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Treatment with the proteasome inhibitor MG132 during the end of oocyte maturation improves oocyte competence for develop...
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0048613 ( Publisher's URL )
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Title: Treatment with the proteasome inhibitor MG132 during the end of oocyte maturation improves oocyte competence for development after fertilization in cattle
Series Title: Plos One 7: e48613
Physical Description: Journal Article
Creator: Hansen, Peter J.
You, Jinyoung
Lee, Eunsong
Bonilla, Luciano
Francis, Jasmine
Koh, Jin
Block, Jeremy
Chen, Sixue
Publisher: Public Library of Science
Place of Publication: San Francisco CA
Publication Date: November 7 2012
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Abstract: Maturation of the oocyte involves nuclear and cytoplasmic changes that include post-translational processing of proteins. The objective was to investigate whether inhibition of proteasomes during maturation would alter competence of the bovine oocyte for fertilization and subsequent development. Cumulus-oocyte complexes were cultured in the presence or absence of the proteasomal inhibitor MG132 from either 0–6 h or 16–22 h after initiation of maturation. Treatment with MG132 early in maturation prevented progression to meiosis II and reduced fertilization rate and the proportion of oocytes and cleaved embryos that became blastocysts. Conversely, treatment with MG132 late in maturation improved the percentage of oocytes and cleaved embryos that became blastocysts without affecting nuclear maturation or fertilization rate. Optimal results with MG132 were achieved at a concentration of 10 mM – effects were generally not observed at lower or higher concentrations. Using proteomic analysis, it was found that MG132 at the end of maturation increased relative expression of 6 proteins and decreased relative expression of 23. Among those increased by MG132 that are potentially important for oocyte competence are GAPDH, involved in glycolysis, TUBA1C, needed for organellar movement, and two proteins involved in protein folding (P4HB and HYOU1). MG132 decreased amounts of several proteins that exert antiapoptotic actions including ASNS, HSP90B1, PDIA3 and VCP. Another protein decreased by MG132, CDK5, can lead to apoptosis if aberrantly activated and one protein increased by MG132, P4HB, is anti-apoptotic. Finally, the pregnancy rate of cows receiving embryos produced from oocytes treated with MG132 from 16–22 h of maturation was similar to that for control embryos, suggesting that use of MG132 for production of embryos in vitro does not cause a substantial decrease in embryo quality.
Acquisition: Collected for University of Florida's Institutional Repository by the UFIR Self-Submittal tool. Submitted by Peter Hansen.
Publication Status: Published
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Source Institution: University of Florida Institutional Repository
Holding Location: University of Florida
Rights Management: All rights reserved by the submitter.
System ID: IR00001360:00001

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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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