Effects of Escherichia coli- and Staphylococcus aureus-induced mastitis in lactating cows
更新时间:2023-05-23 19:33:01 阅读量: 实用文档 文档下载
- effects推荐度:
- 相关推荐
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.
Reproduction(2014)14733–43
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).
Reproduction(2014)14733–43
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.
Reproduction(2014)14733–43
36
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
Reproduction(2014)14733–43
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.
Reproduction(2014)14733–43
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.
Reproduction(2014)14733–43
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
Reproduction(2014)14733–43
42
SAsafandothers
nuclearandcytoplasmicmaturation,aswellasoocytedevelopmentalcompetence;thiseffectseemstobedependentonbacterialtype.Moreover,observedalterationsintranscriptionallevelsintheoocytecarriedovertothefour-cellstageembryo.These ndingsmightexplain,inpart,thereducedfertilityinmastiticcows.
Declarationofinterest
Theauthorsdeclarethatthereisnocon ictofinterestthatcouldbeperceivedasprejudicingtheimpartialityoftheresearchreported.
Funding
ThisworkwassupportedbytheCattleDivisionoftheMinistryofAgriculture,Israel(project#80-0241-08).
References
AliA,CoenenaK,BousquetbD&SirardMA2004Originofbovinefollicular uidanditseffectduringinvitromaturationonthedevelopmentalcompetenceofbovineoocytes.Theriogenology621596–1606.(doi:10.1016/j.theriogenology.2004.03.011)
AravA2001Transilluminationincreasesoocyterecoveryfromovariescollectedatslaughter.Anewtechniquereport.Theriogenology551561–1565.(doi:10.1016/S0093-691X(01)00502-7)
AveryB,StrobechL,JacobsenT,BoghIB&GreveT2003Invitromaturationofbovinecumulus–oocytecomplexesinundilutedfollicular uid:effectonnuclearmaturation,pronucleusformationandembryodevelopment.Theriogenology59987–999.(doi:10.1016/S0093-691X(02)01139-1)
BeereHM,WolfBB,CainK,KuwanaT,TailorP,MorimotoRI,CohenGM&GreenDR2005Heatshockprotein70inhibitsapoptosisbypreventingrecruitmentofprocaspase-9totheapaf-1apoptosome.NatureCellBiology2469–475.
BlumJW,DosogneH,HoebenD,VangroenwegheF,HammonHM,BruckmaierRM&BurvenichC2000Tumornecrosisfactor-aandnitrite/nitrateresponsesduringacutemastitisinducedbyEscherichiacoliinfectionandendotoxinindairycows.DomesticAnimalEndocrinology19223–235.(doi:10.1016/S0739-7240(00)00079-5)
ChristiansE,DavisAA,ThomasSD&BenjaminIJ2000MaternaleffectofHsf1onreproductivesuccess.Nature407693–694.(doi:10.1038/35037669)
ColemanNV,ShagiakhmetovaGA,LebedevaIY,KuzminaTI&GolubevAK2007Invitromaturationandearlydevelopmentalcapacityofbovineoocytesculturedinpurefollicular uidandsupplementationwithfollicularwall.Theriogenology671053–1059.(doi:10.1016/j.therio-genology.2006.10.019)
CordovaB,MoratoR,IzquierdoD,ParamioT&MogasT2010Effectoftheadditionofinsulin–transferrin–seleniumand/orL-ascorbicacidtotheinvitromaturationofprepubertalbovineoocytesoncytoplasmicmaturationandembryodevelopment.Theriogenology741341–1348.(doi:10.1016/j.theriogenology.2010.06.003)
CoticchioG,SereniE,SerraoL,MazzoneS,IadarolaI&BoriniA2004Whatcriteriaforthede nitionofoocytequality?AnnalsoftheNewYorkAcademyofSciences1034132–144.(doi:10.1196/annals.1335.016)DelosReyesM,deLangeJ,MirandaP,PalominosJ&BarrosC2005Effectofhumanchorionicgonadotrophinsupplementationduringdifferentcultureperiodsoninvitromaturationofcanineoocytes.Theriogenology641–11.(doi:10.1016/j.theriogenology.2004.11.008)
DinchukJE,CarBD,FochtRJ,JohnstonJJ,JaffeeBD,CovingtonMB,ContelNR,EngVM,CollinsRJ,CzerniakPMetal.1995Renalabnormalitiesandanalteredin ammatoryresponseinmicelackingcyclooxygenaseII.Nature378406–409.(doi:10.1038/378406a0)
Reproduction(2014)14733–43
DongJW,AlbertiniDF,NishimoriK,KumarTR,LuN&MatzukMM1996Growthdifferentiationfactor-9isrequiredduringearlyovarianfolliculogenesis.Nature383531–535.(doi:10.1038/383531a0)
DuboisRN,AbramsonSB,CroffordL,GuptaRA,SimonLS,VanDePutteLB&LipskyPE1998Cyclooxygenaseinbiologyanddisease.FASEBJournal121063–1073.
