*Author forcorrespondence (e-mail:[email protected]) of Queensland,Brisbane,QLD4072,Australia. Biotechnology andDevelopment,Institute forMolecularBioscience,TheUniversity Division ofMolecularGeneticsandDevelopment, andARCCentre ofExcellencein somatic signalsearmarkasmall cohortofproximalepiblastcellsas has beengleanedfromstudies in mice.Ataround7.2dpcmice, Much ofourknowledge regarding germcell behavior inmammals and maturation Germ cellspecification,proliferation, migration postnatal testis. the regulation ofentrymalegermcellsintomeiosisinthe evidence thatsuggeststheroleofRAmightbereprisedduring theories abouthow germcellsexual fate isspecified. We alsoreview reconcile two substantialbodiesofdatathathave ledtoopposing meiosis inmalesatthattime.We argue herethatthesefindings post coitum(dpc),itsdegradation protectsgermcellsfromentering induces germcellstoentermeiosisintheovary ataround13.5days Koubova etal.,2006).These studiesreportedthat,althoughRA in micebyexposure toRAinthefetalgonad(Bowles etal.,2006; shown thattheinitialchoiceofmaleorfemaleidentityisgoverned environment (McLaren,1995;McLaren,2003).Recentstudieshave not bytheirchromosomalconstitutionbut bycuesfromtheir in thisprocess.Thesex differentiation ofgermcellsisdetermined and theroleofretinoicacid(RA),active derivative ofvitaminA, towards thealternative pathways ofoogenesisorspermatogenesis, reproductive biologists. behavior ofgermcellscontinuetofascinate developmental and meiosis (seeBox1).Notsurprisingly, theorigins,propertiesand generate haploidcellsviaauniquetypeofcelldivision known as are abletopartitiontheirgeneticmaterialinsuchaway asto information betweengenerations.To fulfill thisrole,germcells cells clearlyplayauniqueroleasthecarriersofgenetic depending onwhetherthey endupinanovary oratestis.Germ capacitytodifferentiate intoeitheroocytes orsperm, the long andperilousjourney. Duringthistime,PGCsmustretain gametes, andthecolonizationofgonadsbyPGCsinvolves a remarkably far inadvance ofthefunctional differentiation of population ofprimordialgermcells(PGCs)isestablished As cellsgo,germareundeniablyidiosyncratic.Thefounder Introduction which wediscussinthisreview. in reproductivebiology, italsoposesmanynewquestions, acid. Althoughthisknowledgeclarifiesalong-standingmystery is thepresenceorabsenceofsignalingmoleculeretinoic indicate thatthekeytothissex-specifictimingofmeiosisentry environment donotentermeiosisuntilpuberty. Recentfindings committing tooogenesis.Bycontrast,germcellsinatesticular embryogenesis, germcellsinanovaryentermeiosis,thereby initiated bycuesprovidedthegonadalenvironment.During specific developmentofgermcellsasspermoroocytesis Although mammaliansexisdeterminedgenetically, thesex- Josephine BowlesandPeterKoopman* Retinoic acid,meiosisandgermcellfateinmammals (2007)doi:10.1242/dev.001107 Development 134,3401-3411 This review focusesonhow mammaliangermcellsaredirected et al.,1986;Tam andSnow, 1981). about 26,000germcellsresidein thegonadsby13.5dpc(Donovan become lessmotileandcontinue toproliferatefor1-2days,sothat al., 2001;Molyneauxet2003). PGCsthenchangetheirshape, growth factor kitligand(KITL)(Farini etal.,2007;Molyneaux (also known asCXCL12 –MouseGenomeInformatics)and/orthe that emanatesfromthegenitalridges, possiblythechemokineSDF1 The movements ofPGCs suggestthatthey respondtoanattractant intermediate mesodermandsomaticsex determinationisunderway. 10.5 to11.5dpc,justafterthesestructuresarisefrom the the genitalridges.ThePGCscolonizeridgesataround elongating hindguttowards thefuturesiteofprimitive gonads, 2002). PGCsproliferaterapidlyandmigrateanteriorlythrough the 1994; Ohinataetal.,2005;Saitou2002;Tanaka andMatsui, identifiable asgermcellprecursorsorPGCs(Lawson andHage, selection occurs,resultinginagroupofabout45cellsthat are the baseofallantois,whereasecondroundmolecular 2000). Thisgroupofcellsmoves intotheextraembryonic tissuesat 1999; Lawson andHage,1994;Tam andZhou,1996;Ying etal., potential germcellprecursors(Ginsburg etal.,1990;Lawson etal., (e.g. inhumanmales)todecadesfemales). general, meioticprophase Itakesthelongesttime,rangingfrom days greatly amongspeciesandbetweenthesexesofasinglespecies.In of polarbodies.Thetimerequired foreachstageofmeiosisdiffers ultimately produced, theothergeneticmaterialbeinglostinform spermatozoa but,inthefemale,onlyonefunctionalgameteis genetically different haploidcells.Inthemale,thisamountstofour followed bytworounds ofcelldivision,potentiallyproducing four diploid cellenteringmeiosisundergoesoneround ofDNAreplication produce twocellswithhaploidDNAcontent.To summarize,each align alongasecondspindleandsisterchromatids separateto produced. IndivisionIIofmeiosis(meiosisII),duplicatedchromosomes further DNAreplication, isnecessarybefore haploidgametesare copy ofeachchromosome. Asecondround ofcelldivision,without recombination hasoccurredandamaternal –ratherthanapaternal identical copiesofeachchromosome –differing onlywhere genetic but theydiffer from normaldiploidcellsinthattheyhavetwonear- produced are stilldiploidintermsoftheamountDNAtheycontain, poles ofthespindle,anddivisionImeiosisoccurs.Thetwocells composed oftwosisterchromatids) break apartandmovetoopposite the spindleandatanaphasepairofchromosomal homologs(each four-chromatid structure iscalledabivalent. ‘chiasmata’) andexchangeanalogousfragmentsofDNA.Sucha because non-sisterchromatids ofhomologscancross-over (forming chromosome homologsallowsgeneticrecombination tooccur, replication, producing twosisterchromatids. Thepairingof the caseformitoticdivision,eachchromosome duplicatesbyDNA nucleoprotein structure knownasthesynaptonemalcomplex.Asis chromosomes alignsandisheldtogethertightlybyameiosis-specific the firstwith,andsecondwithout,DNAreplication. meiosis. Meiosisincludestwocelldivisions(meiosisIandmeiosisII), division ofadiploidgermcellinspecializedprocess knownas Gametes are haploid,andsoare produced byreductive nuclear Box 1.Meiosis During metaphaseofmeiosisI,bivalentslineupperpendicularto During prophase ofmeiosisI,eachpairhomologous REVIEW 3401

DEVELOPMENT ~10.5 dpc cycle, resumingmitosisafterbirth(Hilscheretal.,1974;McLaren, life toensure acontinuoussupplyofmature sperm. spermatozoa (sperm).Theprocess isrepeated manytimesthroughout several dayslater. Bothdivisionsare completedrapidlytoproduce birth, germcellsre-enter the mitoticcycleandmeiosisIisinitiated not entermeiosisat13.5dpc,but arrest mitotically. Immediatelyafter completing meiosisIIafterfertilization. Inthetestis(blue),germcellsdo enter meiosisII.Thereleased mature eggisarrested inmetaphaseII, arrested oocytesisstimulated toresume andcompletemeiosisI birth. Oncesexualmaturityisreached, ateachovulationacohortof pachytene stagesbefore enteringdiplonema/dictyatearrest ataround division). Primaryoocytespassthrough theleptotene,zygoteneand (pink), at~13.5dpc,PGCsbegintoentermeiosisI(thefirstmeiotic bipotential gonads,whichtheyoccupyat~10.5dpc.Intheovary ~7.2 dpcandsubsequentlyproliferate andmigratetowards the primordial germcells. Thetimingofmature gameteproduction from mouse Fig. 1. around 13.5dpcmalegermcellsarrestinG completed onlyafterfertilization. meiotic division, begin thesecondandarrestagain.Meiosisis until justbeforeovulation, atwhichpointthey completethefirst known asdictyate(Speed,1982).Theoocytes remaininthisstate time ofbirth,they thenenteraspecialized,prolongedarreststage leptonema, zygonema,pachynemaanddiplonema.Ataroundthe prophase ofthefirst meioticdivision, progressingthrough developing mouseovary, germcellsstopproliferatingandenter development isseenataround13.5dpc(Fig.1).Atthattime,ina 3402 ~13.5 dpc ovulation Periodic nmls emcl eairi akdydfeet nmue at In males,germcellbehavior ismarkedly different. Inmouse, The first morphologicalevidence ofsex-specific germcell Birth REVIEW Ovary of meiosisI–dictyate Arrest inprophase Fertilized egg Mature egg Oogonium 1° oocyte 2° oocyte Embryo Enter meiosisII meiosisI Diplonema Pachynema Zygonema Leptonema Release from andarrestin Resume and metaphase Enter meiosisI complete Oocyte meiosisII Primordial germcells(PGCs)are specifiedat completed ovary PGCs specified body Polar body Polar Proliferate andmigrate Mitotic arrest(G0/G1) Resume proliferation Stop proliferating, Spermatogonium 1° spermatocyte 2° spermatocyte Spermatid Spermatozoon differentiate intogonad and maturation MeiosisI Morphological Meiosis II differentiation 0 or G 1 Testis of themitotic ~10.5 dpc Birth (RARs, whichincludetheRAR diffuses throughtissuesandactsbybindingtonuclearRAreceptors enter meiosisornot.RAisasmall,polarmoleculethateasily indicate thatexposure toRAcontrolswhethermousefetal germcells Two recentstudies(Bowles etal.,2006;Koubova etal.,2006) Exposure toRAregulates entryintomeiosis will bediscussedlater, inthelightofrecentfindings. 