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TreatmentwiththeProteasomeInhibitorMG132during theEndofOocyteMaturationImprovesOocyte CompetenceforDevelopmentafterFertilizationinCattleJinyoungYou1,EunsongLee1,LucianoBonilla2,JasmineFrancis2,JinKoh3,5,JeremyBlock2,4, SixueChen3,5,PeterJ.Hansen2*1 CollegeofVeterinaryMedicine,KangwonNationalUniversity,Chunchon,Korea, 2 DepartmentofAnimalSciencesandD.H.BarronReproductiveandPerinatalBiology ResearchProgram,UniversityofFlorida,Gainesville,Florida,UnitedStatesofAmerica, 3 InterdisciplinaryCenterforBiotechnologyResearch,UniversityofFlorida, Gainesville,Florida,UnitedStatesofAmerica, 4 OvatechLLC,Gainesville,Florida,UnitedStatesofAmerica, 5 Dept.ofBiology,UniversityofFlorida,Gainesville,Florida, UnitedStatesofAmericaAbstractMaturationoftheoocyteinvolvesnuclearandcytoplasmicchangesthatincludepost-translationalprocessingofproteins. Theobjectivewastoinvestigatewhetherinhibitionofproteasomesduringmaturationwouldaltercompetenceofthe bovineoocyteforfertilizationandsubsequentdevelopment.Cumulus-oocytecomplexeswereculturedinthepresenceor absenceoftheproteasomalinhibitorMG132fromeither0–6hor16–22hafterinitiationofmaturation.Treatmentwith MG132earlyinmaturationpreventedprogressiontomeiosisIIandreducedfertilizationrateandtheproportionofoocytes andcleavedembryosthatbecameblastocysts.Conversely,treatmentwithMG132lateinmaturationimprovedthe percentageofoocytesandcleavedembryosthatbecameblastocystswithoutaffectingnuclearmaturationorfertilization rate.OptimalresultswithMG132wereachievedataconcentrationof10mM–effectsweregenerallynotobservedatlower orhigherconcentrations.Usingproteomicanalysis,itwasfoundthatMG132attheendofmaturationincreasedrelative expressionof6proteinsanddecreasedrelativeexpressionof23.AmongthoseincreasedbyMG132thatarepotentially importantforoocytecompetenceareGAPDH,involvedinglycolysis,TUBA1C,neededfororganellarmovement,andtwo proteinsinvolvedinproteinfolding(P4HBandHYOU1).MG132decreasedamountsofseveralproteinsthatexertantiapoptoticactionsincludingASNS,HSP90B1,PDIA3andVCP.AnotherproteindecreasedbyMG132,CDK5,canleadto apoptosisifaberrantlyactivatedandoneproteinincreasedbyMG132,P4HB,isanti-apoptotic.Finally,thepregnancyrateof cowsreceivingembryosproducedfromoocytestreatedwithMG132from16–22hofmaturationwassimilartothatfor controlembryos,suggestingthatuseofMG132forproductionofembryosinvitrodoesnotcauseasubstantialdecreasein embryoquality.Citation: YouJ,LeeE,BonillaL,FrancisJ,KohJ,etal.(2012)TreatmentwiththeProteasomeInhibitorMG132duringtheEndofOocyteMaturationImproves OocyteCompetenceforDevelopmentafterFertilizationinCattle.PLoSONE7(11):e48613.doi:10.1371/journal.pone.0048613 Editor: ShreeRamSingh,NationalCancerInstitute,UnitedStatesofAmerica Received July12,2012; Accepted September27,2012; Published November7,2012 Copyright: 2012Youetal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsunrestricted use,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited. Funding: ThisprojectwassupportedbyAgricultureandFoodResearchInitiativeCompetitiveGrantno.2010-85122-20623fromtheUSDANationalInstituteof FoodandAgriculture.Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript. CompetingInterests: Co-author,JeremyBlock,isaffiliatedwithacompanycalledOvatech.Hehasnootherrelationshipwithanypatents,products,etc.There arenorelevantdeclarationsrelatingtoemployment,consultancy,patents,productsindevelopmentormarketedproducts.Thisdoesnotaltertheau thors’ adherencetoallthePLOSONEpoliciesonsharingdataandmaterials. *E-mail:Hansen@animal.ufl.eduIntroductionTheproteasome,amultisubunitproteolyticcomplexinvolvedin degradationofubiquitinatedproteins,playsacrucialrolein assuringcompletionofmeiosisandformationofadevelopmentally-competentembryo.Earlyinmaturation,completionof meiosisIrequiresinactivationofmaturationpromotingfactor (MPF)throughaprocessmediatedbyproteasomalcleavageof ubiquitinatedcyclinB1[1].Otheraspectsofoocytefunction duringmaturationarealsoundercontroloftheproteasome.In mice,forexample,theproteasomeisrequiredfortheinitiation andmaintenanceoftranslationofmRNAfortheRNAbinding protein SLBP [2].AbundanceofanotherproteininvolvedinRNA processing,CPEB,isundernegativeregulationbyproteasomesin Xenopus [3].Inaddition,cumuluscellsencasingtheoocyterequire proteasomalactivityforoptimalfunctionasindicatedbynegative effectsoftheproteasomalinhibitorMG132onprogesterone productionandexpressionofgenesinvolvedinexpansionofthe extracellularmatrix[4].Thispeptidealdehyde,N-(benzyloxycarbonyl)leucinylleucinylleucinal,functionsasasubstrateanalogand transition-stateinhibitorofthechymotrypsin-likeactivityofthe proteasome[5]. Lateintheprocessofoocytematuration,theproteasomemay contributetoareductioninthefunctionalpropertiesoftheoocyte. TreatmentwithMG132reducedtheeffectofinvitroagingon oocytecompetenceinthemouse[6].Furthermore,treatmentof oocyteswithMG132lateinmaturationincreasedabundanceof specifictranscriptsandimproveddevelopmentalcompetenceof parthenogenetically-activatedoocytesinthepig[7]. Ifinhibitionoftheubiquitin-proteasomepathwaylatein maturationimprovesoocytecompetence,itmaybepossibleto improvethesuccessrateofassistedreproductivetechnologiesthat PLOSONE|www.plosone.org1November2012|Volume7|Issue11|e48613

