreatedwithGC,bodytemperaturewasonlyslightlyhigher(C0.28C,NS)thanthatofcontrols.Inductionofmastitisdidnotaffectmilkyieldfor3daysaftertoxinadministration.IntheGKinducedmastitisgroup,SCCincreasedto5!106cells/ml24hafterLPSadministration,andintheGCgroup,SCCincreasedto2.5!106cells/ml.Localclinicalsymptomsintheudder,suchasswelling,rigidity,andhighsensitivity,weredetectedintheGKquartersbutnotintheGCquarters(datanotshown).

Validationoftheexperimentalmodel

Toexaminethemodel’sreliability,oocytematurationinstandardOMMwascomparedwiththatinundilutedFFaspiratedfromcontroluninfectedcows.Theexperimentincludedthreeto verunswith50oocytesperrunperexperimentalgroup.Cellularandnuclearmaturationcharacteristics,totalcellcount,andapoptoticindexinblastocystsdidnotdifferbetweengroups(Fig.3AandA0).Cleavageintotwo-tofour-cellstageembryosandblastocystformationrates(72vs76%and12.3vs13%respectively)weresimilarinbothgroups(Fig.3BandB0).Inlightofthese ndings,FFaspiratedfromcontrolcowswasusedasacontrolmediumfortheIVMprocedure.Foreachexperimentalgroup,FFswerepooled.Theaverageprogesteroneconcentrationdidnotdifferbetweengroupsandwere96.1,135.5,and236.2ng/mlforGK,GC,andcontrolgroupsrespectively.Theaverageestradiolconcentrationdidnotdifferbetweengroupsandwere1067.4,1109.3,and1060.0ng/mlforGK,GC,andcontrolgroupsrespectively.

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38

SAsafandothers

A

100B100

cit80o)

)

80i%e%m( 60( e60 sgsueaeg40va40lacteuslN20C200

OMM

FF-control

OMM

FF-control

A′

100

B′

)

%Class 320(

s80eClass 2)

luClass 1

%(15 n60stasryg l40c10oatcsitro20al

B5C0

OMM

FF-control

OMM

FF-control

Figure3Comparisonofnuclearandcytoplasmicmaturationanddevelopmentalcompetenceofbovineoocytesmaturedinoocytematurationmedium(OMM)andfollicular uid(FF-control)aspiratedfromnon-treatedcontrolcows.(A)Distributionofoocytestomeioticstages,i.e.,germinalvesicle(GV),germinalvesiclebreakdown(GVBD),metaphaseI(MI),andmetaphaseII(MII),ineachoftheexperimentalgroups.(A0)DistributionofoocytesintocorticalgranuleclassesI,II,andIIIineachoftheexperimentalgroups.Theexaminationincluded50oocytespergroupfromthreedifferentruns.Dataarepresentedasmeans,likelihoodratiotest;*P!0.05.(B)Thepercentageofoocytescleavedtotwo-tofour-cellstageembryos,42–44h

postfertilization,and(B0)thepercentageofembryosdevelopedtotheblastocyststageonday8postfertilization.Theexaminationincluded verunswith50oocytesperrunperexperimentalgroup.DataarepresentedasmeansGS.E.M.;treatmenteffectwithinembryonicstages,*

P!0.05.

Nuclearandcytoplasmicmaturation

Oocytedistributionintonuclearmeioticstages(GV,GVBD,MI,andMII)differed(P!0.05;Fig.4A)betweentheGKandcontrolgroups,characterizedbyareducedproportionofMIIoocytesandincreasedproportionofGVstageoocytesintheformer.Oocytedistributionintothevariouscorticalgranuleclasses(I–III;i.e.cytoplasmicmaturation)differedbetweentheGKandcontrolgroups(P!0.05;Fig.4B),characterizedbyanincreasedproportionofclassI(non-maturedoocytes)anddecreasedproportionofclassIII(maturedoocytes)intheGKgroup.TheGCtreatmentdidnotaffectnuclearorcytoplasmicmaturationoftheoocyte(Fig.4AandB).

