Journal of Reproduction and Development, Vol. 58, No 3 ...
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—Original Article— Mycotoxin Alpha-Zearalenol Impairs the Quality of Preimplantation Porcine Embryos Hongfeng WANG 1)# , Omar CAMARGO RODRIGUEZ 1)## and Erdogan MEMILI 1) 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 mammalian reproduction and development. Previous studies have shown that pigs are sensitive to the estradiol-like effects of α-ZEA. However, the effect of α-ZEA for the early embryonic development has not been fully studied. The objective of this study was to identify the direct toxicity of α-ZEA on porcine preimplantation embryonic development, embryo quality and expression of developmentally important genes. Presumptive zygotes were cultured in porcine zygote medium 3 (PZM-3) in the presence of α-ZEA (n=2,957) or 17β-estradiol (E2) (n=1,333) dissolved in 0.1% Dimethyl Sulfoxide (DMSO) from 24 to 84 h post insemination followed by determination of apoptotic cell numbers and transcript levels of BAX, BCL2L1 and POU5F1 in blastocysts. Cleavage rates on day 2 were significantly decreased in 10, 30 and 60 μM α-ZEA groups; whereas blastocyst rates on day 6 were significantly decreased in the 30 and 60 μM of α-ZEA groups. Only the 100 μM E2 group significantly decreased cleavage and blastocyst rates. Total cell numbers (TCN) in blastocysts were significantly lower in the 10 µM α-ZEA group, but no differences in apoptotic cell rates were found. The expression levels of POU5F1 and BCL2L1 transcripts were similar; however, levels of BAX transcripts and the BAX/BCL2L1 ratio were increased in both α-ZEA groups. Since α-ZEA and E2 did not elicit similar effects, results suggest that α-ZEA might impact porcine preimplantation embryonic development through pathways other than estrogen receptor binding. Key words: α-ZEA, Porcine, Embryonic development, Gene expression, Porcine (J. Reprod. Dev. 58: 338–343, 2012) M ammalian embryogenesis is a fascinating process that is essential for setting the stage for later development. Embryonic development is sensitive to environmental toxicants such as mycotox- ins. There are more than 1,500 existing environmental mycotoxins such as Zearalenone (ZEA), which is a nonsteroidal estrogen. Zearalenone mimics the hormone estrogen and thereby affects early porcine embryonic development. Zearalenone was identified in 1952 and its chemical structure was determined in 1966 [1, 2]. Zearalenone is produced by several Fusarium species found worldwide in corn, wheat, and other cereals stored improperly in wet conditions [3]. Alpha-ZEA has a higher estrogenic potency than ZEA and the other isomeric forms [4]. Alpha-ZEA might bind to estrogen receptors 1 and 2 to decrease fertility, increase embryolethal absorption, reduce litter size, and change the weight of the adrenal and pituitary glands [5]. ZEA and α-ZEA were detected in follicular fluid (FF) from the follicles ≥ 6 mm which may correlated with poor embryonic development [6]. However, the mechanism of α-ZEA toxicity, and its effect on porcine embryogenesis are poorly defined. There is a need for understanding how α-ZEA affects embryonic genome activation and developmental potential of porcine embryos. The objective of this study was to determine the direct toxic effects of α-ZEA on porcine preimplantation embryonic develop- ment, embryo quality and expression of developmentally important genes. Findings from this study are expected to shed light on the molecular details of ZEA’s effects on embryos at the onset of mammalian embryonic development, and how embryonic gene expression is disrupted. Materials and Methods We isolated oocytes from porcine ovaries that were obtained from a local abattoir. All chemicals were purchased from Sigma-Aldrich, St. Louis, MO, except those stated. In vitro production of embryos Pig ovaries were obtained from a local slaughterhouse and transported to the laboratory in saline at 30 C. Cumulus oocyte complexes (COCs) were aspirated from 3–6 mM follicles using 18 g needles. COCs with more than three layers of cumulus cells and having homogeneous cytoplasm were collected, washed three times in TL-HEPES-PVA, and matured in Tissue Culture Medium 199 (TCM-199, Invitrogen, Carlsbad, CA) supplemented with 0.1% polyvinylalcohol, 3.05 mM D-glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 20 mM pyruvate stock, 25 µg/ml gentamycin, and 0.5 µg/ml LH and FSH. Fifty COCs were matured in 500 µl maturation medium under mineral oil at 39.5 C, 5% CO 2 in a 100% humidified atmosphere. These oocytes were transferred into Received: August 12, 2011 Accepted: February 2, 2012 Published online in J-STAGE: March 9, 2012 ©2012 by the Society for Reproduction and Development Correspondence: E Memili (e-mail: [email protected]) Journal of Reproduction and Development, Vol. 58, No 3, 2012
Transcript of Journal of Reproduction and Development, Vol. 58, No 3 ...
—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:[email protected])
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
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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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