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utilizeinvitromaturedoocytes.Thepurposeofthepresentseries ofexperimentswastotestthehypothesisthattreatmentofbovine oocyteswithMG132attheendofmaturationwouldimprove developmentalcompetenceoftheoocytesandresultantembryos whileadditionofMG132atthebeginningofmaturationwould reduceoocytecompetence.Anadditionalgoalwastoassess specificproteinswhoserelativeabundanceintheoocytewas alteredbyMG132lateinmaturationwiththegoalofidentifying candidatemoleculesresponsibleforactionsofMG132onoocyte competence.MaterialsandMethodsUseofanimalswasapprovedbytheUniversityofFlorida InstitutionalAnimalCareandUseCommittee.CultureMediaChemicalswereobtainedfromSigma-AldrichChemicalCompany(St.Louis,MO,USA)orFisher(Pittsburgh,PA,USA)unless otherwisestated.Thebasemediumforoocytematuration(OMM) wasTissueCultureMedium-199(TCM-199;Invitrogen,Carlsbad,CA)withEarle’ssaltssupplementedwith10%(v/v)bovine steerserumcontaining2U/mlheparin(Pel-Freez,Rogers,AR, USA),2 m g/mlestradiol17b ,20 m g/mlbovinefolliclestimulatinghormone(Folltropin-V;BionicheLifeSciences,London,ON, Canada),22 m g/mlsodiumcitrate,50 m g/mlgentamicinsulfate and1mMglutamine.Oocytecollectionmedium(OCM)was TCM-199mediumwithHank’ssalts(Cellgro,Mediatech, Manassas,VA,USA)supplementedwith100U/mlpenicillin-G, 0.1mg/mlstreptomycin,1mMglutamineand2%(v/v)bovine steerserumcontaining2U/mlheparin.HEPES-Tyrodesalbumin lactatepyruvatesolution(TALP)waspreparedasdescribed previously[8].Thefertilizationmediumwasinvitrofertilization (IVF)-TALP[8].PercollwasfromGEHealthcareBio-Sciences AB(Uppsala,Sweden).Frozensemenfrombullsofvariousbreeds wasdonatedbySoutheasternSemenServices(Wellborn,FL, USA).TheembryoculturemediumwasSOF-BE1[9]or,forone experiment,aproprietaryculturemediumcalledBBH7from Minitube(Verona,WI,USA).Hoechst33342waspurchasedfrom Sigma-Aldrich.TheMG132waspurchasedfromSigma-Aldrich.OocyteCollectionandInVitroMaturation(IVM)Bovineovarieswereobtainedfromvariousbreedsatalocal abattoir(CentralPacking,CenterHill,Florida)andtransportedto thelaboratory.Theownerprovidedpermissiontousetheovaries forexperimentalpurposes.Cumulus-oocytecomplexes(COCs) werecollectedbyslicingsuperficialfollicles(2–10mmindiameter) withascalpelbladeandwashingtheovariesintoabeaker containingOCM.TheCOCswereharvestedusingacapillary pipetteandwashedthreetimesinOCM.Groupsof10were placedinto50 m ldropsofOMMcoveredwithmineraloil.The COCswerematuredfor22hat38.5 u Cinahumidified atmosphereof5%(v/v)CO2,withMG132treatmentapplied accordingtotheexperimentaldesign.TheMG132wasdissolved indimethylsulfoxide(DMSO)andwasaddedtomaturationdrops sothatthefinalconcentrationofDMSOwasnotgreaterthan 0.5%(v/v).Thecontroloocyteswereculturedwithmedium supplementedwithasimilaramountofDMSOduringIVMasfor oocytestreatedwithMG132.InVitroFertilizationandCultureAftermaturation,allCOCswerewashedtwiceinHEPESTALPandonceinIVF-TALPandthentransferredingroupsof 30–50oocytesto425 m loffertilizationmediuminwellsofa5-well dish(Minitube,Verona,WI,USA).Oocyteswerefertilizedwitha pooloffrozen-thawedspermfromthreebullspurifiedbyPercoll gradientcentrifugation[8];differentpoolswereusedineach replicate.Oocyteswerefertilized(dayoffertilizationtermedDay 0)byadding30 m lofPercoll-purifiedspermatozoa(finalconcentrationinfertilizationdish=1 6 106spermcells/mlinIVF-TALP and20 m lPHE(0.5mMpenicillamine,0.25mMhypotaurineand 25 mM epinephrinein0.9%(w/v)NaCl).After8hat38.5 u Cand 5%(v/v)CO2inhumidifiedair,putativezygoteswereremoved fromfertilizationwells,denudedofcumuluscellsbyvortexingin hyaluronidase(10,000U/mlin600 m lHEPES-TALP)for4min, washedinHEPES-TALP,andplacedingroupsof20to35 putativezygotesin50 m lmicrodropsofSOF-BE1medium overlaidwithmineraloilat38.5 u Cinahumidifiedatmosphere of5%(v/v)CO2,5%O2andthebalancenitrogen.Cleavageand blastocystformationwereevaluatedonDays3and8afterIVF, respectively.ExaminationofNuclearStatusofOocytesafterIVMAt16hand22hofIVM,COCsweretransferredintoHEPESTALPcontaining0.3%(w/v)hyaluronidaseandthenvortexedfor 5mintoremovecumuluscells.Denudedoocyteswerestained with5 m g/mlHoechst33342in10mMPBS(10mMPO4,0.9% (w/v)NaCl)containing1%(w/v)polyvinylpyrrolidone(PBS-PVP) for1hatroomtemperature.Then,10–15oocytesweremounted onglassslideswithasmallamountofanti-fadesolution(Life Technologies,GrandIsland,NY,USAandcoveredwithacover slip.OocyteswereexaminedusinganAxioplan2epifluorescence microscope(Zeiss,Go ¨ttingen,Germany)withbluefilter(excitation wavelength=365/12nm;emissionwavelengths=395–750nm). Eachoocytewasclassifiedaccordingtostageofnuclear maturationasgerminalvesicle(GV),germinalvesiclebreakdown (GVBD),pre-metaphaseI-metaphaseI(MI),anaphaseI(AI)telophaseI(TI)andmetaphaseII(MII).ExaminationofPronuclearStatusofOocytesafterIVFInseminatedoocyteswereharvestedfromculturedropsofSOFBE1at18hafterfertilization,mountedonglassslidesandnuclei visualizedusingHoechst33342asdescribedaboveforoocytes. Oocyteswereclassifiedasnon-penetratedifthenucleuswasatMI orMIIwithoutthepresenceofaspermheadormalepronucleus. Anoocytewasclassifiedasfertilizedifoneswollenspermheador malepronucleuswasdetectedinsidetheoocyte.Oocyteshaving morethanoneswollenspermheadormalepronucleiwere classifiedaspolyspermy.BlastocystCellNumberBlastocystswerefixedfor1hatroomtemperaturein4%(w/v) paraformaldehydedissolvedinPBS.AfterwashinginPBS-PVP, embryoswereincubatedwith1mg/mlHoescht33342dissolvedin PBS-PVP.EmbryoswerewashedinPBS-PVP,placedona microscopeslideandnumberofnucleicountedusingaZeiss Axioplan2epifluorescencemicroscope(Zeiss,Go ¨ttingen,Germany).ExperimentsonOocyteMaturation,Fertilizationand Development(Experiments1–6)Theconcentration-dependenteffectsofMG132addedatthe endofoocytematurationonembryonicdevelopmentweretested inExperiments1and2.COCswerematuredinOMMthatwas supplementedwith0,1,5,10 m MMG132(Experiment1)or0, 10,20or30 m MMG132(Experiment2)from16hto22hafter initiationofmaturation.TreatmentwasachievedbywashingMG132ImprovesOocyteCompetence PLOSONE|www.plosone.org2November2012|Volume7|Issue11|e48613