Oocytedevelopmentalcompetence

Theproportionofoocytesthatfertilizedandcleavedtotwo-tofour-cellstageembryoswaslower(P!0.02;Fig.5A)intheGKandGCgroupsthaninthecontrolgroup.Thepercentageofembryosdevelopedtotheblastocyststagewaslower(P!0.05;Fig.5B)intheGKvscontrolgroupandnumericallylowerintheGCgroup.

Reproduction(2014)14733–43

TotalcellcountandapoptoticrateinblastocystsEighty-dayblastocystsweresubjectedtoTUNELprocedure.Therangeoftotalcellnumberswas88–95cellsperblastocystanddidnotdifferamonggroups(Fig.6A).However,theproportionofTUNEL-positive(apoptotic)cellsperembryowashigherintheGCembryosthaninthecontrols(P!0.05;Fig.6B)andthatthatintheGKembryosdidnotdifferfromcontrols.Geneexpression

Maturedoocyteswerecollectedattheendof22-hmaturationandembryosatthefour-cellstagewerecollected42–44hpostfertilizationandanalyzedforgeneexpression.TheexpressionofPTGS2andPOU5F1inoocytesmaturedinGKFFwaslowerthanthatofthecontrol,whereastheexpressionofthesetwogenesinoocytesmaturedinGCFFdidnotdifferfromthoseinthecontrol(P!0.05;Fig.7A).TheexpressionofHSF1waslowerinboththeGKandGCgroupsthaninthecontrols(P!0.05;Fig.7A).

IncreasedPTGS2expressionwasdetectedinfour-cellstageembryosofbothGKandGCgroupsrelativetocontrols(P!0.05;Fig.7B).TheexpressionofPOU5F1

A100)

%( 80

MIIseMIgaGVBDts c60GV

itoiem40 suelc20uN0

Control

G+

G–B100)

Class III%(80 Class IIsel60Class I

unarg la40citroC200

Figure4Effectofinducedclinicalmastitisonnuclearandcytoplasmicmaturation.(A)Distributionofoocytestomeioticstages,i.e.germinalvesicle(GV),germinalvesiclebreakdown(GVBD),metaphaseI(MI),andmetaphaseII(MII),ineachoftheexperimentalgroups.(B)

DistributionofoocytesintocorticalgranuleclassesI,II,andIIIineachoftheexperimentalgroups.Theexaminationincluded50oocytespergroupfrom vedifferentruns.Dataarepresentedasmeans,likelihoodratiotest;*P!0.05.

A10080

)

%( e60gaave40lC200

ControlG+G–

B1412

)

%10( tss8yoct6aslB420

Control

G+

G–

Figure5Developmentalcompetenceofbovineoocytesmaturedinfollicular uidaspiratedfrominducedmastiticcows.Presentedisthepercentageofoocytescleavedtothetwo-tofour-cellstageembryo,42–44hpost-fertilization(A),andthepercentageofembryosthatdevelopedtotheblastocyststageonday8postfertilization(B).Theexperimentincludedsixrunswith57–68oocytesperrunperexperimentalgroup.DataarepresentedasmeansGS.E.M.;differentlettersindicatetreatmenteffectwithindevelopmentalstages(P!0.05).

waslowerinGCandhigherinGKgroupsrelativetocontrols(P!0.05;Fig.7B).TheexpressionofHSF1waslowerintheGKgroupthaninthecontrolgroup(P!0.05;Fig.7B).