1974; Byskov, 1975;McLaren,1984).Thesetwo opposingtheories whether thisstepisinducedbysurroundingsomatictissue(Byskov, al., 1986;McLarenandSouthee,1997;Ohkuboet1996),or autonomously, perhapsregulated byamolecularclock(Donovan et initial entryintomeiosisoccursspontaneouslyandcell- debate. Thepointatissuehasbeenwhether, inthedeveloping ovary, meiosis istriggeredintheovary hasbeenasubjectofconsiderable 1990; McLarenandSouthee,1997).Themechanismbywhich likely tobeashort-rangediffusible factor (McLarenandBuehr, the supportingsomaticcellsindeveloping testis,andconsidered supposedly producedataround12.0dpcbyimmatureSertolicells, Francavilla andZamboni,1985;McLaren,1984).Thisfactor was tissue producesameiosis-inhibitingfactor (Buehretal.,1993; environment ongermcellfate hasbeenthatthedeveloping testicular 2003; Taketo-Hosotani etal.,1989). Bradbury, 1983;Burgoyne, 1987;McLaren,1981;Park andTaketo, chromosome compositionofthegermcell(Amlehetal.,2000; formation offunctionalgametesareinfluencedbythesex- McLaren, 2003).However, thecompletionofmeiosisand chromosomal constitution,but byitsenvironment (McLaren,1995; initial sex differentiation ofagermcellisdeterminednotbyits (Evans etal.,1977;Palmer andBurgoyne, 1991).Therefore,the ovary, but avoid doingsointheenvironment ofadeveloping testis XX orXY, entermeiosisifthey find themselves inadeveloping involving XX}XYchimerashave shown thatgermcells,whether What causesthesex-specific behavior ofgermcells?Studies Somatic influencesonsex-specificgermcellfate periphery ofthetestiscords. continuous supplyofspermfromspermatogonialstemcellsatthe male, waves ofmeiosiscontinuethroughoutlife,providing a further intofunctionalgametescalledspermatozoaorsperm.Inthe meiotic divisions promptlytogeneratespermatids, whichmature puberty inhumans.Theprimaryspermatocytes thencompleteboth terms ofgermcelldifferentiation, thisstageinmice isequivalent to (dpn), signifyingthatsomespermatogoniahave enteredmeiosis.In 1984). Primaryspermatocytes areseenasearly5dayspostnatum screens inmice.Insomescreens,thegeneCyp26b1 development camefrommaleversus femalegonadgeneexpression embryogenesis andorganogenesis (Marketal.,2006). in morphogenesis,growth anddifferentiation duringvertebrate RA-responsive genes(Chambon,1996).RA hasmany known roles response elements(RAREs)andtherebycontroltheexpression of (Chambon, 1996;Marketal.,2006).RAR-RXRdimersbindto RA- heterodimerize withnuclearretinoidXreceptors(RXR regulate locallevels ofRAinseveral developmental contexts, such (White etal.,2000;Yashiro etal.,2004).CYP26 enzymesactto that actsbycatabolizingall-trans RAintoinactive metabolites encodes aP450enzyme,CYP26B1 (alsoknown asP450RAI-2), meiosis (Bowles et al.,2000;Menke andPage, 2002). dpc, thecriticaltimeatwhichfemale, but notmale,germcellsenter highly expressed inmale thaninfemalegonads,particularlyat13.5 A well-acceptedhypothesistoexplain theeffect oftissue The first cluethatRAmightplayaroleinfemale-specific gonadal ␣ , ␤ and Development 134(19) ␥ isotypes), which ␣ was more , Cyp26b1 ␤ and ␥ )

DEVELOPMENT ( Sycp3 Stra8 cniee atr ngr elseii ul,X n X mitcpohs . etal.,2004;Nichols meioticprophaseI. Ingermcell-specificnulls, XYandXX consideredamaster Oct3/4) ( Pou5f1 plppie1 40 eet,gr el eeomna ohsxsfo t Bowlesetal.,2006; bothsexesfromat developmental defects,germcells polypeptide1;P450 P450RAI-2) ( Dmc1 condensed meiotic nucleiandtheexpression ofthreemeiotic Koubova etal.,2006),asjudgedbythehistologicaldetection of mouse fetaltestistoentermeiotic prophase(Bowles etal.,2006; Furthermore, exogenous RAcaninduceXYgermcellsinacultured the malemousegonadat13.5 dpc(Bowles etal.,2006). Koubova etal.,2006).First,RAlevels arehigherinthefemalethan factor (McLarenandSouthee,1997). dpc, andmatchesthatexpected ofthepostulatedmeiosis-inhibiting protect malegonadsfromtheactionsofRAbetween12.5and 14.5 2002). Thisprofile ofexpression suggeststhatCYP26B1might and peaksataround13.5dpc(Bowles etal.,2006;Menke andPage, Expression isassociatedwithSertolicellsandsomeinterstitialcells, undetectable infemalegonadsbut isvery highinmalegonads. (Bowles etal.,2006).After11.5dpc, gonads at11.5dpc,higherexpression levels inmalesthanfemales Yashiro etal., 2004). (Hernandez etal.,2007;Romand2006;Sakai2004; as duringcentralnervous system,innerear, eye andlimbpatterning A/P; anterior/posterior;dpc,dayspostcoitum;DSB,double-strandbreak; ss,single-stranded;ds,double-stranded. Expressionpattern in Mousemutant Mousemutant Encodedprotein ( Cyp26b1 aliases) (common Gene Table 1.Detailsofgenesinvolvedinthemeioticdivisiongermcellsmouse Development 134(19) Scp3, rncito atr cyststageanddieatimplantation. transcriptionfactor; Oct4, DsutdmitccN reta yoee arse tpe mitclyi 2001;Menkeetal., meioticallyin arrestedatpre- arrestatzygotene- DisruptedmeioticcDNA Dmc1h) aiy2,sbaiyb eeomna various developmental family26,subfamilyb, CP26, It isnow clearthat RA actstoinitiatemeiosis(Bowles etal.,2006; poen tutrl eeet alt elaepod eoial n Koopman,2004; meioticallyin cellaneuploidy elementsfailto protein;structural Cor1) yatnmlcmlx Sterile,axial Synaptonemal complex tmltdb eioc Ifrie r-eoi netl,gr Expressed Infertile,germ Infertile,pre-meiotic Stimulated byretinoic O oan ls , Ml n eaenl eeo obat- nfml Gs (Bullejosand InfemalePGCs: Maleandfemalenulldeveloptoblasto- POU domain,class5, ooo fyat Seie emcls Seie emcls Epesdpe (ChumaandNakatsuji, Expressedpre- Sterile,germcells Sterile,germcells Homolog ofyeast yohoeP5, Dea it,vros Dea it, Epesdi Mcene l,2001; (MacLeanetal., Expressedin Dieat birth, Dieatbirth,various Cytochrome P450, synaptonemal complex protein, main yolsi rti, apa n hw pre-meiotic appearandshow cytoplasmic protein, osiun fteail deb ppoi mnsm)de sxsa o ee, 2001;Nakatsuji and sexesatalowlevel, monosomy)due diebyapoptosis elements ofthe constituent oftheaxial fmitcpohs pohs fmissI eoi .B spermatocytes).In al.,1996) andpossibly (spermatogonia meiosisI.By prophaseofmeiosisI. donotenter morphologybut donotenter condensationbut of meioticprophase the subsequentevents chromosomal DNA replicationand required forpre-meiotic acid, gene8; iblt fmmain thegonad germ line viability ofmammalian euao fpui gr el neg ppoi ewe nml Gs al.,1998;Pesceet InmalePGCs: expressed 9.5and10.5dpc,beforecolonization of germcellsundergoapoptosisbetween potency, maintains regulator ofpluri- n sN idn yassi o eeeae wavefrom degenerates initiated synapsisisnot protein, repairsDSBs and dsDNAbinding metabolites nye erds express RA toinactive enzyme, degrades pcfchmlgo neg ppoi. aots n h hnurgltd etal.,1998;Yoshida et ovary thenupregulated apoptoseandthe Chromosomal undergoapoptosis. germcells bacterial RecA;ssDNA meiosisIandthen specific homologof likestageof mck1 homolog,meiosis- 1, dosagesuppressorof and function Cyp26b1 transcripts aredetectableinmouse Cyp26b1 ne eoi rmtrl tsi n on MacLeanetal.,2007) testisanddown- prematurely enter meiosis emcl ubr brh vre r embryonicovaryan birth,ovariesare reduced Germ cellnumber during fetallife Scp3 praoye el eeo xlsvl npe Menkeetal.,2003; exclusively inpre- cellsdevelop spermatocytes omadgr el (trisomyor form andgermcells hntp ml) hntp fml) mousefetalgonads phenotype(female) phenotype (male) coial n cellsexpress ectopically and Stra8 expression is n eet,gr es 15dcte Whiteetal.,2000; least11.5dpcthen defects,germ and u-etl,gr xrse r- (Bullejosand Expressed pre- Sub-fertile, germ ahtn tg, gr el fbt 2003;Nakatsujiand germcellsofboth pachytene stage, meiotic odfcie te peuae Chuma,2001;Yao et then upregulated to defective hoooe wavefrom chromosome segregation fgr el at~12.5dpc of germcells eeeydpee A/Pwaveinitiates severely depleted rpaeo preleptotene prophase of levels having increased inthemalegonad(Bowles etal.,2006; upregulation oftheRA-responsive geneStra8, indicative ofRA inhibitors, orwithmore-specific CYP26inhibitors,causedastrong male mousefetalgonadswith broad-spectrumcytochrome P450 fetal mousetestis.Insupportof thistheory, treatmentofcultured developing gonadshadsuggestedthatRAisactively degraded inthe culture acceleratestheirentryintomeiosis(Livera etal.,2000). that exogenous RAaddedtoratXXgermcellsinex vivo organ al., 2006).Thesefindings complementearlierstudies thatshowed germ cellsfromenteringmeiosis(Bowles etal.,2006;Koubova et antagonizing RARsinculturedmousefetalovaries prevents XX et al.,2003;NakatsujiandChuma,2001).Finally, chemically Sycp3 (Sycp3)anddosagesuppressorof synaptonemal complex protein3 (Stra8), stimulatedbyretinoic acidgene8 markers, named: Untreated XYgermcellsdonotnormallyexpress Stra8, andexpress Chuma, 2001;Oulad-Abdelghanietal.,1996)(seeTable 1). mck1 homolog(Dmc1)(ChumaandNakatsuji,2001;Nakatsuji As mentionedabove, themale-specific expression of and Stra8 Dmc1 weakly only(ChumaandNakatsuji,2001;Menke hogotftl 2006) throughout fetal erse uig Koopman,2004;Kehler repressed during noayi nAP al.,1998) in ovaryanA/P ee talwlvl Chuma,2001;Pittman sexes atalowlevel, noayi nAP al.,2003;Yuan etal., in ovaryanA/P ~13.5 dpc eoi emcls Oulad-Abdelghani et meiotic germcells oai el f MenkeandPage,2002; somatic cellsof regulated inovary emclso oh ChumaandNakatsuji, germ cellsofboth ~13.5 dpc life peuae n Yashiro etal.,2004; upregulated in (Baltus etal.,2006; 1998; Wang etal., 2000; Yuan etal.,2002) References REVIEW Cyp26b1 3403 in

