In germ cells of mouse embryonic ovaries, the decision to enter meiosis precedes premeiotic DNA replication
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- Anastasia McDowell
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1 26 Ntur Pulishing Group In grm lls o mous mryoni ovris, th ision to ntr miosis prs prmioti DNA rplition Anrw E Bltus 1,2,4, Dougls B Mnk 1,2,4, Yuh-Ching Hu 1,2, Mry L Goohrt 1,2, Ann E Crpntr 2, Dirk G Rooij 3 & Dvi C Pg 1,2 Th trnsition rom mitosis to miosis is ining juntur in th li yl o sxully rprouing orgnisms. In yst, th ision to ntr miosis is m or th singl roun o DNA rplition tht prs th two mioti ivisions 1.W prsnt gnti vin o n nlogous ision point in th grm lin o multillulr orgnism. Th mous Str8 gn is xprss in grm lls o mryoni ovris, whr miosis is initit, ut not in thos o mryoni tsts, whr miosis os not gin until tr irth 2. Hr w rport tht in ml mryos lking Str8 gn untion, th rly, mitoti vlopmnt o grm lls is norml, ut ths lls thn il to unrgo prmioti DNA rplition, mioti hromosom onnstion, ohsion, synpsis n romintion. Comin with prvious inings, ths gnti t suggst tht tiv irntition o ovrin grm lls ommns t rgultory point upstrm o prmioti DNA rplition. Mhnisms y whih iploi lls trnsition rom mitosis to miosis hv n invstigt most thoroughly in th uing yst Shromys rvisi n in th ission yst Shizoshromys pom 1. In ths two spis, th molulr pthwys govrning mioti initition r issimilr, n thr is no vin tht ithr initition pthwy is onsrv in multillulr orgnisms. Nonthlss, gnti stuis o mioti initition in S. rvisi n S. pom unovr n unrlying ommonlity: in oth spis, th ision to ntr miosis is m or th roun o DNA rplition tht prs th mioti ivisions. An nlogous ision point upstrm o prmioti DNA rplition might, in prinipl, xist in multillulr orgnisms, ut gnti vin o this hs n lking. Th mryoni ovry o th mous prsnts n opportunity to xplor gnti ontrol o mioti initition in vivo 3 6. In mi, th timing o mioti initition irs mrkly twn th sxs: ovrin grm lls ntr miosis uring mryognsis, whrs tstiulr grm lls gin miosis only tr irth. Nonthlss, grm lls o mryoni ovris rmin unommitt to miosis until out h or mioti prophs is osrv thr 4. Within this in tmporl winow, th ision to initit th mioti progrm is m in grm lls o th ovry ut not in thos o th tstis. Th mous Str8 gn is xprss xlusivly in prmioti grm lls in oth sxs. In mls, Str8 is xprss in th mryoni ovry in n ntrior-to-postrior wv tht prs similr wv o E16.5 E14.5 E13.5 Wil-typ Figur 1 Photomirogrphs o hmtoxylin n osin stin ovrin stions rom wil-typ n Str8-iint littrmt mryos. (,) E13.5. (,) E14.5. (,) E16.5. Arrows init rprsnttiv grm lls. 1 Howr Hughs Mil Institut. 2 Whith Institut n Dprtmnt o Biology, Msshustts Institut o Thnology, 9 Cmrig Cntr, Cmrig, Msshustts 2142, USA. 3 Dprtmnts o Enorinology, Fulty o Biology, Utrht Univrsity n o Cll Biology, Univrsity Mil Cntr Utrht, 3584 CH Utrht, Th Nthrlns. 4 Ths uthors ontriut qully to this work. Corrsponn shoul rss to D.C.P. (pg_min@wi.mit.u). Riv 28 August; pt 6 Otor; pulish onlin 19 Novmr 26; oi:1.138/ng VOLUME 38 [ NUMBER 12 [ DECEMBER 26 NATURE GENETICS