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COCsafter16hofmaturationandplacingtheminfreshmedium containingMG132orvehicle.Endpointswerecleavagerateatday 3afterinsemination,theproportionofoocytesandcleaved embryosthatbecameblastocystsatDay8,andblastocystcell number.Theexperimentswerereplicatedsixtimeswith20–50 COCspertreatmentforeachreplicate(Experiment1)andfour timeswith20–30COCspertreatmentforeachreplicate (Experiment2). Experiment3wasconductedtodeterminewhethertimingof MG132treatmentalteredeffectsoftheinhibitoronembryonic development.COCswereuntreatedortreatedwith10 m M MG132attwotimes[0–6hofmaturation(duringtheinitiationof maturation)or16–22hofmaturation(attheendofmaturation)] usinga2 6 2factorialarrangementoftreatments.TheCOCswere placedinappropriatetreatmentsat0h(vehicleorMG132), washedat6h,placedinfreshmediumwithoutMG132,washedat 16hofmaturation,andplacedinfreshmediumwithappropriate treatment.Thus,someculturesreceivedvehicleat0–6hand16– 22h,somereceivedMG132from0–6hand16–22h,some receivedMG132from0–6handvehiclefrom16–22h,andsome receivedvehiclefrom0–6handMG132from16–22h.Endpoints werecleavagerateatday3afterinseminationandtheproportion ofoocytesandcleavedembryosthatbecameblastocystsatDay8. Theexperimentwasreplicatedsixtimeswith20–50COCsper treatmentforeachreplicate. Experiments4–6wereconductedtodetermineeffectsof MG132onoocytenuclearmaturation(Experiments4and5) andfertilizationrate(Experiment6).ForExperiment4,COCs weretreatedwithvehicleor10 m MMG132forthefirst6hof maturationandnuclearmaturationwasexaminedat16hafter initiationofmaturation.Theexperimentwasreplicatedthree timeswith20–35COCspertreatmentforeachreplicate.For Experiments5and6,COCswereuntreatedortreatedwith 10 m MMG132attwotimes(0–6hofmaturation,16–22hof maturation,oratbothtimes)usinga2 6 2factorialarrangementof treatmentsandproceduresasdescribedforExperiment3.The endpointswerenuclearmaturationat22hofmaturation (Experiment5)orspermpenetrationat18hafterexposureto sperm(Experiment6).Experiment5wasreplicatedthreetimes with20–35COCspertreatmentforeachreplicate.Experiment6 wasreplicatedfourtimeswith20–50COCspertreatmentforeach replicate. Datawereanalyzedstatisticallyasfollows.Foreachreplicate, percentagedata(forexample,percentageofoocytesthatcleaved andpercentageofcleavedembryosthatbecameblastocysts)were calculatedforalloocytesorembryoswithinthesametreatment. Thus,thegroupofoocytestreatedalikewithineachreplicatewas theexperimentalunit.Statisticalanalyseswereperformedusing theStatisticalAnalysisSystem(version9.2;SASInstituteInc., Cary,NC,USA).DatawereanalyzedusingtheGeneralLinear Modelsprocedure.Formaineffectswithmorethan1degreeof freedom,thepdiffmeanseparationprocedurewasusedwhen maineffectsorinteractionsdifferedat P 0.10.Percentagedata werearcsine-transformedpriortoanalysistomaintainhomogeneityofvariance.Resultsareexpressedasleast-squaresmeans 6 standarderrorofthemean(SEM)oftheuntransformeddata.EffectofMG132ontheOocyteProteome(Experiment7)Oocyteswerematuredasdescribedabove.After16hof maturation,COCswereplacedinfreshmediumcontaining 10mMMG132orvehicle.TheCOCsweredenudedafter22hof maturationbyvortexingaftertreatmentwithhyaluronidase. Thoseoocytesinwhichapolarbodywasevidentbylight microscopywereretainedandprocessedforproteinextraction. Thezonapellucidawasremovedbytreatmentfor5minwith 0.1%(w/v)proteasefrom Streptomycesgriseus followedbymechanicalshearing. Threebiologicalreplicateswereincludedforbothvehicleand MG132groups.Abiologicalreplicaterepresentedapoolofpolarbody-extrudedoocytescollectedfromseveraloocytematuration procedures.Thenumberofoocytesperpoolwas225forreplicate 1,225forreplicate2and1000forreplicate3.Oocyteswere suspendedin10mMKPO4,pH7.4containing1mg/ml polyvinylalcoholand1%(w/v)proteaseinhibitorcocktail(Sigma) andstoredat 2 70 u Cuntilprocessing.Totalproteinwasisolated frompooledoocytesandpurifiedasdescribedelsewhere[10].The proteinconcentrationwasdeterminedusingtheBCA H Protein Assay(Thermo,Rockford,IL,USA). Foreachsample(regardlessofthenumberofstartingoocytes), 100mgproteinwasdissolvedinproteinbuffer[0.2%(w/v)sodium dodecylsulfate,8Murea,and10mMTritonX-100).The sampleswerereduced,alkylated,trypsin-digested,andlabeled followingthemanufacturer’sinstructionsfortheiTRAQReagents 4-plexkit(ABSciexInc.,FosterCity,CA,USA).Toverifythetag efficiencyoftheiTRAQmethod,iTRAQtags114and115were usedtolabelcontrolsamplesandtags116and117wereusedto labelMG132groups.TwoiTRAQprocedureswereconducted.In Set1,onecontrolandoneMG132samplewereanalyzedtwiceto determinetechnicalreplication.InSet2,twobiologicalreplicates ofeachtreatmentwereanalyzed.Proteinswereidentifiedusingan off-line2Dliquidchromatography-MS/MSmethodwithstrong cationexchange(SCX)chromatographyasafirststepto fractionatetheoocyteproteome(FigureS1).Thetrypticpeptide mixtureswerelyophilized,dissolvedinSCXsolventA[25%(v/v) acetonitrile,10mMammoniumformate,and0.1%(v/v)formic acid,pH2.8],andfractionatedusinganAgilentHPLCsystem 1260withapolysulfoethylAcolumn(2.1 6 100mm,5mm,300 A ;PolyLC,Columbia,MD,USA).Trypticpeptideswere separatedwithaLCPackingC18PepMapHPLCcolumn (Dionex,SanFrancisco,CA,USA),andahybridquadrupoleTOFQSTAREliteMS/MSsystem(ABSciexInc.,Framingham, MA,USA)wasusedfordataacquisition. TheMS/MSdatawereprocessedbyathoroughsearch consideringbiologicalmodificationandaminoacidsubstitution againsttheNationalCenterforBiotechnologyInformationnonredundant Bostaurus fastadatabase(83,655entries)anduniprot B. taurus database(33,808entries)undertheParagonTMalgorithm [11]usingProteinPilotv.4.2software(AppliedBiosystems).After