EdwardsRG1974Follicular uid.JournalofReproductionandFertility37189–219.(doi:10.1530/jrf.0.0370189)
FeigeU&vanEdenW1996Infection,autoimmunityandautoimmunedisease.EXS77359–373.
GendelmanM&RothZ2012aSeasonaleffectongerminalvesicle-stagebovineoocytesisfurtherexpressedbyalterationsintranscriptlevelsinthedevelopingembryoassociatedwithreduceddevelopmentalcompetence.BiologyofReproduction861–9.(doi:10.1095/biolreprod.111.092882)GendelmanM&RothZ2012bInvivovs.invitromodelsforstudyingtheeffectsofelevatedtemperatureontheGV-stageoocyte,subsequentdevelopmentalcompetenceandgeneexpression.AnimalReproductionScience134125–134.(doi:10.1016/j.anireprosci.2012.07.009)
GiriSN,StabenfeldtGH,MoseleyTA,GrahamTW,BrussML,BonDurantRH,CullorJS&OsburnBI1991Roleofeicosanoidsinabortionanditspreventionbytreatmentwith unixinmeglumineincowsduringthe rsttrimesterofpregnancy.JournalofVeterinaryMedicine38445–459.
GuiLM&JoyceIM2005RNAinterferenceevidencethatgrowthdifferentiationfactor-9mediatesoocyteregulationofcumulusexpansioninmice.BiologyofReproduction72195–199.(doi:10.1095/biolreprod.104.033357)
HarmanJL,GrohnYT,ErbHN&CasellaG1996Event-timeanalysisoftheeffectofseasonofparturition,parity,andconcurrentdiseaseonparturitiontoconceptionintervalindairycows.AmericanJournalofVeterinaryResearch57640–645.
HisaedaK,HagiwaraK,EguchiJ,YamanakaH,KirisawaR&IwaiH2001Interferon-gandtumornecrosisfactoralevelsinseraandwheyofcattlewithnaturallyoccurringcoliformmastitis.JournalofVeterinaryMedicalScience/theJapaneseSocietyofVeterinaryScience631009–1011.(doi:10.1292/jvms.63.1009)
HlaT&NeilsonK1992Humancyclooxygenase-2cDNA.PNAS897384–7388.(doi:10.1073/pnas.89.16.7384)
HuangJC,WunWA,GoldsbyJS,WunIC,FalconiSM&WuKK2003Prostacyclinenhancesembryohatchingbutnotspermmotility.HumanReproduction182582–2589.(doi:10.1093/humrep/deg490)
HuangJC,WanWS,GoldsbyJS,Matijevic-AleksicN&WuKK2004Cyclooxygenase2derivedendogenousprostacyclinenhancesmouseembryohatching.HumanReproduction192900–2906.(doi:10.1093/humrep/deh524)
HuszeniczaG,Ja
´nosiS,Kulcsa´rM,Ko´ro´diP,ReiczigelJ,Ka´taiL,PetersAR&DeRensisF2005Effectsofclinicalmastitisonovarianfunctioninpost-partumdairycows.ReproductioninDomesticAnimals40199–204.(doi:10.1111/j.1439-0531.2005.00571.x)