2 Wil-typ Wil-typ Wil-typ Wil-typ 26 Ntur Pulishing Group Figur 2 Tsting ovrin grm lls or molulr mrkrs o mioti prophs. (,) Immunohistohmil stining or () REC8 protin or () SCP3 protin in grm lls rom wil-typ n Str8-iint E15.5 ovris. () Whol-mount immunohistohmil stining or g-h2ax protin in wil-typ n Str8-iint E15.5 ovris. () Whol-mount in situ hyriiztion or Dm1 mrna in wil-typ n Str8-iint E15.5 ovris. () RT-PCR nlysis o Spo11 n Dm1 trnsription in wil-typ n Str8-iint E14.5 ovris; -tin srvs s ontrol. xprssion o rly mioti gns 2,7,8.Inmls,Str8 is xprss in purtl n ult tsts, in prmioti sprmtogni lls 9.Thus, Str8 might hv rol rly in miosis or prhps in th trnsition rom mitosis to miosis, in on or oth sxs. To xplor th rol o Str8 in grm ll vlopmnt, w us gn trgting to riv mi tht lk Str8 untion (Supplmntry Fig. 1 onlin). W oun tht oth ml n ml homozygots or th trgt Str8 lll wr invrily inrtil; htrozygots wr rtil. Insption o th rproutiv orgns o Str8-iint ults show mrk rution in th siz o ovris n tsts (Supplmntry Fig. 2 onlin). Aprt rom th gons, w tt no phnotypi normlitis in Str8-iint nimls. Although th ovris o htrozygous mls show norml istriution o mturing ovrin ollils t 8 wks o g, th ovris o Str8- iint mls ontin no ooyts or ollils. Although htrozygous mls show norml sprmtognsis t 8 wks o g, with ull rng o prmioti, mioti n postmioti lls, th tstiulr grm lls o Str8-iint nimls wr svrly ru in numr, n nrly ll ppr to prmioti. Hving oun no ooyts in 8-wk-ol Str8-iint mls, w ompr histologilly th gonl grm lls o wil-typ n Str8- iint ml mryos (Fig. 1). W i not osrv ny irns through mryoni y 13.5 (E13.5). In wil-typ ovris, grm lls prolirt mitotilly until out E13.5, y whih tim thy quir istintiv morphology, with pths o onns hromtin t th priphry o th nulus 3,1 (Fig. 1). Str8-iint grm lls lso Wil-typ Str8 / + RT RT + RT RT Dm1 Spo11 β-tin show this prmioti morphology t E13.5 (Fig. 1). Notly, tstiulr grm lls show similr morphology t out this tim, whih, s som stuis hv suggst, my pivotl prio o grm ll snsitivity to xtrinsi miosis-inuing or inhiiting tors tht ir twn ml n ml gons 3,11. W onlu tht Str8 untion is not rquir or grm lls to vlop th prmioti morphology osrv in wil-typ mls t E13.5. In ovris o wil-typ mryos, grm lls ntr mioti prophs in sptiotmporl wv tht gins t E13.5 E14.5 (rs. 2,7,8,1,12,13). By E14.5, mny grm lls in wil-typ ovris show th thr-lik hromosom onnstion tht ins lptotn, th initil stg o mioti prophs (Fig. 1). By E16.5, most grm lls in wil-typ ovris show th mor vn nulr morphologis tht hrtriz th zygotn n phytn stgs o mioti prophs (Fig. 1). By ontrst, th grm lls o Str8-iint ovris o not vn yon th prmioti morphology osrv in wil-typ ovris t E13.5 (Fig. 1,). Inst, Str8-iint grm lls rtin this morphology until t lst E16.5, y whih point most o thir nuli r nlrg (Fig. 1). (Ooyts with similr morphology r somtims osrv in g-mth wiltyp ovris, ut t