searchingMS/MSspectraagainstthesedatabases,resultswere combinedintoeachgroup.Animalspecies,fixedmodificationof methylmethanethiosulfate-labeledcysteine,fixediTRAQmodificationofaminegroupsintheN-terminusandlysine,and variableiTRAQmodificationsoftyrosinewereconsidered.The ProteinPilotcutoffscorewassetto1.3(aconfidencelevelof95%), andthefalsediscoveryrate(FDR)wasestimatedbyperforming thesearchagainstconcatenateddatabasescontainingboth forwardandreversesequences(TableS1). Forproteinquantification,weonlyconsideredMS/MSspectra thatwereuniquetoaparticularproteinandwherethesumofthe signal-to-noiseratioforallofthepeakpairswas 9(software defaultsettings,ABSciexInc.).Theaccuracyofeachproteinratio wascalculatedbytheProGroupanalysisinthesoftwareto determinewhethertheproteinissignificantlydifferentially expressed[12].Tobeidentifiedasbeingsignificantlydifferentially expressed,aproteinmusthavebeenquantifiedwithatleastthree spectra,thefoldchangewas 1.2or 0.8,andthePvaluefor vehiclevsMG132was 0.05asdeterminedwithFisher’s combinedprobabilitytest[13](Fisher,1948).ThestrengthofMG132ImprovesOocyteCompetence PLOSONE|www.plosone.org3November2012|Volume7|Issue11|e48613

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theproteinsignalisreferredtoasrelativeexpressionbecausethe totalamountofproteinanalyzedwassimilarforMG132and vehicle-treatedoocytes.Differentiallyexpressedproteinswere analyzedforGOtermsbyBlast2GO[14]and,afterconversionto officialgenesymbols,bytheDatabaseforAnnotation,VisualizationandIntegratedDiscovery[DAVID;(DAVIDBioinformatics Resources6.7,http://david.abcc.ncifcrf.gov/)][15].ForDAVID, geneswereannotatedusingthebovinegenomeasareference.In addition,functionalpropertiesofdifferentially-abundantproteins weredeterminedbyminingPUBMEDusingtheChilibotprogram (www.chilibot.net)[16].PregnancyRatesafterTransferofEmbryosProduced usingMG132DuringOocyteMaturation(Experiment8)EmbryoswereproducedinvitrousingHolsteinCOCsthat werecollectedfromabattoir-derivedovaries(CentralPacking, CenterHill,FL).Maturationwascarriedoutusingconditions similartothoseforotherexperiments.At16hofmaturation, COCswerewashedandthenplacedinfreshmediumcontaining 10mMMG132orvehicle.Fertilizationwascarriedoutfor8hin SOF-IVF[17]usingX-sortedsemenfromasingleHolsteinbull (AcceleratedGenetics,Baraboo,WI,USAandSelectSires,Plain City,OH,USA).Atotalof4differentbullswereusedinthe experiment.Spermwerepurifiedbeforefertilizationasdescribed elsewhere[18].Thefinalspermconcentrationinthefertilization wellwas1 6 106sperm/ml.Followingfertilization,presumptive zygoteswereculturedin50 m lmicrodropsofBBH7culture mediumoverlaidwithmineraloilingroupsof25–30ina humidifiedatmosphereof5%CO2and5%O2(balanceN2)at 38.5 u C. Grade1expandedblastocysts[19]wereharvestedatd7after inseminationandvitrifiedusingtheopen-pulledstrawmethodas describedelsewhere[18].Onthedayoftransfer,open-pulled strawswerethawedandcontentsemptiedintoa2-wellplate (Agtech,Manhattan,KS,USA)filledwiththawmedium[Tissue CultureMedium199withHank’ssaltsandsupplementedwith 10%(v/v)fetalbovineserumand50mg/mLgentamicin] containing0.33Msucrose.Immediatelyafterwards,embryos weretransferredtoafreshwellofthesamemedium.Embryos werethenloadedindividuallyinto0.25mLembryotransferstraws andtransferredimmediatelythereafter( 5minafterthawing). EmbryosweretransferredtolactatingfemaleHolsteinsonfour occasionsbetweenJune10,2011andAugust19,2011atthe UniversityofFloridaDairyUnit(Hague,FL;29.77904N, 82.48001W).Cowswerehousedinfree-stallbarnsequippedwith fansandsprinklers.Theywerefedatotalmixedrationandmilked twotimesperday.Cowswereeitherfirst-servicecowsorcowsthat hadpreviouslybeeninseminatedorreceivedanembryoduring thatlactationandhadbeendiagnosednon-pregnant.Cowswere subjectedtotheOvsynch-56timedovulationprotocol[20]. Specifically,cowsreceived100mggonadotropinreleasinghormone(GnRH),i.m.,ond–10;25mgprostaglandinF2 a(PGF), i.m.,ond–3;and100mgGnRH,i.m.,at56hafterPGF.For first-servicecowsonly,thetimedovulationprotocolwaspreceded byapresynchronizationprotocol(twoinjectionsof25mgPGF, i.m.,14dapart),withthelastinjection12dbeforeinitiationofthe Ovsynch-56protocol. Embryosweretransferredonday7oftheabove-mentioned synchronizationprotocoltocowsdiagnosedbyultrasonographyas havingacorpusluteumonthescheduleddayforembryotransfer. Eachcowreceivedasingleembryointheuterinehornipsilateral tothecorpusluteum.Cowswerepairedandrandomlyassigned withinpairtoreceiveanembryoproducedwithvehicleor MG132.Transferwasachievedtranscervicallyandcowsreceived anepiduralblock(5mL2%lidocaine,w/v)beforetransfer. Pregnancywasdiagnosedbyultrasoundatd32andbyrectal palpationatd46and71.Atotalof24embryosproducedwith vehicleand30embryosproducedwithMG132weretransferred. Dataoncleavageandblastocystdevelopmentwereanalyzedby least-squaresanalysisofvarianceasdescribedforExperiments1–6 (n=10replicates)whiledataonpregnancyratewereanalyzedby chi-squareanalysis.Results Concentration-dependentEffectofMG132from16–22h ofMaturationonSubsequentEmbryonicDevelopment (Experiments1and2)Inthefirstexperiment,COCsweretreatedfrom16to22hof maturationwith0,1,5or10 m MMG132(Table1).Thehighest concentrationofMG132increased( P 0.05)thepercentageof oocytesthatcleaved(i.e.,thatwere $ 2cells)andthepercentageof oocytesthatbecameblastocysts.Therewas,however,noeffectof 10 m MMG132onthepercentageofcleavedembryosthatbecame blastocystsoronthenumberofcellsperblastocyst.Therewere alsonoeffectsoflowerconcentrationsofMG132onanyendpoint. InExperiment2,COCsweretreatedwith0,10,20or30 m M MG132(Table2).AsshowninExperiment1,treatmentofCOCs with10 m MMG132increasedcleavagerateandthepercentageof