DEVELOPMENT an RARpanantagonist,Stra8 When malegonadswereculturedwithbothaCYP26inhibitorand early stagesofmeiosisI(Bowles etal.,2006;Koubova etal.,2006). indicating thattheseXYgermcellsinthetreatedgonadsare mesonephric ductandtubules. germ cellsare present andretinoic acid(RA)isproduced inthe developing gonads. Fig. 2.Regulationofgermcellentryintomeiosisinthe Koubova etal.,2006). 3404 dpc), Cyp26b1 during thistime.(Ba)Once are physicallyconnectedwiththeanterior(Ant)endofgonad the gonadofbothsexes.Themesonephrictubules,whichproduce RA, demonstrating thatgermcellsintheXYmutantembryosare upregulation ofStra8 2004). XYgonadsfrom with multipleabnormalities,includinglimbdefects(Yashiro etal., (Bowles etal.,2006). endogenously, expression (Koubova etal.,2006). whichwould otherwiseactviaRARstoupregulate RA, that, inadeveloping malegonad,CYP26B1functionstodegrade dpc; (Cb)by13.5dpc,latermarkersofmeiosis, suchas cells attheanteriorendofgonad begintoexpress expression isdetectableat11.5dpc,butdisappearsby12.5dpc.Germ pluripotency markerPou5f1.(Ca)Inthefemalegonad, do notentermeiosisat13.5dpc,andcontinuetoexpress the germ cellsfrom theactionsofRA.(Bb)Germcellsinmalegonad might concentratetheenzymeintheseregions, thereby protecting interstitial cells.Thetestiscords, whichformaround germ cellclusters, anterior endthan theposteriorend.Seetextformore details. the posteriorend,orRAconcentration mightbegreater atthe anterior endofthegonadmightbe exposedtoRAearlierthanthoseat expressed bygermcellsonce theyentermeiosis.Germcellsatthe anterior-to-posterior (Post)wave over~3days. are expressed strongly. Meiosismarkersare upregulated inan Dmc1, Mesonephros To prove thatCYP26B1functionsasameiosisinhibitor A Bipotential(11.5dpc) duct Mesonephric tubules Mesonephric REVIEW Key o-eoi emcl Meioticgermcell Non-meiotic germcell expression isupregulated, probably inbothSertoliand Cyp26b1 Post Ant ( Cyp26b1-null micedieimmediatelyafterbirth A Sycp3 ) Inthebipotential(11.5dpc)mousegonad, CYP26B1 SRY CYP26B1 Sry -knockout micewerealsoexamined and expression was notinduced,indicating is expressed inthemalegonad(at11.5 Cyp26b1 and Cyp26b1 Sycp3 a 12.5dpcb13.5 B Male C Female a 12.5dpcb13.5 Dmc1 is expressed atlowlevelsin cords Testis expression at13.5dpc, Stra8 expression alsoincreased, -null embryosshowed Pou5f1 is nolonger Stra8 Cyp26b1 Sycp3 RA at 12.5 Pou5f1 Dmc1 Sycp3 Pou5f1 express continue to Germ cells and Stra8 driven byanRARE(RARE- Using reportermiceandcellsinwhichthegene two organs remainincontactforsometimeduringdevelopment. begins togrow outfromthemesonephrosataround10dpc,and to whichthegonadsareattached(Bowles etal.,2006).Thegonad synthesized inthegonadsthemselves, but rather bythemesonephroi provided several linesofevidence indicatingthatRAisnot elsewhere inthedeveloping urogenitalsystem.Bowles etal.have produced inthedeveloping fetalgonadsorwhetheritcomesfrom One questionraisedbytheseobservations iswhetherRA development? What isthesource ofRAduringurogenital cells. into meiosishasany detrimentaleffect on cells ofbothsexes. Itwillbeinterestingtoseewhetherearlyentry expression mightberelevant tothetimingofmeioticentryingerm level earlierthaninwild-typefemalegonads.Therefore, CYP26B1 mightallow RAtoaccumulateameiosis-inducing we surmisethat,inCyp26b1-nullXXgonads,completeabsenceof initially inthegonadsofbothsexes (Bowles etal.,2006)(Fig.2), is normal(Bowles etal.,2006).BecauseCyp26b1 ovaries, thewave of limited topreventing meiosisinthefetaltestis.In metabolite. preventing itfromsignaling,ratherthantoproduceanactive RA that theroleofCYP26B1inthissystemistodegrade RA,hence retinoid thatcannotbedegraded byCYP26B1,itwas demonstrated embryonic testeswhenCYP26B1isnotpresent.Usingasynthetic studies confirmed independentlythatRAlevels areincreasedin progressive apoptosis(MacLeanetal.,2007).Importantly, these virtually allgermcellswerelostin histological stainingandchromosomespreadanalysis.Bybirth, asjudgedby prophase, withsomereachingzygotene/pachytene, cells entermeiosisby13.5dpcandproceedthroughmeiotic al., 2007);itwas foundthat,intheabsenceofCYP26B1,XYgerm by theanalysisofasecondCyp26b1-nulllinemice(MacLeanet embryo (Bowles etal.,2006).Thesefindings wererecentlyextended entering meioticprophaseIatthisearlystage,asifinanormalXX encodes amajor structuralproteinofthesynaptonemal complex (see (Bullejos andKoopman, 2004;Yao etal.,2003)(Fig.3B). POU domain,class5,transcription factor 1(Pou5f1 marker also seen,about1daylater, withtheupregulation ofthemeiosis at theposteriorend(Menke etal.,2003)(Fig.3A).Thispatternis dpc intheanteriorpartofgonad andextinguishes at~16.5dpc This wave isevident from (Bullejos andKoopman, 2004;Menke etal.,2003;Yao etal.,2003). might have beenexpected, simultaneously orstochastically ovary entermeiosisinananterior-to-posterior wave andnot,as Romand etal.,2006). that inthedeveloping retinaandinnerear (McCaffery etal.,1999; gonad indicatesthatanRAsource/sinksystemexists, analogousto mesonephros andanRA-catabolyzingenzymeintheattachedmale complementary patternsofanRA-synthesizingenzymein the mesonephros fromatleast10.5dpc(Bowles etal.,2006).The with RAproductionintheembryo,ishighlyexpressed inthe encodes theenzymeRALDH2andismostcommonlyassociated (Bowles etal.,2006).Inconcordancewiththis, ducts andtubules ofthemesonephroibothsexes produceRA The roleofCYP26B1ingermcelldevelopment mightnotbe Several studieshave shown thatgermcellsinthedeveloping Sycp3 and thedownregulation ofthepluripotency marker Stra8 expression appearstoinitiateearlierthan Stra8 lacZ expression, whichbegins at~12.5 mice), itwas demonstratedthatthe Cyp26b1-null testesbecauseof Cyp26b1-null XXgerm Development 134(19) Aldh1a2 Cyp26b1 is expressed , Cyp26b1 Oct3/4 , which lacZ Sycp3 -null is )

DEVELOPMENT expression hasbeendownregulated attheanterior(top)endthisstage(seetext).g,gonad;m,mesonephros. structures andthedispersalofgermcellsinfemalesample.Germare present throughout thelengthoffemalegon meiotic germcellsexpress thisgeneatstageofdevelopment.Staininghighlightstheclusteringgermcellsinmale sampleintestiscord end ofthegonad.( Only germcells,justpriortoentryintomeiosis,express thismarkergeneandhigherexpression isseeningermcellslocated total ablationofAldh1a2 mesonephros-specific the sourceofRAwould requiretheproductionandanalysis of mesonephros inregulating meiosis.Proofthatthemesonephrosis mesonephros earlyandcleanlyenoughtotesttheimportanceof the the developing ovary can beseparatedexperimentally fromthe at 12.5-14.5dpc(Bowles etal.,2006).Itremainstobeseenwhether concentrated attheanteriorthanposteriorendofgonads mouse embryos,␤ markers. that mightleadtotheobserved wave ofexpression ofmeiosis into theanteriortipofgonadandemitRA,producingagradient Alternatively, cellsmightmigratefromthemesonephrictubules If so,theanteriorgermcellswould bethefirst tobeexposed toRA. their anteriorends(Byskov, 1978b;KarlandCapel,1995)(Fig.2). mesonephric tubules thatconnectthemesonephrosandgonadat propose thatRAentersthegonadpredominantlythroughopen than beingautomatic,ingermcells. of entryintomeiosisisconsistentwithbeinginduced,rather first (Molyneauxetal.,2001),thisslow anterior-to-posterior wave no evidence thatgermcellscolonizetheanteriorendofgonad promotes germcellsurvival (Kehler etal.,2004).Becausethereis differentiation genes(Pesceetal.,1998).Inaddition,Pou5f1 whereas cells (ChumaandNakatsuji,2001;NakatsujiChuma,2001), Box 1)andisconsideredtobeaspecific marker ofmeioticgerm ( Fig. 3.Markergeneexpression in13.5dpcmousegonads. Development 134(19) findings. main theoriesandthenre-evaluate theminthelightofrecent data. Here,weexamine theevidence supportingeachofthetwo because botharguments aresupportedbyconvincing experimental spontaneously andcell-autonomously hasbeencontentious, The