muh lowr rquny.) By irth, Str8-iint ovris r svrly plt o grm lls (t not shown). Tkn s whol, ths histologil inings suggst tht, in ml mryos, Str8-iint grm lls vlop normlly to th rink o miosis ut o not unrgo th hromosom onnstion initiv o rly mioti prophs. Wil-typ Cll ount 3,5 2,8 2,1 1,4 7 E14.5 E15.5 7, Wil-typ , 4, 3, 2, 1, 2 4 All lls Grm lls 5,6 4,2 2,8 1,4 7, 5,6 4,2 2,8 1,4 2 4 g DNA ontnt (C) DNA ontnt (C) Cll ount h Figur 3 Histogrms o llulr DNA ontnt in mryoni ovris. ( ) All lls rom () wil-typ E14.5, () wil-typ E15.5, () Str8-iint E14.5 or () Str8-iint E15.5 ovris. ( h) Grm lls rom () wil-typ E14.5, () wil-typ E15.5, (g) Str8-iint E14.5 or (h) Str8-iint E15.5 ovris. Totl numrs o lls plott:, 12,255;, 27,626;, 18,16;, 27,711;, 1,532;, 4,329; g, 2,493; h, 3,224. NATURE GENETICS VOLUME 38 [ NUMBER 12 [ DECEMBER
3 26 Ntur Pulishing Group Our histologil inings suggst tht Str8 might rquir or ml grm lls to ntr mioti prophs. I so, thn th hromosoms o Str8-iint ml grm lls shoul not ort with th ohsion or synptonml omplxs hrtristi o mioti prophs. To tst this, w immunostin hromosoms rom ovrin grm lls o wil-typ n Str8-iint E15.5 littrmts with ntiois to ithr REC8, mioti ohsin 14,15,orSCP3, synptonml omplx protin 16. As xpt, REC8 n SCP3 protins wr oun long th lngths o hromosoms in most wil-typ ooyts t this tim (Fig. 2,). In ll Str8-iint grm lls xmin, REC8 n SCP3 wr loliz not long th lngths o hromosoms ut in th pttrns rntly rport in prmioti grm lls o th mryoni ovry (Fig. 2,) 17 (Supplmntry Not onlin). W onlu tht Str8 is rquir in ml grm lls or mioti ohsion n synptonml omplx ormtion. I Str8 is rquir or ml mioti prophs, thn Str8-iint lls shoul not ngg in mioti romintion. Aoringly, w sk whthr Str8-iint ml grm lls orm DNA oulstrn rks (DSBs), hllmrk o mioti romintion. Whn DNA DSBs r orm, lls rspon y phosphorylting H2AX, histon H2A isoorm, to yil g-h2ax 18. As xpt, immunostining or g-h2ax monstrt th prsn o DNA DSBs throughout th ovris o wil-typ mryos t E15.5 (Fig. 2). By ontrst, th ovris o Str8-iint mryos wr ngtiv or g-h2ax, initing tht DNA DSBs h not orm (Fig. 2). In itionl tsts o nggmnt in mioti romintion, w sk whthr Str8-iint ml grm lls xprss Spo11 n Dm1, whih r gns rquir to orm n to rpir mioti DSBs, rsptivly As prviously monstrt y whol-mount in situ hyriiztion, Str8 is xprss shortly or Dm1 in wil-typ mryoni ovris 2.Bythissmssy,wounthtStr8-iint grm lls o not xprss Dm1 (Fig. 2), rsult onirm y RT- PCR nlysis (Fig. 2). Likwis, Spo11 ws not xprss in Str8- iint ml grm lls (Fig. 2). Tkn togthr, th sn o g-h2ax stining n th sn o Spo11 n Dm1 xprssion provi strong vin tht Str8-iint ml grm lls o not unrtk mioti romintion. Immitly or mioti prophs, grm lls rplit thir DNA n thus inrs thir DNA ontnt rom 2C to 4C, whih thn rmins onstnt throughout th xtn prophs o th irst mioti ivision. Givn th wight o vin tht Str8 is rquir or ml grm lls to ntr mioti prophs, th qustion riss whthr Str8 is lso rquir or prmioti DNA rplition. Although no mrkrs spii to prmioti DNA