Discussion

Mastitisisoneoftheriskfactorsfordisruptedreproduc-tiveperformanceindairycows,butthemechanismbywhichitimpairstheovarianpoolofoocytesisnotentirelyclear.Here,weprovideevidenceforthedifferentialimpairmentofmaturationanddevelopmentalcompe-tenceofoocytesinassociationwithalterationsinbothcellularandmolecularfeaturesbyLPSofE.coli(GK)vsS.aureusextract(GC)mastitis.Theexperimentalapproachwasbasedoninducedmastitis(invivo)followedbyIVMofoocytesinthepreovulatoryFFaspiratedfromthetreatedcows.Giventhelimitedabilitytoidentifyclinicalmastitisinatimelyfashion,thiswasfoundtobeagoodsimulationapproach.Oneofthemainadvantagesofthismodelwasthatmastitiseventswerewellcontrolled:cowsweresynchronizedandtoxinadministrationwasperformedatthesamestageoftheestrouscycle.AnotheradvantagewasthatIVMintheFF

Mastitisimpairsoocytecompetence

39

enabledtheuseofalargenumberofoocytesratherthanasingleoocytepercowaspiratedfromasinglepreovula-toryfollicle.Moreover,maturationofoocytesintheFFdidnothaveanydeleteriouseffectsonoocytedevelopmentalcompetence,astheproportionofoocytesthatcleavedanddevelopedtotheblastocyststagedidnotdifferfromthatofoocytesmaturedinthestandardmaturationmedium.Similarly,previousstudieshavereportedthatoocytematurationinFForadditionofacertainconcentrationofFFcanevenimproveembryonicdevelopment(Alietal.2004,Colemanetal.2007).Bycontrast,anotherstudyshowedthatmaturationofoocytesinFFreducesdevelopmentalcompetence(Averyetal.2003).DifferencesbetweenstudiesmightbeduetotheuseofFFaspiratedatdifferentstagesoffolliculargrowthortheestrouscycle.Itshouldbenoted,however,thatmaturationofoocytesaspiratedfrom3to8mmfollicles(i.e.immatureoocytes),usedinthecurrentstudy,differfromthatofpreovulatoryfollicle-enclosedoocytes.

Oocytedevelopmentalcompetenceisacquiredinaprogressivemannerthroughoutfolliculardevelopment.The ndingsofthecurrentstudysuggestthatmastitisnotonlyimpairsfollicularfunction(Lavonetal.2011b)butcanalsoaffectthefollicle-enclosedoocyte.MastitisinducedbyGKaffectedbothnuclearandcytoplasmicmaturation.Theproportionofoocytesthat

resumed

A

120

)

100

n( tn80uoc ll60e clat40oT200

Control

G+G–

B

3530)

%( 25slle20 ccito15tpop10A50

Control

G+

G–

Figure6Effectofinducedmastitisonblastocystfeatures.(A)Totalnumberofcells.(B)Percentageofapoptoticcells.Theexaminationincluded8-dayblastocysts,20embryosperexperimentalgroup,fromthreedifferentruns.ResultsarepresentedasmeansGS.E.M.;differentlettersindicatetreatmenteffect,P!0.05.

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40

SAsafandothers

AControl

G+

G–

1.5e

gna1.0hc dlo0.5F0

B3.5e

3.0gn2.5ah2.0c d1.5loF1.00.50

Figure7Effectofinducedmastitisongeneexpression.MIIstage

oocyteswerecollectedattheendof22-hmaturation(A)andfour-cellstageembryoswerecollected42hpostfertilization(B).PresentedarerelativetranscriptlevelsofPTGS2,HSF1,POU5F1,GDF9,and

SLC2A1.Quanti cationrelativetoYWHAZispresentedasmeansGS.E.M.;differentlettersindicatetreatmenteffectwithinaspeci cgene,P!0.05.Theexaminationincluded100oocytesand50embryospergroupfrom vedifferentruns.

meiosisandreachedtheMIIstage(i.e.nuclearmaturation)waslowerintheGKgroup.Similarly,theproportionofoocytesde nedaccordingtotheircorticalgranuledistributionasclassI(i.e.cytoplasmicmatu-ration)wasalsoaffectedintheGKgroupcomparedwiththecontrolgroup.Ontheotherhand,GCmastitisdidnotaffecteithernuclearorcytoplasmicmaturationbutloweredthecleavagerate.These ndingssuggestthatdifferentmechanismsforeachtypeoftoxin,resultingindifferentialeffectsonoocytematuration,couldberelatedtothedegreeofthein ammatoryresponse,rgerinthemastiticgroupinducedbyGKtoxinthaninthatinducedbyGCtoxin.

