Zona pellucida-mediated acrosomal exocytosis in mouse spermatozoa: Characterization of an intermediate stage prior to the completion of the acrosome reaction☆

摘要

Thezonapellucida(ZP)-inducedacrosomereactioninmousespermproceedsintwosteps,identifiedbythreespermfluorescencepatternsobservedsequentiallywiththefluorescentprobechlortetracycline.Capacitated,acrosome-intactspermdisplayingaBpatternproceedtoanintermediateSpattern,andthenprogressfromtheSpatterntothefullyacrosome-reactedARpattern.Previously,itwasnotfeasibletocharacterizethenatureofthetransientintermediateSpattern.Recently,itwasdemonstratedthatspermbindtotheZPofeggstreatedwith12-O-tetradecanoylphorbol-13-acetate(TPA)andundergoaBtoStransition,butdonotcompletetheacrosomereaction.ThesecellsaccumulateintheSpatternandfailtoundergotheStoARtransition(Endo,Y.,Schultz,R.M.,andKopf,G.S.1987a.Dev.Biol.119,199a€“209).ThepresentstudyutilizedZPfromTPA-treatedeggstoassessthestateofSpatternsperm.ThekineticsoftheBtoStransitionofspermincubatedwitheitherstructurallyintactorsolubilizedZPfromuntreatedorTPA-treatedeggsareidentical.AdditionofeithersolubilizedZPfromuntreatedeggsorA-23187toSpatternspermboundtointactorsolubilizedZPfromTPA-treatedeggsinducestheStoARtransition,whileZPfromTPA-treatedorfertilizedeggsdoesnot.LossofthetransmembranepHgradientintheanteriorportionofthespermhead,monitoredbythefluorescentpHprobe9-N-dodecylaminoacridine,followstheBtoStransitioninspermincubatedwithZPfromunfertilizedeggs,butnolossisobservedwhentheBtoStransitionisinducedusingZPfromTPA-treatedeggs.SubsequentadditionofsolubilizedZPfromuntreatedeggsorA-23187resultsinthelossofthetransmembranepHgradientoftheseSpatternsperm.AdditionofnigericintoSpatternspermboundtoZPfromTPA-treatedeggsdischargesthetransmembranepHgradientandcausestheStoARtransition.Incontrast,nigericinaddedtoBpatternspermdischargesthepHgradientbutdoesnotinduceaBtoStransition.ElectronmicroscopicevaluationofSpattern-arrestedspermusingZPfromTPA-treatedeggsrevealsintactplasmaandouteracrosomalmembranes.TheseresultssuggestthatZPfromTPA-treatedandfertilizedeggsaremodifiedsuchthattheZPligandsinducingtheStoARtransitionarelostorareinactivated.TheplasmamembranesofSpatternspermboundtotheseZPretaintheircationpermeabilitybarriers,butthespermhaveattainedametastablestateactivatablebynigericin.ThisSstatehasbeendesignatedS1andistakenastheendpointoftheBtoStransitionandstartingpointoftheStoARtransition.