rplition r known in mmmls, this rplition hs n shown to our uring th 15 h or mioti prophs 23. By img ytomtry, w ompr th DNA ontnt o lls rom wil-typ n Str8-iint ovris t E14.5 n E15.5, whn wil-typ 4C grm lls shoul umult in mioti prophs. (Th numrs o grm lls in mryoni ovris r insuiint or DNA ontnt nlysis vi low ytomtry.) W irst nlyz DNA ontnt in lls o th ntir ovry, whr grm lls r grtly outnumr y somti lls. In wil-typ ovris, th proportion o 4C lls inrss rom E14.5 (Fig. 3) to E15.5 (Fig. 3), rlting th inrsing numr o grm lls tht hv omplt prmioti DNA rplition. By ontrst, Str8-iint ovris ontin w i ny 4C lls, n thir numrs o not inrs (Fig. 3,), implying tht Str8-iint grm lls hv not unrgon prmioti DNA rplition. To orroort n rin this mol, w us grm ll spii ntioy (-mous Vs homolog (MVH)) 24 to ll n istinguish th grm ll popultion in our img-ytomtri nlyss o DNA g Wil-typ Figur 4 Photomirogrphs o hmtoxylin n osin stin stions rom wil-typ n Str8-iint tsts t 7 or 8 tr irth. In h stion, rrows init th most vn sprmtogni ll typ; rrowhs init poptoti lls. (,) B-typ sprmtogoni in wil-typ n Str8-iint tsts. (,) Prlptotn sprmtoyts in wil-typ n Str8-iint tsts. () Lptotn sprmtoyts in wil-typ tsts. () Prlptotn sprmtoyts in Str8-iint tsts, with no mioti hromosom onnstion. (g) Zygotn sprmtoyts in wil-typ tsts. (h) Prlptotn sprmtoyts n poptoti sprmtoyts in Str8-iint tsts. ontnt in wil-typ n Str8-iint ovris. W osrv tht sustntil rtion o grm lls in wil-typ ovris r 4C t E14.5 (Fig. 3), n th grt mjority r 4C t E15.5 (Fig. 3), gin rlting th inrsing numr o grm lls tht hv omplt prmioti DNA rplition y E15.5. In Str8-iint ovris, y ontrst, only vry smll rtion o grm lls r 4C t E14.5 (Fig. 3g), n vn this smll rtion hs isppr y E15.5 (Fig. 3h). W suggst tht, in Str8-iint (n wil-typ) ovris, smll popultion o (4C) grm lls hs not omplt th lst mitoti ivision y E14.5 (Fig. 3g) ut hs on so y E15.5 n thus rturns to 2C stt (Fig. 3h). Tkn togthr, our inings init tht in th ml grmlin, Str8 is not rquir or ompltion o th inl mitoti ivision, ut it is rquir or prmioti DNA rplition n th susqunt vnts o mioti prophs. Although mny gns in ition to Str8 r known to rquir or mmmlin miosis, ths gns untion uring or tr mioti prophs n r not rquir or prmioti DNA rplition. For xmpl, grm lls lking ithr R8, Sp3, Spo11 or Dm1 rily progrss into mioti prophs in oth mls n mls 19 22, Is Str8 rquir or ml miosis? In wil-typ ml mi, sprmtognsis is initit shortly tr irth (Fig. 4). Sprmtogoni (Fig. 4) unrtk sris o mitoti ivisions, vntully giving ris to popultion o sprmtoyts, whih unrgo miosis. In wiltyp mi o th strin tht w us, sprmtoyts irst ppr 7 or 8 tr irth (Fig. 4) n quikly progrss into mioti prophs. In h 1432 VOLUME 38 [ NUMBER 12 [ DECEMBER 26 NATURE GENETICS
