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发育生物学.pdf

发育生物学

杨三十二郎
2011-11-04 0人阅读 0 0 0 暂无简介 举报

简介:本文档为《发育生物学pdf》,可适用于自然科学领域

TaiwanJObstetGynecol•June•Vol•No■SHORTCOMMUNICATION■IntroductionCatechins,afamilyofpolyphenolsfoundintea,haveantioxidative,antiallergic,antimutagenicandanticarcinogenicproperties–Recentreportsdemonstratethatcatechinspreventcancerprogressionbyinhibitingcarcinogenesis,tumorgrowth,cancercellinvasion,andtumorangiogenesis,Atthecellularlevel,themajormechanismsunderlyingtheantitumoreffectsofcatechinsinvolveapoptosisinductionandcellcyclearrest–Epicatechingallate(ECG),oneofthemajorpolyphenolsingreentea,reportedlyinducesapoptosisinhumancolorectalcancerThiscompoundmaythusbeanimportantcontributortotheantitumorigenicactivityofgreenteaECGsignificantlysuppressescyclinDexpressioninheadandnecksquamouscellcarcinoma(HNSCC)cellsAdditionally,cellgrowthandapoptosisaretriggeredbythepresenceofECGinHNSCCcellsThesefindingssupporttheuseofECGasaEPICATECHINGALLATEDECREASESTHEVIABILITYANDSUBSEQUENTEMBRYONICDEVELOPMENTOFMOUSEBLASTOCYSTSHsiaoChenTu,ChihPingChen,,,,,,WenHsiungChan*DepartmentofBioscienceTechnologyandCenterforNanotechnology,ChungYuanChristianUniversity,ChungLi,DepartmentsofObstetricsandGynecologyandMedicalResearch,MackayMemorialHospital,Taipei,DepartmentofBiotechnology,AsiaUniversity,SchoolofChineseMedicine,CollegeofChineseMedicine,ChinaMedicalUniversity,Taichung,InstituteofClinicalandCommunityHealthNursing,NationalYangMingUniversity,DepartmentofObstetricsandGynecology,SchoolofMedicine,NationalYangMingUniversity,Taipei,TaiwanSUMMARYObjective:Catechins,afamilyofpolyphenolsfoundintea,evokevariousresponsesincludingcelldeathWeexaminedthecytotoxiceffectsofepicatechingallate(ECG),apolyphenolextractfromgreentea,ontheblastocyststageofmouseembryos,subsequentembryonicattachment,andinvitroandinvivooutgrowthimplantationafterembryotransferMaterialsandMethods:MouseblastocystswereincubatedinmediumwithorwithoutECG(μM,μMorμM)forhoursCellproliferationandgrowthwereinvestigatedusingdualdifferentialstaining,apoptosiswasanalyzedwithterminaldeoxynucleotidyltransferasemediateddUTPnickendlabeling,andimplantationandpostimplantationdevelopmentofembryosweremeasuredbyinvitrodevelopmentanalysisandinvivoembryotransfer,respectivelyResults:BlastocyststreatedwithμMECGexhibitedasignificantincreaseinapoptosisandacorrespondingdecreaseintotalcellnumberImportantly,theimplantationsuccessrateofblastocystspretreatedwithμMECGwaslowerthanthatofcontrols,andinvitrotreatmentwithμMECGwasassociatedwithincreasedresorptionofpostimplantationembryosanddecreasedfetalweightConclusion:OurresultscollectivelyindicatethatinvitroexposuretoECGinducesapoptosisandretardsearlypostimplantationdevelopmentaftertransfertohostmiceThedegreeofteratogenicpotentialexertedbyECGinearlyhumandevelopmentisunknownatpresentandrequiresfurtherinvestigationTaiwanJObstetGynecol():–KeyWords:apoptosis,blastocyst,embryonicdevelopment,epicatechingallate*Correspondenceto:DrWenHsiungChan,DepartmentofBioscienceTechnologyandCenterforNanotechnology,ChungYuanChristianUniversity,ChungLi,TaiwanEmail:whchancycuedutwAccepted:March,potentialchemopreventiveagentinhumancancers,suchasHNSCCHowever,todate,fewstudieshaveinvestigatedthepotentialofECGasacytotoxicagentinembryonicdevelopmentApoptosisplaysanimportantroleindevelopmentanddiseaseAlthoughseveralstudieshaveshownthatapoptosisfunctionsduringnormalembryonicdevelopment–,mechanisticallydiverseteratogenscaninduceexcessiveapoptosisinearlyembryos,leadingtodevelopmentalinjury–RecentinvestigationshaveshownthatECGinducesorinhibitscellapoptosis,–Inthepresentstudy,weinvestigatedwhetherECGhascytotoxiceffectsonmouseblastocystsMaterialsandMethodsCollectionofmousemorulasandblastocystsImprintingcontrolregionmicewerefromtheNationalLaboratoryAnimalCenter,TaiwanThisresearchwasalsoapprovedbytheanimalresearchethicsboardofChungYuanChristianUniversity,TaiwanAllanimalsreceivedhumanecare,asoutlinedintheGuidelinesforCareandUseofExperimentalAnimalsAllmiceweremaintainedonbreederchow(HarlanTekladchowHarlan,Madison,Wisconsin,USA)withfoodandwateravailableadlibitumHousingwasastandard××cmpolypropylenecagewithwiregridtopsandkeptunderahourdayhournightregimeniparousfemales(–weeksold)weresuperovulatedbyinjectionofIUofpregnantmareserumgonadotropin(Sigma,StLouis,MO,USA)hourslater,theywereinjectedwithIUofhumanchorionicgonadotropin(NVOrganon,Oss,TheNetherlands),andthenmatedovernightwithasinglefertilemaleofthesamestrainThedayavaginalplugwasfoundwasdefinedasdayofgestationPlugpositivefemaleswereseparatedforexperimentationMorulaswereobtainedbyflushingtheuterinetubesontheafternoonofgestationday,andblastocystswereobtainedbyflushingtheuterinehornondayinbothcases,theflushingsolutionconsistedofCMRLculturemedium(GibcoLifeTechnologies,GrandIsland,NY,USA)containingmMglutamineandmMsodiumpyruvateTheconcentrationofglucoseinthismediumwasmMExpandedblastocystsfromdifferentfemaleswerepooledandrandomlyselectedforexperimentsECGtreatmentandterminaldeoxynucleotidyltransferasemediateddUTPnickendlabeling(TUNEL)assayBlastocystswereincubatedinmediumcontainingtheindicatedconcentrationsofECGforhoursForapoptosisdetection,embryoswerewashedinECGfreemedium,fixed,permeabilizedandsubjectedtoTUNELlabelingusinganinsitucelldeathdetectionkit(Roche,Mannheim,Germany)accordingtothemanufacturer’sprotocolPhotographicimagesweretakenusingafluorescencemicroscope(OlympusBXOlympus,Tokyo,Japan)ECGtreatmentandcellproliferationBlastocystswereincubatedwithorwithoutculturemediumcontainingμM,μMorμMECG(Sigma)Afterhours,blastocystswerewashedwithECGfreemedium,anddualdifferentialstainingwasusedtofacilitatecountingofcellnumbersintheinnercellmass(ICM)andtrophectoderm(TE)BlastocystswereincubatedinpronaseinMmediumcontainingbovineserumalbumin(BSA)(Sigma)forremovalofthezonapellucidaThedenudedblastocystswereexposedtomMtrinitrobenzenesulfonicacidinBSAfreeMmediumcontainingpolyvinylpyrrolidoneat°Cforminutes,thenwashedwithMmediumTheblastocystswerefurthertreatedwithantidinitrophenolBSAcomplexantibodyatμgmLinMBSAat°Cforminutes,andthenwithMmediumsupplementedwithwholeguineapigserumasasourceofcomplement,alongwithbisbenzimideatμgmLandpropidiumiodide(PI)atμgmLat°CforminutesTheimmunolysedblastocystsweregentlytransferredtoslidesandprotectedfromlightbeforeobservationUnderultravioletlightexcitation,theICMcells,whichtookupbisbenzimidebutexcludedPI,appearedblueTheTEcells,whichtookupbothfluorochromes,appearedorangeredSincemultinucleatedcellsarenotcommoninpreimplantationembryos,thenumberofnucleiwasconsideredtorepresentanaccuratemeasureofthecellnumberAnnexinVstainingBlastocystswereincubatedinμM,μMorμMECGforhours,washedwithECGfreeculturemediumandthenstainedusinganAnnexinVFLUOSstainingkit(Roche),accordingtothemanufacturer’sinstructionsBriefly,theblastocystswereincubatedinMBSAforremovalofthezonapellucida,washedwithphosphatebufferedsalinecontainingBSA,andthenincubatedforminuteswithamixtureofμLofbindingbuffer,μLoffluoresceinisothiocyanateconjugatedAnnexinVandμLofPIAfterincubation,theembryoswerewashedandphotographedusingafluorescencemicroscopeCellsthatwereAnnexinVPI−wereconsideredapoptotic,whilethosethatwereAnnexinVPIwereconsiderednecroticTaiwanJObstetGynecol•June•Vol•NoEffectsofCatechinonBlastocystsMorphologicanalysisofembryonicdevelopmentBlastocystswereculturedaccordingtoapreviouslyreportedmethodwithsomemodificationBriefly,embryoswereculturedinfourwelldishesat°CForgroupculture,fourembryoswereculturedperwellThebasicmediumconsistedofCMRLsupplementedwithmMglutamineandmMsodiumpyruvatepluspenicillinatIUmLandstreptomycinatmgmL(hereaftercalledculturemedium)Fortreatments,theembryoswereculturedwiththeindicatedconcentrationsofECGforhoursinserumfreemediumTheembryoswerethenculturedfordaysinculturemediumsupplementedwithfetalcalfserum,andfordaysinculturemediumsupplementedwithheatedinactivatedhumanplacentalcordserum,foratotalculturetimeofdaysfromtheonsetoftreatmentEmbryoswereinspecteddailyunderaphasecontrastdissectingmicroscope,anddevelopmentalstageswereclassifiedaccordingtoestablishedmethods,Underthesecultureconditions,eachhatchedblastocystattachedtofibronectinandgrewtoformaclusterofICMcellsoverthetrophoblasticlayerviaaprocesscalledTEoutgrowthAfteratotalincubationperiodofhours,morphologicscoresforoutgrowthwereestimatedGrowingembryoswereclassifiedaseither“attached”or“outgrowth”,withthelatterdefinedbythepresenceofaclusterofICMcellsoverthetrophoblasticlayerAsdescribedpreviously,ICMclusterswerescoredaccordingtoshape,rangingfromcompactandroundedICMtoafewscatteredcellsoverthetrophoblasticlayerBlastocystdevelopmentfollowingembryotransferToexaminetheabilityofexpandedblastocyststoimplantanddevelopinvivo,thegeneratedembryosweretransferredtorecipientmiceImprintingcontrolregionfemales(whiteskincolor)werematedwithvasectomizedmales(CBLJ,blackskincolorNationalLaboratoryAnimalCenter,Taiwan)toproducepseudopregnantdamsasrecipientsforembryotransferToensurethatallfetusesinthepseudopregnantmicecamefromembryotransfer(whitecolor)andnotfromfertilizationbyCBLJ(blackcolor),weexaminedtheskincolorofthefetusesatdaypostcoitusToassesstheimpactofECGonpostimplantationgrowthinvivo,blastocystswereexposedtotheindicatedconcentrationsofECGforhours,andtheneightembryosweretransferredinparalleltothepaireduterinehornsofdaypseudopregnantmiceThesurrogatemicewerekilledondaypostcoitus,andthefrequencyofimplantationwascalculatedasthenumberofimplantationsitespernumberofembryostransferredTheincidenceratesofresorbedandsurvivingfetuseswerecalculatedasthenumberofresorptionsorsurvivingfetuses,respectively,pernumberofimplantationsTheweightsofthesurvivingfetusesandplacentasweremeasuredimmediatelyafterdissectionStatisticsDatawereanalyzedusingonewayanalysisofvarianceandttests,andarepresentedasthemean±standarddeviation,withsignificancetakenatp<ResultsEffectsofECGonmouseblastocystsToinvestigatethepossibilityofECGinducedcytotoxicity,wetreatedmouseblastocystswithμM,μMorμMECGat°CforhoursandmonitoredapoptosisusingtheTUNELassayCellapoptosiswasclearlyevidentinblastocyststreatedwithμMECG(FigureA)QuantitativeanalysisrevealedabouttenfoldmoreapoptoticcellsinμMECGtreatedblastocystscomparedwithuntreatedcontrolcells(FigureB)OurresultsclearlydemonstratethatECGinducesapoptosisinmouseblastocystsEffectsofECGoncellproliferationDifferentialstainingfollowedbycellcountingwasusedtoassesscellproliferationinblastocyststreatedwithμM,μMorμMECGforhours,orthoseleftuntreatedWeobservedsignificantlylowercellnumbersinμMECGtreatedblastocystscomparedwithcontrolcells(FigureA)AnnexinVstainingrevealedmarkedlyhighernumbersofAnnexinVPI–(apoptotic)cellsintheICMoftreatedblastocystsversuscontrols,butnosuchdifferencesintheTE(FigureB)ItseemsthatμMECGsignificantlyinducesapoptosisintheICMbutnottheTEofmouseblastocysts,furthersupportingthetheorythatECGimpairsthedevelopmentalpotentialofblastocystsEffectsofECGonmouseembryonicdevelopmentalpotentialinvitroUntreatedcontrolmorulasdisplayedaboutdevelopmentintoblastocysts,whereasonlyoftheμMECGtreatedmorulasdevelopedintoblastocystsunderourexperimentalconditions(FigureA)TofurtherdeterminetheeffectsofECGonpostimplantationeventsinvitro,wetreatedblastocystswithorwithoutμM,μMorμMECG,andanalyzedsubsequentdevelopmentfordaysincultureImportantly,therateofembryoonlyattachmenttofibronectincoatedcultureddisheswasmarkedlyhigherinblastocystspretreatedwithμMECG(FigureB)Additionally,thesepretreatedblastocystsdisplayedalowerincidenceofpostimplantationdevelopmentalmilestones(FigureB)TaiwanJObstetGynecol•June•Vol•NoHCTu,etalTaiwanJObstetGynecol•June•Vol•NoEffectsofCatechinonBlastocystsECG(μM)ECG(μM)ECG(μM)ECG(μM)AApoptoticcellsperembryo∗ECG(μM)BFigureEpicatechingallate(ECG)inducesapoptosisinmouseblastocysts(A)MouseblastocystsweretreatedwithECG(μM,μMorμM)forhoursorleftuntreated,andapoptosiswasexaminedviatransferasemediateddUTPnickendlabeling(TUNEL)stainingCellswerevisualizedusinglightmicroscopywherebyTUNELcellsaredepictedinblack(B)Themeannumberofapoptotic(TUNEL)cellsperblastocystwascalculatedValuesarepresentedasmean±standarddeviationoftendeterminations*p<versusthecontrolgroupApoptoticcellsperembryo∗TEICMECG(μM)TotalTEICMECG(μM)Cellnumberperembryo∗∗ABFigureEffectsofepicatechingallate(ECG)onblastocystviabilityMouseblastocystsweretreatedwithorwithoutECG(μM,μMorμM)forhours(A)Thetotalnumberofcellsperblastocystandcellnumbersintheinnercellmass(ICM)andtrophectoderm(TE)werecountedDataarebasedonatleastblastocystsamplesfromeachgroup*p<vsthecontrolgroup(B)ThepercentagesofAnnexinVpropidiumiodide−cellsintheblastocystsofeachgroupwereexaminedDataarebasedonatleastblastocystsamplesfromeachgroup*p<vsthecontrolgroupTheseresultsindicatethatECGaffectsimplantationaswellastheinvitropotentialofblastocyststodevelopintopostimplantationembryosEffectsofECGonthedevelopmentalpotentialofblastocystsinvivoTodeterminetheeffectsofECGonblastocystdevelopmentinvivo,wetransferredcontrolandECGpretreatedmouseblastocystsandexaminedtheuterinecontentatdaysposttransfer(daypostcoitus)TheimplantationratiointheμMECGpretreatedgroupwassignificantlylowerthanthatoftheuntreatedcontrolgroup(FigureA)EmbryosthatimplantedbutfailedtodevelopweresubsequentlyresorbedHowever,theproportionofimplantedembryosthatfailedtodevelopnormallywasmarkedlyhigherinthegrouptreatedwithμMECG(FigureA)Interestingly,therewasnomarkeddifferenceinplacentalweightbetweentheμMECGtreatedanduntreatedgroups(FigureB)However,fetalweightwaslowerintheμMECGtreatedgroupversusthecontrolgroup(±mgversus±mg,respectively)Previousstudies,includingarecentreportbyourgroup,haveshownthat–offetusesweighed≥mg,andtheaverageweightoftotalsurvivingfetuseswasabout±mgintheuntreatedcontrolgroupatdayofpregnancyinamouseembryotransferassay,–Fetalweightisanimportantindicatorofdevelopmentalstatus,andtheaveragefetalweightoftheuntreatedcontrolgroupisusedasakeyindicatorofthedevelopmentofblastocyststreatedwithμMECGInterestingly,onlyofthefetusesintheμMECGpretreatedgroupweighed≥mg,animportantindicatorofsuccessfulembryonicandfetaldevelopment,whereasofcontrolfetusesexceededthisthreshold(FigureC)Together,theseobservationsindicatethatECGexposureattheTaiwanJObstetGynecol•June•Vol•NoHCTu,etalFigureInvitrodevelopmentofmouseembryosexposedtoepicatechingallate(ECG)attheblastocyststage(A)MousemorulasweretreatedwithECG(μM,μMorμM)forhoursorleftuntreatedandculturedforanadditionalhoursat°CBlastocystswerecountedandpercentagescalculatedValuesarepresentedasmeans±standarddeviationofeightdeterminations*p<versusthecontrolgroup(B)MouseblastocystsweretreatedwithECG(μM,μMorμM)forhoursorleftuntreatedandculturedfordaysposttreatmentBlastocystswereidentifiedashatched,innercellmassICM(),ICM()andICM()viamorphologicassessment,whereICMrangedfromcompactandrounded()toafewscatteredcells()overthetrophoblasticlayerValuesarepresentedasmeans±standarddeviationofeightdeterminationsAttachedonlyICMICMICMBMorphologicdevelopment()APercentageofdevelopedblastocystsfrommorulas∗ECG(μM)ECG(μM)(n=)(n=)(n=)(n=)(n=)(n=)(n=)(n=)blastocyststagereducedembryoimplantationandthepotentialforpostimplantationdevelopmentDiscussionDuringthecomplexandpreciselyorchestratedembryonicdevelopmentalprocess,chemicalorphysicalinjurycanaffectnormaldevelopmentandleadtomalformationormiscarriageoftheembryoThus,itisimportanttoestablishthepossibleteratogeniceffectsofvariousagents,includingnaturalchemicalcompoundscontainedinfood,suchasECGPreviousstudieshaveshownthatECGinducesapoptosisinvariouscancers,includinghumanbladdercancercellsandhumanprostatecancerDUcells,,andinhibitsgrowthImplantationsResorptionsSurvivingfetusesECG(μM)APercentage()††∗Weightofplacenta(mg)ECG(μM)BControlECGμMECGμMECGμMC≤–≥Distribution()Fetalweight(mg)∗∗FigureEffectsofepicatechingallate(ECG)oninvivoimplantation,resorption,fetalsurvivalandfetalweightofmouseblastocysts(A)MouseblastocystsweretreatedwithECG(μM,μMorμM)forhoursorleftuntreatedImplantations,resorptions,andsurvivingfetuseswereanalyzedThepercentageofimplantationsrepr

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