expressionsamh

meioticspindle)ifthoseoocyteswereexposedinanwrongwindowofexposure.
Therefore,itcanbeexpectedthat,achronicexposureofwomantoenvironmentalestrogensmayaffectspindleformationandchromosomesegregationinvivoaswell.
Ourhypothesisisthattheinvitrooocytematurationsystemcouldcontributeforanintegratedtestingstrategy(ITS)forscreeningchemicalsthataffectsfemalefertility.
Furthermore,byusingovariesfromslaughterhousewaste,itcouldalsocontributetoreducethenumberofexperimentalanimalsbeingusedforthosestudies.
Wehaveselectedcompoundswithaknownmechanismofaction(i.
e.
cycloheximide,estradiol,DES,cadmiumchloride)tobetestedinthissystem.
Asthefirststeptoinvestigatethisproofofprinciple,weculturedbovineoocytes,harvestedfromslaughterhouseovaries,inaM199mediaat398Cduring22h,under5%CO2andinthepresenceofthefollowingconcentrationsofcycloheximide(CHX),aknownproteinsynthesisinhibitor:0.
75;0.
60;0.
45;0.
30;0.
15and0lM.
ThetoxicologicalendpointwastheabilityoftheoocytetoreachthemetaphaseIIstage(i.
e.
thefinalstageofmaturation).
Inaddition,weinvestigatedthegeneralcytotoxicityofCHXbyusingMTT-test.
OurresultsshownogeneralcytotoxicityforCHXinalloftheconcentrationused.
Themeiosiswasinhibitedinadose-responsemannerandtheEC50forCHXwas0.
55lM,muchlowerthantheconcentrationusedtoblockproteinsynthesisinsomaticcells,indicatingthehighsensivityoftheoocytetoexpresstoxicity.
Thefollowingstepistoextendthisinvestigationtotheotherselectedcompoundsandtocombinefunctionalparameterswiththeuseofproteomicsandtranscriptomicstoobtainmechanisticinsightsinoocytematurationandtheeffectofchemicalshereon.
Itisexpectedthatmarkersderived,alongwithfunctionalparameters,canformapowerfulassayfortheinvitropredictionofreproductivetoxicity.
Inconclusion,wedobelievethattheuseoftheIVMassaycangrantapowerfulandphysiologicalmodelinanITSofeffectsofchemicalsonfemalefertility.
ResearchsupportedbyTNOResearchProgramonHazardousSubstancesandOccupationalSafety;andTheNetherlandsToxicogenomicsCenter.
275.
Calbindin-D9kExpressioninGH3CellsIsaBiomarkerofXenoestrogenicPotentialofParabens.
ThuyT.
B.
VoandEui-BaeJeung.
ChungbukNationalUniversity,Cheongju,RepublicofKoreaThepotentialadverseeffectsofparabenswerereportedinbothinvivoandinvitrosystems,butthemolecularmechanism(s)andlong-termconsequencesofparabensexposurearelargelyunknown.
Inthisstudy,wefurtherexaminedtheinductionofanestrogenicbiomarkergene-calbindin-D9k(CaBP-9k)toinvestigatetheestrogenicactivityofparabens(methyl-,ethyl-,propyl-,isopropyl-,butyl-,andisobutylparabens)intheratpituitaryGH3cellline.
Following24hexposure,significantincreasesinCaBP-9ktranscriptandproteinwereobserveddependingontheconcentrationtreatedandthelinearlengthofthealkylchainfrommethyl-toisobutylparabens,whereasco-treatmentwithfulvestrant,apureantiestrogen,largelyreversedtheparaben-inducedexpressionsofCaBP-9kmRNAandproteininGH3cellline.
Tobetterunderstandthemechanism(s)ofCaBP-9kinductionbytheseendocrinedisruptingcompounds,wemeasuredthelevelsofestrogenreceptor(ERalpha)andprogesteronereceptor(PR)expressionfollowingparabensexposure.
IntheGH3cells,agreatincreaseinPRmRNAandproteinwasobservedinaconcentration-dependentmannerafterparabenstreatment.
Paraben-inducedexpres-sionofCaBP-9kwaseffectivelyblockedinthepresenceofantagonistof17beta-estradiol(fluvestrant).
ToconfirmwhetherprogesteronereceptorsignalingisinvolvedinparabensderivedinductionofCaBP-9kmRNAandprotein,wetreatedGH3cellswithantiprogesterone(mifepristone).
Inthestudy,parabens-inducedup-regulationofCaBP-9kwascompletelyreversedbymifepristone.
Takentogether,theseresultsindicatethatCaBP-9kmaybeinducedbyparabensviathePR-involvedpathwayinadditiontoERapathwayinGH3cellline.
