—Original Article—

Mycotoxin Alpha-Zearalenol Impairs the Quality of Preimplantation Porcine EmbryosHongfeng WAng1)#, Omar CAMArgO rOdrIguEZ1)## and Erdogan MEMIlI1)

1)Department of Animal and Dairy Sciences, Mississippi State University, MS, USA#Present: College of Engineering, Marquette University, Wisconsin, USA##Present: Department of Animal Production, National University of Colombia, Medellín, Colombia

Abstract. Alpha-Zearalenol (α-ZEA) is one of derivatives from Zearalenone (ZEA) which impacts mammalianreproductionanddevelopment.Previousstudieshaveshownthatpigsaresensitivetotheestradiol-likeeffectsofα-ZEA.However,theeffectofα-ZEAfortheearlyembryonicdevelopmenthasnotbeenfullystudied.Theobjectiveofthisstudywas to identify thedirect toxicityofα-ZEAonporcinepreimplantation embryonicdevelopment, embryoquality andexpressionofdevelopmentallyimportantgenes.Presumptivezygoteswereculturedinporcinezygotemedium3(PZM-3)inthepresenceofα-ZEA(n=2,957)or17β-estradiol(E2)(n=1,333)dissolvedin0.1%DimethylSulfoxide(DMSO)from24to84hpostinseminationfollowedbydeterminationofapoptoticcellnumbersandtranscriptlevelsofBAX,BCL2L1 andPOU5F1inblastocysts.Cleavageratesonday2weresignificantlydecreasedin10,30and60μMα-ZEAgroups;whereasblastocystratesonday6weresignificantlydecreasedinthe30and60μMofα-ZEAgroups.Onlythe100μME2groupsignificantlydecreasedcleavageandblastocystrates.Totalcellnumbers(TCN)inblastocystsweresignificantlylowerinthe10µMα-ZEAgroup,butnodifferencesinapoptoticcellrateswerefound.TheexpressionlevelsofPOU5F1 andBCL2L1transcriptsweresimilar;however,levelsofBAXtranscriptsandtheBAX/BCL2L1ratiowereincreasedinbothα-ZEAgroups.Sinceα-ZEAandE2didnotelicitsimilareffects,resultssuggestthatα-ZEAmightimpactporcinepreimplantationembryonicdevelopmentthroughpathwaysotherthanestrogenreceptorbinding.Key words: α-ZEA,Porcine,Embryonicdevelopment,Geneexpression,Porcine

(J. Reprod. Dev. 58: 338–343, 2012)

Mammalianembryogenesis isafascinatingprocess that isessentialforsettingthestageforlaterdevelopment.Embryonic

developmentissensitivetoenvironmentaltoxicantssuchasmycotox-ins.Therearemorethan1,500existingenvironmentalmycotoxinssuchasZearalenone (ZEA),which isanonsteroidalestrogen.Zearalenonemimics thehormoneestrogenandtherebyaffectsearlyporcineembryonicdevelopment.Zearalenonewasidentifiedin1952anditschemicalstructure

wasdeterminedin1966[1,2].ZearalenoneisproducedbyseveralFusariumspeciesfoundworldwideincorn,wheat,andothercerealsstoredimproperlyinwetconditions[3].Alpha-ZEAhasahigherestrogenicpotencythanZEAandtheother isomericforms[4].Alpha-ZEAmightbindtoestrogenreceptors1and2todecreasefertility,increaseembryolethalabsorption,reducelittersize,andchangetheweightof theadrenalandpituitaryglands[5].ZEAandα-ZEAweredetectedinfollicularfluid(FF)fromthefollicles≥6mmwhichmaycorrelatedwithpoorembryonicdevelopment[6].However,themechanismofα-ZEAtoxicity,anditseffectonporcineembryogenesisarepoorlydefined.There isaneedforunderstandinghowα-ZEAaffectsembryonicgenomeactivationanddevelopmentalpotentialofporcineembryos.

Theobjectiveof thisstudywastodeterminethedirect toxiceffectsofα-ZEAonporcinepreimplantationembryonicdevelop-ment,embryoqualityandexpressionofdevelopmentallyimportantgenes.FindingsfromthisstudyareexpectedtoshedlightonthemoleculardetailsofZEA’seffectsonembryosat theonsetofmammalianembryonicdevelopment,andhowembryonicgeneexpressionisdisrupted.