oocytesbecomingblastocysts( P 0.05).Inaddition,thepercentageofcleavedembryosbecomingblastocystswasincreased (P 0.05)bytreatmentwith10 m MMG132.AsinExperiment 1,therewasnoeffectof10 m MMG132onblastocystcellnumber. Treatmentwith20 m MMG132increased(P 0.05)cleavagerate butdidnotaffectotherendpointsexamined.Treatmentwith 30 m MMG132hadnoeffectonsubsequentdevelopment.ActionsofMG132DuringtheFirstSixorLastSixHoursof Maturation(Experiment3)ResultsofExperiments1and2indicatedthattheresponseof COCstoMG132occurredoveranarrowrangeandthatoptimal effectsonmaturationwereachievedwith10 m MMG132. Consequently,subsequentexperimentswereperformedwith MG132atthisconcentration.ForExperiment3,itwastested whetherMG132wouldaffectmaturationdifferentlywhenadded at0–6hofmaturation,whenproteasomesarenecessaryfor completionofmeiosisI,thanwhenaddedat16–22hof maturation(Table3).Whenaddedfrom0–6h,additionof MG132reducedtheproportionofoocytesthatcleavedandthe proportionofoocytesandcleavedembryosthatbecameblastocysts(maineffectofMG132,P 0.01orless).AdditionofMG132 from16–22ofmaturationincreased(P 0.05)thepercentageof oocytesundergoingcleavage.MG132from16–22halsoincreased thepercentageofoocytesandcleavedembryosdevelopingtothe blastocyststageprovidedCOCswerenotalsotreatedwith MG132from0–6h(interaction,P 0.05).AdditionofMG132 from16–22hincreased(P 0.02)blastocystcellnumberslightly butdifferenceswerenotdetectedusingthepdiffmeanseparation test.NuclearMaturationofBovineOocytesTreatedwithor withoutMG132from0–6hafterMaturation(Experiment 4)or16–22hafterMaturation(Experiment5)ItwashypothesizedthatMG132treatmentfrom0–6hof maturationreducedcleavagerateandthepercentageofoocytes becomingblastocystsbecauseitblockedprogressionthrough meiosisI.ThishypothesiswasexaminedinExperiment4(Table4).MG132ImprovesOocyteCompetence PLOSONE|www.plosone.org4November2012|Volume7|Issue11|e48613

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Indeed,MG132treatmentfrom0–6hofmaturationincreased (P 0.05)theproportionofoocytesthatwereatmetaphaseIat 16haftermaturationandtended(P 0.10)todecreasethe numberofoocytesthatwereatmetaphaseII.Asecond experiment(Experiment5)wasconductedinwhichMG132was addedateither0–6or16–22hofmaturation(Table5).In general,treatmenteffectswerenotsignificantexceptthatthere wasaninteraction(P 0.09)affectingthepercentageofoocytesat metaphaseI.Inparticular,thepercentageofoocytesatmetaphase IwasincreasedbytreatmentwithMG132at0–6hwhile treatmentfrom16–22hincreasedthepercentageofoocytesatMI whenMG132wasnotalsoaddedat0–6h.Whilenotsignificant, MG132treatmentfrom0–6halsotendedtoreducethe percentageofoocytesthatwereatmetaphaseII.FertilizationRatesofOocytesTreatedwithMG132from 0–6or16–22hofMaturation(Experiment6)ResultsareinTable6.AdditionofMG132from0–6hof maturationreducedfertilizationrateregardlessofwhether MG132wasalsoaddedat16–22hofmaturation(P 0.05). TherewasnoeffectofMG132from16–22honfertilizationrate. Therewasatendency(P 0.07)foradditionofMG132from0– 6htodecreasethepercentageofoocyteswithpolyspermy.TheEffectofMG132TreatmentonProteinExpressionof MaturedOocytes(Experiment7)UsingiTRAQlabelingandthe2DLC-MSMSmethod,atotal of669proteinswasidentifiedinmaturedoocyteswith653having areporterionregion.Alistoftheseproteinsanddifferencesin relativeamountbetweenoocytestreatedwithMG132andvehicle areshowninFileS1.Relativeexpressionof7distinctproteins increasedinresponsetoMG132whereasrelativeexpressionof24 distinctproteinswasdecreased(Table7).Representativeresultsfor onedifferentially-expressedprotein,CAND1,isshowninFigure1, includingmean 6 SEMofCAND1expressionforcontroland MG132-treatedoocytes(Figure1A),exampleofreporterion expressionfortheCpeptidefragmentofCAND1fromone iTRAQprocedure(Figure1B),andanexampleofbandyions andaminoacidsequencefromonepeptidefragmentofCAND1 (Figure1C). AnalysisofmolecularfunctionGOtermsusingDAVID revealedthatsixproteins(alllowerinMG132treatedoocytes) wereclassifiedintheregulationofapoptosisterm(HSP90B1, PDIA3,VCP,ALB,ASNS,CDK5),5inthemacromolecule catabolicprocessterm(HSP90B1,VCP,UBA1,andCDK5lower forMG132andCAND1higherforMG132)and5inthe proteolysisterm(HSP90B1,VCP,UBA1,andTHOP1lowerfor MG132andCAND1higherforMG132).OtherGOtermswere synonymoustothesetermsorincludedfewerproteinsthatwere affectedbyMG132. Todeterminethedegreetowhichtheproteomeofthebovine oocytematchespublishedoocyteproteomes,weevaluated whetherasubsetofrandomly-chosenproteins(minimumof2 peptidesdetected)inthepresentdatabasewaspresentina databaseofproteinsidentifiedinmouseoocytes[21].Ofthe125 proteinsexamined,73(58%)wereidentifiedinthemouse. Table1. EffectsofMG132(1–10mM)addedfrom16–22hofmaturationonsubsequentembryonicdevelopment(Experiment1).aMG132,mMNo.ofoocytes Percentageofoocytesdevelopingto Percentageofcleaved embryosdevelopingto theblastocyststageNo.ofcellsinblastocyst $ 2-cellBlastocyst 024174.5 6 1.3b35.9 6 2.8b48.6 6 3.4b146.5 6 1.7b123275.9 6 1.3b32.9 6 2.7b43.9 6 3.2b147.2 6 1.7b522475.6 6 1.3b31.7 6 2.7b42.7 6 3.2b146.6 6 1.7b1025986.6 6 1.3c49.8 6 2.7c54.8 6 3.2b146.9 6 1.7b aDataareleast-squaresmeans 6 SEMofvaluesfromsixreplicates.b,cValuesinthesamecolumnwithdifferentsuperscriptlettersaresignificantlydifferent( P 0.05). doi:10.1371/journal.pone.0048613.t001 Table2. EffectsofMG132(10–30mM)addedfrom16–22hofmaturationonsubsequentembryonicdevelopment(Experiment 2).aMG132,mMNo.ofoocytes Percentageofoocytesdevelopingto Percentageofcleaved embryosdevelopingto theblastocyststageNo.ofcellsinblastocyst $ 2-cellBlastocyst 024160.9 6 2.4b21.3 6 1.6b35.0 6 2.4b154.9 6 1.5b1023274.3 6 2.3c35.0 6 1.5c46.8 6 2.3c155.4 6 1.5b2022469.1 6 2.3cd24.4 6 1.5b34.9 6 2.3b154.2 6 1.5b3025964.0 6 2.3bd22.0 6 1.5b34.2 6 2.3b153.5 6 1.5b aDataareleast-squaresmeans 6 SEMofvaluesfromfourreplicates.b,c,dValuesinthesamecolumnwithdifferentsuperscriptlettersaresignificantlydifferent( P 0.05). doi:10.1371/journal.pone.0048613.t002 MG132ImprovesOocyteCompetence PLOSONE|www.plosone.org5November2012|Volume7|Issue11|e48613

