Transcriptome profiling of the murine testis during the first wave of spermatogenesis

T ansc ip ome P o iling o he Mu ine Tes is du ing he Fi s Wa e o Spe ma ogenesis As a Laiho 1 , Noo a Ko aja 3 , A ila Gyenesei 1 , Anu Si onen 2 * 1The Finnish Mic oa ay and Sequencing Cen e, Tu ku Cen e o Bio echnology, Uni e si y o Tu ku and A ˚bo Akademi Uni e si y, Tykis o ¨ka u 6, Tu ku, Finland, 2Ag i ood Resea ch Finland, Bio echnology and Food Resea ch, Animal Genomics, Jokioinen, Finland, 3Depa men o Physiology, Ins i u e o Biomedicine, Uni e si y o Tu ku, Tu ku, Finland Abs ac Co ec gene exp ession pa e ns o m he basis o male ge m cell di e en ia ion and male e ili y. Al hough p e ious s udies ha e elucida ed he impo ance o es is speci ic gene exp ession, he exac ansc ip s and comp ehensi e gene exp ession pa e ns emain unknown. La ge scale sequencing echniques ha e enabled cos e ec i e analysis o gene exp ession and iso o m s udies. Using he SOLiD 4 nex -gene a ion sequencing pla o m we ha e in es iga ed he gene exp ession pa e ns a i e di e en ime poin s du ing he i s wa e on mu ine spe ma ogenesis. Ou esul s highligh he up egula ion o spe ma ogenesis ela ed biological p ocesses and associa ed cellula componen s. Elucida ion o di e en ial gene exp ession a impo an ime poin s du ing he spe m de elopmen emphasizes he impo ance o co ec iming o gene exp ession wi hin biological p ocesses. Di e en ial gene le el exp ession was analyzed wi h R/Bioconduc o ’s Limma package and iso o m analysis was conduc ed wi h he Cu links pipeline. A gene le el o al o 2494 di e en ially exp essed genes we e iden i ied and Cu links cha ac e ized o e 160 000 gene iso o ms, o which 29% we e no el ansc ip s assigned o known genes. Iso o ms we e de ec ed o 57% o exp essed genes and in a o al o e 26 000 genes we e exp essed in he es is. Di e en ial p omo e and ansc ip ion s a si e usage appea s also o play a ole in egula ion o gene exp ession du ing spe ma ogenesis. Fu he mo e, we iden i ied 947 up egula ed long non-coding RNAs du ing he i s wa e o spe ma ogenesis. These RNAs appea ed o be highly speci ic o di e en ime poin s. T ansc ip omic analysis o es is issue samples is highly in o ma i e due o he la ge numbe o exp essed genes and iden i ied iso o ms. Ou s udy p o ides a e y aluable basis o in es iga ion o gene iso o ms and egula ion and ac o s con ibu ing o male e ili y. Ci a ion: Laiho A, Ko aja N, Gyenesei A, Si onen A (2013) T ansc ip ome P o iling o he Mu ine Tes is du ing he Fi s Wa e o Spe ma ogenesis. PLoS ONE 8(4): e61558. doi:10.1371/jou nal.pone.0061558 Edi o : S e an Schla , Uni e si y Hospi al o Mu ¨ns e , Ge many Recei ed Oc obe 29, 2012; Accep ed Ma ch 11, 2013; Published Ap il 17, 2013 Copy igh : ß2013 Laiho e al. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed. Funding: This s udy was unded by he Academy o Finland (h p://www.aka. i/eng). The unde s had no ole in s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he manusc ip . Compe ing In e es s: The au ho s ha e decla ed ha no compe ing in e es s exis . * E-mail: [email p o ec ed] In oduc ion Spe ma ogenesis is a complex p ocess, whe e spe ma ogonia de elop in o highly di e en ia ed spe ma ozoa h ough se e al s ic ly con olled s eps. Mouse spe ma ogenesis begins by mi o ic p oli e a ion o ype A, In e media e and ype B spe ma ogonia. Type B spe ma ogonia hen di ide o o m ea ly spe ma ocy es, which ma ks he beginning o he meio ic phase o spe ma ogen- esis. A e he long-las ing p ophase o he i s meio ic di ision, he emainde o he cell di ision p ocess is comple ed apidly and wo subsequen di isions p oduce haploid ound spe ma ids. In he las di e en ia ion phase (spe miogenesis), spe ma ids unde go d a- ma ic cell ans o ma ion ha includes ch oma in condensa ion and nuclea shaping, emo al o excess cy oplasm, and he ac osome and spe m ail o ma ion. Finally, ma u e spe ma ozoa a e eleased in o he lumen o semini e ous epi