Materials and Methods

Weisolatedoocytesfromporcineovariesthatwereobtainedfromalocalabattoir.AllchemicalswerepurchasedfromSigma-Aldrich,St.Louis,MO,exceptthosestated.

In vitro production of embryosPigovarieswereobtained froma local slaughterhouseand

transportedtothelaboratoryinsalineat30C.Cumulusoocytecomplexes(COCs)wereaspiratedfrom3–6mMfolliclesusing18gneedles.COCswithmorethanthreelayersofcumuluscellsandhavinghomogeneouscytoplasmwerecollected,washedthreetimesinTL-HEPES-PVA,andmaturedinTissueCultureMedium199(TCM-199,Invitrogen,Carlsbad,CA)supplementedwith0.1%polyvinylalcohol,3.05mMD-glucose,0.91mMsodiumpyruvate,0.57mMcysteine,20mMpyruvatestock,25µg/mlgentamycin,and0.5µg/mlLHandFSH.FiftyCOCswerematured in500µlmaturationmediumundermineraloilat39.5C,5%CO2ina100%humidifiedatmosphere.Theseoocytesweretransferredinto

Received:August12,2011Accepted:February2,2012PublishedonlineinJ-STAGE:March9,2012©2012bytheSocietyforReproductionandDevelopmentCorrespondence:EMemili(e-mail:em149@ads.msstate.edu)

Journal of Reproduction and Development, Vol. 58, No 3, 2012

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maturationmediumwithouthormonesafter20–22huntil40–44h.Fertilizationwasperformedin500µlmodifiedTris-bufferedmediumwhichcontained2.0mMcaffeine,2mg/mlBSA,25µg/mlgentamycin,and10µl/mlpenicillin-streptomycin(Invitrogen,Carlsbad,CA).Freshspermsampleswereseparatedbydensitygradientcentrifugation[7]andaddedintofertilizationdropsatafinalconcentrationof1×106sperm/ml.

Effect of α-ZEA on porcine embryonic developmentFertilizedporcinezygoteswererandomlyassignedtotheculture

media,PZM-3(Chemicon,Temecula,CA).In vitrocultureswereperformedin50µldropscoveredbymineraloil.Sinceα-ZEAandE2weredifficulttobesolvedintheculturemedium,DMSOwasusedasasolvent.ThefinalconcentrationofDMSOwasadjustedto0.1%inculturemedium.Toaccountfortheinfluenceof0.1%DMSO,anothergroupofembryoswereonlyexposedto0.1%DMSOinculturemedium.Thefirstexperimentincludedcontrol,0.1%DMSO,3,10,30,and60µMα-ZEAgroups.Thesecondexperimentcontainedcontrol,0.1%DMSO,0.3,3,30,and100µME2groups.Allthesechemicalswereaddedfrom24to84hpostinsemination(HPI).At84hpi,allembryoswerewashedandreculturedinthefreshPZM-3.Cleavagerateswerecountedat48hpi,and10%FBSwasaddedintoeachdroponday4postinsemination(PI).Blastocystrateswerecountedatday6pi.

Detection of apoptosis in blastocystsTotalcellnumberandthepresenceofapoptoticcellsinporcine

blastocystswereassessedusingterminaldeoxynucleotidyltrans-ferasedUTPnickend labeling(TUNEL)(Promega,Madison,WI)[8].Briefly,blastocystsfromcontrol,0.1%DMSO,and3and10µMα-ZEAtreatmentgroupswerefixedinfreshlyprepared4%methanol-freeformaldehydesolutionfor1hat25C.Afterincubationina50µldropofpermeabilizationsolution[0.5%(v/v)TritonX-100,0.1%(w/v)sodiumcitrate]for15–30minat25Cinahumidifiedenvironment,blastocystswerewashedtwicein50µlPBS/PVPdropsandincubatedin50µldropsofrecombinantTerminalDeoxynucleotidylTransferaseincubationbufferfor1hat37Cinthedark.Reactionswereterminatedbyincubatingembryosin50µldropsof2×saline-sodiumcitratesolutionfor15minat25C.Blastocystswerewashed3timesfor2minin50µlPBS/PVPdrops,stainedin1µg/mlHoechst33342,andwashed3times(2mineach)in50µlPBS/PVPdropstoremoveunincorporatedHoechst33342.Blastocystsweretransferredontoslidesandsealedbyclearnailpolish.Totalcellnumbersandapoptoticcellnumberswerecountedundera40×objectiveofanepifluorescentmicroscopeequippedwitha450–490nmexcitationand520nmemissionfilter(Nikon,Tokyo,Japan).