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PregnancyRatesafterTransferofEmbryosProducedwith MG132(Experiment8)TreatmentofoocyteswithMG132from16–22hofmaturation increased(P 0.06)cleavageratefrom48.8%to62.6% (SEM=4.8%).Whilenumericallygreater,theeffectofMG132 onpercentageofoocytesbecomingblastocystswasnotsignificant (12.4%vs19.2%forvehicleandMG132,SEM=3.3%).Notethat thereducedcleavageandblastocystratesinthisexperimentreflect theuseofX-sortedspermforfertilization. AsshowninTable8,therewasnosignificanteffectofMG132 onpregnancyrateat32,46or71dofgestation.DiscussionOocytecompetencefornuclearmaturation,fertilizationand abilitytosupportembryonicdevelopmentwasaffectedbyaddition oftheproteasomalinhibitorMG132duringthematuration process.ActionsofMG132dependedonthetimeofaddition. OocytecompetencewasimprovedwhenMG132wasadded duringthelast6hofmaturation(from16–22hofmaturation) andreducedwhenaddedduringthefirst6hofmaturation. Itiswellestablishedthatproteasomalactivityisrequiredfor completionofmeiosisI.Proteasomalcleavageofubiquitinated cyclinB1leadstotheinactivationofMPFrequiredforcompletion ofmeiosisI[1].Inhibitionofmeiosisislikelyamajorcausefor reducedoocytecompetencecausedbyadditionofMG132from 0–6hofmaturationbecauseMG132treatmentatthistime tendedtoreducetheproportionofoocytesthatreachedMIIatthe endofmaturation.Inhibitionofotherproteasome-mediated eventsearlyinmaturationmayalsobeinvolvedinreduced oocytecompetence.Forexample,inthepig,MG132canaffect cumuluscellsbyreducingprogesteroneproductionandexpression ofgenesinvolvedinexpansionoftheextracellularmatrix[4]. ThefindingthattreatmentwithMG132lateinmaturation improvesoocytecompetenceisconsistentwithotherresults showingbeneficialeffectsofMG132onagedmouseoocytes fertilizedbyintracytoplasmicsperminjection[6]andparthenogeneticallyactivatedpigoocytes[7].BeneficialeffectsofMG132 onnuclearremodeling,transcriptabundanceandembryonic developmenthavealsobeenshownforembryosconstructedby somaticcellnuclearcloninginmice[22,23],rats[24,25],goats [23]andpigs[7,26,27].Unlikeforadditionfrom0–6h,MG132 addedfrom16–22hdidnotimproveoocytecompetenceby improvingnuclearmaturationbecausethepercentageofoocytes thatwereMIIattheendofmaturationwasnotaffectedby MG132laterinmaturation.Rather,someofthebeneficialeffect ofMG132from16–22honthepercentageofoocytesthat Table3. Effectoftreatmentwith10 m MMG132from0–6or16–22hofmaturationonsubsequentembryonicdevelopment(Experiment3).aMG132, 0–6hMG132,16–22hNo.ofoocytes Percentageofoocytesdevelopingto Percentageofcleavedembryos developingtotheblastocyst stageNo.ofcellsinblastocyst $ 2-cellBlastocyst NoNo16458.8 6 2.3b22.4 6 1.9b38.0 6 2.4b142.4 6 2.2bNoYes14368.4 6 2.3c34.8 6 1.9c49.3 6 2.4c148.7 6 2.2bYesNo16134.5 6 2.3d10.9 6 1.9d29.9 6 2.4bd141.3 6 2.5bYesYes16641.0 6 2.3d10.2 6 1.9d24.1 6 2.4d144.1 6 2.2bProbabilityoftreatmenteffectseMG132,0–60.0010.010.01N.S. MG132,16–220.05N.S.N.S.P 0.02 InteractionN.S.0.050.05N.S.aDataareleast-squaresmeans 6 SEMofvaluesfromsixreplicates.b,c,dValuesinthesamecolumnwithdifferentsuperscriptlettersaresignificantlydifferent( P 0.05).eN.S.=non-significant(P 0.10). doi:10.1371/journal.pone.0048613.t003 Table4. EffectoftreatmentwithMG132from0–6hof maturationonmeioticmaturationat16hafterinitiationof maturation(Experiment4).aMG132,mM No.of oocytes Nuclearstatus,%bGVBDMIAna-TeloMII 0671.3 6 2.0c34.8 6 5.0c14.7 6 2.7c49.3 6 6.0c10764.9 6 2.0c53.4 6 5.0d11.0 6 2.7c30.8 6 6.0c aDataareleast-squaresmeans 6 SEMofvaluesfromthreereplicates.bGVBD:germinalvesiclebreakdown;MI:metaphaseI;Ana-Telo:anaphase– telophase;MII:metaphaseII.c,dValuesinthesamecolumnwithdifferentsuperscriptlettersaresignificantly different( P 0.05). doi:10.1371/journal.pone.0048613.t004 MG132ImprovesOocyteCompetence PLOSONE|www.plosone.org6November2012|Volume7|Issue11|e48613

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becameblastocystswasdueto1)increasedcleavageratethrough actionsnotinvolvingfertilizationrateand2)increasedcompetence ofthefertilizedoocytetodeveloptotheblastocyststage.Indeed, thepotentialofanewlyformedembryotobecomeablastocystwas improvedbyadditionofMG132from16–22hintwoofthree experimentsevaluated,asindicatedbyasignificantimprovement inthepercentageofcleavedembryosthatbecameblastocysts. ThemechanismbywhichMG132lateinmaturationimproves competenceoftheoocytetosupportdevelopmentislikelyto involvearrestofprocessesmediatedbyproteasomesthat ordinarilycompromisetheoocyte.Oneresultislikelytobe increasedtranscriptabundanceforgenesrequiredforembryonic development,asshowninthepigoocyte[7].Inthemouse, MG132improvedoocytecompetenceinagedoocytesbutdidnot affectnon-agedoocytes[6].ItmightbethatMG132blocked proteasome-mediateddegenerativechangesinaportionof maturingoocytesofinferiorqualitycausedbyprolongedculture duringmaturationorotherreasons. Proteomicanalysiswasperformedtodeterminepossibletargets ofproteasomalcleavagewhoserelativeexpressionwasalteredby MG132treatmentfrom16–22h.Suchproteinsmightbeinvolved inthebeneficialeffectsofMG132onoocytecompetenceandmay beimportantmoleculesfordeterminingtheabilityofanoocyteto completethefirstcleavagedivisionandsupportdevelopmentof theembryototheblastocyststage.Onelimitationtothe experimentalapproachwasthatlessabundantproteinswereless