helium and anspo ed o he epididymis o u he ma u a ion. De elop- men o spe ma ogonia o haploid spe ma ids akes app oxima ely 35 days in mice [1], o which he spe miogenic phase las s abou 2–3 weeks [2]. The i s wa e o spe ma ogenesis in mouse is ini ia ed only a ew days a e bi h and p oceeds in a synch onized manne . Key ime poin s o appea ance o pa icula ge m cell ypes a e well de ined du ing his i s cycle o spe ma ogenesis. Se en-day-old mouse es es con ain only Se oli cells (SCs) and spe ma ogonia in he semini e ous ubules. A pos na al day (PND) 9 ea ly spe ma ocy es appea , a PND 14 he pachy ene s age o he i s meio ic p ophase is ini ia ed, and a PND 18 la e pachy ene and diplo ene spe ma ocy es a e also p esen . As a esul o meio ic di isions, ound spe ma ids appea a ound PND 20 and a PND 30 spe ma ids ha e al eady eached he elonga ion phase and spe m ail accesso y s uc u es a e being cons uc ed [3]. Due o he synch onized p og ession o he i s wa e o spe ma ogenesis, i p o ides an excellen model sys em o s udy gene exp ession du ing spe m de elopmen . Gene exp ession du ing spe ma ogenesis is highly o ches a ed and s ic ly egula ed a ansc ip ional and pos ansc ip ional le el. In o de o comple e he e y complex de elopmen o spe ma ozoa, se e al speci ic ansc ip ional egula o s a e needed. T ansc ip ion is egula ed bo h by epigene ic ch oma in modi i- ca ions and ans-ac ing ac o s ha bind o he speci ic DNA sequences in he p omo e egions [4–6]. In la e elonga ing spe ma ids ansc ip ion is la gely silenced as a consequence o he ch oma in condensa ion by his one-p o amine ansi ion. Mos o he RNAs ound in male ge m cells du ing his phase a e p oduced PLOS ONE | www.plosone.o g 1 Ap il 2013 | Volume 8 | Issue 4 | e61558 ea lie and a e ansla ionally egula ed in speci ic ibonucleop o- ein pa icles [2]. P e ious s udies o la ge scale gene exp ession in spe ma ogen- esis ha e been conduc ed wi h mic oa ay expe imen s. Se e al s udies ha e been dedica ed o he iden i ica ion o gene exp ession di e ences in no mal e sus e ili y de ec ed pa ien s ( e iewed in [7]). In addi ion, se ial analysis o gene exp ession (SAGE) and mic oa ay p o iling o oden es is samples and en iched cell popula ions ha e iden i ied a la ge numbe o genes impo an o spe ma ogenesis [8–15]. The mos comp ehensi e map o he mammalian es icula exp ession p og am a a cell- ype speci ic le el iden i ied 7066 genes, which we e classi ied in o soma ic (1,684), mi o ic (2,581), meio ic (1,809), and pos meio ic (1,435) exp ession clus e s, howe e his classi ica ion does no necessa ily e lec cell- ype speci ic exp ession [16]. Al hough hese and single gene exp ession s udies ha e been able o elucida e he s age speci ici y and complexi y o es icula ansc ip ion machine y [10], he o e all pic u e o he gene exp ession pa e ns and ansc ip a ie y du ing spe ma ogenesis is s ill la gely unknown. O e 50% o all known mouse genes ha e been shown o be exp essed du ing es icula de elopmen and o e 47% o he exp essed ansc ip s a e uncha ac e ized [17]. In addi ion, i has been e alua ed ha ,30% o he mu ine genome is di e en ially exp essed du ing es is de elopmen . Majo pe iods o exp essional change occu du ing he i s ew days a e bi h (PND 0–6), a he beginning o meiosis (PND 12–14), and along wi h he appea ance o haploid game es (a ound PND 20) [14]. Mic oa ay expe imen s a e limi ed o known ansc ip s and he analysis is hinde ed by c oss-hyb idiza ion and high signal o noise a io. Nex -gene a ion sequencing (NGS) has enabled he ex en- si e in es iga ion o gene exp ession wi hou any p io knowledge o ansc ip con en in he sample. NGS can also iden i y and quan i y a e ansc ip s and p o ides in o ma ion ega ding al e na i e splicing and sequence a ia ion in iden i ied genes. In his s udy, we ha e analyzed he gene exp ession di e ences du ing he i s wa e o mu ine spe ma ogenesis. To al es is issue samples a c i ical ime poin s du ing ge m cell