4 26 Ntur Pulishing Group Str8-iint mls, sprmtogonil ivisions sm to pro normlly (Fig. 4), with prmioti sprmtoyts ppring s xpt 7 or 8 tr irth (Fig. 4). Ths Str8-iint sprmtoyts (Fig. 4) show prmioti hromtin onnstion s in wiltyp tsts (Fig. 4), ut hr th vlopmnt o Str8-iint sprmtoyts sms to stll. Whrs wil-typ lls rily progrss through lptotn (Fig. 4) n zygotn (Fig. 4g) stgs o mioti prophs, most Str8-iint sprmtoyts unrgo poptosis or rhing ths stgs (Fig. 4,h). Amixtur with mitotilly tiv sprmtogoni prlu our trmining whthr Str8- iint sprmtoyts rplit thir DNA or unrgoing poptosis. Nonthlss, w onlu tht ml grm lls, lik ml grm lls, rquir Str8 or norml progrssion into mioti prophs. Our nlyss o Str8-iint ml grm lls sh light on two unmntl ut poorly unrstoo prosss in mmmlin rproution: mioti initition n th ginnings o ovrin irntition. Clssil sriptions o mmmlin miosis gin with prophs o th irst ivision. W suggst tht, in mryoni mous ovris, th ision to ngg in miosis is m onsirly rlir. W hv shown tht Str8 untion is rquir or prmioti DNA rplition, s wll s or th nsuing mioti prophs, in mryoni ovris. Rnt stuis hv monstrt tht Str8 xprssion in mryoni ovris is th rsult o inution y rtinoi i, whih is prsnt thr t highr lvls thn in mryoni tsts 5,6. W onlu tht, in ovrin grm lls, th ision to initit miosis, m in rspons to rtinoi i, is m or or rly in th roun o DNA rplition tht prs th mioti ivisions. Th t tht Str8 is not rquir or mitoti DNA rplition in ithr grm or somti lings unrsors th spiiity with whih prmioti DNA rplition is rgult in mmmls. Ths insights hv rmiitions or mmmlin sx trmintion n th ivrgnt pths y whih ovry n tstis ris rom ommon prursor. Gnti unrstning o ovry trmintion lgs hin tht o tstis trmintion 28, n rly ovrin vlopmnt is somtims portry s rltivly unvntul pross. To th ontrry, our prsnt rsults imply tht prmioti DNA rplition rgult t o trminl irntition pnnt on Str8 istinguishs grm lls o th rly mryoni ovry rom thos o its sxully unirntit prursor s wll s rom thos o th rly mryoni tstis. Prmioti DNA rplition, in gonl grm lls, my mong th rlist mnisttions o tiv miniztion uring mmmlin mryognsis. W not tht th rgultory logi, i not th molulr spiis, o th mitosis-to-miosis trnsition r shr twn ysts n mi. In mous ovris, s in uing n ission ysts, th ision to ngg th mioti progrm is tkn upstrm o prmioti DNA rplition 1. In mous ovris, s in ysts, grmlin lls initit th mioti progrm in rspons to molulr us or y thir nvironmnt: rtinoi i in mmmls n nutrint pltion in yst. To sur, th moluls involv in th mitosis-to-miosis trnsition r not wily onsrv. Th Str8 gn n th protin it nos, or xmpl, sm to rstrit to vrtrts (Supplmntry Fig. 3 onlin), n rtinoi i is limit to horts 29. Nonthlss, th ommon logi o mioti initition rom singlll ysts to sxully imorphi niml with physilly n mryologilly sgrgt grmlin ttsts to th unity o sxul rproution mong ukryots. METHODS Trgt isruption o th Str8 lous. A Str8 IRES-LZ/PGK-No trgting onstrut ws gnrt using PCR prouts mplii with Avntg HF2 polymrs (Clonth). All PCR prouts wr squn to nsur th sn o point muttions. Th v6.5 mryoni stm (ES) ll lin ws ltroport with 4 mg o linriz trgting onstrut DNA, n sltion ws prorm with 3 mg/ml G418 (GIBCO-BRL). ES ll olonis wr pik n srn y long-istn PCR spii or homologous trgting