(发育生物学）III 生殖细胞发生与受精

(发育生物学）III生殖细胞发生与受精SpecificationanddeterminationofthePGCsGermplasmcanbeidentifiedmorphologicallybythepresenceofconspicuousmembrane-unboundorganelleswithanelectron-densegranulofibrillarappearancecalledgermgranulesPosteriorgranules(Pgranules)inC.elegansPolargranules(Pgranules)inDrosophilaGerminalgranulesinXenopusThegermcellfateofC.elegansisdeterminedatthe16-cellembryoP4blastomereisthePGCOriginofthePGCsinDrosophilaThePGCsknownaspolecellsbecomedistinctattheposteriorpoleoftheeggabout90minutesafterfertilization.Thecytoplasmattheposteriorpoleiscalledpoleplasm(极质)andisdistinguishedbylargeorganelles,thepolargranules.OriginofthePGCsinDrosophilaThePGCsknownaspolecellsbecomedistinctattheposteriorpoleoftheeggabout90minutesafterfertilization.Thecytoplasmattheposteriorpoleiscalledpoleplasm(极质)andisdistinguishedbylargeorganelles,thepolargranules.TheroleofpoleplasminPGCdetermination:NogermcellsdevelopiftheposteriorendoftheeggisirradiatedwithultravioletlightIfpoleplasmofaneggistransferredtotheanteriorpoleofanotherembryo,thenucleisurroundedbythepoleplasmattheanteriorpolearespecifiedasgermcellsThepoleplasmattheposteriorpoleiscriticalfordeterminationofthePGCsThepoleplasmincludesseveralcomponentsthatarecrucialforthePGCdetermination.ThoseareOskar,Nanos,Vasa,mRNAofgcl(germcellless)gene,mitochondrialribosomalRNA(mtrRNA)etcSofar,ithasbeenreportedthatseveralmaternalgene(atleast8genes)arefunctionallynecessaryandsufficientforthepoleplasmformation,andthegermcellspecification/determinationMutationsinanyofthosegenes,e.goskar,resultintheaffectedhomozygousfemalesbeing“grandchildless”.Themutantfemaledoesnotproducefunctionalpoleplasminhereggs,thushavingoffsprings(F1)wholackgermcellsandthereforearesterile(noF2offsprings).ExpressionofoskaraloneissufficientforectopicallyspecifyingthegermcellsThemolecularmechanismsunderlyingthespecificationanddeterminationofthePGCsinDrosophilaThegeneoskarissufficienttospecifythegermcellsinDrosophilaFunctionallyconservedgenesinGerm-CellDevelopmentOriginofthePGCsinmammalsThereisnoobviousgermplasminmammals,andmammaliangermcellsarenotmorphologicallydistinctduringearlydevelopment.Rather,germcellsareinducedingastrulatingembryosInmice,thegermcellsformattheposteriorregionoftheepiblast,atthejunctionoftheextraembryonicectoderm,epiblast,primitivestreak,andallantois(尿囊).ThemammalianPGCsappeartobeinducedintheearlygastrulatingembryosThemolecularmechanismsunderlyingthespecificationanddeterminationofthePGCsinMammalsThereisnoevidenceforgermplasmbeinginvolvedingermcellformationinthemouseorothermammals.Instead,germcellspecificationinthemouseinvolvescellinduction(cell-cellinteractions).Inmice,Atday6.5ofembryonicdevelopment,BMP4/BMP8bfromtheextraembryonicectodermgivecertaincellsinthisareatheabilitytoproducegermcells.Germcellspecificationinthemouseinvolvescell-cellinteractionsFig11.3Germ-cellformationinthemouse.Theprecursors(white)ofprimordialgermcells(PGCs)andextra-embryonicmesodermareinducedintheproximalepiblastbysignalsfromtheextra-embryonicectodermthatincludeBMP-4.Duringgastrulation,thesecellsmovetotheposteriorendoftheembryoabovetheprimitivestreak.HeretheyformaclusterinwhichthecentralcellsbecomesspecifiedasPGCsandtheperipheralcellsasextra-embryonicmesoderm.Aftertheirformation,thePGCsmigratetothegonads.PGCs:Fragilis(transmembraneprotein),StellaandOct-4BMP4isrequiredforthegenerationofprimordialgermcellsinthemouseembryosA:Wildtypeembryo;B:HighmagnificationofpartofAshowingindividualPGCsinthehindgut;C:EmbryosheterozygousforBMP4mutant.TherearefewerPGCscomparedwiththewildtype;D:Homozygousembryo.PGCsareentirelyabsentinthehindgutAmolecularmarkerofthePGCs:AP(alkalinephosphatase):Thegermcelldevelopmentandfertilization1Thegermcellfatedeterminationanddevelopmentofgonads1.1Specificationanddeterminationoftheprimordialgermcells(PGCs)1.2Thegermcellmigrationanddevelopmentofgonads2Gametogenesis2.1Spermatogenesis2.2Oogenesis3Fertilization3.1Therecognitionofeggandsperm3.2Thepreventionofpolyspermy3.3TheactivationofeggmetabolismInmanyanimals,germcellsdevelopatsomedistancefromthegonads,andonlylatermigratetothem,wheretheydifferentiateintoeggsandsperm.Inthemousegastrula,germcellsfirstbecomedetectableattheposteriorregionoftheepiblast(上胚层).Theybecomeincorporatedintothehindgutandthenmovefromguttubeintothegenitalridge(生殖嵴).GermcellmigrationandtheunderlyingmechanismsPathwayforthemigrationofmammalianPGCsThePGCsareshownbypositivestainingforhighlevelsofalkalinephosphatase(AP)Duringthemigration,thePGCshaveproliferatedfromaninitialpopulationof10-100cellstothe2500-5000PGCspresentinthegonadsbyday12mouseembryos.2genesWhitespottingandSteel,areinvolvedincontrollingproliferationofmigratinggermcells.WhitespottingcodesforthecellsurfacereceptorKit(RTK),whichisexpressedinthemigratingPGCs.Steelencodesastemcellfactor,aligandfortheRTK,whichisproducedbythecellsliningthemigrationpathway.MutationsthatinactivateeitherofWhitespottingandSteelcauseadecreaseingerm-cellnumber.GermcellmigrationandtheunderlyingmechanismsInDrosophila,thePGCsmovefromtheposteriorpoletothegonadsinamannersimilartothatofmammaliangermcells.DrosophilaisamodelorganismforunderstandingthemechanismsunderlyingthegermcellmigrationMigrationofPGCsintheDrosophilaembryoThePGCsarelabelledbystainingofVasa,aspecificmarkerforthegermlinecells(A-F)ThesegmentationisshownbystainingwithantibodiestoEngrailedproteins(D)InDrosophila,thePGCsmovefromtheposteriorpoletothegonadsinamannersimilartothatofmammaliangermcells.GeneticstudieshavefoundthatanumberofgenesfunctionincontrollingthePGCmigration.wunengenewhichisexpressedintheendoderm(midgut)appearstoberesponsiblefordirectingthemigrationofthePGCsfromtheendodermtothemesodermcolumbusandhedgehog,whichareexpressedinthemesodermalcellsofthegonads,appeartobecriticalfortheattractingtheDrosophilaPGCstothegonads.Drosophilaisamodelorganismforunderstandingthemechanismsunderlyingthegermcellmigrationcolumbus(clb)geneisnecessaryforPGCstomigratetogonadsa-d:VasalabelsthePGCse,f:VasalabelsthePGCs(purple);Cliftlabelsgonadalmesodermalcells(brown)Incolumbusmutantembryos,somePGCsfailtomigratefromtheendoderm(midgut)tothemesoderm,andinsteadremainassociatedwiththeendoderm(midgut)(a).Inaddition,manyPGCsthatdomigratetothemesodermfailtoassociatewiththegonadalmesoderm(e),andinsteadscatterwidelyintheembryo(c)EctopicexpressionofcolumbusgeneissufficienttoattractPGCstonewlocationsa:theembryowasimmunostainedwithanti-beta-galactosidasetoshowtheabdominalsegments.b-e:theembryoswereimmunostainedwithanti-VasatoshowthePGCsTheGal4/UasbinarysystemwasemployedtoectopicallyexpresscolumbusgeneinspecifictissuesofDrosophilaembryosincludingabdominalsegments(b),epidermis(c),thenervoussystem(d)andthePGCs(e);a:embryosectopicallyexpressingUAS-lacZintheabdominalsegments.Thegermcelldevelopmentandfertilization1Thegermcellfatedeterminationanddevelopmentofgonads1.1Specificationanddeterminationoftheprimordialgermcells(PGCs)1.2Thegermcellmigrationanddevelopmentofgonads2Gametogenesis2.1Spermatogenesis2.2Oogenesis3Fertilization3.1Therecognitionofeggandsperm3.2Thepreventionofpolyspermy3.3TheactivationofeggmetabolismSpermatogenesis(I)Spermatogenesisistheproductionofspermfromtheprimordialgermcells.Basically,thereare3majorphasesforspermatogenesisinmammals:PGCSpermatocyteSpermatidOncethevertebratePGCsarriveatthegenitalridgeofamaleembryo,theybecomeincorporatedintothesexcords(生殖索).ThePGCsremainthereuntilmaturity,atwhichtimethesexcordshollowouttoformtheseminiferoustubules(生精小管),andtheepitheliumofthetubulesdifferentiatesintotheSertolicells(支持细胞)Theinitiationofspermatogenesisduringpuberty(青春期)isprobablyregulatedbythesynthesisofBMP8binthespermatogonia(精原干细胞),thestemcell-likespermatogenicgermcells.MicelackingBMP8bdonotinitiatespermatogenesisatpubertySpermatocyteSpermatidSpermDiagramofhumanmalegermcelldevelopmentThetransitionbetweenspermatogoniaandspermatocytesAtmaturityDiagramofhumanmalegermcelldevelopmentThetransitionbetweenspermatogoniaandspermatocytesSertolicellsGDNFAtmaturitySpermatogenesis(II)Levelsoftheglialcellline-derivedneurotrophicfactor(GDNF),whichissecretedbytheSertolicells,determinewhetherthedividingspermatogoniaremainspermatogoniaorenterthepathwaytobecomespermatocytes.LowlevelsofGDNFfavorthedifferentiationofthespermatogoniaintospermatocytesHighlevelsofGDNFfavorself-renewalofthestemcellsMiceheterozygousforGDNFmutantalleleshowdepletionofstemcellreservesduetodefectiveself-renewalofspermatogoniaGDNF+/-mice(A,B)at5weeksofageWildtype(D)andGDNF+/-(E)miceat8weeksofageD,E:BrdUincorporationassayA,B:InyoungerGDNF+/-mice,partofseminiferoustubulesaredegenerated.Inthedegeneratingtubules,spermatocytesandspermatidsarevisible,butspermatogoniahavedisappearedD,E:InolderGDNF+/-mice,decreasedexpressionofGDNFoftenresultedinSertolicell–onlyseminiferoustubuleswithoutspermatogonia,andthecellproliferationratewasreduced,reflectingdepletionofspermatogonia,duetodefectsintheself-renewalofspermatogonia.MiceoverexpressingGDNFshowaccumulationofundifferentiatedspermatogoniaB:Largeclustersofspermatogonia(spermatogonialmarker,EE2)areevidentinthetransgenicgonadsE:Thespermatogoniacellclustergraduallydegenerateafterpuberty,resultingintubularatrophyA,D:wildtypeB,E:transgenicmiceat3or10weeksofagerespectivelyA-E:EE2labelsspermatogoniaOogenesis(I)Oogenesisdiffersfromspermatogenesisinseveralways.Whereasthegameteformedbyspermatogenesisisessentiallyamotilenucleus,thegameteformedbyoogenesiscontainsallmaterialsneededtoinitiateandmaintainmetabolismanddevelopment.Therefore,oogenesisneedstobuildupastoreofcytoplasmicenzymes,mRNAs,organellesandmetabolicsubstrates,aswellasformingahaploidnucleusThemechanismsofoogenesisvaryamongspeciesmorethanthoseofspermatogenesis.Insomespecies,suchasseaurchinsandfrogs,thefemaleroutinelyproduceshundredsorthousandsofeggsatatime,whereasinotherspecies,suchashumansandmostmammals,onlyafeweggsareproducedduringthelifetimeofanindividual.OverviewoftheDrosophilaoogenesisOogenesis(II)Inthehumanembryo,oogonia(卵原细胞),theself-renewinggerm-linestemcellsdividerapidlyfromthesecondtotheseventhmonthofgestationtoformroughly7milliongermcells.Sincethen,mostoogoniadie,whiletheremainingoogoniaenterthefirstmeioticprophase.Atthisstage,thegermcellsbecomeprimaryoocytes.Thoseoocytesarearresteduntilthefemalematures.Withtheonsetofpurberty,groupsofoocytesperiodicallyresumemeiosis.Inthehumanfemale,thefirstpartofmeiosisbeginsintheembryo,andthesignaltoresumemeiosisisnotgivenuntilroughly12yearslater.Infact,