Withrespecttooocytedevelopmentalcompetence,maturationinFFobtainedfrombothmastiticgroups(inducedbyGKandGC)reducedtheproportionofoocytesthatfertilizedandcleavedtotwo-andfour-cellstageembryos.Moreover,bothtypesofbacteriadeleteriouslyaffectedtheproportionofembryosthatdevelopedtotheblastocyststage,withaprominenteffectintheGKmastiticgroup.BecauseinthecurrentstudyoocyteswerematuredinFFaspiratedfrommastiticcows,thedeleteriouseffectonoocytedevelopmentalcompetenceshouldbeconsideredadirecteffectonthepreovulatoryenclosedoocytethatiscarriedovertotheblastocyststage.Suchalterationsmightexplain,inpart,thereducedfertilityinmastiticcows(Lavonetal.2011b).Anepidemiologicalstudy(Lavonetal.2011b)showedalong-termeffectofE.coli-inducedmastitisonconceptionrate.Inparticular,apastclinicaleventoccurringupto10dayspriortoAIsigni cantlyreducedtheprobabilityofconception.Furthermore,wehave

Reproduction(2014)14733–43

recentlyshownthatE.colimastitisoccurringduringa90-dayperiodpriortoperformingIVM/IVFdisruptstheovarianpoolofGVstageoocytes,resultinginadecreaseinblastocystformationrate(Rothetal.2013).Takentogether,the ndingssupporttheviewthatnotonlythedevelopingembryobutalsotheoocyteishighlysusceptibletopathogenic(mastitis)stress.

Giventheprominenteffectonoocytematurationandthelong-lastingeffectonembryonicdevelopment,onemightexpectthatblastocystsdevelopedfromoocytesmaturedinFFaspiratedfrommastiticcowswillbeofinferiorquality.However,the ndingsarecomplexandnotentirelyclear.Whilethetotalcellcountfortheblastocystsdidnotdifferbetweengroups,theapoptoticcellcountwashigherintheGCbutnotintheGKgrouprelativetocontrols.Modestapoptosisisessentialforembryonicsurvival(Paula-Lopes&Hansen2002),whereasahighlevelofapoptoticblastomeresmightdecreasetheinnercellmass,reduceembryonicsurvival,andincreaseembryonicdeath(Pampferetal.1994,Wuuetal.1999).Thisphenomenonhasbeenwelldocumen-tedforpreimplantationembryosexposedtothermalstress(Paula-Lopes&Hansen2002).Nevertheless,inthecurrentstudy,neitheroocytesnorembryoswereexposedtohyperthermiaandthetransienthyperthermiadevelopedinvivointheGKtreatedcowshasnothingtodowithoocytesmaturedinGKFF.Ontheotherhand,astheoocyteswerematuredinFF,itispossiblethatimmune-activatedfactorspassviathecirculationintotheFFandinturnimpairoocytedevelopmentalcompetence.Supportingthisassumptionarereportsthatmastitisimpairstheconcentrationsofin ammationmediatorssuchasIL6,TNFa,andNOinmilkandblood(Nakajimaetal.1997,Blumetal.2000,Hisaedaetal.2001).Althoughnotexaminedinthecurrentstudy,anincreaseinin ammationmediatorsmightalsobeexpressedintheFF.Previousstudieshavereportedanincreaseinthein ammatorycomponentintheplasmawithin6–12hofmastitisinduction(Mehrzadetal.2007).Inthecurrentstudy,maturationinFFaspirated6hafterinductionofmastitisimpairedoocytedevelop-mentalcompetence,whichmightsupportthisassump-tion.Itisalsopossiblethatmastitisinducedchangesinthehypothalamus–pituitary–ovarianaxismightaffectsteroidconcentrationinthepreovulatoryfollicle(Lavonetal.2011b),whichinturnmightaffecttheoocytemicroenvironment.