t th 5 rm. Homologous trgting t th 3 rm ws onirm y DNA lot nlysis. Corrtly trgt ES ll lons wr injt into Bl/ or C57Bl/6 lstoysts n trnsrr to psuoprgnnt Swiss Wstr mls. Grmlin trnsmission ws otin with thr inpnnt lons. Primrs Str8_p1, Str8_p2 n Str8_p3 (Supplmntry Tl 1 onlin) wr us or PCR gnotyping. Th wil-typ lll givs ris to PCR prout o 28 p. Th mutnt lll givs ris to PCR prout o 18 p. Histology. Disst tissus wr ix ovrnight in Bouin s solution, m in prin, stion n stin with hmtoxylin n osin. Imgs wr tkn with Nikon Optiphot 2 mirosop, Kok DC29 Zoom igitl mr n Ao Photoshop 7. Cll sprs or mioti hromosom nlysis. Gons rmov rom mryos twn E14.5 n E15.5 n isst r o msonphroi wr inut in 5 ml o trypsin-edta solution t 37 1C or 5 min n thn wsh rily in PBS. To otin singl lls, ths trypsin-igst gons wr piptt rptly n thn ntriug, ollow y rsuspnsion in 3 ml o hypotoni solution (.5% soium hlori in PBS). Cll suspnsions wr pl on poly-l-lysin-ot slis n kpt in humi hmr t room tmprtur (22 1C) until th lls h sttl. Th slis wr thn ix in 2% prormlhy n.3% SDS or 1 h t room tmprtur, wsh thr tims in.4% Photolo (Kok) or 1 min n ir ri. Ths slis wr stor t 8 1C or us. Cll-spr immunohistohmistry. For luorsn immunostining, slis wr rmov rom storg t 8 1C, wsh twi in PBS n trt with loking ur (3% ovin srum lumin,.5% Twn-2 n.5% Triton X-1 in PBS). Slis wr thn inut with 1:5 ilution o rit -SCP3 or -REC8 ovrnight t 4 1C, wsh with PBS n inut with Txs r or luorsin isothioynt (FITC)-onjugt sonry ntioy (1:2 ilutions; Jkson ImmunoRsrh Lortoris) or 1 h t room tmprtur. Atr xtnsiv wshing with PBS, slis wr mount using VECTASHIELD mium with 4,6-imiino-2-phnylinol (DAPI) (Vtor Lortoris). Imgs wr tkn using n Olympus BX51TF Fluorsn Mirosop, Spot RT igitl mr n Spot 4.1 sotwr (Dignosti Instrumnts). Whol-mount immunohistohmistry. Whol-mount inirt luorsnt immunohistohmistry ws prorm s prviously sri 3. Mous monolonl g-h2ax ntioy (Upstt) ws us t 1:1, ilution, n onky nti-mous Txs r onjugt sonry ntioy (Jkson) ws us t 1:2 ilution. Imgs wr mrg z-stks tkn using Ziss LSM 51 mt onol mirosop. Whol-mount in situ hyriiztion. Gons or in situ hyriiztion wr ollt t E15.5 n ix in 4% prormlhy t 4 1C ovrnight. Tissus wr hyrt in 1% mthnol n stor t 2 1C or us. Digoxignin whol-mount in situ hyriiztion or Dm1 ws prorm s prviously rport 2. RT-PCR. Totl RNA ws isolt rom gons using n RNA miniprp kit (Qign). Totl RNA (1 mg) ws rvrs trnsri with oligo-(t)18n using First-Strn DNA Synthsis Kit or RT-PCR (Roh) in totl rtion volum o 2 ml, o whih 1 ml ws us s tmplt or PCR with primr sts or Dm1, Spo11 n -tin, list in Supplmntry Tl 1. DNA ontnt nlysis o ovrin lls. Cll sprs wr prpr s sri ov or mioti hromosom nlysis xpt tht lls wr not trt with hypotoni solution. Immunostining with rit ntioy to MVH (1:1, ilution) ws prorm s sri ov. DAPI (lu) n MVH (r) imgs wr tkn using n Olympus BX51TF Fluorsn Mirosop, Spot RT igitl mr n Spot 4.1 sotwr (Dignosti Instrumnts). For h NATURE GENETICS VOLUME 38 [ NUMBER 12 [ DECEMBER