likelytobedetectedbymassspectrometry.Nonetheless,atotalof 653proteinscouldbeanalyzedfordifferencesinamountbetween oocytestreatedwithvehicleorMG132.Surprisingly,therewerea greaternumberofproteinswhoserelativeexpressionwas decreasedbyMG132thantherewereproteinsthatwere increased.Regulationofintracellularproteinsinthepresenceof MG12iscomplex.InHEK293Tcells,MG132canincrease ubiquitinationofsomeproteinsanddecreaseubiquitinationof others[28].Someproteinsinthebovineoocyteincreasein abundanceduringoocytematurationwhereasothersdeclinein amount[29].Itispossiblethatinhibitionoftheproteasomeby MG132lateinmaturationprotectedsomeproteinsfrom proteolysis,whichinturnhastenedorexaggeratedthematuration-dependentdeclineinotheroocyteproteins.Sixoftheproteins thatweredecreasedbyMG132(ADSL,AHCY,CDK5,GSTM3, STIP1,andTHOP1)andtwothatwereincreasedbyMG132 (CAND1andGAPDH)areencodedforbytranscriptsthat decreaseduringnuclearmaturationofbovineoocytes[30]. Table5. Effectoftreatmentwith10 m MMG132from0–6or16–22hofmaturationonmeioticmaturationat22hafterinitiation ofmaturation(Experiment5).aMG132,0–6hMG132,16–22hNo.ofoocytes Nuclearstatus,%bGVBDMIAna-TeloMII NoNo910.9 6 1.2c14.2 6 0.8c0.0 6 2.9c84.9 6 3.5cNoYes790.0 6 1.2c19.1 6 0.8d6.0 6 2.9c74.8 6 3.5cYesNo690.0 6 1.2c34.8 6 0.8e3.2 6 2.9c62.0 6 3.5cYesYes691.6 6 1.2c34.7 6 0.8e3.6 6 2.9c60.2 6 3.5cProbabilityoftreatmenteffectsfMG132,0–6N.S.N.S.N.S.N.S. MG132,16–22N.S.N.S.N.S.N.S. InteractionN.S.P 0.09N.S.N.S.aDataareleast-squaresmeans 6 SEMofvaluesfromthreereplicates.bGVBD:germinalvesiclebreakdown;MI:metaphaseI;Ana-Telo:anaphase–telophase;MII:metaphaseII.c,d,eValuesinthesamecolumnwithdifferentsuperscriptlettersaresignificantlydifferent( P 0.05or,for).fN.S.=non-significant(P 0.10). doi:10.1371/journal.pone.0048613.t005 Table6. Effectoftreatmentwith10 m MMG132from0–6or16–22hofmaturationonfertilizationrate(Experiment6).aMG132,0–6hMG132,16–22hNo.ofoocytes Percentageof oocytesfertilized Percentage polyspermy NoNo13672.1 6 2.5bcd12.1 6 2.9bNoYes13881.7 6 2.5c14.2 6 2.9bYesNo11059.3 6 2.5d8.1 6 2.9bYesYes12057.8 6 2.5d7.9 6 2.9bProbabilityoftreatmenteffectseMG132,0–6 P 0.05P 0.07 MG132,16–22 N.S.N.S. Interaction N.S.N.S.aDataareleast-squaresmeans 6 SEMofvaluesfromfourreplicates.eN.S.=non-significant(P 0.10). doi:10.1371/journal.pone.0048613.t006 MG132ImprovesOocyteCompetence PLOSONE|www.plosone.org7November2012|Volume7|Issue11|e48613

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Amongtheoocyteproteinsregulatedbytheproteasomeare proteinsinvolvedinRNAprocessing[2,3]soinhibitionof proteasomalactivitywithMG132couldaffectstabilityand translationofavarietyofmRNA. Therewere6annotatedproteinsidentifiedwhoserelative expressionwasincreasedbyMG132(ACAT1,CAND1,TUBACA1C,P4HB,HYOU1,andGAPDH).TheincreaseinGAPDH maybeadirectresultofinhibitionoftheproteasomebecause intracellularamountsofGAPDHareregulatedbyubiquitination [31,32].Anothermechanismmaybeinvolvedinregulationof CAND1byMG132.Thisproteininterfereswithubiquitinligase activity[33].Perhaps,inhibitionofcleavageofubiquitinated proteinsleadstoincreasedsynthesisordecreaseddegradationof CAND1throughfeedbackmechanisms.Otherproteinsinvolved intheubiquitinpathwayweredecreasedbyMG132,notably HSP90B1,THOP1,UBA1,andVCP. Noneofthe6annotatedproteinsincreasedbyMG132have beenidentifiedasamarkerofoocytecompetence.Nonetheless,an increaseinamountsoftheseproteinscouldpotentiallyaffect oocytecompetence.GAPDH,forexample,catalyzesanimportant stepinglycolysis.Glycolysisinthebovineoocyteislowandmost pyruvatefortheoocyteissuppliedbythesurroundingcumulus cells[34].Thereissomeevidence,though,thatrateofglycolysisin thebovineoocyteisproportionaltodevelopmentalcompetence [35].AnotherproteinincreasedbyMG132wasTUBA1C. Tubulinsareimportantfororganellemovementintheoocyte andcompletionofmeiosis[36,37].Twootherupregulated proteins,P4HBandHYOU1,functioninproteinfolding[38,39]. Thematuringoocyteiscapableofapoptosis[40].While MG132affectedrelativeexpressionofseveralproteinsinvolvedin apoptosis,itisnotclearwhethersucheffectswouldmakethe oocytemoreorlesssusceptibletopro-apoptoticsignals.MG132 decreasedamountsofseveralproteinsthatexertanti-apoptotic actionsincludingASNS[41],HSP90B1[42],PDIA3[43],and VCP[44].AnotherproteindecreasedbyMG132,CDK5,can leadtoapoptosisifaberrantlyactivated[45]andoneprotein increasedbyMG132,P4HB,isanti-apoptotic[46]. OneproteindecreasedbyMG132,VCP,hasbeenimplicated asanoocyte-derivedspermattractantinascidians[47].Itremains tobedeterminedwhetherthisproteinplaysasimilarrolein mammals.Inanycase,additionofMG132from16–22hof maturationdidnotaffectfertilizationoraltertherateof polyspermy. Thedose-responsecurveforoocytesexposedtoMG132from 16–22ofmaturationwasunusual.Theoptimalbeneficialeffect wasachievedwith10mMandlowerorhigherconcentrationswere notgenerallyeffective.Similareffectshavebeenseeninmouse, goatandpigoocytesusedforsomaticcellnucleartransfer[22,26] Figure1.ExpressionlevelsanddetectionofpeptideofCullin-associatedNEDD8-dissociatedprotein1(CAND1). PanelA:Mean 6 SEM ofCAND1expressionforcontrolandMG132-treatedoocytes.Therewasadifference(P=0.004)betweentreatments.PanelB:Reporterionexpression fortheCpeptidefragmentofCAND1.114and115representtwoseparatebiologicalreplicatesofcontroloocyteswhile116and117representtwo separatebiologicalreplicatesofMG132-treatedoocytes.PanelC:bandyionsandaminoacidsequencefromonepeptidefragmentofCAND1. doi:10.1371/journal.pone.0048613.g001 MG132ImprovesOocyteCompetence PLOSONE|www.plosone.org8November2012|Volume7|Issue11|e48613