di e en ia ion we e collec ed a PND 7, 14, 17, 21 and 28. Since p ope de elopmen o male ge m cells equi es co ec unc ion o es icula soma ic cells and di e ences in gene exp ession may be a ec ed by cell-cell in e ac ions, he whole es is is a desi able sample ype o in es iga ion o gene exp ession du ing spe ma ogenesis. Replica e samples we e sequenced wi h he SOLiD 4 pla o m and analyzed o de ec ion o di e en ial gene exp ession and splicing a ian s. In addi ion, unc ional anno a ion o iden i ied gene exp ession di e ences highligh ed biological p ocesses impo an o co ec spe m de elopmen . Ma e ials and Me hods E hics S a emen All mice we e handled in acco dance wi h he ins i u ional animal ca e policies o he Uni e si y o Tu ku. Mice we e main ained in a speci ic pa hogen- ee s age a he Cen al Animal Labo a o y o he Uni e si y o Tu ku and sac i iced by CO 2 inhala ion. The s udies we e app o ed by he Labo a o y Animal Ca e and Use Commi ee o he Uni e si y o Tu ku. Animals and Sample P epa a ion Whole es es om wo C57BL/6NHsd mice (Mus musculus) o each ime poin we e collec ed and snap ozen in liquid ni ogen and s o ed a 280uC p io o RNA ex ac ion. The p esence o speci ic cell ypes in he es is samples was con i med wi h squash p epa a ions o he pieces o he semini e ous ubules ollowed by phase con as mic oscopy. A ac ion o he es es was dissec ed and sho ubule segmen s we e cu in PBS [18]. Tubule segmen s we e ans e ed wi h a pipe e on mic oscope slides in 15 mlo PBS. A co e slip was placed ca e ully on o he ubule segmen , and he excess luid was emo ed by blo ing, which allowed he cells o loa ou om he ubule. RNA Ex ac ion and Lib a y P epa a ion To al RNA was ex ac ed using RNeasy Midi ki (Qiagen) ollowing he manu ac u e ’s ins uc ions. The quali y and concen a ions o he RNA was checked wi h Agilen ’s 2100 Bioanalyze (Agilen ) and Nanod op ND-2000 spec opho ome e (The mo Scien i ic). Ribosomal RNA was emo ed wi h Ribomi- nusTM Euka yo e Ki o RNA-Seq (In i ogen) and ibosomal RNA-deple ed o al RNA was agmen ed using RNaseIII, o con e he whole ansc ip ome sample o RNA o a size app op ia e o SOLiD TM Sys em sequencing. A e cleanup using he Pu elinkTM RNA Mic o ki , agmen ed RNA samples wi h su icien yield and an app op ia e size dis ibu ion we e eady o p epa a ion o ampli ied cDNA lib a ies. Quali y o he agmen- a ion was checked wi h Bioanalyze . The agmen ed RNA sample was hyb idized and liga ed wi h he Adap o Mix. RNA popula ion wi h liga ed adap o s was e e se ansc ibed o gene a e single-s anded cDNA copies o he agmen ed RNA molecules. A e a cleanup s ep using he MinElu eHPCR Pu i ica ion Ki , he sample was subjec ed o dena u ing gel elec opho esis, and gel slices con aining cDNA in he desi ed size ange we e excised. The size-selec ed cDNA was ampli ied using 15 cycles o PCR ha akes place in he gel slices. This s ep appends equi ed e minal sequences o each molecule and gene a es su icien empla e o SOLiD sequencing. A e he PCR, he ampli ied cDNA was cleaned up using Pu eLinkTM PCR pu i ica ion ki . Lib a ies we e quan i a ed wi h wo di e en me hods; Qubi luo ome e (In i ogen) and quan i a i e PCR o wa an he accu acy. SOLiD Lib a y TaqMan Quan i a ion Ki was used o de e mining he mola concen a ion o ampli ied empla e in a SOLiD lib a y. In qPCR, he s anda d and unknown lib a y empla e a e ampli ied using wo sequence-speci ic p ime s wi h a TaqMan luo ogenic p obe labeled wi h FAM TM dye and a dye quenche . The uni o mi y o agmen size o lib a ies was con i med wi h Bioanalyze . Templa ed bead p epa a ion was pe o med by emulsion PCR (ePCR). SOLiD TM EZ Bead TM ins umen a ion was used o empla ed bead p epa a ion. T ansc ip omic Da a Analysis The colo space eads ob ained om he SOLiD sequence we e aligned agains he mouse e e ence genome (mm9 assembly) using he s anda d whole ansc ip ome pipeline and he colo space alignmen ool p o ided by Applied Biosys ems and dis ibu ed wi h he