5 26 Ntur Pulishing Group sli, mny nonovrlpping ils wr img to nl nlysis o s mny inpnnt lls s possil. All img sts wr pross utomtilly using th sotwr progrm CllProilr ( to nsur onsistnt, unis rsults. Bor prossing, lump lls n nonllulr rtits wr mnully rmov using Ao Photoshop 7.. For purposs o nlysis, kgroun luorsn in MVH imgs low n solut thrshol o.1 ws st to. (Th ynmi rng o inoming imgs is 1.) DAPI n MVH imgs wr lign, n nuli wr intii in DAPI imgs using lol intnsity mxim with mxim supprssion nighorhoo o 7 n lur rius o 3. Egs o nuli wr intii using thrshol lult using Otsu s mtho n n justmnt tor o.7. In this nlysis, ojts touhing th img orr or thos with r smllr thn 35 pixls wr isr. As MVH is ytoplsmi protin, MVH-positiv lls wr intii using nuli s ss or th propgt untion, with rgulriztion tor o.5. For h ll, th primtr o th MVH-positiv rgion ws trmin using thrshol lult using Otsu s mtho n n justmnt tor o.8. Intnsitis o DAPI n MVH signls wr thn lult or h ll. As th mjority o mryoni gonl lls r nonrpliting somti lls (with 2C DNA ontnt), w normliz th intgrt intnsity o DAPI (irtly proportionl to DNA ontnt) o h ll to th min intgrt intnsity o DAPI (st to 2C) ross th il. Cll t rom svrl gons t h tim point n gnotyp wr pool, n histogrms wr prpr with lls sort y DNA ontnt into 4 ins rnging rom C to 8C t.2c inrmnts. Aitionlly, histogrms wr gnrt or grm ll nrih popultions y inning only thos lls with n intgrt intnsity o MVH ov thrshol trmin mpirilly or h t st. Mi. All xprimnts involving mi wr pprov y th Committ on Animl Cr t th Msshustts Institut o Thnology. Not: Supplmntry inormtion is vill on th Ntur Gntis wsit. ACKNOWLEDGMENTS W thnk R. Jnish (Whith Institut or Biomil Rsrh) or v6.5 ES lls; A. Bortvin, T. Hssol n T. Ashly or vi; C. Hyting (Dprtmnt o Gntis, Agriulturl Univrsity, Wgningn) or SCP3 n REC8 ntisr; T. No (Mitsuishi Kgku Institut o Li Sin) or MVH ntisr n E. Anrson, G. Bltus, M. Cplson, M. Gill, J. Kouov, J. Lng, Y. Lim, J. Mullr n J. Potsh or ommnts on th mnusript. Mirosopy n img ptur wr onut in prt t th W.M. Kk Fountion Biologil Imging Fility t th Whith Institut. A.E.C. is Novrtis Fllow o th Li Sins Rsrh Fountion. This work ws support y th Howr Hughs Mil Institut. COMPETING INTERESTS STATEMENT Th uthors lr tht thy hv no ompting innil intrsts. Pulish onlin t Rprints n prmissions inormtion is vill onlin t rprintsnprmissions/ 1. Mrston, A.L. & Amon, A. Miosis: ll-yl ontrols shul n l. Nt. Rv. Mol. Cll Biol. 5, (24). 2. Mnk, D.B., Kouov, J. & Pg, D.C. Sxul irntition o grm lls in XX mous gons ours in n ntrior-to-postrior wv. Dv. Biol. 262, (23). 3. MLrn, A. & South, D. Entry o mous mryoni grm lls into miosis. Dv. Biol. 187, (1997). 4. Ams, I.R. & MLrn, A. Sxully imorphi vlopmnt o mous primoril grm lls: swithing rom oognsis to sprmtognsis. Dvlopmnt 129, (22). 5. Kouov, J. t l. Rtinoi i rgults sx-spii timing o mioti initition in mi. Pro. Ntl. A. Si. USA 13, (26). 6. Bowls, J. t l. Rtinoi signling trmins grm ll t in mi. Sin 312, (26). 