Single-Cell Pulsed-Field Gel Electrophoresis to Detect the Early Stage of DNA Fragmentation in Human Sperm Nuclei 英文参考文献.docVIP

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Single-Cell Pulsed-Field Gel Electrophoresis to Detect the Early Stage of DNA Fragmentation in Human Sperm Nuclei 英文参考文献.doc

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Single-Cell Pulsed-Field Gel Electrophoresis to Detect the Early Stage of DNA Fragmentation in Human Sperm Nuclei 英文参考文献

Single-CellPulsed-FieldGelElectrophoresistoDetectthe EarlyStageofDNAFragmentationinHumanSperm Nuclei SatoruKaneko1*,JojiYoshida1,HiromichiIshikawa2,KiyoshiTakamatsu1 1ReproductionCenter,Gynecology,IchikawaGeneralHospital,TokyoDentalCollege,Ichikawa,Chiba,Japan,2ReproductionCenter,Urology,IchikawaGeneralHospital, TokyoDentalCollege,Ichikawa,Chiba,Japan Abstract Single-cellpulsed-fieldgelelectrophoresis(SCPFGE)withdualelectrodepairswasdevelopedtodetecttheearlystageof DNA fragmentation in human sperm. The motile sperm were purified by the commonly used density-gradient centrifugationtechniqueandsubsequentswim-up.Thespermwereembeddedinathinfilmofagarosecontainingbovine trypsin(20mg/mL)andwerethenlysed.PriortoSCPFGE,proteolysisofDNA-bindingcomponents,suchasprotamineand thenuclearmatrixwasessentialtoseparatethelongchainfibersfromthefibrousandgranularfragmentsderivedfroma single nucleus. The overall electrophoretic profiles elucidated the course of DNA fragmentation. A few large fibrous fragmentswereobservedatthebeginningoftheprocess,however,asthefragmentationadvanced,thelongchainfibers decreasedandshortened,and,conversely,thegranularfragmentsincreaseduntilfinallyalmostalltheDNAwasshredded. Althoughtheejaculatecontainedspermwithheterogeneousstages,thepurifiedmotilespermexhibitedseveraldozensof uniformlyelongatedfibersarisingfromthetangledDNAattheorigin,whereasapartofthesefibersgaverisetofibrous fragmentsbeyondthetipoftheelongatedfibers,andtheirnumbersandsizesvariedamongthesperm.Conventionalintra- cytoplasmicsperminjection(ICSI)usuallydependsonintra-operativelightmicroscopicobservationtoselectaspermfor injection.ThepresentresultsrevealedthatspermmotilitycouldnotgivefullassuranceofDNAintegrity.SCPFGEislikelyto serveanimportantroleinthepreoperativedifferentialdiagnosistodeterminethecompetenceofthespermpopulation providedforinjection. Citation:KanekoS,YoshidaJ,IshikawaH,TakamatsuK(2012)Single-CellPulsed-FieldGelElectrophoresistoDetecttheEarlyStageofDNAFragmentationin HumanS