issueofwhethermeiosis isinducedorwhetheritinitiates theories Reconciling newfindingswithestablished mouse fetalgonads,isshown inFig.2. of germcellentryintomeiosis,andtheroleRAinthisprocess in 1999). A Expression ofthepre-meiotic marker ) When gonadsandmesonephroiwereexplanted fromRARE- What causesthewave ofentryintomeiosisinthefetalovary? We A modelsummarizingourcurrentunderstandingoftheregulation Pou5f1 B -galactosidase stainingconfirmed thatRAismore encodes atranscriptionfactor thatrepresses ) Expression ofthepluripotencymarker Aldh1a2 is lethalby10.5dpc(Niederreitheretal., -deleted mice(Vermot etal.,2006); Stra8 is absentinmale(M,left)butpresent infemale(F, right)13.5dpcmouseurogenital ridgesamples. Pou5f1 Urogenital ridgetissuesampleswere dissectedfrom wild-typemouseembryos. lacZ ( Oct4) inmaleandfemale13.5dpcmouseurogenital ridgesamples.Onlypre- germ cellstoenterintomeiosis(McLarenandSouthee,1997). dissociation andre-aggregation offetalurogenital ridges allows depended onthestructuralintegrity ofthetestiscords,because 1984). Theproductionofthismeiosis-inhibitingfactor evidently (Buehr etal.,1993;Francavilla andZamboni,1985; McLaren, must operateinthefetaltestistoactively block entryintomeiosis (Chuma andNakatsuji,2001). that asignalfromthegonadalcellsisnotrequiredtoinitiatemeiosis the gonadsalsoentermeiosiswhencultured,supportingtheory (Chuma andNakatsuji,2001).Germcellsisolatedpriortoentryinto either sex canreachtheleptotenestageoffirst meioticdivision cultured onfeedercellswith20%fetalcalfserum,germof mesonephros complexes, or11.5dpcgonads,aredissociatedand When10.5dpcmousegonad- Nakatsuji andChuma,2001). by studiesofculturedgermcells(ChumaandNakatsuji,2001; (McLaren, 2003;McLarenandSouthee,1997;Ohkuboetal.,1996). after thegermcelllineagewas establishedorhadstartedtomigrate meiosis mightbedeterminedbythenumberofmitosesoccurring automatic ingermcellsofacertainage.Thus,timingentryinto environment, itwas hypothesizedthatthispathway mightbe Because itappearedthatmeiosiscouldoccurinalmostany tissue enter meiosis(Byskov, 1978c;McLaren,1983;1984). In theectopiclocationofmesonephros,XYgermcellstendto several days,alsocommencemeiosis(McLarenandSouthee,1997). combined withdissociatedembryoniclungcellsandculturedfor 1983). Similarly, XYgermcellsisolatedat10.5or11.5dpc, ovary (UpadhyayandZamboni,1982;ZamboniUpadhyay, as theadrenalgland,entermeiosisthey would inthedeveloping Germ cellsthataccidentallycolonizenon-gonadallocations,such The ‘intrinsicclock’theory and Saxen, 1976)orinfollicularfluid taken frompre-ovulatory or testesinwhichmeiosisisoccurring (Byskov etal.,1993;Byskov fetal gonadsfor6dayswithconditioned mediafromculturedovaries example, meiosisinXY germcellscanbeinducedbyculturingmale existence ofasomatically derived, meiosis-inducingsubstance.For and long-standingbodyofexperimental evidence hassupportedthe Despite theobvious attractions oftheintrinsicclocktheory, alarge The meiosis-inducing-substancetheory A corollaryofthishypothesisisthatanoverriding mechanism This ‘intrinsicclock’ theory(Donovan etal.,1986)was supported attheanterior(top) ad, butPou5f1 REVIEW 3405

DEVELOPMENT concentration ofall-transRAis around 4 mesonephroi (HortonandMaden, 1995;Rossantetal.,1991).The in many embryonictissues, includingthelungs,adrenalsand cells entermeiosisbecauseofexposure toRA.First,RAispresent basis oftheintrinsicclocktheory?Itseemslikely thatthose germ ectopic orculturedfetalgermcellsintomeiosis,whichformed the entry intomeiosisintheovary. just theavoidance ofmeiosisinthefetaltestis, but alsothetimingof previously predicted–thatthemeiosis-inhibitingfactor controlsnot inhibiting factor. Thenew modelalsoincludesafeaturenot Hence, CYP26B1isameiosis-inhibitingfactor, ifnotthemeiosis- cells entermeiosis(Bowles etal.,2006;MacLean2007). mechanism. WhenCyp26b1 rather thanoccurringspontaneouslyviaanintrinsicclock Koubova etal.,2006),indicatingthatmeiosisisactively induced normally does,inducefetalgermcellmeiosis(Bowles etal.,2006; that includeselementsofboththeories(Fig.2).RAcan,and recent identification oftheRA-CYP26B1systemsuggestsamodel by ameiosis-inhibitingfactor? Theanswerisneitherandboth:the spontaneous entryoffetalgermcellsintomeiosisunlessprevented So, whichtheoryiscorrect–active inductionofmeiosis,or A modelforsex-specificregulation ofgermcellfate male-specific meiosis-inhibitingsubstance. largely discredited.Researchersinsteadconcentratedonfinding the in recentdecades,theexistence ofameiosis-inducingsubstance was theory, andgiven thatnomeiosis-inducingsubstancewas identified meiosis (Byskov etal.,1998). biosynthetic pathway: theseappearnottotriggerinitialentryinto occurring sterolsthatareintermediatesofthecholesterol initiation ofmeiosis,theirexperiments ledthemtoisolate naturally 1995). Perhapsbecausethey assayedresumptionrather thanthe substances fromhumanfollicularfluidandbull testes(Byskov etal., this effect inthedeveloping testes(Byskov, 1978c;Byskov, 1986). can inducemeiosisbut thatgermcellsarenormallyprotectedfrom meiosis, leadingtotheconclusionthatmale,aswellfemale,rete instead enduplocatedneartheconnectingretecordscanenter germ cellsthathappentohave beenexcluded fromtestiscordsand contact withmesonephrictissue(Byskov, 1975).Fetaltesticular ferrets, meiosisbegins inthepartofovary thatisinclosest oogonia (Byskov, 1974).Inotherspecies,suchascats,minksand tubules, isgrafted,germcellsdonotentermeiosisandremainas normally. Bycontrast,when the caudalpart,withoutassociatedrete nude mice,thegermcellsentermeiosisandcontinuetodevelop ovarian retetubules andmesonephrosstillattached,isgraftedinto When thecranialhalfof12.0dpcfetalmouseovaries, withtheextra- connections withtheanteriorendofgonad(Byskov, 1978b). developing gonad,andincludesthemesonephrictubules andtheir ovarii develops frommesonephriccellsthatmigrateintothe from aregion known asthereteovarii (Byskov, 1974).Therete fetal germcellstoundergo meiosis. humans (GrinstedandByskov, 1981),canalsoinducemouseXY pubertal miceorbulls (Grinstedetal.,1979),orfetalandadult from culturedfetalepididymidesofmice(Byskov, 1978a), follicles (Westergaard etal.,1984).Similarly, conditionedmedia 3406 dpc germcellsin vitro(J.B.andP.K., unpublisheddata).Secondly, little as1 at 10.5and13.5dpc(Horton Maden,1995),andwefind thatas How doesthismodelexplain theapparently spontaneousentryof In view oftheconsiderableevidence supportingtheintrinsicclock Byskov etal.attemptedtocharacterizemeiosis-inducing In particular, meiosisappearedtobeinducedbyafactor secreted REVIEW ϫ 10 –8 M RAcaninduceStra8 is absentfromthefetaltestis,germ ϫ expression inisolated11.5 10 –8 M invisceraltissues progress throughmeiosis:inCyp26b1 mechanisms fordealingwithgermcellsthatinadvertently enterand from enteringmeiosis.Certainly, thedeveloping malegonadshave in additiontoRAdegradation byCYP26B1,toprevent germcells male developing gonadshave asupportingorback-upmechanism, exist, orareinsufficient toinducemeiosis.Itremainspossiblethat suggesting thatRAisessentialandotherpathways eitherdonot entering meiosis(Bowles etal.,2006;Koubova etal.,2006), in culturedmousefetalovaries, germcellsareprevented from into meiosisinthemousefetus?BychemicallyantagonizingRARs system ofeliminatingRAonlyinmalegonads. RA levels increase withtime,because in theovary have notbeenmade.However, itispossibletoinferthat Direct measurementsofRAlevels atvarious timesofdevelopment meiotic induction)andthenpresent (whenmeiosisisinduced). 2006). IfRAisinstructive, thenitshouldfirst beabsent(before protein known tobeessentialformeioticinitiation(Baltusetal., Stra8 of RARsresultsindownregulation ofmeiosis markers, especiallyof meiosis? Thedatasuggestthatitisinstructive, becausetheblocking subsequently undergo apoptosis (MacLeanetal.,2007). markers areupregulated ingermcells,but meioticgermcells and leadingtoCyp26b1 ovary. Instead,apathway ofgeneregulation setinmotionbySRY germ cellstoRAensurethatentryintomeiosisoccursonlyinthe and itwould seemimprudenttorelyonsex-specific exposure of and regulates many developmental processes(Rossantetal.,1991), produced inmaleandfemalereproductive tissue?RAiswidespread several new questions. previous theoriesongermcell-fate specification. Italsoraises to prevent thisinductioninmales,combineselementsofboththe meiosis, combinedwiththetestis-specific expression ofCYP26B1 regulation ofgermcellfate inmicebyasystemofRAinduction (Bowles etal.,2006;Koubova etal.,2006).So,thenew paradigmof not asecretedsignalbut ratherthecytosolic enzymeCYP26B1 a testis-derived meiosisinhibitoractsinfetalmice,thismoleculeis developing testisandprevents meiosis.Althoughwenow know that molecule emanatesfromsomatic(probablySertoli)cellsofthe shield germcellsfromany RAintheinterstitium. cells inwhichitisproduced,thecordstructurecouldphysically al., 2006)and,becausethisenzymeactswithinthecytoplasm ofthe from RA(Fig.2).CYP26B1isproducedbySertolicells(Bowles et Sertoli cellsincords,andthisarrangementmightprotectgerm hypothesized. Intheintactfetaltestis,germcellsaresurroundedby and notbythelossofameiosis-inhibitingfactor, aswas originally cells withRA-producingmesonephric(Bowles etal.,2006), 1997) ismostlikely tobeexplained bythejuxtaposition ofgerm urogenital ridgesispermissive formeiosis(McLarenandSouthee, meiosis incellculturecanbeinhibitedbyblockingRARs. 