7. Yo, H.H., DiNpoli, L. & Cpl, B. Mioti grm lls ntgoniz msonphri ll migrtion n tstis or ormtion in mous gons. Dvlopmnt 13, (23). 8. Bulljos, M. & Koopmn, P. Grm lls ntr miosis in rostro-ul wv uring vlopmnt o th mous ovry. Mol. Rpro. Dv. 68, (24). 9. Oul-Alghni, M. t l. Chrtriztion o prmioti grm ll-spii ytoplsmi protin no y Str8, novl rtinoi i-rsponsiv gn. J. Cll Biol. 135, (1996). 1. Hrtung, M. & Sthl, A. Prlptotn hromosom onnstion in mous oognsis. Cytognt. Cll Gnt. 18, (1977). 11. Dvitor, M., Luini, J.M. & Sthl, A. Do ml grm lls gin miosis uring tl li? J. Gnt. Hum. 27, (1979). 12. Borum, K. Oognsis in th mous. A stuy o th mioti prophs. Exp. Cll Rs. 24, (1961). 13. Sp, R.M. Miosis in th otl mous ovry. I. An nlysis t th light mirosop lvl using sur-spring. Chromosom 85, (1982). 14. Eijp, M., Onrg, H., Jssrgr, R., Rvnkov, E. & Hyting, C. Mioti ohsin REC8 mrks th xil lmnts o rt synptonml omplxs or ohsins SMC1t n SMC3. J. Cll Biol. 16, (23). 15. L, J., Iwi, T., Yokot, T. & Ymshit, M. Tmporlly n sptilly sltiv loss o R8 protin rom mioti hromosoms uring mmmlin miosis. J. Cll Si. 116, (23). 16. Mons, P.B. & Spyropoulos, B. Immunoytology o hismt n hromosoml isjuntion t mous miosis. Chromosom 14, (1995). 17. Prito, I. t l. Cohsin omponnt ynmis uring mioti prophs I in mmmlin ooyts. Chromosom Rs. 12, (24). 18. Rogkou, E.P., Pilh, D.R., Orr, A.H., Ivnov, V.S. & Bonnr, W.M. DNA oulstrn rks inu histon H2AX phosphoryltion on srin 139. J. Biol. Chm. 273, (1998). 19. Pittmn, D.L. t l. Mioti prophs rrst with ilur o hromosom synpsis in mi iint or Dm1, grmlin-spii RA homolog. Mol. Cll 1, (1998). 2. Yoshi, K. t l. Th mous RA-lik gn Dm1 is rquir or homologous hromosom synpsis uring miosis. Mol. Cll 1, (1998). 21. But, F., Mnov, K., Yun, J.P., Jsin, M. & Kny, S. Chromosom synpsis ts n sxully imorphi mioti progrssion in mi lking Spo11. Mol. Cll 6, (2). 22. Romninko, P.J. & Cmrini-Otro, R.D. Th mous Spo11 gn is rquir or mioti hromosom synpsis. Mol. Cll 6, (2). 23. Cron, M., Lvy, E. & Ptrs, H. Th urtion o th prmioti DNA synthsis in mous ooyts. Exp. Cll Rs. 39, (1965). 24. Toyook, Y. t l. Exprssion n intrllulr loliztion o mous Vs-homologu protin uring grm ll vlopmnt. Mh. Dv. 93, (2). 25. Bnnistr, L.A., Rinholt, L.G., Munro, R.J. & Shimnti, J.C. Positionl loning n hrtriztion o mous mi8, isrupt llll o th mioti ohsin R8. Gnsis 4, (24). 26. Yun, L. t l. Th murin SCP3 gn is rquir or synptonml omplx ssmly, hromosom synpsis, n ml rtility. Mol. Cll 5, (2). 27. Xu, H., Bsly, M.D., Wrrn, W.D., vn r Horst, G.T. & MKy, M.J. Asn o mous REC8 ohsin promots synpsis o sistr hromtis in miosis. Dv. Cll 8, (25). 28. Brnnn, J. & Cpl, B. On tissu, two ts: molulr gnti vnts tht unrli tstis vrsus ovry vlopmnt. Nt. Rv. Gnt. 5, (24). 29. Fujiwr, S. & Kwmur, K. Aquisition o rtinoi i signling pthwy n innovtion o th hort oy pln. Zoolog. Si. 2, (23). 3. Alrht, K.H. & Eihr, E.M. Evin tht Sry is xprss in pr-srtoli lls n Srtoli n grnulos lls hv ommon prursor. 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