276.
PotentialEstrogenicEffect(s)ofParabensattheNeonatalStageofanImmatureFemaleRatMode.
Kyung-ChulChoiandEui-BaeJeung.
ChungbukNationalUniversity,Cheongju,RepublicofKoreaThisstudywasperformedtoexaminetheestrogeniceffectsofparabensonhormonalresponsivenessandonthemorphologyofreproductivetissuesduringacriticaldevelopmentalstageoffemalerats.
TwohundredimmaturefemaleSprague-Dawleyrats(n=10/group)wereorallytreatedwithmethyl-,ethyl-,propyl-,isopropyl-,butyl-,andisobutylparabenfrompostnatalday21to40inadose-dependentmannerbasedonourpreviousstudy[62.
5,250,and1000mg/kgofbodyweight(BW)perday].
Ahighdoseofmethyl-andisopropylparaben(1000mg/kgofBWperday)resultedinasignificantdelayinthedateofvaginalopeningandadecreaseinlengthoftheestrouscycle.
Inmeasurementsoforganweightandbodyweight,weobservedsignificantweightchanges(i.
e.
,decresedinovaries,kidneysandincreasedinadrenalglands,thyroidglands,liver)conversely,bodyweightwasnotalteredfollowingparabentreatment.
Inallgroupsexposedtoparabentreatment,histologicalanalysisoftheovariesfromtheimmatureratsrevealedinterstitialcelldisorders,decreasedcorporalutea,anincreaseinthenumberofcysticfollicles,andthinningofthefollicularepithelium,whichoccurredinadose-dependentmanner.
Inaddition,morphologicalstudyoftheuterusrevealedthemyometrialdysplasiasuchasmyometrialhyperplasiainthehighestdoseofpropyl-andisopropylparabenandinalldoseofbutyl-andisobutylparabens.
WealsoobservedasignificantdecreaseinserumestradiolandT4concentrationsinmethyl-,ethyl-,propyl-,isopropyl-,andisobutylparaben-treatedgroups(P,0.
01and0.
05).
Takentogether,long-termexposuretoparabenscanproducesuppressiveeffectsonhormonalresponsivenessandcandisruptthemorphologyofreproductivetargettissuesduringthiscriticalstageofdevelopmentinimmaturefemalerats.
277.
EvidenceThatMammaryGlandInfection/InjuryDuringPreg-nancyinDairyCowsMayHaveaNegativeImpactonSizeoftheOvarianReserveinTheirDaughters.
JamesJ.
Ireland,DanielleM.
Scheetz,FerminJimenez-Krassel,JosephK.
Folger,GeorgeW.
Smith,FrancescaMossa,andAlexanderC.
O.
Evans.
MichiganStateUniversity,EastLansing,MI,USA;UniversityCollegeDublin,Dublin,IrelandTheeffectofdiseaseonmaternalenvironment,whichhasacrucialroleinembryodevelopmentandsubsequenthealthofoffspring,hasnotbeenexaminedextensivelyincattle.
Therefore,thepurposeofthisstudywastodetermineifapersistentmammaryglandinfectionsuchasmastitisandcorrespondinglyachronicallyhigh(.
200,000units)somaticcellcount(SCC)inmilkofpregnantdairycowshasapotentialnegativeimpactonsizeoftheovarianreserve(totalnumberofhealthyfolliclesandoocytesinovaries)andpotentiallyfertilityoftheiroffspring.
ThisstudywasconductedatGreenMeadowFarmsInc.
inElsie,MIandused192,12-mo-oldHolsteinheifers.
Eachanimalwasinjectedwith2injectionsofprostaglandinF2alpha(PG)spaced11dapart.
Toassessrelativesizeoftheovarianreserveineachheifer,asinglebloodsamplewasremoved4dafterthe2ndPGinjectionandassayedforconcentrationofanti-Mu¨llerianhormone(AMH),andovarianultrasonographywasusedtodeterminenumberoffollicles!
3mmindiameterandovarysize.
ResultsconfirmpreviousobservationsthatAMHconcentrationswerehighlyvariableamonganimalsbutpositivelycorrelated(P,0.
001)withbothovarysizeandnumberofantralfollicles.
TodetermineifachronicmammaryglandinfectionduringpregnancyofdairycowswasassociatedwithalterationsinAMHconcentrationsandcorrespondinglysizeoftheovarianreserveintheiroffspring,wenexttestedwhethernumberofSCCmeasurements.
200,000foreachheifer'sdamwasassociatedwithalterationsintheserumAMHconcentrationineachheifer.
NumberofSCCmeasurements.
200,000perindividualcowisanaccurateindexofrecurrentudderinfections,injuryand/orinflammation.