IzadyarF,HageWJ,ColenbranderB&BeversMM1998Thepromotoryeffectofgrowthhormoneonthedevelopmentalcompetenceofinvitromaturedbovineoocytesisduetoimprovedcytoplasmicmaturation.MolecularReproductionandDevelopment49444–453.(doi:10.1002/(SICI)1098-2795(199804)49:4!444::AID-MRD12O3.0.CO;2-U)
KaloD&RothZ2011Involvementofthesphingolipidceramideinheat-shockinducedapoptosisofbovineoocytes.Reproduction,Fertility,andDevelopment23876–888.(doi:10.1071/RD10330)
KnowltonAA2006NFkB,heatshockproteins,HSF-1,andin ammation.CardiovascularResearch697–8.(doi:10.1016/j.cardiores.2005.10.009)LangenbachR,MorhamSG,TianoHF,LoftinCD,GhanayemBI,ChuladaPC,MahlerJF,LeeCA,GouldingEH,KluckmanKDetal.1995ProstaglandinsynthaseIgenedisruptioninmicereducesarachidonicacid-inducedin ammationandindomethacin-inducedgastriculcera-tion.Cell83483–492.(doi:10.1016/0092-8674(95)90126-4)
LavonY,LeitnerG,GoshenT,Braw-TalR,JacobyS&WolfensonD2008Exposuretoendotoxinduringestrusaltersthetimingofovulationandhormonalconcentrationsincows.Theriogenology70956–967.(doi:10.1016/j.theriogenology.2008.05.058)
LavonY,EzraE,LeitnerG&WolfensonD2011aAssociationofconceptionratewiththepatternandlevelofsomaticcellcountelevationrelativetotimeofinseminationindairycows.JournalofDairyScience944538–4545.(doi:10.3168/jds.2011-4293)
LavonY,LeitnerG,MoallemU,KlipperE,VoetH,JacobyS,GlickG,MeidanR&WolfensonD2011bImmediateandcarryovereffectsofGram-negativeandGrampositivetoxin-inducedmastitisonfollicularfunctionindairycows.Theriogenology76942–953.(doi:10.1016/j.theriogenology.2011.05.001)
LeitnerG,KrifucksO,YounisA,HellerED&SaranA2002In uenceofStaphylococcusaureusexosecretionsisolatedfrombovinemastitisonleukocyteactivityinvitro.JournalofVeterinaryMedicine.B,InfectiousDiseasesandVeterinaryPublicHealth49354–360.(doi:10.1046/j.1439-0450.2002.00575.x)
LeitnerG,KrifucksO,MerinU,LaviY&SilanikoveN2006Interactionsbetweenbacteriatype,proteolysisofcaseinandphysico-chemicalpropertiesofbovinemilk.InternationalDairyJournal16648–654.(doi:10.1016/j.idairyj.2005.10.020)
Leppens-LuisierG,UrnerF&SakkasD2001Facilitatedglucosetransporterplayacrucialrolethroughoutmousepreimplantationembryodevelopment.HumanReproduction16229–1236.(doi:10.1093/humrep/16.6.1229)
LewisDS1989Prostaglandinsecretionbytheblastocyst.JournalofReproductionandFertility37261–267.