1981). Itremainstobeseenwhether‘spontaneous’ entryinto serum isestimatedtocontain3.6ϫ drive meioticinduction.Mediasupplementedwith10%fetalbovine the bovine seruminthemediaprobablyprovides sufficient RAto in isolation,orco-culturedwithdissociatedembryoniclungcells, in experiments inwhichdissociatedgonadalcellshave beencultured 2006). Moreover, thepatternofentryintomeiosis ofXXgermcells fetal ovary disappears between11.5dpcand12.5(Bowles etal., Does RAprovide theonlysignalthatstimulatesgerm cell entry Is RAinstructive ormerelypermissive forgermcellentryinto Why mightameiosis-inducingsubstance,suchasRA,be A pillaroftheintrinsicclocktheoryisthatashort-rangesignaling The observation thatdissociation/re-aggregation offetalmale (Bowles etal.,2006;Koubova etal.,2006),whichencodesa expression provides arobust, male-specific 10 –8 Cyp26b1 -null fetaltestes,meiotic M RA(FuchsandGreen, Development 134(19) expression inthe

DEVELOPMENT each ofthethreeRARisotypescaninduce 2006; Koubova etal.,2006).Moreover, RARagonistsselective for RA mustsignalthroughRARstoinducemeiosis(Bowles etal., of meioticmarkers andthedownregulation Because theupregulation of How doesRAinducemeiosis? engagement ofthemeioticmachinery, towhichwenow turn. molecular detailsoftherelationshipbetweenRAand distinction betweeninductionandpermissionmightbefoundinthe the anteriorendofgonad-mesonephroscomplex. Afurther is consistentwithaninstructive signalcomingpredominantlyfrom Development 134(19) Abdelghani etal.,1996). meiosis byinducingthecytoplasmic proteinSTRA8 (Oulad- meiosis inStra8-null mice(Baltusetal.,2006),soRAmusttrigger orchestration ofameioticresponse?Germcellsdonotinitiate molecular stepsbetweenthereceptionofRAligandand signal togermcellsinducemeiosis. possible thatRAactsonsomaticcells,whichthensendasecondary Morita andTilly, 1999;Vernet etal.,2006)soitistheoretically (Boulogne etal.,1999;Bowles etal.,2006;Li and Kim,2004; development, but somaticcellsalsoexpress thesereceptors RXRs areexpressed bygermcellsattherelevant stageof RAR inthissystem(Koubova etal.,2006).AtleastsomeRARsand cultured ex vivo testes,suggestingthatRAcansignalthroughany associated genes,suchas during fetaldevelopment, thelow level expression ofmeiosis- 1997). AlthoughXYgermcellsdonotnormallyentermeiosis whether they beinductive orpreventative (McLarenand Southee, of eithersex arepoisedtorespondsignalsfromtheenvironment, condensation supportstheideathatgermcellsinadeveloping gonad observed (Baltusetal.,2006).Thepresenceofsuchchromosome repair. However, pre-meioticchromosomecondensationsare does notformandDNA double-strandbreaks appearnottoformor proteins arenotproduced,thesynaptonemalcomplex (see Box1) regulation ofStra8 (Weston etal.,2003).Thismechanismmightalsounderliethe the transcriptionoftargets untilanRAligandbecomesavailable circumstances, unligandedRAR-RXRcomplexes bindandrepress some intermediatetranscriptionfactor(s) remainspossible.Insome (Chiba etal.,1997).Nonetheless,anindirectpathway involving combination playsthemajorroleinmediatingStra8 RARE sequenceupstreamofStra8. TheRXR-RAR ␥ to anRAR-RXRheterodimerthatthenrecognizesandbinds 2 hours(Oulad-Abdelghanietal.,1996),RAmightbindasaligand teratocarcinomas) withRAupregulates Stra8 Because treatmentofembryonalcarcinoma(EC)cells(stem whether itisregulated directlyorindirectlybyRAisnot known. gene (Bouilletetal.,1995;Oulad-Abdelghani1996),although independent ofany RAeffect becauseCyp26b1 2000; NakatsujiandChuma,2001).Thispreparationmust be ridge (Byskov, 1978c; ChumaandNakatsuji,2001;DiCarloetal., germ cellspreparetoentermeiosisaftertheirarrival inthegenital cellular function oftheproteinisunknown. necessary forentryintomeiotic prophase(Baltusetal.,2006),the genes. Althoughwenow have goodevidence that STRA8is expression, leadingtothe upregulation ofothermeiosis-associated dpc orearlier. RAmightdirectlyorindirectlyupregulate Stra8 no reportedexpression of gonads ofbothsexes fromanearlytime-pointandbecausethereis Assuming thatRAactsdirectlyongermcells,whatarethe In germcellsoftheStra8-null mousefetalovary, cohesion Pou5f1 are inhibitedbyRARantagonistsinfetalovary culture, and ofany othertarget genesinthissystem. Stra8 Stra8 Sycp3 in themaledeveloping gonadat12.5 was first identified asanRAtarget and mayindicatethatall Dmc1, expression inlessthan Stra8 is expressed inthe expression in induction isotype al., 1996).Possibly, RAisonly abletoupregulate that they downregulate thepluripotency marker gonadal environment. XY germcellstosurvive andprogresspastpachyteneinanXX (Matsui andHayashi,2007)underlying,forexample, thefailure of modification mightbemediatedbyadditionalsex-specific factors In addition,maleandfemalepatternsofDNA methylationorhistone if acellisinspecific epigeneticconfiguration (Sekietal.,2005). rounds, aretriggered.Theexpression profile of cells. Itisnotknown how theinitialroundofmeiosis,orlater by aself-perpetuatingcycle ofmeioticinitiationingermline stem both sexes. al., 1993)furthersupportsideaofacommonmeiosisinducerin induce XYgermcellsinafetaltestistoentermeiosis(Byskov et conditioned for24hourswithmincedadultmousetestescan of meiosisI(Baltusetal.,2006).Also,theobservation thatmedia Stra8 cells. Thismightindeedbethecase,becausegeneticdeletionof mechanisms inprenatalXXgermcellsandpostnatalXY Parsimony suggeststhatmeiosismightbeinducedbysimilar Does RAinducemeiosisinthepubertaltestis? migratory germcells(Yeom etal.,1996). is thoughttoregulate Pou5f1 promoter ofPou5f1 distal andproximalenhancers(DE/PE)theTATA-less proximal Minucci etal.,1996;Pikarsky etal.,1994).Thefurtherstudyofthe affect nuclear factor (GCNForNR6A1,anorphannuclearreceptor),can RA, directlyviaRARsorindirectlymoleculessuchasgerm-cell al., 1990;RosneretScholer1989).Itappearsthat with thedownregulation of transcription; various studieshave linked RA-induceddifferentiation 2003). PerhapsRAhasadirectorindirecteffect onPou5f1 events inmales:bothpre-pubertalandadultmalemice, and adultmousetestesisconsistentwithRAbeinginvolved inboth from theirwild-type counterpartsinthatthey donotexpress type Aspermatogoniathatremain inVAD adultmalemicediffer 1990a; van Peltand deRooij,1991).Recently, it was notedthatthe synchronously (MoralesandGriswold, 1987;van PeltanddeRooij, are provided toVAD ratsandmice,meiosisresumespromptly Pelt anddeRooij,1990b), whenretinolorlarge doses ofRA arealsofound (HuangandHembree,1979;van spermatocytes – differentiated forms–typeBspermatogoniaandpreleptotene but, inadditiontotypeAspermatogonia,theslightly more (van PeltanddeRooij,1990a).ThesituationissimilarinVAD rats, germline stemorearlydifferentiating pre-meioticcells)remain mice, germcellsdegenerate andonlytypeAspermatogonia(male male infertilityiswell-known. Intheseminiferoustubules ofVAD al., 1964),anassociationbetweenvitaminAdeficiency (VAD) and cycle (Oulad-Abdelghanietal.,1996). expression isrestrictedtoapre-meioticstageofthespermatogenic tubules, andweaklyornotatallinothers,suggesting thatits testes, Abdelghani etal.,1996).Furthermore,insectionsofadultmouse spermatocytes immediatelypriortoentryintomeiosis (Oulad- expressed byspermatogoniaand,possibly, bypreleptotene-stage Even thoughRAiswidespreadthroughouttheembryo,itseems When germcellsinafetalmouseovary begin toexpress In malemice,thefirst meioticentryatmalepubertyisfollowed In mice(McCarthyandCerecedo,1952)rats(Thompson et Stra8 precludes germcellsofeithersex progressingintoprophase Pou5f1 Stra8 is never expressed insomaticcells(Oulad-Abdelghaniet is highlyexpressed inbasalnon-somaticcellsofsome transcription (Fuhrmannetal.,2001;Gu2005; might clarifythispoint;inparticular, theDEsite Pou5f1 expression inmigratoryandpost- in ESandECcells(Okamotoet Pou5f1 Stra8 REVIEW Stra8 (Menke etal., in postnatal expression Stra8 Stra8, Stra8 3407 is

