Determination of the time course of capacitation in mouse spermatozoa using a chlortetracycline fluorescence assay☆

摘要

Theheadsofmousespermatozoaobtained5minafterreleasefromtheexcisedcaudaeepididymidesshowedacharacteristicfluorescencepatterninthepresenceofthefluorophorechlortetracycline(CTC).Therewasuniformfluorescenceovertheentireheadwithabouthalfthespermpopulationshowingabrighterlineoffluorescenceacrosstheequatorialsegment;thisfluorescencepatternwasdesignateda€?F.a€?After90-minincubationinculturemedium(CM)containing2%(w/v)bovineserumalbumin,mostofthespermheadsshowedadarkbandofnonfluorescenceovertheequatorialandpostequatorialsegment,whiletheanteriorportionoftheheadshowedbrightfluorescence.Thisfluorescencepatternwasdesignateda€?B.a€?ThetimecourseforthedisappearanceofpatternFmatchedthetimecourseoftheappearanceofpatternB,withahalf-timeof30min.Thetransformationwascompletein90min.AtlongertimesofincubationinCM,thepercentageofspermatozoashowingpatternBdeclined;fluorescenceovertheentireheadwaslost,characteristicofthepatternforacrosome-reactedsperm(P.M.SalingandB.T.Storey(1979).J.CellBiol.83,544a€“555).MousespermshowingpatternBwereabletoundergotheacrosomereaction,eitherspontaneouslyorbyinductionwithacid-solubilizedzonaepellucidaefrommouseeggs(H.M.FlormanandB.T.Storey(1982).Dev.Biol.91,121a€“130).Thelatterreactionwasblockedbyitsspecificinhibitor3-quinuclidinylbenzilate(QNB).MousespermshowingpatternFcouldnotbeinducedtoundergotheacrosomereactionbyexposuretosolubilizedzonae.ThisimpliesthatthechangefromfluorescencepatternFtofluorescencepatternBcorrespondswithchangesinthespermwhichmakethemsusceptibletoundergotheacrosomereaction.ThischangeoccursduringthetimeintervalpreviouslydeterminedtobeneededforcapacitationofmousesperminvitroinCM(M.InoueandD.P.Wolf(1975).Biol.Reprod.13,340a€“346).TheseresultsimplythatspermatozoashowingCTCfluorescencepatternBcanbeconsideredtobecapacitatedandthatafunctionaldefinitionforcapacitationistheacquiredabilitytoundergotheacrosomereactionrapidlywhentreatedwithacid-solubilizedzonaepellucidae.TheCTCfluorescenceassayprovidesforthefirsttimeameanstomonitorthetimecourseofepididymalmousespermcapacitationinvitro.