Atotalof5to7measurementsofSCCweremadeforeachdairycowbeginning2mobeforepregnancyandcontinuingthroughoutpregnancy.
NumberofSCCmeasurements.
200,000rangedfrom0to5perindividualcows,thuscowswereseparatedintogroupsbasedontheirnumberofSCCmeasurements.
200,000.
Cowswith4or5SCCmeasurements.
200,000were~2yearsolder(P,0.
01,6.
1vs4.
4yrsold)andtended(P,0.
09)toproducelessmilkcomparedwithcowsthathad0to3SCCmeasurements.
200,000.
Resultsshowedthatthedaughtersofcowswith4and5SCCmeasurements.
200,000alsohadmarkedlylower(P,0.
02)AMHconcentrationsasyoungadultscomparedwithdaughtersofthecowswith0to3SCCmeasurements.
200,000.
Althoughmechanismsareunclear,ourresultsimplythatachronicmammaryinfectionorinjuryduringpregnancyofoldercows(aspredictedbyahighnumberofSCCmeasurements.
200,000)isassociatednotonlywithlowmilkproduction,butalsowithdiminishedsizeoftheovarianreserveandperhapsreducedreproductiveperformance(fertilityandreproductivelifespan)oftheirdaughters.
ResearchsupportedbyUSDA-NRI2004-01697,2007-01289toJJI.
278.
TheEndocrineDisruptorTCDDModulatesmicroRNAExpres-sioninPreimplantationMouseEmbryosandSpheroidsAttachmentonHumanEndometrialEpithelialCellsInVitro.
Kai-FaiLee,Wei-MinLiu,HildaTsang,Tsz-YanCheung,SurangaP.
Kodithuwakku,WilliamS.
B.
Yeung,andChrisK.
C.
Wong.
TheUnviversityofHongKong,HongKong,China;HongKongBaptistUniversity,HongKong,ChinaTheendocrinedisruptor(ED)isanexogenoussubstancethatactsontheendocrinesystemandmodulatesnormalphysiologicalfunctionsofthebody.
AlthoughEDssuchas2,3,7,8-Tetrachlorodibenzodioxin(TCDD)affectnormalreproductivefunctioninhumansandaffectsthegrowthandreproductivefunctionsinrodents,theunderlyingmechanismthatmodulatesthesechangesremainsunclear.
AccumulatingevidencesuggestedpreimplantationembryodevelopmentiscontrolledbyconcertedexpressionofmicroRNAs(miRNA)thatregulatemRNAstabilityandproteintranslation.
WehypothesizedthatEDsaffectpre-implantationembryodevelopmentbymodulatingmiRNAexpression,aswellasattachment(aninitialsteponimplantationprocess)ofembryoontoendometrialepithelium.
Treatmentofmousezygotewith10nMTCDDdidnotaffecttheblastulationrateofmouseembryodevelopedinvitro.
Yet,analysisoftheexpressionof238-plexmiRNAbyRT-PCRattheblastocyststagerevealed9and11,up-anddown-regulatedmiRNA(.
2-foldchanges;p,0.
05)betweentheTCDD-treatedandcontrolembryos.
RT-PCRconfirmedthedifferentialexpressiononsomeoftheselectedmiRNAincludingmiR-133aand199aexpressioninsingleblastocyst.
Computationanalysis(picTar)identifiedputativeimplantation-relatedtargetsforthesemiRNAsincludingcadherin,integrinandGSK3beta.
Aninvitrochoriocarcinoma(Jeg-3andBeWo)andendometrialepithelial(IshikawaandRL95-2)cellsco-culturesystemwasestablishedandusedtostudytheeffectofTCDDonspheroidsattachment.
ThereceptorofTCDD,arylhydrocarbonreceptor(AhR),aswellasestrogenreceptor(ERalpha)alphaand(ERbeta)betaweredetectedinallthecelllines.
TCDDdose-dependently(1pM-10nM,for24hours)inducedCYP1A1(abiomarkergeneforTCDDexposure)transcriptandproteinexpressioninthesecelllines.
Inaddition,TCDDdose-dependentlysuppressedattachmentofBeWospheroidontotheRL95-2monolayer(from82%to53%,n=6).
Yet,TCDDhasnoeffectoncellproliferation.
OurresultsarethefirstdemonstratingthatEDmodulatesthemiRNAexpressionofpreimplantationembryos,andmayinturnaffectthenormalexpressionofgenesthatareimportantforembryoattachmentandimplantationinvitro.
ThisresearchwassupportedinpartbyaRGCCollaborativeResearchfund(CRF)GroupResearchProject(HKBU1/CRF/08).
ABSTRACTS62