someoocytesaremaintainedinmeioticprophasefornearly50yearsPrimaryoocytescontinuetodieevenafterbirth.Ofmillionsofprimaryoocytespresentatbirth,only400matureduringawoman’slifetime.SchematicrepresentationofXenopusoocytematurationThemeioticcelldivisionsareregulatedbyprogesterone(孕酮)andfertilizationThegermcelldevelopmentandfertilization1Thegermcellfatedeterminationanddevelopmentofgonads1.1Specificationanddeterminationoftheprimordialgermcells(PGCs)1.2Thegermcellmigrationanddevelopmentofgonads2Gametogenesis2.1Spermatogenesis2.2Oogenesis3Fertilization3.1Therecognitionofeggandsperm3.2Thepreventionofpolyspermy3.3TheactivationofeggmetabolismFertilizationAlthoughthedetailsoffertilizationvaryfromspeciestospecies,itinvolves3majorevents:Contactandrecognitionbetweenspermandegg.Thisensuresthatfertilizationisspecies-specific.Regulationofspermentryintotheegg.Onlyonespermcanultimatelyfertilizetheegg.Thatistopreventpolyspermywhichleadstoabnormaldevelopmentinmostorganisms.Activationofeggmetabolismtoinitiateembryonicdevelopment.FertilizationTherecognitionofeggandspermThepreventionofpolyspermyTheactivationofeggmetabolismTheplasmamembraneontheheadofthespermcontainsvariousspecializedproteinsforbindingtotheeggcoatsandfacilitatingentryGametebindingandrecognitioninmammalsFertilizationofamammalianegg.Thespecies-specificrecognitionofspermandegginmammalsandtheunderlyingmolecularbasis.ZP3:aglycoproteininthezonapellucidafunctioningasareceptorforspecies-specificbindingofthespermZP3isthesperm-bindingglycoproteinofthemousezonapellucidaB:PurifiedZP3canbindtothespermandpreventthespermfrombindingtothezonapellucida(Theinhibitionassay)A:ThespermswerestainedbyantibodyagainstZP3-bindingproteinGametebindingandrecognitioninmammalsFertilizationofamammalianegg.Thespecies-specificrecognitionofspermandegginmammalsandtheunderlyingmolecularbasis.ZP3:aglycoproteininthezonapellucidafunctioningasareceptorforspecies-specificbindingofthespermThemolecularmechanismsunderlyingthefusionofspermandeggmembrane.Fertilin:onthespermsurfaceIntegrin-likereceptorCD9:ontheeggplasmamembrane.FemalemicecarryinggeneknockoutsforCD9areinfertilebecausetheireggsfailtofusewithspermFertilizationTherecognitionofeggandspermThepreventionofpolyspermyTheactivationofeggmetabolismThepreventionofpolyspermyDepolarizationoftheseaurchineggplasmamembraneatfertilization(rapidblock).Thecorticalreactionatfertilizationintheseaurchin(slowblock).Polyspermycanbeinducedifdepolariztionisartificiallyblocked.Itisnotknownhowthechangeinmembranepotentialactsonblockingpolyspermy.FertilizationTherecognitionofeggandspermThepreventionofpolyspermyTheactivationofeggmetabolismTheactivationofeggatfertilizationinitiatesaseriesofeventsthatresultinthestartofdevelopment.Themaineventsarethattheeggcompletesmeiosis,andthefertilizedeggentersmitosis.TheexplosivereleaseoffreeCa2+ionswithintheegg,triggeredbyspermentry,isbothnecessaryandsufficienttoinitiatetheonsetofdevelopmentinbothmammalandseaurchins.However,themechanismfortheCa2+releaseisnotknown.Whatarethemechanismsunderlyingtheactivationoftheeggandonsetofdevelopmentofthefertilizedegg,initiatedbytheCa2+wave?InXenopusInseaurchinsTheactivationofeggmetabolismMPFisacomplexofacyclin-dependentkinase(Cdk)anditspartnercyclininXenopus.FreeCa2+Cyclindegradation/ReductionofMPFactivityCompletionofmeiosis/OnsetofmitosisCalmodulin-dependentproteinkinaseIICalciumreleaseatfertilizationactivatesaseriesofmetabolicreactionsforinitiatingdevelopmentoftheembryointheseaurchin