DEVELOPMENT cells couldactin aparacrinemanneronadjacent pre-meioticgerm RALDH1/RALDH2 (retinalto RA).RAproducedbytheSertoli alcohol dehydrogenases(retinol toretinal)(Deltouretal.,1997)and steps ofRAproductioncould be carriedoutunderthecontrolof peritubular myoidcells intotheSertolicells.There,final two enzymes) passesfreelyfromthe circulationthroughthebarrierof postulate thatretinol(but notRA,whichisdegraded byCYP26 to inducemeiosisinthepre-pubertalmaletestis(Fig.4). We (Vernet etal.,2006). to theSertolicellswerefoundexpress theRAtarget gene et al.,2006).Undifferentiated spermatogoniathatarefoundclosest seminiferous tubules, separatingthemfromthecirculation (Vernet produced inperitubular myoidcellsthatlinetheouter surface ofthe RA-degrading enzymes(CYP26A1,CYP26B1andCYP26C1)are consistent witharoleforRAindriving meiosispostnatally(Fig.4). and RARsRA-bindingproteinsareexpressed bygermcells, (1-20 dpn),RA-synthesizingenzymesareexpressed inSertolicells, of various isotypes(Vernet etal.,2006).Intheearlypostnatalperiod synthesizing andRA-degrading enzymes,andofRARs andRXRs 2006). VAD micearegiven RA(Ghyselincketal.,2006;Koubova etal., (Ghyselinck etal.,2006).Expressionof meiosis. Fig. 4.AmodelofRAregulation duringprenatal andpostnatal 3408 germ cellsand,hence,triggersentryintomeiosis. locally withintheseminiferous tubules,triggers enter meiosistheyproduce STRA8.We postulatethatRA,produced (RALDH1 andRALDH2)germcellsexpress RARs.Asgermcells theseminiferous tubules, SertolicellsproduceWithin RALDHenzymes surround andisolateseminiferous tubulesfrom therest ofthebody. and CYP26C1)are produced byperitubularmyoid(PM)cells,which 10 dayspostpartum(10dpp).CYP26enzymes(CYP26A1,CYP26B1 production. (C they are notexposedtosufficient amountsofRAtoinduceSTRA8 that, althoughallgermcellsexpress RARsandthuscanrespond toRA, degrading enzymeCYP26B1isproduced bysomaticcells.We postulate expressing STRA8.(B in thefemalegonadexpress RAreceptors (RARs)andrespond toRAby adjacent gonadthrough openmesonephrictubules.Germcellsresident postulate thatretinoic acid(RA)movesfrom themesonephros intothe The mesonephros produces anRA-synthesizingenzyme(RALDH2).We Mesonephros These observations leadustoproposeamodelinwhichRAacts A recentstudyhasexamined theexpression intestesofRA- AFemaleUGRBMaleCSeminiferous REVIEW 25dc12.5dpc 12.5 dpc ( A Key ) Schematicofa12.5dpcfemaleurogenital ridge(UGR). ) Schematizedcross-section ofaseminiferous tubuleat tubule Y2 (erdsR)STRA8(RAtarget) RAR(RA receptor) CYP26 (degradesRA) RALDH (synthesizesRA) Germ cells ) Schematicofa12.5dpcmaleUGR.TheRA- Germ cells Stra8 Stra8 10 dpp is restoredwhen expression in PM cells Stra8 to thesequestions willbekeenly sought. behavior amongreproductive anddevelopmental biologists,answers With thecurrent intenseinterestintheregulation ofgermcell and how isRAsignaling usedtoengagethemeioticmachinery? defines thewindow ofcompetencegermcellstorespondRA questions ofwhyismeioticdivision specific togermcells,what lines ofinvestigation. Asyet,therearenoclearanswerstothe new questionshave arisenbased onthesefindings, openingupnew to studythemechanismofmeiosisanditssomaticcontrol.Many development inmammals,andwillinfluence theapproachesused meiosis addsmuchtoourunderstandingofsex-specific germcell meiosis would maximizefertility. because meioticgermcellsapoptoseinthefetaltestis,avoidance of for ovarian but nottesticulardevelopment (McLaren,1991).Also, meiotic germcellsprobablyproducesignalingfactors appropriate for ensuringthenormalsomaticdevelopment ofthetestes,because guards againstmeiosisinfetalmalegermcellsmightbeimportant (MacLean etal.,2007;Yao etal.,2003).Amulti-level systemthat experimental situations,thesecellsareeliminatedby apoptosis developing testis,whetheroccasionallyinnatureorinduced compartmentalization. Ifandwhengermcellsdoentermeiosisina degradation isassistedbythephysicalshieldingprovided bythis possible thatthesystemofpreventing meiosisinfetaltestesbyRA whereas thereisfar lessorganization intheovary. Hence,itis structure; inthemalegonad,germcellsareenclosedtestiscords, indirect upregulation ofStra8 appropriate maturity. Evidently, RAinducesmeiosisviadirector the initialtransitionfrommitosistomeiosisingermcellsof gonads indicatethatRAisbothnecessaryandsufficient totrigger addition orfunctionalblockadeofRAfunctioninmousefetal meiosis-inhibiting factor. Experimentalobservations involving the embryogenesis, CYP26B1actstoremove RAandsofunctionsasa regulate meioticprogressioninthepubertaltestis.During male meiosis inthedeveloping mouseovary, andthatitmightalso Available evidence indicatesthatRAstimulatesgermcellstoenter Conclusions conserved. entry intomeiosisviatheupregulation of germ cellsdiffers inmalesandfemales,even thoughtheeffect – the timing,siteofproductionandmeansdelivery ofRAtothe fashion tospermatogonia,whichthenentermeiosis.Ifthisiscorrect, cells inthepostnatalmousetestis,anddelivered inajuxtacrine Rooij, 1991). fertility, but treatmentwithRAisfar lesseffective (van Peltandde observation thatthetreatmentofVAD micewithretinolrestores isolate theseminiferoustubule (seeFig.4G),mightexplain the enzymes producedbyperitubular myoidcells,whichsurroundand to storeretinol(Zhaietal.,1997).ThecatabolicbarrierofCYP26 protein (CRBP1)inSertolicellssuggeststhatthey have thecapacity meiotic prophase.Bycontrast,expression ofcellularretinol-binding a functioninoneormoreofthetransitionsuptoandincluding type ofthetestis(Vernet etal.,2006)supportsthetheorythatRAhas binding proteinCRABP1inallspermatogoniaandnoothercell meiosis. Theobservation ofrobust expression ofthecellularRA- to RAbyexpressing (Vernet etal.,2006).Thepre-meioticgermcellscouldthenrespond through topreleptotenespermatocytes, allofwhichexpress RARs cells, ranginginstagesofmaturityfromtypeAspermatogonia The recentidentification ofaroleforRAintheinitiation of Mouse fetaltestesandovaries areremarkablydifferent in In summary, wesuggestthat RA isprobablyproducedbySertoli Stra8, therebytriggeringthefirst roundof expression infetalgermcells. Development 134(19) Stra8 expression –is

DEVELOPMENT Amleh, A.,Smith,L.,Chen,H.andTaketo, T. References Professorial Research FellowoftheARC. and NationalHealthMedicalResearch CouncilofAustralia.P.K. isa was supportedbyresearch grantsfrom theAustralianResearch Council(ARC), We dedicatethisreview toourfriendandmentorAnneMcLaren. Thiswork Development 134(19) Byskov, A.G.,Baltsen,M.andAndersen,C.Y. Byskov, A.G.,Andersen,C.Y., Nordholm, L.,Thogersen,H.,Guoliang,X., Byskov, A.G.,Fenger, M.,Westergaard, L.andAnderson,C.Y. Byskov Baltus, A.E.,Menke,D.B.,Hu,Y.-C., Goodheart,M.L.,Carpenter, A.E.,de Chiba, H.,Clifford, J.,Metzger, D.andChambon,P. Chambon, P. Byskov, A.G. Bowles, J.,Bullejos,M.andKoopman,P. Boulogne, B.,Levacher, C.,Durand,P. andHabert,R. Bouillet, P., Qulad-Abdelghani,M.,Vicaire, S.,Garnier, J.M.,Schuhbaur, B., Chuma, S.andNakatsuji,N. Byskov, A.G. Byskov, A.G. Byskov, A.G. Byskov, A.G. Bowles, J.,Knight,D.,Smith,C.,Wilhelm,Richman,J.M.,Mamiya,S., Deltour, L.,Haselbeck,R.J.,Ang,H.L.andDuester, G. Byskov, A.G. Burgoyne, P. S. Bullejos, M.andKoopman,P. Buehr, M.,Gu,S.andMcLaren, A. Bradbury, M.W. Di Carlo,A.D.,Travia, G. andDeFelici,M. Donovan, P. L.A.,Heasman,J.andWylie,C. J.,Stott,D.,Cairns, fetal malegermcells. Forskolin andthemeiosisinducingsubstancesynergisticallyinitiatein vitro. Dev. Biol. female mouse. cytoplasmic componentsare defectiveinoocytesoftheB6.Y(TIR)sex-reversed FASEB J.10,940-954. sterols: background, discovery, andpossibleuse. structure ofsterols thatactivateoocytemeiosis. Wassmann, O.,Andersen,J.V., Guddal,E.andRoed,T. Rev. of gonocytes.Biol.Reprod. development: immunolocalizationandimplicationinthecontrol ofthenumber receptors andretinoid Xreceptors intherattestisduringfetalandpostnatal 38, 1430-1434. the decisiontoentermeiosisprecedes premeiotic DNAreplication. Rooij, D.G.andPage,C. Mol. Cell.Biol. control ofexpression ofretinoic targetgenesinF9embryonalcarcinoma cells. receptor-retinoic acidreceptor heterodimers are differentially involvedinthe the cat,minkandferret. J.Reprod.Fertil.45,201-209. 334. rete cellsinthefetalmousegonad. fetal mouseovary. Biol.Reprod. screen suggestsarole forvanin-1intestisdevelopmentmice. novel mousegene,Stra1(mouseLERK-2/Eplg2). responsive genesinP19embryonalcarcinoma cellsandcharacterizationofa Dolle, P. andChambon,P. Dev. Biol. mouse fetalgermcellsinvitro anditsinhibitionbygp130-mediatedsignaling. Androl. Nature differentiation inthefetalmouse. determines germcellfate. Sex-specific regulation ofretinoic acidlevelsindeveloping mousegonads Yashiro, K.,Chawengsaksophak,Wilson,M.J.,Rossant,J.etal. 124-135. Reprod. potential retinol dehydrogenases forendogenousretinoic acidsynthesis. class IandIValcoholdehydrogenases inmousetestisandepididymis: 422-428. caudal waveduringdevelopmentofthemouseovary. Mol.Reprod.Dev. 68, chimera. J.Exp.Zool.226,315-320. germ cellsasshownbyprogeny derivedfrom XX,Sxr/+ oocytes ofafemale spermatogenesis. culture. Cell Migratory andpostmigratorymouseprimordial germcellsbehavedifferently in germ cellsofthemouseembryo. synaptonemal complexprotein SCP3isexpressed byfemaleandmaleprimordial 66, 71-117. , A.G.andSaxen,L. 252, 396-397. Suppl. 