RNA isolationQIAGENRNeasyMicrokit(Qiagen,Valencia,CA)wasusedto

isolatetotalRNAfromeachgroupaccordingtopreviousstudies[9,10].Briefly,3–5blastocystswerepooledinRLTlysisbuffer(Qiagen,Valencia,CA)andfrozenat–80CuntilmRNAextraction.Thelysisbuffercontainingblastocystswastransferredontosilica-gelmembranespincolumns,andwashedwithRW1bufferfollowedby80%ethanolwashing.FinalRNAelutionwasconductedusing14

µlofRNase-freewater.RNAconcentrationwasdetectedusingtheNanodropND-1000spectrophotometer(NanoDropTechnologies,Wilmington,DE,USA),andRNAintegrityandqualitywereas-sessedusingaBioanalyzer2100RNA6000Picochipkit(AgilentTechnologies,PaloAlto,CA,USA).

Real-time PCR analysisReal-timequantitativePCRwasperformedtoanalyzelevels

ofrelativetranscriptsforBAX, BCL2L1,andPOU5F1genes[11].PrimersweredesignedbyPrimerPremier5software(PremierBiosoftInt,PaloAlto,CA,USA)(Table1).Primerconcentrationwasadjustedto10μMandallprimersweretestedusingcDNAfromin vitro-producedembryos.ASuperscriptIIIPlatinumTwo-StepqRT-PCRkit(Invitrogen)wasusedtosynthesizethefirststrandofcDNA.Briefly,totalRNAfromeachsamplewasnormalizedto3.5ng/μlandincubatedat25Cfor10min,42Cfor50min,and85Cfor5min.Then,2UofE. coliRNaseHwasaddedandincubatedat37Cfor20mintoeliminateRNA.Real-timePCRreactions included558.41ng templatecDNA

foreachsample,primers,andSYBRGreenERqPCRSuperMix(Invitrogen).TheiCycleriQReal-timePCR(Bio-Rad,Hercules,CA,USA)machinewiththefollowingsetupwasused:50Cfor2minforuracilDNAglycosylaseincubation,95Cfor8min30sforinitialdenaturation,40cyclesof15sat95C,30sat60C,and30sat72C.Themeltingcurveanalysiswasperformedattheend,whichstartedat55Candincreased0.5Cpercyclereachingto95Cattheendof80cycles.Betaactin(ACTB)wasusedastheendogenous internalhousekeepinggene.Standardcurvesweregeneratedusing10-foldserialdilutionsfortheendogenouscontrolACTBandall the targetgenesBAX,BCL2L1,andPOU5F1,bymeasuringthecyclenumberatwhichexponentialamplificationoccurred.RelativedifferentialmRNAexpressionlevelsofBAX,BCL2L1,andPOU5F1geneswerecalculatedbynormalizingtheirvaluestothatofthereferencegeneACTB.

Statistical analysisDifferencesamongexperimentalgroupswereanalyzedwith

one-wayanalysisofvariance(ANOVA)usingSAS9.1software(SASInstituteInc.Carey,NC,USA).GeneexpressionanalyseswereperformedusingtherelativeexpressionsoftwaretoolREST-MCSbetasoftwarev2availableathttp://www.gene-quantification.info/,whichisbasedonanefficiencycorrectedmathematicalmodelfordataanalysis.ThemathematicalmodelusedwasbasedonthePCRefficiencies(E)andthecrossingpointdeviation(ΔCP)betweentargetandreferencegenes[12–14].DifferenceswereconsideredtobesignificantwhenP<0.05.