ins umen (Li eScope 2.1). Reads associa ed wi h ibosomal RNA, ans e RNA, epea s and o he unin o ma i e eads we e il e ed ou du ing he p ocess as well as eads wi h mo e han 10 po en ial alignmen s. A e alignmen o he e e ence genome, he unique eads we e associa ed wi h known genes based on Ensembl anno a ions, and he numbe o eads aligned wi hin each gene was coun ed. The coun s a e epo ed as RPKM (Reads Pe Kilobase o exon model pe Million mapped eads) alues, which a e used o no malizing he coun alues by aking he gene leng hs in o accoun . The alues p oduced a e also independen o he o al numbe o he eads in each sample and hus make he da a compa able ac oss he sample se . Quali y con ol o he da a was pe o med using R/ Bioconduc o and R-package Limma was used o pe o ming T ansc ip ome o he Mouse Tes is PLOS ONE | www.plosone.o g 2 Ap il 2013 | Volume 8 | Issue 4 | e61558 he s a is ical es ing be ween he g oups. Limma was es ed wi h a ious da a no maliza ion and ans o ma ion me hods, bu he choice o p ep ocessing me hods did no ha e a signi ican e ec in he anking o he esul genes. The sample g oups we e compa ed in ch onological o de . P- alue o a leas 0.01 and an absolu e old-change o a leas wo was equi ed o a gene o be called signi ican ly di e en ially exp essed be ween he g oups. I was also equi ed ha he samples had a leas 10 uni di e ence be ween he g oup mean exp ession alues. TopGO and GOs a s R/Bioconduc o packages we e used o unc ional en ichmen analysis o he di e en ially exp essed il e ed gene lis s owa ds KEGG pa hways and Gene On ology ca ego ies. Hea maps compa ing he en ichmen esul s ac oss compa isons we e p oduced wi h GENE-E ool (h p://www.b oadins i u e.o g/ cance /so wa e/GENE-E/) using pea son’s co ela ion me ics and a e age linkage dis ance me ics. Gene a ian de ec ion and di e en ial ansc ip exp ession analysis we e ca ied ou using he Cu links pipeline [19]. The Li eScope mapping esul s we e used as an inpu also o he Cu links analysis. In he Cu links analysis he coun s a e epo ed as FPKM (F agmen s Pe Kilobase o exon model pe Million mapped eads) alues. GO en ichmen analyses o Cu links esul s we e ca ied ou using GO illa [20,21]. Resul s Da a E alua ion The samples we e sequenced wi h he SOLiD 4 ins umen a 50 bp ead leng h. In o al 10 samples we e used o he s udy. Samples we e collec ed a di e en ime poin s du ing he i s wa e o mouse spe ma ogenesis. Each ime poin co esponds o speci ic cell con en in he es is. Fi s ime poin was a PND 7, when only soma ic cells and spe ma ogonia a e p esen in he es is. The ea e addi ional cell ypes a e p esen in selec ed samples in ch onological o de : PND 14 con ains ea ly spe ma o- cy es, PND 17 la e spe ma ocy es, PND 21 ound spe ma ids and inally PND 28 elonga ing spe ma ids. The assumed cell con en in each sample was also con i med by phase con as mic oscopy o squash p epa a ions (Figu e S1A). The analysis o squash p epa a ions con i med he expec ed and simila cell con en in each sample g oup. In addi ion, he es icula weigh s wi hin he g oups we e highly compa able (Figu e S1B). Each ime poin was analyzed in wo biological eplica es and PND 7 samples we e di ided o wo ba codes o echnical easons (Table 1). To al numbe o eads a ied om 18 o 54 million, he mapping a e o il e ed eads being 32–52% (Table 1). Mos o he eads we e mapped on exons (50%), al hough a high numbe o eads was also mapped on in ons o in e genic egions (Table 1). Some di e ences in ead coun s we e obse ed be ween biological eplica es, especially be ween PND_17_s1 and s2. The s a ing ma e ial (amoun o o al RNA) was iden ical in hese samples, hus he di e ences a ise possibly om he sample p epa a ion and Solid un. Howe e , he di e ence in o al ead coun s do no signi ican ly a ec he exp ession analysis, since he RPKM and FPKM alues a e co ec ed wi h he o al ead coun o each sample. Visualiza ion wi h he IGV con i med he e en dis ibu- ion o eads h oughou he