2 56, 102-109. 229, 468-479. (1996). Adecadeofmolecularbiologyretinoic acidreceptors. 44, 831-838. (1986). Differentiation ofmammalianembryonicgonad. (1978a). Regulationofinitiationmeiosisinfetalgonads. (1975). Therole oftherete ovariiinmeiosisandfollicleformation in (1978c). Themeiosis-inducinginteractionbetweengermcellsand (1978b). Theanatomyandultrastructure oftherete system inthe (1974). Doestherete ovariiactasatriggerfortheonsetofmeiosis? (1987). Therole ofthemammalianYchromosome in 17, 3013-3020. Dev. Biol. 52, 193-200. (1983). Functionalcapacityofsex-reversed (XX,Sxr/+)mouse , 29-38. Dev. Suppl. Mol. Reprod.Dev. 219, 277-286. Science 61, 1548-1557. (1976). Inductionofmeiosisinfetalmousetestis (1995). Efficient cloningofcDNAsretinoic acid- (2001). Autonomoustransitionintomeiosisof 101, 133-141. (2006). Ingermcellsofmouseembryonicovaries, (2004). Germcellsentermeiosisinarostro- 19, 720-735. Int. J.Dev. Biol.44,241-244. Development 312, 596-600. (1993). Mesonephriccontributiontotestis Ann. Biol.Anim.Biochim.Biophys. 34, 47-52. (2000). Asubtractivegeneexpression (2000). Themeioticspecific (2000). Bothnuclearand (1998). Meiosis-activating Nature 117, 273-281. Dev. Biol. J. Mol.Med.76,818-823. (1997). Distinctretinoic X (1999). Retinoicacid 374, 559-562. (1997). Localizationof 170, 420-433. (1995). Chemical Genesis (1993). Nat. Genet. Physiol. (1986). 18, 327- Int. J. Biol. 27, (2006). Grinsted, J.,Byskov, A.G.andAndreasen, M.P. Kehler, J.,Tolkunova, E.,Koschorz,B.,Pesce, M.,Gentile,L.,Boiani, Karl, J.andCapel,B. Huang, H.F. S.andHembree, W. C. Gu, P., LeMenuet,D.,Chung,A.C.,Mancini,M.,Wheeler, D.A.andCooney, Grinsted, J.andByskov, A.G. MacLean, G.,Abu-Abed,S.,Dolle,P., Tahayato, A.,Chambon,P. and Li, H.andKim,K. Lawson, K.A.,Dunn,N.R.,Roelen,B.Zeinstra,L.M.,Davis,A. Horton, C.andMaden,M. Hilscher, B.,Hilscher, W., Bulthoff-Ohnolz, B.,Kramer, U.,Birke,A.,Pelzer, H. R.E.,Purzke,A.P.,Hernandez, Myers,J.P., Margaretha, L.andMoens,C.B. Farini, D.,LaSala,G.,Tedesco, M.andDeFelici, Ginsburg, M.,Snow, M.H.andMcLaren, A. MacLean, G.,Li,H.,Metzger, D.,Chambon,P. andPetkovich,M. Livera, G.,Rouiller-Fabre, V., Valla, J.andHabert,R. Lawson, K.A.andHage,W. J. Koubova, J.,Mencke,D.B.,Zhou,Q.,Capel,Griswold,M.andPage, Francavilla, S.andZamboni,L. Ghyselinck, N.B.,Vernet, N.,Dennefeld,C.,Giese,Nau,H.,Chambon,P., Evans, E.P., Ford, C.E.andLyon, M.F. Mark, M.,Ghyselinck,N.B.andChambon,P. Fuhrmann, G.,Chung,A.C.,Jackson,K.J.,Hummelke,Baniahmad,A., Fuchs, E.andGreen, H. McCarthy, P. T. andCerecedo, L.R. McCaffery, P., Wagner, E.,O’Neil,J.,Petkovich, M.andDrager, U.C. Matsui, Y. and Hayashi, K. McLaren, A. A in Vitamin Adeficientrats. A inVitamin Reprod. Fertil.56,653-656. fetal mousetestisinvitro byrete testistissuefrom pubertalmiceandbulls. substances inhumanmalereproductive organs. required forprimordial germcellsurvival. Lomeli, H.,Nagy, A.,McLaughlin,K.J.,Scholer, H.etal. mouse genitalridge. 312-323. differentiation. Mol.Cell.Biol. pluripotency genesduringretinoic acid-inducedembryonicstemcell A. J. mouse embryoduringgastrulation. murine development. P450, Cyp26B1,andcomparativeexpression analysiswithCyp26A1duringearly Petkovich, M. 231. on themeiosisinfetalratovaryculture. Biol. Reprod. blocking mesonephriccellmigrationanddecreasing thenumberofgonocytes. germ cellsinthemouse. 436. generation ofprimordial germcellsinthemouseembryo. Wright, C.V., Korving,J.P. andHogan,B.L. normal developmentandteratogenesisinthemouseembryo. germ cells.Dev. Biol. action andmolecularsignalingpathwaysofKitligandonmouseprimordial during oogenesisandprespermatogenesis. and autoradiographicstudiesofoocytestransitionalprospermatogonia and Gauss,G. for hindbraindevelopment. (2007). Cyp26enzymesgeneratetheretinoic acidresponsenecessary pattern the XYgermcellinmousetobecomeanoocyte. 1622. Endocrinology Apoptotic extinctionofgermcellsintestes mice. Proc.Natl.Acad.Sci.USA D. C. human keratinocytesbyvitaminA. germinal cellsinintra-andperigonadallocations. mutant micelackingtheplasmaretinol bindingprotein. S.andMark,M. Viville, 377-387. down andnuclearreceptor expression. Dorsal andventralretinal territoriesdefinedbyretinoic acidsynthesis,break- meiosis. Cell.Mol.LifeSci. Pharmacol. Toxicol. retinoic acidsignalingpathway duringmouseembryogenesis. nuclear receptors: lessonsfrom geneticandpharmacologicaldissectionsofthe germline restriction ofOct4expression bygermcellnuclearfactor. Sutter, J.,Sylvester, I.,Scholer, H.R.andCooney, A.J. Nutr. 46,361-376. J. Reprod.Fertil.61,461-467. (2005). Orphannuclearreceptor GCNFisrequired fortherepression of (2006). Retinoicacidr (1981). Thefateofgermcellsinthetestis offetalSex-reversed mice. 70, 687-693. doi:10.1210/en.2007-0492. (2001). Cloningofanovelretinoic-acid metabolizingcytochrome (1974). Kineticsofgemetogenesis.I.Comparativehistological 46, 451-480. (2004). RetinoicacidinhibitsratXYgonaddevelopmentby (1995). Three-dimensional structure ofthedeveloping Philos. Trans. R.Soc.Lond.BBiol.Sci. 306, 572-583. Mech. Dev. (1981). Regulationofterminaldifferentiaiton ofcultured Ciba Found.Symp. (2006). Retinoidsandspermatogenesis:Lessonsfrom (2007). Epigeneticregulation fortheinductionof (1995). Endogenousdistributionofretinoids during 64, 257-262. egulates sex-specifictimingofmeioticinitiationin Development Biol. Reprod. (1981). Meiosis-inducingandmeiosis-preventing 25, 8507-8519. (1994). Clonalanalysisoftheoriginprimordial (1985). Differentiation ofmouseectopic 103, 2474-2479. 107, 195-201. (1952). Vitamin Adeficiencyin themouse. (1952). Vitamin Cell Development (1979). Spermatogenicresponse toVitamin (1977). Direct evidenceofthecapacity Mech. Dev. 25, 617-625. EMBO Rep. 134, 177-187. 21, 891-904. Cell Tissue Res. 182, 84-91. (1990). Primordial germcellsinthe Cyp26b1 Mol. Cell.Endocrinol. (2006). Functionofretinoid (1999). Bmp4isrequired forthe Fertil. Steril.35,199-204. (1979). Inductionofmeiosisin J. Exp.Zool. 110, 521-528. 82, 119-130. Nature (2007). Chemoattractant 5 (2000). Effects ofretinoids , 1078-1083. knockout mice. Dev. Dyn. Genes Dev. 13,424- (2001). Mouse 154, 443-470. 350, 235-242. REVIEW 267, 430-431. (2004). Oct4is Annu. Rev. 233, 101-109. Dev. Dyn.202, 235, 1608- Dev. Cell (2007). 165, 225- (1999). 3409 J. 1 , J.

DEVELOPMENT Rosner, M.A.,Ozato,K.,Timmons,P. M.H.,Vigano, M.,Poirier, F., Rigby, P. Romand, R.,Dolle,P. and Hashino, E. Pittman, D.L.,Cobb,J.,Schimenti,K. J.,Wilson,L.A.,Cooper, D.M., Pikarsky, E.,Sharir, H.,Ben-Shushan,E.andBergman, Y. Pesce, M.,Wang, X.,Wolgemuth, D.J.and Scholer, H. Park, E.H.andTaketo, T. Palmer, S.J.andBurgoyne,P. S. Okamoto, K.,Okazawa,H.,Pkuda,A.,Sakai,M.,Muramatsu,M.and Ohkubo, Y., Shirayoshi,Y. andNakatsuji,N. Ohinata, Y McLaren, A. 3410 Oulad-Abdelghani, M.,Bouillet,P., Decimo,D.,Gansmuller, A.,Heyberger, McLaren, A. McLaren, A. McLaren, A. McLaren, A.andBuehr, M. McLaren, A. McLaren, A.andSouthee,D. Menke, D.B.,Koubova,J.andPage,C. Menke, D.B.andPage,C. Minucci, S.,Botquin,V., Yeon, Y. I.,Dey, A.,Sylvester, I.,Zand,D.J.,Ohbo, Molyneaux, K.A.,Stallock,J.,Schaible,andWylie,C. Molyneaux, K.A.,Zinszner, H.,Kunwar, P. S.,Schaible,K.,Stebler, J., Morales, C.andGriswold,M.D. Nakatsuji, N.andChuma,S. Morita, Y. andTilly, J.L. Nichols, J.,Zevnik,B.,Anastassiadis,K.,Niwa,H.,Klewe-Nebenius,D., Niederreither, K.,Subbarayan,V., Dolle,P. andChambon,P. differentiation. Mech.Dev. expression oftheOct-4transcription factorduringmousegermcell cells andgerm of themammalianembryo. W. andStaudt,L.M. development. J.Neurobiol.66,687-704. germline-specific RecAhomolog. arrest withfailure ofchromsome synapsisinmice deficientforDmc1,a Brignull, E.,Handel,M.A.andSchimenti, J.C. 1038. elements locatedinthepromoter-enhancer region. represses Oct-3/4geneexpression through severalretinoic acid-responsive 1889. the oocytesinB6.YTIRsex-reversed mouseovary. exclusively, XY. Development adult XX expressed inmouseembryoniccells. Hamada, H. and clonalcultures. proliferation andgrowth arrest inmouseprimordial germcellsstudiedbymixed 207-213. Blimp1 isacriticaldeterminantofthegermcelllineageinmice.Nature S. C.,Obukhanych,T., Nussenzweig,M.,Tarakhovsky, A.etal. Exp. Zool. retinoic acid-responsive gene. of apremeiotic germcell-specific cytoplasmicprotein encodedbyStra8,anovel S., Dolle,P., Bronner, S.,Lutz,Y. andChambon,P. Cambridge: CompanyofBiologists. Experimental Biology Controlling EventsinMeiosis,Thirty-eighthSymposiumoftheSociety supporting celllineage. Biol. Sci. of fetalgonads. meiosis. Dev. Biol. developing mousegonad. EMBO J. regulation ofOct3/4coincideswiththelosspromoter occupancyinvivo. K., Ozato,K.andScholer, H.R. Biol. 262,303-312. germ cellsinXXmousegonadsoccursananterior-to-posterior wave. analysis oflivingmousegermcellmigration. cellm igrationandsurvival. The chemokineSDF1/CSCL12anditsreceptor CXCR4regulate mousegerm Sunshine, M.J.,O’Brien,W., Raz,E.,Littman,D.,Wylie,C.etal. in seminiferous tubulesoftherat. cells intofemaleormalegermcells. macromolecular synthesis.Endocrinology ovarian germcellmitosisversusapoptosisbyrequirement fornew development. Nat.Genet.21 Embryonic retinoic acidsynthesisisessentialforearlymousepost-implantation Cell cells inthemammalianembryodependsonPOUtranscriptionfactorOct4. Chambers, I.,Scholer, H.andSmith,A. 95, 379-391. REVIEW ϫ 350, 229-233. 