Results

Alpha-ZEA impact of porcine embryonic developmentAtotalof300to500porcineembryosfrom11replicateswere

usedintheα-ZEAexperiment.Thecontrol(non-treated)and0.1%DMSOtreatedembryoshadsimilarcleavagerates(44.3±2.5vs.48.8±2.6)andblastocystrates(25.1±3.6vs.23.2±3.6).Nosignificantcleavageratedifferenceswerefoundamongcontroland3μMα-ZEAgroups(44.3±2.5vs.43.9±1.9),whilethecleavageratebeganto

WANGet al.340

decreasein10μMα-ZEAgroup(44.3±2.5vs.36.5±2.2,P<0.05).Inaddition,cleavageratesinthe30μMand60μMα-ZEAgroups(23.6±2.6and20.7±3.2)decreasedcomparedtothecontroland10μMα-ZEAgroups(P<0.05)(Table2).Nosignificantdifferenceswerefoundinblastocystratesamongcontrol,0.1%DMSO,3μM,and10μMgroups.However,blastocystdevelopmentdecreasedin30μMand60μMα-ZEAgroup(P<0.05)(Table2).Atotalof100to300porcineembryosfrom4replicateswere

usedintheE2experiment.Differencesincleavagerateswerefoundinthe100μME2group,comparedtocontrol(19.3±2.1vs.30.3±2.2,P<0.05)andotherE2groups(P<0.05).Theblastocystratein100μME2groupwassignificantlylowerthanthecontrolgroup(22.7±5.2vs.36.3±3.5)(Table3).

Detection of apoptosis in blastocystsAtotalof22,23,23,and21blastocystswerecollectedfrom11

replicatesincontrol,0.1%DMSO,3μM,and10μMα-ZEAgroups,respectively.InFig.1Aand1B,bluefluorescenceindicates thetotalcellnumbers(TCN)inablastocyst,andthegreen-blueortealfluorescenceindicatesapoptoticcells.TherewerenosignificantdifferencesinaverageTCN(42.4±4.3vs.40.7±3.9)andapoptoticcellrates(0.7±0.3vs.0.7±0.3)betweenthecontroland0.1%DMSOgroups.Nosignificantdifferenceswerefoundinthecontroland3μMα-ZEAgroupforaveragetotalcellsandapoptoticcellnumbers.TheTCNinthe10μMα-ZEAgroupwas26.3±1.9,whichwaslowerthanothergroups(P<0.05)(Table4).Apoptosisdatawerecalculatedfromapoptoticcellsdivided

bytotalcellsinblastocysts.Thepercentapoptosiswasincreasedfrom0.74%incontrolsto1.27%inthe10μMα-ZEAgroup,but

Table 1. PrimersusedforReal-timeRCR

Gene Accessionnumber Primersequences(5′-3′) Tm Fragmentsize(bp)

BAX AJ606301F:TTTCTGACGGCAACTTCAACTG

60 236R:AGCCACAAAGATGGTCACTGTCT

BCL2L1 NM_214285 F:TGAATCAGAAGCGGAAACCC 60 416R:GCTCTAGGTGGTCATTCAGGTAAG

POU5F1 NM_001113060 F:AGGTGTTCAGCCAAACGACC 60 334R:GATCGTTTGCCCTTCTGGC

ACTB U07786 F:ACTGGCATTGTCATGGACTCTG 60 397R:AGTTGAAGGTGGTCTCGTGGAT

Table 2. Effectofalpha-Zearalenolonporcineembryonicdevelopment

Culturemedium Numberofoocytes

Cleavagerate(%±SEM;n)

Blastocystrate%±SEM;n)

Control 551 44.3±2.5(240)a 25.1±3.6(59)a

DMSO,0.1%v/v 520 48.8±2.6(256)a 23.2±3.6(57)a

α-ZEA,3μM 693 43.9±1.9(303)a 27.2±3.3(79)a

α-ZEA,10μM 543 36.5±2.2(200)b 21.8±3.7(44)a

α-ZEA,30μM 326 23.6±2.6(78)c 4.4±2.5(4)b

α-ZEA,60μM 324 20.7±3.2(67)c 2.9±2.0(2)b

Theresultswerecombinedatleast4replications.Differentletters(a,bandc)meansignificantlydifferent(P<0.05).