Corticalgranuledistributionhasbeenshowntobeaffectedbyenvironmentalcultureconditions(Liuetal.2005).Forinstance,supplementationofantioxidantandgrowthfactorstothematurationmediumincreasestheproportionsofclass-Ioocytesanddevelopingblastocysts(Izadyaretal.1998,Cordovaetal.2010).Exposingoocytestoelevatedtemperatureduringmaturationimpairscorticalgranuledistribution(Paytonetal.2004).Therefore,passageofimmune-activatedfactorsintotheFF,assuggestedabove,mightaffectoocyte

cytoplasmicmaturationuponinductionofmastitisbyGK.Increasedin ammatoryfactorsintheFFmightalsodisruptthematernalmRNAtranscriptsstoredintheoocyte.OocytematurationinFFaspiratedfrommastiticcowswasassociatedwithalteredgeneexpressioninastage-dependent(MIIstageoocytesandfour-cellstageembryos)andbacterial-type-dependent(GKandGC)manner.Moreover,thealterationsdocumentedintheoocytethroughthefour-cell-stageembryos(i.e.beforeembryonicgenomeactivation)indicatealong-lastingeffectonthematernaltranscriptionallevel.Inparticular,maturationinGKFFreducedtheexpressionofPOU5F1inmaturedoocytes,whereasmaturationinGCFFfurtherreducedPOU5F1expressioninfour-cellstageembryos.Thisisanimportant ndingbecausePOU5F1isamemberofthePOUfamilyoftranscriptionalactivators(Ryan&Rosenfeld1997)andessentialformaintenanceoftotipotency/pluripotencyofembryonicstemcellsandprimordialgermcells.ThelevelofPOU5F1governsembryofate,andacriticallevelofPOU5F1isrequiredtomaintainembryonicstemcellrenewal.Up-ordownregulationofPOU5F1hasbeenfoundtoinducechangesindevelopmentalprograms(Niwaetal.2000).Therefore,theimpaired(i.e.increasedordecreased)POU5F1expressionnotedinthecurrentstudymightexplain,inpart,thereducedoocytedevelopmentalcompetenceinmastiticgroups.Whetherthesealterationsarefurtherexpressedinadvancedstagesofembryonicdevelopmentisnotclear.COX2ishighlyinduciblebydiversestimuli,includingcytokines,growthfactors,andmitogenandtumorpromoters(Hla&Neilson1992,Smithetal.1994).Inmice,Ptgs2-de cientfemalesareinfertileduetoabnormalprocessesofovulation,fertilization,implan-tation,anddecidualization(Dinchuketal.1995,Langenbachetal.1995,Limetal.1997).ItisthereforepossiblethatthealteredPTGS2expressionnotedhereresultedfromexposuretomediatorssecretedintothecirculationuponmastitisinductionandeventuallyendingupintheFF.Inparticular,thereducedPTGS2expressioninoocytesmaturedinGKFFandtheincreasedPTGS2expressioninfour-cellstageembryosinbothGKandGCgroupsaresuggestedtounderliethereduceddevelopmentalcompetenceofoocytesinmastiticcows.COXisaninitiatorenzymeinthecascadeofprostaglandin(PG)formationandplaysanimportantroleinearlyembryonicdevelopment(Lewis1989).Invivostudieshavereportedthatmastitisand/orendotoxinadministrationareassociatedwithincreasedPGF2ainthecirculation(Girietal.1991,Huszeniczaetal.2005).Short-terminvitroexposureofoocytesandearly-stageembryostoPGs,mimickingtheacutephaseofclinicalintramammaryinjection,disruptsbothoocytematurationandembryonicdevelopment(Sotoetal.2003a,2003b).Inaddition,COX2-derivedPGI2playsanimportantroleinblastocystdevelopment(Pakrasi&Jain2007)andhasapartthroughembryo

Mastitisimpairsoocytecompetence

41

hatching(Huangetal.2003,2004).Althoughnotexaminedhere,involvementofPGviaCOXactivationindisruptionofoocyteandembryodevelopmentcannotberuledout.