LimH,PariaBC,DasSK,DinchukJE,LangenbachR,TrzaskosJM&DeySK1997Multiplefemalereproductivefailuresincyclooxygenase2-de cientmice.Cell91197–208.(doi:10.1016/S0092-8674(00)80402-X)
LiuXY,MalSF,MiaoDQ,LiuDJ,BaoS&TanJH2005Corticalgranulesbehavedifferentlyinmouseoocytesmaturedunderdifferentconditions.HumanReproduction203402–3413.(doi:10.1093/humrep/dei265)Loef erSH,deVriesMJ&SchukkenYH1999Theeffectsoftimeofdiseaseoccurrence,milkyield,andbodyconditiononfertilityofdairycows.JournalofDairyScience822589–2604.(doi:10.3168/jds.S0022-0302(99)75514-1)
MaizonDO,OltenacuPA,Gro
¨hnYT,StrawdermanRL&EmanuelsonU2004EffectsofdiseasesonreproductiveperformanceinSwedishRedandWhitedairycattle.PreventiveVeterinaryMedicine66113–126.(doi:10.1016/j.prevetmed.2004.09.002)
McPherronAC&LeeSJ1993GDF-3andGDF-9:twonewmembersofthetransforminggrowthfactor-bsuperfamilycontaininganovelpatternofcysteines.JournalofBiologicalChemistry2683444–3449.
MehrzadJ,DosogneH,DeSpiegeleerB,DuchateauL&BurvenichC2007Bovinebloodneutrophilacyloxyacylhydrolase(AOAH)activityduringendotoxinandcoliformmastitis.VeterinaryResearch5655–668.(doi:10.1051/vetres:2007024)
MetchatA,AkerfeltM,BierkampC,DelsinneV,SistonenL,AlexandreH&ChristiansES2009Mammalianheatshockfactor1isessentialforoocytemeiosisanddirectlyregulatesHsp90aexpression.JournalofBiologicalChemistry2849521–9528.(doi:10.1074/jbc.M808819200)
MorimotoRI&SantoroMG1998Stress-inducibleresponsesandheatshockproteins:newpharmacologictargetsforcytoprotection.NatureBiotechnology16833–838.(doi:10.1038/nbt0998-833)
NakajimaY,MikamiO,YoshiokaM,MotoiY,ItoT,IshikawaY,FuseM,NakanoK&YasukawaK1997Elevatedlevelsoftumornecrosisfactor-a(TNF-a)andinterleukin-6(IL-6)activitiesintheseraandmilkofcowswithnaturallyoccurringcoliformmastitis.ResearchinVeterinaryScience62297–298.(doi:10.1016/S0034-5288(97)90209-5)
NiwaH,MiyazakiJ&SmithAG2000QuantitativeexpressionofOct-3/4de nesdifferentiation,dedifferentiationorself-renewalofEScells.NatureGenetics24372–376.(doi:10.1038/74199)
OliverSP,AlmeidaRA,GillespieBE,HeadrickSJ,DowlenHH,JohnsonDL,LamarKC,ChesterST&MoseleyWM2004Extendedceftiofurtherapyfortreatmentofexperimentally-inducedStreptococcusuberismastitisinlactatingdairycattle.JournalofDairyScience873322–3329.(doi:10.3168/jds.S0022-0302(04)73468-2)
PakrasiPL&JainAK2007Evaluationofcyclooxygenase-2derivedendogenousprostacyclininmousepreimplantationembryodevelopmentinvitro.LifeSciences801503–1507.(doi:10.1016/j.lfs.2007.01.044)
PampferS,WuuYD,VanderheydenI&DeHertoghR1994Expressionoftumornecrosisfactor-a(TNF-a)receptorsandselectiveeffectofTNF-aontheinnercellmassinmouseblastocyst.Endocrinology134206–212.(doi:10.1210/en.134.1.206)
ParrishJJ,Susko-ParrishJL,CristerES,EyestoneWH&FirstNL1986Bovineinvitrofertilizationwithfrozen–thawedsemen.Theriogenology25591–600.(doi:10.1016/0093-691X(86)90143-3)
Mastitisimpairsoocytecompetence
43
Paula-LopesFF&HansenPJ2002Heat-shockinducedapoptosisinbovinepreimplantationembryosisadevelopmentally-regulatedphenomenon.BiologyofReproduction661169–1177.