genome and a exonic egions (da a no shown). Fo gene exp ession analysis, only hose ea u es we e used, which had non-ze o coun alues among he sample se (n = 26 789 genes). The esul s o o al and mapped eads, mapping a e, o al eads passing il e s, coun ed exons, in ons and in e genic egions a e p esen ed in Table 1. Sample Co ela ions Pea son’s me ics was used o measu e he simila i y be ween samples. Co ela ions be ween eplica e samples a ied be ween 0.97 and 0.99 depic ing e y high ep oducibili y. The samples clus e ed acco ding o he sample g oups. PND 14 and 17 sample g oups we e highly simila o each o he . Sample g oups PND28 and PND7 we e mo e dis an o o he g oups. Figu e 1A shows he hie a chical clus e ing o he indi idual samples. Compa isons o he Sample G oups The sample g oups we e compa ed in ch onological o de using he R-package Limma. Compa ison o PND 14 and PND 17 samples had he lowes amoun o di e en ially exp essed (DE) genes (Table 2) consis en wi h he esul s ob ained wi h hie a chical clus e ing (Figu e 1A). A high numbe o DE genes we e p esen be ween PND 7/14 and 17/21 and 21/28 (Table 2) and mos o he iden i ied DE genes we e speci ically egula ed in one compa ison (Figu e 1B). Highes di e ence in gene exp ession was de ec ed be ween samples PND21 and PND28 du ing spe ma id di e en ia ion. To al numbe o 906 DE genes we e ound be ween hese ime poin s, and 612 o hem we e di e en ially exp essed only in his compa ison (Table 1 and Figu e 1B). These esul s co ela e wi h he expec ed pa e n o gene exp ession du ing spe ma ogenesis. The mos p ominen Table 1. SOLiD 4 ead coun s and mapping s a is ics o indi idual samples. Sample To al eads Mapped eads Mapping a e To al eads passing il e s Coun ed on exons Coun ed on in ons Coun ed on in e genic egions PND_7_s1 45,706,071 21,230,399 47% 14,518,593 9,476,631 4,001,849 1,040,113 PND_7_s2 46,592,933 16,249,678 35% 10,811,100 6,911,439 3,074,225 825,436 PND_14_s1 33,787,664 16,249,484 48% 11,373,935 7,657,560 2,599,696 1,116,679 PND_14_s2 26,776,971 13,525,537 50% 8,652,432 6,946,490 1,037,577 668,365 PND_17_s1 54,374,927 25,471,965 46% 15,825,273 13,133,830 1,490,487 1,200,956 PND_17_s2 16,733,599 7,235,322 43% 4,611,709 3,890,612 404,87 316,227 PND_21_s1 26,409,512 8,475,148 32% 5,700,864 3,961,522 1,006,336 733,006 PND_21_s2 23,544,136 11,445,588 49% 7,403,489 6,150,493 635,138 617,858 PND_28_s1 29,895,585 15,482,573 52% 10,513,142 8,709,227 814,463 989,452 PND_28_s2 23,640,573 9,106,849 39% 6,141,624 5,064,489 467,358 609,777 doi:10.1371/jou nal.pone.0061558. 001 T ansc ip ome o he Mouse Tes is PLOS ONE | www.plosone.o g 3 Ap il 2013 | Volume 8 | Issue 4 | e61558 mo phological and biochemical changes in spe m de elopmen occu du ing spe ma id di e en ia ion s a ing om PND 20. T ansc ip ion o genes equi ed o he cons uc ion o spe m- speci ic s uc u es s a s al eady in la e spe ma ocy es and con inues h oughou he ound spe ma id phase and in ea ly elonga ing spe ma ids. Mos o he ansc ip s needed du ing he la e s eps o spe miogenesis ha e o be p esen in ea ly elonga ing spe ma ids a PND 28 be o e he cease o ansc ip ion in condensing nucleus. Highes co ela ion in gene exp ession di e ences was de ec ed be ween sample compa ison o PND 17/21 and PND 21/28 ( o al numbe o common DE genes 255, Figu e 1B), whe e almos all common DE genes we e egula ed in he same di ec ion in bo h compa isons (99%, Figu e 1C and D). High numbe o o e lapping (n = 203) and ag eeing (98%) DE genes we e also p esen in sample compa isons o PND 7/14 and PND 17/21 indica ing a change in he ansc ip ion o same genes a ea ly spe ma ocy e and spe ma id le el (Figu e 1B–D). Compa ison o PND 14/17 appea ed o ha e mo e disag eeing DE genes when compa ed o o he compa isons. Also a low numbe o sha ed genes wi h o he compa isons we e de ec ed, p obably due o he low o al numbe o DE genes in his g oup. Di e en DE genes appea ed also o be p esen in compa ison o PND 