HSF1isamemberoftheheat-shocktranscriptionfactorfamilyresponsibleforstress-inducedexpressionofheat-shockproteins(HSPs).HSPlevelrapidlyincreasesinresponsetostress(Santoro2000)orundersomephysiologicalandpathogenicconditionssuchasfever,in ammation,cellortissuetrauma,aging,andinfection(Feige&vanEden1996,Morimoto&Santoro1998).Inturn,HSPshaveprotectivepropertiesincludingproteinsynthesis,refoldingofdenaturedproteins,protectionoffoldedproteinsandtargetingirreversiblydenaturedproteinsforremoval(Knowlton2006).Moreover,HSPshavedirectanti-in ammatoryandprotectiveeffectsonmembraneintegrityandmitochondrialfunction,aswellasaninhibitoryeffectonapoptosis(Beereetal.2005,Vossetal.2005).However,inthecurrentstudy,theexpressionofHSF1mRNAisolatedfrombothoocytesandfour-cellstageembryoswasgenerallylowerinthemastiticgroupsthaninthecontrol,withaprominentreductionintheGKgroup.ReducedexpressionofHSF1mRNAmightleadtoreducedlevelsofHSP,whichinturnmightimpairoocytecompetencetocopewithstress.Inaddition,HSF1isessentialforoocytemeiosis(Metchatetal.2009)andplaysaroleinreproductivesuccess(Christiansetal.2000).Itisthereforesuggestedtobeinvolvedinthemechanismunderlyingdecreasedembryonicdevelopmentinthemastiticgroups.SupportingthisassumptionisthefactthatreducedHSF1geneexpressioninculturedcellsdecreasestheirresponsetostressinassociationwithdelayedHSPactivation(Westerheideetal.2009).

Unliketheimpairedgeneexpressiondiscussedabove,GDF9transcriptionleveldidnotchangeinoocytesorembryosuponinductionofmastitis.GDF9isagermcellmarkerandamemberofthelargetransforminggrowthfactorb(TGFb)superfamily(McPherron&Lee1993),whichplaysapivotalroleinfolliculogenesis.Homozygousknockoutfemalemicearesterileduetoblockageoffolliclesattheprimarystage(Dongetal.1996).Italsostimulatesproliferationofthecacellsderivedfrombovinesmallfolliclesandisthereforede nedasamitogenicfactor(Spiceretal.2008).GDF9isinvolvedinoocytematurationviaregulationofcumuluscellfunctioninthepreovulatoryfollicle(Gui&Joyce2005).ArecentstudyreportedlowerGDF9expressioninprimordial,primary,andsecondaryfolliclesincowswithsubclinicalmastitis(Rahmanetal.2012).ItisthereforepossiblethattheeffectonGDF9expressionisassociatedwithsubclinicalmastitis,ratherthantheacutemastitisexaminedhere.

Insummary,themodelusedinthecurrentstudyenabledexaminingtheeffectoffollicularmicroenviron-ment(i.e.FFaspiratedfrommastiticcows)onoocytecompetence.Findingsindicatethatmastitisimpairsboth

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SAsafandothers

nuclearandcytoplasmicmaturation,aswellasoocytedevelopmentalcompetence;thiseffectseemstobedependentonbacterialtype.Moreover,observedalterationsintranscriptionallevelsintheoocytecarriedovertothefour-cellstageembryo.These ndingsmightexplain,inpart,thereducedfertilityinmastiticcows.

Declarationofinterest

Theauthorsdeclarethatthereisnocon ictofinterestthatcouldbeperceivedasprejudicingtheimpartialityoftheresearchreported.

Funding

ThisworkwassupportedbytheCattleDivisionoftheMinistryofAgriculture,Israel(project#80-0241-08).

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Received15August2013

Firstdecision23September2013

Revisedmanuscriptreceived11October2013Accepted14October2013

Reproduction(2014)14733–43

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