PaytonRR,RomarR,CoyP,SaxtonAM,LawrenceJL&EdwardsJL2004Susceptibilityofbovinegerminalvesicle-stageoocytesfromantralfolliclestodirecteffectsofheatstressinvitro.BiologyofReproduction711303–1308.(doi:10.1095/biolreprod.104.029892)
RahmanMM,MazzilliM,PennarossaG,BreviniTA,ZecconiA&Gandol F2012Chronicmastitisisassociatedwithalteredovarianfollicledevelopmentindairycattle.JournalofDairyScience951885–1893.(doi:10.3168/jds.2011-4815)
RothZ,DvirA,KaloD,LavonY,KrifucksO,WolfensonD&LeitnerG2013Naturallyoccurringmastitisdisruptsdevelopmentalcompetenceofbovineoocytes.JournalofDairyScience966499–6505.(doi:10.3168/jds.2013-6903)
RyanAK&RosenfeldMG1997POUdomainfamilyvalues: exibility,partnerships,anddevelopmentalcodes.GenesandDevelopment111207–1225.(doi:10.1101/gad.11.10.1207)
SantoroMG2000Heatshockfactorsandthecontrolofthestressresponse.BiochemicalPharmacology5955–63.(doi:10.1016/S0006-2952(99)00299-3)
SantosJE,CerriRL,BallouMA,HigginbothamGE&KirkJH2004Effectoftimingof rstclinicalmastitisoccurrenceonlactationalandreproductiveperformanceofHolsteindairycows.AnimalReproductionScience8031–45.(doi:10.1016/S0378-4320(03)00133-7)
SchrickFN,HockettME,SaxtonAM,LewisMJ,DowlenHH&OliverSP2001In uenceofsubclinicalmastitisduringearlylactationonreproductiveparameters.JournalofDairyScience841407–1412.(doi:10.3168/jds.S0022-0302(01)70172-5)
SmithWL,MeadeEA&DewittDL1994InteractionsofPGHsynthaseisozymes-1and-2withNSAIDs.AnnalsoftheNewYorkAcademyofSciences74450–57.(doi:10.1111/j.1749-6632.1994.tb52723.x)
SotoP,NatzkeRP&HansenPJ2003aIdenti cationofpossiblemediatorsofembryonicmortalitycausedbymastitis:actionsoflipopolysaccharide,prostaglandinF2a,andthenitricoxidegenerator,sodiumnitro-prussidedihydrate,onoocytematurationandembryonicdevelopmentincattle.AmericanJournalofReproductiveImmunology50263–272.(doi:10.1034/j.1600-0897.2003.00085.x)
SotoP,NatzkeRP&HansenPJ2003bActionsoftumornecrosisfactor-aonoocytematurationandembryonicdevelopmentincattle.AmericanJournalofReproductiveImmunology50380–388.(doi:10.1034/j.1600-0897.2003.00101.x)
SpicerLJ,AadPY,AllenDT,MazerbourgS,PayneAH&HsuehAJ2008Growthdifferentiationfactor9(GDF9)stimulatesproliferationandinhibitssteroidogenesisbybovinethecacells:in uenceoffolliclesizeonresponsestoGDF9.BiologyofReproduction78243–253.(doi:10.1095/biolreprod.107.063446)
VossMR,GuptaS,SticeJP,BaumgartenG,LuL,TristanJM&KnowltonAA2005Effectofmutationofaminoacids246251(KRKHKK)inHSP72onproteinsynthesisandrecoveryfromhypoxicinjury.AmericanJournalofPhysiology.HeartandCirculatoryPhysiology289519–525.(doi:10.1152/ajpheart.00872.2004)