Table 3. Effectofestrogenonporcineembryonicdevelopment

Culturemedium Numberofoocytes

Cleavagerate(%±SEM;n)

Blastocystrate(%±SEM;n)

Control 248 30.3±2.2(75)a 36.3±3.5(28)a

DMSO,0.1%v/v 304 31.6±2.5(96)a 31.5±3.8(30)ab

E2,0.3μM 117 35.4±9.0(42)a 27.2±9.0(13)ab

E2,3μM 132 34.9±7.0(47)a 24.9±9.2(15)ab

E2,30μM 316 27.1±3.3(85)a 31.5±4.7(29)ab

E2,100μM 216 19.3±2.1(42)b 22.7±5.2(10)b

Theresultswerecombinedatleast4replications.Differentletters(aandb)meansignificantlydifferent(P<0.05).

Fig. 1. Apoptotic cell results from TUNEL. A: blue fluorescenceindicatesnon-apoptoticcells.B:greenish-blueortealindicatesapoptoticcells.

Fig. 2. BioanalyzergelimageoftotalRNA.Representativebioanalyzergel image was to present the quality of isolated RNA. 0.1%DMSO added did not affect quality of RNA comparing toother experimental groups. Rat: positive control, nt: size innucleotidesorbases,and theupperand lowerbandswere28sand18sribosomalRNAbands.

ALPHA-ZEARALENOLAFFECTSPORCINEEMBRYOGENESIS 341

differenceswerenotsignificant(Table4).

Expression of BAX, BCL2L1 and POU5F1 genesTotalRNAwithclearbandsof28Sand18SrRNAwithoutany

signofdegradationwereusedforexpressionanalysis (Fig.2).Real-timePCRexpressionanalysisindicatedthattheexpressionlevelofPOU5F1andBCL2L1didnotchangebetweencontrolandtreatmentgroups(Fig.3and4).Similarly,nodifferenceswerefoundfortheBAXexpressionbetweencontrolandE2groups.AlthoughtheexpressionofBAXandtheBAX/BCL2L1ratioinbothα-ZEAgroupswerenotsignificantlydifference(P>0.05),weobservedaclearincreasingtrendfromcontrolto3µMand10µMα-ZEAgroups.(Fig.5and6).Therewerethreereplicates,andtheseresultswerefromthreedifferentcDNAsamplesforeachreplicate.

Discussion

IthasbeenreportedthatreproductivesystemisamajortargetofZEA[15].ZEAmaycausereproductivesystemalteration,decreasefertility,reducelittersize,andlowertheprogesteroneandoestradiollevel.Previously,ZEAhasbeenstudiedinimmunotoxic,inducingapoptosis,DNAfragmentation,andenhancingpolymorphonuclear(PMNs)[16–19].Oocytesprovidenumerousmaternalproteinsandtranscriptsthatareessentialforsustainingearlyembryonicdevelopment.Thatis,followingfertilization,whilelargeamountsofmaternalmacromoleculesareusedanddegraded,zygoticandembryonictranscriptsandproteinsaresynthesizedintheprocessknownas“maternaltoembryonictransition”incontrolofearlydevelopment.Thus,environmentalcontaminantsinfluencingoocytequalityarethereforeexpectedtoperturbembryonicdevelopmentaswell.Analysisofthedirectimpactofα-ZEAonporcinepreimplantation

embryonicdevelopmentandgeneexpressionintheblastocystsstageindicatedthatthedevelopmentofearlyembryoswasaffectedbyα-ZEAinadose-dependentmanner.Comparedtothenon-treatmentcontrolgroup,10μMα-ZEAsignificantlydecreasedcleavagerates,while30μMα-ZEAresultedindecreasedblastocystrates(Table2).TheseresultswereinagreementwithAlmet al.[20],where7.5µMα-ZEAstartedtosignificantlydecreasethematurationrateofporcineoocytesascomparedtothecontrolgroup.AccordingtoAlmet al.[20]15µMα-ZEAstartedtodecreaseblastocystrates(%)andtotalnumberofnuclei(N)inblastocystscomparedtothecontrolgroupwhenin vivo-derivedzygoteswereculturedin vitro (26.5±9.2vs.61.9±10.0and15.2±1.9vs.48.2±1.9,P<0.05).In