7/14 and 21/ 28, which is expec ed. The numbe s o o e lapping di e en ially Figu e 1. Clus e ing o sample g oups and o e lapping DE genes. A. Hie a chical clus e ing o he no malized da a. Biological eplica e samples clus e closely oge he . Sample g oups PND28 and PND7 a e mo e dis an o o he g oups and PND14 and PND17 a e simila o each o he based on he gene exp ession di e ences. B. To al numbe o o e lapping DE genes be ween compa isons. C. Ag eeing and o e lapping up egula ed genes be ween compa isons. D. Ag eeing and o e lapping down egula ed genes be ween compa isons. doi:10.1371/jou nal.pone.0061558.g001 Table 2. To al numbe o Ensemble anno a ed DE genes and up- and down egula ed gene numbe s be ween sample g oups. To Up Down PND7/PND14 724 632 92 PND14/PND17 136 100 36 PND17/PND21 728 665 63 PND21/PND28 906 838 68 doi:10.1371/jou nal.pone.0061558. 002 T ansc ip ome o he Mouse Tes is PLOS ONE | www.plosone.o g 4 Ap il 2013 | Volume 8 | Issue 4 | e61558 exp essed ea u es be ween he compa isons a e p esen ed in igu e 1B, ag eeing up egula ed genes in Figu e 1C and down egula ed genes in 1D. A gene le el highes anked genes included se e al ibosomal and mi ochond ial genes and non-coding RNAs, which exp ession was high in all sample g oups. Howe e , he amoun o highly exp essed es is speci ic genes and he le el o exp ession inc eased du ing he p og ess o spe ma ogenesis. The wen y highes exp essed genes a each ime poin a e shown in Table S1 and he ull gene lis s ha e been submi ed o GEO-da abase (h p:// www.ncbi.nlm.nih.go /geo/) wi h accession numbe GSE39970. In ou expe imen al se up, he whole es is RNA was used o sequencing, and he p oduced da a includes bo h ge m cell and soma ic es icula cell de i ed ansc ip s. In o de o unde s and he con ibu ion o soma ic cells in he gene exp ession di e ences, we analysed he exp ession pa e ns o p e iously iden i ied SC and Leydig cell age speci ic genes [22,23]. Ve y low exp ession o SC speci ic genes Pla2g4a, Gap43 (Figu e S2A) and no exp ession o Pap3 we e iden i ied and no change in he exp ession pa e n was de ec ed. In addi ion, exp ession o SC sec e ed an i- Mu¨lle ian ho mone (Amh), ollicle-s imula ing ho mone ecep o (Fsh ) and SC speci ic cance -associa ed genes [22] showed low le el exp ession ha dec eased du ing he i s wa e o spe ma o- genesis, possibly e lec ing he inc ease in ge m cell popula ion (Figu e S2A). The analysis o gene exp ession o p e iously iden i ied Leydig cell speci ic genes [24,25] showed some di e ences in pos -na al gene exp ession pa e n (Figu e S2B). Two o he analyzed s e oidogenic genes (S a and Vcam1) had highes exp ession a PND17 and Cy och ome P450 genes Cyp11a1 and Cyp17a1 a PND 28. De elopmen o he adul popula ion o Leydig cells occu s a e PND 10 and he exp ession o s e oidogenic genes inc eases eaching adul le els in he mouse by PND 25 [25,26]. Thus, i is possible ha some o he iden i ied gene exp ession di e ences de i e om soma ic cells. Func ional Analysis TopGO and GOs a s R/Bioconduc o packages we e used o en ichmen analysis o he di e en ially exp essed il e ed gene lis s owa ds KEGG pa hways and Gene On ology ca ego ies. In gene al, a high numbe o en iched biological p ocesses and cellula componen s we e ound. Analysis o molecula unc ion GO e ms esul ed wi h only ew signi ican en ichmen s. In o al 97 GO e ms unde biological p ocesses we e signi ican ly (p,0.01) en iched du ing he i s wa e o spe ma ogenesis (Figu e 2A). Al hough mino pa o he di e en ial gene exp ession may come om he soma ic cells (Figu e S2), ge m cell de elopmen associa ed e ms we e highligh ed in he en ichmen analysis. DE genes du ing he ansi ion om spe ma ogonia o ea ly spe ma ocy es (PND7_ s_PND 14) we e highly en iched in biological p ocessed in ol ed in mi o ic and meio ic pa hways. Also exp ession o genes in ol ed in RNA p ocessing and anspo we e signi ican ly al e ed. Du ing meio ic p og ess (PND 14_ s_PND 17) genes