WesterheideSD,AnckarJ,StevensSMJr,SistonenL&MorimotoRI2009Stress-inducibleregulationofheatshockfactor1bythedeacetylaseSIRT1.Science3231063–1066.(doi:10.1126/science.1165946)
WuuYD,PampferS,BecquetP,VanderheydenI,LeeKH&DeHertoghR1999Tumornecrosisfactoradecreasestheviabilityofmouseblastocystinvitroandinvivo.BiologyofReproduction60479–483.(doi:10.1095/biolreprod60.2.479)
YounisA,KrifucksO,HellerED,SamraZ,GlickmanA,SaranA&LeitnerG2003Staphylococcusaureusexosecretionsandbovinemastitis.JournalofVeterinaryMedicine.B,InfectiousDiseasesandVeterinaryPublicHealth501–7.(doi:10.1046/j.1439-0450.2003.00613.x)
Received15August2013
Firstdecision23September2013
Revisedmanuscriptreceived11October2013Accepted14October2013
Reproduction(2014)14733–43
正在阅读:
Effects of Escherichia coli- and Staphylococcus aureus-induced mastitis in lactating cows05-23
上海海关进出口结汇联、退税联签发申请表(含填表说明及示范文本03-07
noip2010提高组解题报告10-24
在全镇教职工作风整顿会议上的讲话01-21
让反思成为一种习惯,在反思中促进成长论文04-07
1.5.2 科学计数法08-14
电工实作(教材) - 图文01-16
劳动力持续转移过程中的通胀陷阱及治理对策03-14
《老年人权益保障法》法制讲座讲话稿09-09
- 12005-Characterization of Apoptosis induced by fucoxanthin=FX
- 2Iron_and_the_Effects_of_Exercise1
- 3Effects of salinity on the growth, physiology and relevant
- 4Cumulative Effects Assessment and Sustainability Diamond Min
- 5Luminol chemiluminescence induced by silver nanoparticles in the presence of nucleophiles and Cu2+
- 6E.coli genetype 大肠杆菌基因型
- 7E.coli genetype 大肠杆菌基因型
- 8Effects of Temperature and Atmosphere on Pellets Reduction Swelling Index
- 9Effects of receptor clustering on ligand dissociation Theory
- 10六下M3U2 The cows are drinking water
- 教学能力大赛决赛获奖-教学实施报告-(完整图文版)
- 互联网+数据中心行业分析报告
- 2017上海杨浦区高三一模数学试题及答案
- 招商部差旅接待管理制度(4-25)
- 学生游玩安全注意事项
- 学生信息管理系统(文档模板供参考)
- 叉车门架有限元分析及系统设计
- 2014帮助残疾人志愿者服务情况记录
- 叶绿体中色素的提取和分离实验
- 中国食物成分表2020年最新权威完整改进版
- 推动国土资源领域生态文明建设
- 给水管道冲洗和消毒记录
- 计算机软件专业自我评价
- 高中数学必修1-5知识点归纳
- 2018-2022年中国第五代移动通信技术(5G)产业深度分析及发展前景研究报告发展趋势(目录)
- 生产车间巡查制度
- 2018版中国光热发电行业深度研究报告目录
- (通用)2019年中考数学总复习 第一章 第四节 数的开方与二次根式课件
- 2017_2018学年高中语文第二单元第4课说数课件粤教版
- 上市新药Lumateperone(卢美哌隆)合成检索总结报告
- Staphylococcus
- Escherichia
- lactating
- mastitis
- Effects
- induced
- aureus
- coli
- cows
- 别忽视面试时让你丢分的身体语言
- 保险代理人管理流程
- 机修钳工(高级)技师题
- 大学英语教学指南(教育部2017版)
- 2014中考圆的专题练习 (绝对经典真题)
- 精品解析:山东省济南市平阴县2020-2021学年八年级(上)期末考试物理试题(解析版)
- 中信证券金融行业周刊 第6期 20120220
- 《纵横四海》观后感
- 电气运行规程2014915
- 金融本科生进入麦肯锡的超长记录-2002
- 城市河道综合治理中存在的问题及对策研究
- 1969年属鸡的人每月运势
- 如何发掘隐形新闻线索
- 医院门诊一卡通系统流程设计
- 2014新人教版第八章第3节摩擦力(系列版)
- 教你如何做一个知性女人
- PHSJ-5型实验室pH计使用说明书
- 2013-14第二学期高一历史教学工作总结
- 电路的串联和并联以及电流的测量
- 开学典礼主持会议讲话