anotherstudy,0.312μMα-ZEAsignificantlydecreasedmaturationofporcineoocytes,andincreasedtherateofaberrantnucleiofoocytesin vitro[21].DifferentconcentrationsofE2wereaddedintheculturemedium

toinvestigatewhetherα-ZEAhasthesameeffectasE2.Only100μME2significantlydecreasedthecleavageandblastocystratescomparedtothecontrolgroup(Table3).Thisconcentrationisabout10timesgreaterthanα-ZEAwhichdecreasedembryocleavagerates.AccordingtoMalekinejadet al.[21],aberrantnucleiofoocyteswerenegativelyimpactedby0.312µME2,andreachedthegreatestlevelby31.2µME2.Malekinejadet al.[21]alsofoundthattheembryocleavageandblastocystratesweresignificantlydecreasedwhentheseoocyteswerematuredinthemediumcontaining3.12µME2.Totalcellnumbersandapoptoticcellratesinblastocystsareimportantcharacteristics,usedtoevaluateforthequalityofin vitroculturesystemsandin vitroembryos[22].Theresultsofthisstudyshowedthat10μMα-ZEAsignificantlydecreasedtheTCNinblastocysts,whiletheapoptoticcellratesweresimilar(Table4).Apreviousstudyreportedsimilarresults,thatwhenin vivo-derivedporcineembryoswereculturedinNCSU-23, theadditionof15µMα-ZEAinitiatedadecreaseintheblastocystrateandTCNinblastocystscomparedtothecontrolgroup[20].Weusedvehicle,0.1%DMSO,sothatα-ZEAandE2canbe

solvedinculturemedium.Toensuretherewasnosideeffectfromthesechemicalsoncleavageorblastocystrates,weaddeda0.1%DMSOgroupasasecondcontrol:Thecleavageandblastocystrateswerenotsignificantlydifferentcomparingto thecontrolgroup(Tables2–4).Themajordifferencesofthisstudywerethat:1)Wefocusedtodeterminethesideeffectsofenvironmentaltoxicantssuchasα-ZEAtotheembryonicearlydevelopment,whiletheotherresearchersmainlystudiestheeffectsofα-ZEAontotheoocytematuration,cleavageandblastocystrates.Wetreatedoocytesinthesameconditionsandexposedzygotesfrom24to84hpi(embryonicgenomeactivationoccursduringthiscriticalwindowofdevelop-ment)todeterminetheeffectsondevelopmentalcompetencyandapoptosis.Therationalwasthatenvironmentaltoxicitymayexertdeleteriouseffectsontotheearlyembryonicgenomeactivationandfurtherdevelopment.2)Theconcentrationsofα-ZEAandE2usedinourstudyweredifferent.Thiswasbecauseourpreliminarydatashowedthatα-ZEAstartedtoaffectcleavageandembryonicdevelopmentonlyat10μMorhigherconcentrations;wethenusedseveraldilutionsaboveandbelow10μMα-ZEA(Table2).Inthetest forE2,wefound thatonly100μME2showedsignificantdifferences(Table3).Thequalityofembryosstartedtodecrease,sothatweonlyusedseveralE2concentrationsupto100μM.Regulationbetweenanti-apoptotic(BCL2,BCL-W,BCL-XL)and

pro-apoptotic(BAX,BAK,BAD)genesplaysacriticalroleduringtheembryopreimplantationstage[23].TheratiobetweenBAXandBCL2L1determinessurvivalordeathofcells[24].ItwasfoundthattherelativegeneexpressionofPOU5F1andBCL2L1weresimilaramongcontrol,α-ZEAandE2groups.ThegeneexpressiontestswerebasedonthedatainTables2and3;wecouldexaminegeneexpressiononlyupto10μMα-ZEAand100μME2becauseofthequalityoftheembryos.AlthoughtheexpressionofBAXandtheBAX/BCL2L1ratioswerenotsignificantlydifferentbetween3µMand10µMα-ZEAgroupscomparedtothecontrolgroup,

Table 4. Totalcellnumberandapoptoticcellratesofporcineblastocysts

Culturemedium Totalcellnumber(±SEM;n)

Apoptoticcellrates(%±SEM;n)

Control 42.4±4.3(22)a 0.7±0.3(22)a

DMSO,0.1%v/v 40.7±3.9(23)a 0.7±0.3(23)a

α-ZEA,3μM 39.8±3.1(23)a 1.0±0.4(23)a

α-ZEA,10μM 26.3±1.9(21)b 1.3±0.9(21)a

Theresultswerecombinedatleast4replications.Differentletters(aandb)meansignificantlydifferent(P<0.05).