in ol ed in pa hways o ch omosome o ganiza ion and nucleus packing we e u he highligh ed. A he onse o pos -meio ic spe ma id phase (PND 17_ s_PND 21) cell wall associa ed pa hways, py imidine p ocessing and cilia y mo ili y we e highligh ed. Finally, he ansi ion om ound o elonga ing spe ma ids induced di e en ial exp ession o genes impo an o usion o spe m o egg. Th oughou he i s wa e o spe ma ogenesis en iched GO e ms in biological p ocess we e associa ed wi h spe ma ogenesis, ep oduc ion, meiosis and e iliza ion (Figu e 2A). En iched GO e ms unde cellula componen s included 60 di e en e ms (p,0.01, Figu e 2B). Be ween PND 7 and 14 gene exp ession in RNA p ocessing associa ed wi h ch oma oid body, pi-body, p-g anule and RNA g anule was al e ed. Also mic o u- bule and cilia ela ed GO e ms we e en iched al eady in ea ly spe ma ocy es (PND14) including cen osome, mic o ubule o ga- nizing cen e , mic o ubule cy oskele on, p ima y cilium and dynein complex. Fu he mo e, GO e ms ela ed o ch omosome o ganiza ion appea ed o be en iched al eady in ea ly spe ma o- cy es (Figu e 2B). Nucleosomal genes we e also di e en ially exp essed al eady in ea ly spe ma ocy es and u he in la e spe ma ocy es and elonga ing spe ma ids. En iched GO e ms be ween PND 17 and 21 included cilium, lagellum and ac osomal esicle ela ed e ms, which we e u he en iched be ween PND 21 and 28 consis en wi h he spe m ail and ac osome o ma ion in spe ma ids. Ou analysis o he en ichmen o DE genes in KEGG pa hways e ealed 13 pa hways, which we e en iched al eady be ween PND 7 and 14. p53 signalling, TCA cycle, cell cycle, basal ansc ip ion ac o s, oocy e meiosis and RNA ansc ip ion we e mainly en iched in ea ly spe ma ocy es (Figu e 2C). mRNA su eillance pa hway and glycolysis we e en iched in all compa isons, bu mo e signi ican ly be ween PND 7/14 and 14/17 o mRNA and 7/14 and 17/21 o glycolysis. Glyce olipid me abolism and p o ein p ocessing in endoplasmic e iculum we e highly en iched be ween PND 7/14 and PND 21/28. F uc ose and mannose me abolism and pen ose phospha e pa hway we e en iched un il PND28. In addi ion, me abolic pa hways we e en iched h ough- ou he i s wa e o spe ma ogenesis (Figu e 2C). Func ional Compa ison o Up- and Down egula ed Genes En ichmen s o GO e ms we e also analyzed sepa a ely o up- and down egula ed genes in o de o iden i y di ec ion o gene exp ession in di e en e ms. This analysis was ca ied ou using GO illa web ool [20,21]. Figu e S3 shows he o e all dis ibu ion o iden i ied GO e ms among up- and down egula ed gene lis s. Consis en ly wi h he high numbe o up egula ed genes in each compa ison, mos o he en iched GO e ms o DE genes we e iden i ied in he up egula ed gene g oups. Howe e , in he compa ison o PND 14/17 en iched GO e ms we e s a is ically signi ican (p,0.01) only in he down egula ed genes. In his compa ison, almos all up egula ed genes we e non-coding o spliceosomal RNA genes (Table S2). As mos o he DE genes we e up egula ed, also he GO e ms iden i ied wi hin he up egula ed DE genes co esponded wi h he GO e ms iden i ied wi hin he ull se o DE genes (Figu e 3). Game e gene a ion, ge m cell de elopmen , spe m-egg ecogni- ion, e iliza ion and spe m mo ili y ela ed GO e ms we e up egula ed in all compa isons (excep PND 14/17) (Figu e 3A,C and E). GO e ms speci ic o spe ma ogonia ea ly spe ma ocy e ansi ion we e cell cycle p ocesses, ch omosome o ganiza ion, RNA anspo , DNA me abolic p ocesses (me hyla ion and epai ) and ATP and piRNA me abolic p ocesses. Also, speci ic GO e ms we e iden i ied o spe ma id elonga ion phase be ween PND 21 and 28 and we e mainly ela ed o e iliza ion; egg ac i a ion, ac osome eac ion and memb ane usion. En iched cellula componen e ms o up egula ed genes co ela ed wi h he BP e ms (Figu e 3B,D and F). The iden i ied gene exp ession changes a e consis en wi h he de elopmen al p ocesses in male ge m cell ma u a ion. Venn diag ams o sample g oup