Structure and function of the zona pellucida: Identification and characterization of the proteins of the mouse oocyte's zona pellucida☆

摘要

Thezonapellucidaisanacellularcoatwhichsurroundstheplasmamembraneoffullygrownmammalianoocytesandwhichperformsavarietyofimportantfunctionsduringoogenesis,fertilization,andpreimplantationdevelopment.Inthisinvestigationtheproteinsofthemouseoocyte'szonapellucidahavebeenidentifiedandcharacterizedbyusingzonaepellucidaeisolatedindividuallyfromfullygrownoocyteswithmouth-operatedmicropipets.Variousmorphologicalandbiochemicalcriteriawereemployedtoassessthepurityoftheisolatedzonaepellucidaeand,inmostcases,theywerefoundtobevirtuallyfreeofcontaminationbyotheroocyteproteins.Itwasdeterminedthateachzonapellucidacontains4.8ngofprotein,whichrepresents80%ormoreofthedryweightofthezonapellucidaandabout17%oftheoocyte'stotalprotein.Electrophoreticanalysesofasfewasfiveisolatedzonaepellucidaetreatedwithdiazotized[125I]iodosulfanilicacidrevealedthepresenceofonlythreeradiolabeledproteins,designatedZP1,ZP2,andZP3.ThesamethreeproteinswereidentifiedbyCoomassiebluestainingwhenlargenumbersofisolatedzonaepellucidae(approximately750)weresubjectedtoSDS-polyacrylamidegelelectrophoresis.ThesethreeproteinsmigrateasbroadbandsonSDS-polyacrylamidegels,consistentwiththeirbeingglycoproteins,withapparentmolecularweightsof200,000(ZP1),120,000(ZP2),and83,000(ZP3).Thesameproteinswereradiolabeledwhenintactoocytesweretreatedwithdiazotized[125I]iodosulfanilicacid,areagentwhichdoesnotpenetratetheoocyte'splasmamembrane,orwhenisolatedzonaepellucidaeweretreatedwith3H-labeled1-dimethylaminonaphthalene-5-sulfonylchloride.Resultsofaminoacidanalysisandhigh-resolutiontwo-dimensionalelectrophoresisoftheindividualproteinssuggestthateachproteinrepresentsauniquepolypeptidechain.TheproteinsZP1,ZP2,andZP3representabout36,47,and17%,respectively,ofthetotalproteinofthezonapellucida.Inthepresenceofreducingagentswhichcausedissolutionofthezonapellucida,ZP1isconvertedintoaspecieswhichmigrateswithanapparentmolecularweightof130,000,suggestingthatitexistsasanoligomer,stabilizedbydisulfidebonds,intheunreducedstate.Theresultsoftheseexperimentsarediscussedintermsofthepropertiesofthezonapellucidabeforeandafterfertilizationandarecomparedwithresultsobtainedusingvitellineenvelopesofeggsfromnonmammaliananimalspecies.