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Fig. 3. RelativegeneexpressionratioofPOU5F1.Allgroupswereculturedinporcinezygotemedium3(PZM-3)(Chemicon,Temecula,CA);from24to84hpi.TherewerenosignificantdifferencesoftheexpressionforPOU5F1amonggroups.

Fig. 4. RelativegeneexpressionratioofBCL2L1.TherewerenosignificantdifferencesoftheexpressionforBCL2L1amonggroups.

Fig. 5. RelativegeneexpressionratioofBAX.ThereweresignificantdifferencesoftheexpressionBAXbetweencontrolandbothα-ZEAgroups.Differentlettersmeansignificantlydifferent(P<0.05).

Fig. 6. TheratioofBAX/BCL2L1.Therewasnosignificantdifferencebetween0.1%DMSOgroupcomparingwithcontrol,andnosignificantdifferencebetweenE2andcontrolgroupsaswell.However,significantdifferencesoftheratioofBAX/BCL2L1betweencontrolandbothα-ZEAgroupshavebeenobserved.Differentlettersmeansignificantlydifferent(P<0.05).

ALPHA-ZEARALENOLAFFECTSPORCINEEMBRYOGENESIS 343

therewasanincreasingtrendwhichmightrepresentthatblastocystswereprogressingtowardsapoptosisalthoughthemorphologyoftheminα-ZEAappearednormal(Figs.5and6).Inaddition,itwasfoundthateven100µME2didnotincreasetheexpressionofBAX andtheBAX/BCL2L1ratiocomparedtothecontrolgroup,andthisconcentrationwasdramaticallygreaterthan0.3µM,whichisthelevelinthefluidofantralfollicles[25].EstrogencanbindtothenuclearreceptorsESR1andESR2,and

thisbindingmightcauseestrogenreceptors(ERs)transactivationandoverexpressionoftheTATAboxbindingprotein,whichregulatesDNAtranscription[26].ItwasfoundthatgeneexpressionofERsdecreasedgraduallyfromoocytestothe5- to8-cellstage.Theestrogenreceptorsgenewasnotdetectedduringthemorulastagebutbecamedetectablebytheblastocyststage[27].Inthisstudy,porcinezygoteswerecarefullyexposed inα-ZEAatdifferentconcentrationsfrom24to84hpi,whichcoversthe4-cellstageandcontemporarywithlowerERsexpression.Inaddition,duringthisstage,porcineembryosprocessadegradingmaternalgenomeandactivatetheembryonicgenome(embryonicgenomeactivation;EGA),acriticalperiodofmammalianearlypreimplantationembryonicdevelopment[28].Sowecarefullyestimatethathighertoxicityofα-ZEAcomparedtoE2maybecausedbydifferentmechanismofactionofα-ZEAduringearlyembryonicdevelopment.Tofurtherstudythisphenomena,oocyteswithknockingouttheER1andER2receptorswillbebettermethodtofullydetecttheα-ZEApathways.Limitationsofthepresentstudyinclude1)Weexpectheterogeneity

amongthethreepooledporcineembryospergroupthetotalRNAswereisolatedfrom.2)TheE2pathwayinhibitormighthavebeenusefulhadweincludeditinthisstudy.3)Examiningtheeffectsofα-ZEAontolaterdevelopment,i.e.,fetalandlivebirthratewouldbehelpfulindeterminingthefullscopeofα-ZEA’sinfluenceondevelopment.

Acknowledgments

Thismanuscript was developed byHongfengWang inADS/CVM/FO8993SpecialTopic:“ScientificCommunication”classatMississippiStateUniversity.Thisstudywasfunded inpartbyTheLifeSciencesandBio-

technology Institute, Mississippi Agricultural and Forestry Ex-perimentStation.

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