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aswellasforagedmouseoocytesfertilizedusingintracytoplasmic sperminjection[6].Onepossibilityisthatresidualamountsof MG132inoocytestreatedwithhighconcentrationsofMG132 interferewithfertilizationorsubsequentembryonicdevelopment. Indeed,functionalproteasomesarerequiredforfertilization[48]. OnepotentialuseofMG132istoimproveembryoyieldfrom systemsofembryoproductionbasedoninvitromaturationof Table7. Proteinswhoseabundancewasalteredby10mMMG132from16–22hofmaturation.AccessionnumberGenesymbolNameFoldchangeaP gi|85682743GAPDHRecName:Full=Glyceraldehyde-3-phosphate dehydrogenase 3.010.027 gi|332634826HYOU1hypoxiaup-regulatedprotein12.300.014 tr|E1B748|E1B748_BOVINUncharacterizedprotein1.990.004 tr|A6H7J6|A6H7J6_BOVINP4HBP4HBprotein1.720.027 sp|Q3ZCJ7|TBA1C_BOVINTUBA1CTubulinalpha-1Cchain1.600.018 sp|A7MBJ5|CAND1_BOVINCAND1Cullin-associatedNEDD8-dissociatedprotein11.440.004 sp|Q29RZ0|THIL_BOVINACAT1Acetyl-CoAacetyltransferase,mitochondrial1.360.016 gi|74355032SLC25A5Solutecarrierfamily25(mitochondrialcarrier;adenine nucleotidetranslocator),member5 0.370.039 gi|78369310STIP1stress-induced-phosphoprotein10.450.002 gi|296470781UBA1ubiquitin-activatingenzymeE10.460.010 tr|F1MHP6|F1MHP6_BOVINUncharacterizedprotein0.480.013 sp|Q8SQH5|ADT2_BOVINADT2ADP/ATPtranslocase20.480.021 tr|F1MF89|F1MF89_BOVINUncharacterizedprotein(Fragment)0.490.010 gi|296486956ADSLadenylosuccinatelyase0.490.024 sp|Q3MHL4|SAHH_BOVINAHCYAdenosylhomocysteinase0.520.039 tr|F1N785|F1N785_BOVINUncharacterizedprotein0.530.044 tr|Q1JPA2|Q1JPA2_BOVINEEF1GEukaryotictranslationelongationfactor1gamma (Fragment) 0.530.001 gi|3336842ALBbovineserumalbumin0.540.030 gi|296475166PDIA3proteindisulfide-isomeraseA3precursor0.540.021 gi|296484906PLAAphospholipaseA2-activatingprotein0.560.043 gi|95767537THOP1thimetoligopeptidase10.580.011 sp|Q02399|CDK5_BOVINCDK5Cyclin-dependentkinase50.590.021 tr|Q3T0K7|Q3T0K7_BOVINMFGE8MFGE8protein0.630.021 sp|Q1LZA3|ASNS_BOVINASNSAsparaginesynthetase[glutamine-hydrolyzing]0.630.016 tr|F1MEN8|F1MEN8_BOVINUncharacterizedprotein0.640.014 gi|7545448MGP57/53MGP57/53glycoproteinantigen0.640.021 tr|A5D7E8|A5D7E8_BOVINPDIA3PDIA3protein0.660.005 sp|Q3ZBT1|TERA_BOVINVCPTransitionalendoplasmicreticulumATPase0.700.016 gi|75775556HSP90B1Tumorrejectionantigen(gp96)10.710.002 tr|E1BBC4|E1BBC4_BOVINUncharacterizedprotein0.730.021 tr|Q2KIV8|Q2KIV8_BOVINGSTM3GlutathioneS-transferasemu3(Brain)0.750.000aMG132/vehicle. doi:10.1371/journal.pone.0048613.t007 Table8. Effectoftreatmentwith10mMMG132from16–22hofmaturationabilityontheabilityoftheresultantblastocyststo establishpregnancyaftertransfertorecipientfemales.MG132(mM) Pregnancyrateatvariousdaysofgestation,fractionandpercentage Day32Day46Day71 09/24(38%)7/24(29%)6/20(30%) 1012/30(40%)12/30(40%)7/23(30%) doi:10.1371/journal.pone.0048613.t008 MG132ImprovesOocyteCompetence PLOSONE|www.plosone.org9November2012|Volume7|Issue11|e48613

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oocytes.Resultsoftheembryotransferexperimentreportedhere indicatesthatembryosproducedfromoocytestreatedwith MG132from16–22hofmaturationhavetheabilitytoestablish pregnancyaftertransfertorecipientsthatisgenerallysimilarto controlembryos.Thus,eventhoughMG132didrescuesome oocytesthatmightotherwisemightnothavebeenfertilized,there wasnonoticeabledecreaseinembryocompetenceforestablishmentofpregnancy.Alargerstudywithmoreembryosisneededto verifythisobservation. Inconclusion,ourresultsconfirmpreviousfindingsthat inhibitionofproteasomalactivityearlyinoocytematurationcan blockprogressionthroughmeiosisandprovidenewinformation thatinhibitionofproteasomeslateinmaturationcanimprovethe competenceoftheoocytetocleaveandtheresultantembryoto developtotheblastocyststage.Suchresultsimplythataging-like effectsontheoocytemediatedbyproteasomesattheendof maturationcancompromisethefunctionoftheoocyteandimplies thatyieldofembryosfrominvitroembryoproductionsystemscan beimprovedbyappropriately-timedtreatmentwithMG132. Resultsfromtheembryotransferexperimentwouldsuggestthat embryoyieldcanbeincreasedwithoutalossofcompetenceto establishpregnancyaftertransfertorecipients.SupportingInformationFigureS1Chromatograms(280nmdetection)for strongcationexchangechromatographyofiTRAQ labeledpeptides. PanelAisthechromatogramfromanalysis ofSet1whereonecontrolandoneMG132samplewereanalyzed twicetodeterminetechnicalreplicationPanelBisthechromatogramfromSet2inwhichtwobiologicalreplicatesofeach treatmentwereanalyzed.Inbothanalyses,controlwaslabeled withiTRAQtags114and115andMG132withiTRAQtags116 and117.Atotalof12fractionsweresubmittedtoanalysisusinga quadrupoleTOFMS/MSsystem.Theareacoverageofeach fractionisshowninpanelC. (TIF)FileS1Resultsofanalysisoftheoocyteproteome. The firsttabcontainsdatafromallproteinsdetectedwhilethesecond tabisasubsetofdatafromproteinsthatweredifferentially expressedbetweenMG132andvehicle.Cellsinwhichtherewas significantincreaseinrelativeexpressioncausedbyMG132are highlightedinorangewhereascellsinwhichtherewasadecrease inrelativeexpressionarehighlightedinblue. (XLSX)TableS1Numberofproteinsidentifiedatcriticalfalse discoveryrates(FDR)fromtwodatabases. (PDF)AcknowledgmentsTheauthorsthankWilliamRembert,forcollectingoocytes,theChernin familyandCentralPacking(CenterHillFlorida),fordonatingovarian tissueandScottA.RandellofSoutheasternSemen(WellbornFlorida),for donatingsemen.TheauthorsalsothankEricDieperslootandthestaffof theUniversityofFloridaDairyUnitforinvaluableassistancewiththe embryotransferexperiment.AuthorContributionsConceivedanddesignedtheexperiments:JYELPJH.Performedthe experiments:JYELLBJFJKJBSC.Analyzedthedata:JYJKPJH. 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