15, 888-899. ., Payer, B.,O’Carroll, D.,Ancelin,K.,Ono,Y., Sano,M.,Barton, 228, 167-171. XY chimaericmousetestes:Sertolicellsare predominantly, butnot (1983). Studiesonmousegermcellsinsideandoutsidethegonad. (1984). Meiosisanddifferentiation ofmousegermcells.In (1991). Developmentofthemammaliangonad:fate (1995). Germcellsandgermcellsex. (2003). Primordial germcellsinthemouse. (1990). Anoveloctamerbindingtranscriptionfactorisdifferentially Cell Differ. Dev. 187, 107-113. Exp. CellRes. (ed. C.W. EvansandH.G.Dickinson), pp.1-23. (1990). APOU-domaintranscriptionfactor inearlystem BioEssays 13,151-156. (1999). Aggregation ofretinoic acideffects onfetal (2003). Onsetandprogress ofmeioticprophase in Gene Expr. Patterns (1990). Developmentofmousegermcellsincultures 71, 89-98. Development (2001). Differentiation ofmouseprimoridialgerm , 444-448. 112, 265-268. (2002). Sexuallydimorphicgeneexpression inthe (1997). Entryofmouseembryonicgermcellsinto J. CellBiol.135,469-477. 31, 185-195. (1991). Insitu (1987). Retinol-inducedstagesynchronization Mol. Cell 222, 291-297. (1996). Retinoicacid-mediateddown- Endocrinology Int. J.Dev. Biol.45,541-548. Cell (2006). Retinoidsignalingininnerear 60, 461-472. 130, 4279-4286. 140, 2696-2703. (1998). Formationofpluripotentstem 1 (2003). Sexualdifferentiation of , 697-705. Dev. Biol. (1996). Autonomousregulation of 2 analysis offetal,prepuberal and Nature , 359-367. Philos. Trans. R.Soc.Lond.B 121, 432-434. (1998). Meioticprophase Mol. Cell.Biol. (1996). Characterization 240, 488-498. Dev. Biol. Biol. Reprod. 345, 686-692. (1998). Differential (2001). Time-lapse (1994). Retinoicacid (1999). 262, 1-15. 14, 1026- 69, 1879- (2005). Dev. (2003). 436, J. Taketo-Hosotani, T., Nishioka,Y., I.and Nagamine,C.M.,Villalpando, Speed, R.M. Seki, Y., Hayashi,K.,Itoh,Mizugaki,M.,Saitou,M.andMatsui,Y. Sakai, Y., Luo,T., McCaffery, P., Hamada,H.andDrager, U.C. Saitou, M.,Barton,S.C.andSurani,M.A. Yeom, Y. I.,Fuhrmann,G.,Ovitt,C.E.,Brehm, A.,Ohbo,K.,Gross, M., Ying, Y. L., Liu,X.M.,Marble,A.,Lawson,K.A. andZhao,G.Q. Tam, P. P. andSnow, M.H. Scholer, H.,Hatzopoulos,A.,Balling,R.,Suzuki,N.andGruss,P. Rossant, J., Zirngibl, R.,Cado,D.,Shago,M.andGiguere,Rossant, J.,Zirngibl, V. Tam, P. P. andZhou,S.X. Tanaka, S.andMatsui,Y. van Pelt,A.M.anddeRooij,D.G. Upadhyay, S.andZamboni,L. Thompson, J.N.,Howell,M.andPitt,G.A. van Pelt,A.M.anddeRooij,D.G. van Pelt,A.M.anddeRooij,D.G. Vernet, N.,Dennefeld, C.,Rochette-Egly, C.,Oulad-Abdelghani,M., Vermot, J.,Garnier, J.M.,Dierich,A.,Niederreither, K.,Harvey, R.P., White, J.A.,Ramshaw, H.,Taimi, M.,Stangle,W., Zhang,A.,Everingham, Yashiro, K.,Zhao,X.,Uehara,M.,Yamashita, K.,Nishijima,M.,Nishino,J., Yao, H.H.,DiNapoli,L.andCapel,B. Weston, A.D.,Blumberg, B.andUnderhill,T. M. Westergaard, L.,Byskov, A.G.,Anderson,C.Y., Grinsted,J.and McNatty, K. Wang, J.,Rao,S., Chu,J.,Shen,X.,Levasseur, D.N.,Theunissen,T. W. and microscope levelusingsurface-spreading. B6.Y Merchant-Larios, H. Biol. 278,440-458. associated withspecificationandearlydevelopmentofgermcellsinmice. Extensive andorderly reprogramming ofgenome-widechromatin modifications the specificationofgermcellfateinmice. 2550. evidence forgermline-specificexpression ofanOctfactor. family ofoctamer-specific proteins present duringmouseembryogenesis: equatorial retina duringlatereyedevelopment. CYP26A1 andCYP26C1cooperateindegradingretinoic acidwithinthe 1333-1344. domains oftranscriptionalactivityduringmouseembryogenesis. Expression ofaretinoic acidresponse element-hsplacZtransgenedefinesspecific Sakai, Y. andHamada, H. specific forthetotipotent cycleofembryonalcells. Hubner, K.andScholer, H.R. limb. Dev. Cell required forproximodistal andoutgrowth patterning ofthedevelopingmouse 64, 133-147. cells duringcompensatorygrowth inmouseembryos. gastrulating mouseembryo. and germ-linelineagesisinfluencedbythepositionofcellsin reproduction inrats. during formaitonanddifferentiation ofprimordial germcells. mil-1 andmil-2,mouseinterferon-induced transmembraneprotein likegenes, 43, 363-367. epithelium aftervitaminAreplacement invitaminA-deficientmice. 6588. study ofgermcellsexualdifferentiation. Suppl. 1,S261-S267. replacement. Biol.Reprod. the seminiferous epitheliuminvitaminA-deficientratsafterA full developmentofspermatogeniccells. spermatogenesis invitaminA-deficientratsandhighreplicate dosessupportthe Endocrinology metabolism andsignalingpathwaysintheadultdevelopingmousetestis. Chambon, P., Ghyselinck,N.B.andMark,M. (RALDH2). Genesis encoding theretinoic acid-synthesizingenzymeretinaldehyde dehydrogenase 2 Chambon, P. andDolle,P. retinoic acidmetabolism.Proc.Natl.Acad.Sci.USA predominantly expressed intheadultcerebellum andisresponsible forall-trans- Identification ofthehumancytochrome P450,P450RAI-2,whichis S., Creighton, S.,Tam, S.P., Jones,G.andPetkovich,M. Biol. 161,223-228. unliganded retinoid receptors indevelopment:lessissometimesmore. Development mesonephric cellmigrationandtestiscord formationinmousegonads. 377-384. by ameiosis-inducingsubstance(MIS)inthefollicularfluid? P. embryonic stemcells. Orkin, S.H. (1984). Isresumption ofmeiosis inthehumanpreovulatory oocytetriggered DOM sex-reversed femalemouse. (1982). Meiosisinthefoetalmouseovary. I.Ananalysisatthelight (2006). Aprotein interactionnetworkforpluripotencyof 130, 5895-5902. 6 147, 96-110. , 411-422. 44, 155-158. Proc. R.Soc.Lond.BBiol.Sci. Nature (1989). Developmentandfertilityofovariesinthe (1996). Theallocationofepiblastcellstoectodermal (1981). Proliferation andmigrationofprimordial germ 42, 677-682. (2002). Developmentallyregulated expression of (2004). Regulationofr (2006). Conditional(loxP-flanked)alleleforthegene Dev. Biol. 444, 364-368. (1982). Ectopicgermcells:naturalmodelforthe (1996). Germlineregulatory elementofOct-4 (1990a). Synchronization oftheseminiferous (1990b). Theoriginofthesynchronization of (1991). Retinoicacidisabletoreinitiate Development (2003). Meioticgermcellsantagonize 178, 124-132. Proc. Natl.Acad.Sci.USA Endocrinology Chromosoma Nature (2002). Amolecularprogramme for (1964). Vitamin Aand (1964). Vitamin Dev. Biol. (2006). Retinoicacid 418, 293-300. etinoic aciddistributionis (2003). Activerepression by Development 159, 510-535. 107, 95-105. 97, 6403-6408. Development 134(19) J. Embryol. Exp.Morphol. J. Embryol. 128, 697-704. 85, 427-437. 276, 143-157. EMBO J. Fertil. Steril.41, (2000). Mech. Dev. (1991). (2004). 122, 881-894. Genes Dev. 5 79, 6584- 8 (2000). (1989). A Biol. Reprod. , 2543- J. Cell (2005). 119 Dev. ,

DEVELOPMENT Development 134(19) Yuan, L.,Liu,J.,Zhao,Brundell, E.,Daneholt,B.andHoog,C. Yoshida, K.,Kondoh,G.,Matsuda,Y., Habu,T., Nishimune,Y. andMorita,T. mouse. Mol.Endocrinol. Requirement ofBmp8bforthegenerationprimoridalgermcellsin synapsis, andmalefertility. murine SCP3geneisrequired forsynaptonemalcomplexassembly, chromosome chromosome synapsisduringmeiosis. (1998). ThemouseRecA-likegeneDmc1isrequired forhomologous 14, 1053-1063. Mol. Cell 5 , 73-83. Mol. Cell 1 , 707-718. (2000). The Yuan, L.,Liu,J.,Hoja,M.,Wilbertz, J.,Nordqvist, K.andHoog,C. Zhai, Y., Higgins,D.andNapoli,J.L. Zamboni, L.andUpadhyay, S. specific protein SCP3. Female germcellaneuploidyandembryodeathinmicelackingthemeiosis- glands. Cell. Physiol. encoding retinol dehydrogenase isozymesandcellularretinol-binding proteins. J. Exp.Zool.228,173-193. 173, 36-43. Science 296, 1115-1118. (1983). Germcelldifferentiation inmouseadrenal (1997). Coexpression ofthemRNAs REVIEW (2002). 3411 J.

DEVELOPMENT