compa - isons wi h en iched biological p ocess and cellula componen GO e ms o up egula ed genes a e shown in igu e S4. GO e ms, which we e en iched in all sample g oup compa - isons (excep PND 14/17) we e in es iga ed a gene le el in o de o see, i same genes a e up o down egula ed a di e en ime T ansc ip ome o he Mouse Tes is PLOS ONE | www.plosone.o g 5 Ap il 2013 | Volume 8 | Issue 4 | e61558 T ansc ip ome o he Mouse Tes is PLOS ONE | www.plosone.o g 6 Ap il 2013 | Volume 8 | Issue 4 | e61558 poin s. Some o he up egula ed genes we e de ec ed in se e al sequen ial g oup compa isons, bu in gene al, highly speci ic gene exp ession was iden i ied be ween he speci ic ime poin compa isons. PND 7/14 and 21/28 appea ed o ha e he mos di e en up egula ed gene exp ession pa e ns (Figu es 1 and 4). We analyzed he DE genes en iched unde he GO e m game e gene a ion mo e ca e ully and showed ha de elopmen al phase speci ic genes we e up egula ed in each compa ison (Figu e 4). The cellula compa men GO e ms iden i ied o hese genes demons a ed ha genes speci ically up egula ed in game e Figu e 2. Highes anked GO e ms (p , 0.01) wi h di e en ial exp ession be ween sample g oups o GO biological p ocesses (A) and cellula componen s (B) and o KEGG pa hways (C). The ela i e signi icance o e m en ichmen is indica ed by le els o ed shading ( he lowe he p- alue he da ke shade o ed colou ). Hea map was p oduced wi h GENE-E (h p://www.b oadins i u e.o g/cance /so wa e/GENE-E/) using pea son’s co ela ion me ics and a e age linkage dis ance me ics. doi:10.1371/jou nal.pone.0061558.g002 Figu e 3. En iched biological p ocess and cellula componen GO e ms among up egula ed genes. Compa ison o PND14/17 is no p esen ed due o low numbe o DE genes. A. PND 7/14 biological p ocesses, B. PND 7/14 cellula componen , C. PND 17/21 biological p ocesses, D. PND 17/21 cellula componen , E. PND 21/28 biological p ocesses and F. PND 21/28 cellula componen . Only lowe le el GO e ms a e shown. doi:10.1371/jou nal.pone.0061558.g003 T ansc ip ome o he Mouse Tes is PLOS ONE | www.plosone.o g 7 Ap il 2013 | Volume 8 | Issue 4 | e61558 gene a ion be ween samples PND 7 and 14 (n = 28) we e localized o nucleus, cy oplasm, ch oma oid body, piP body, polysome, synap onemal complex and condensed nuclea ch omosome (Figu e 4). Speci ic up egula ed genes be ween PND 17 and 21 (n = 15) we e localized in he lagellum and mi ochond ial inne memb ane pep idase complex and in compa ison o PND 21/28 (n = 22) in he ac osomal esicle, Ca Spe complex, lagellum and ou e dense ibe (Figu e 4). Genes up egula ed in all compa isons (n = 6, P m1, Tnp2, Tcp11, Spa a24, Spa a19 and Tnp1) we e associa ed wi h nucleus, nucleosome and ch omosome. Gene Va ian Analysis Gene a ian de ec ion and di e en ial ansc ip exp ession analysis was ca ied ou using he Cu links pipeline [19]. On a e age 17 000 genes, 50 000 known iso o ms and 25 000 no el iso o ms we e exp essed in each sample g oup (FPKM.1, Figu e 5A). Di e en ial p omo e and ansc ip ion s a si e usage we e also analyzed be ween all sample g oups highligh ing 500– 800 and 7 000 - 15 000 a ian s be ween sample compa isons, espec i ely (Figu e 5B and C). Al hough he di e en ial gene exp ession analysis wi h Limma showed a low numbe o DE genes be ween PND 14 and 17, a high di e ence in p omo e and ansc ip ion s a si e usage was de ec ed wi h Cu links (Figu e 5B and C). The compa ison be ween ansc ip s iden i ied by Cu links and he Ensembl e e ence ansc ip s elucida ed he p e iously unknown splicing e en s compa ed o known ansc ip s. Mos o he Cu links ansc ip s (46%) had a comple e ma ch wi h a e e ence ansc ip (Table 3). 28% o he iden i ied ansc ip s we e desc ibed as po en ially no el iso o ms and 25% had no ma ch in gene o ansc ip le el o he e e ence ansc ip s. Exonic o e lap wi h e e ence on he opposi e s and was ound in 937 ansc ip s (Table 3), which may co espond o po en ial an isense egula o s o sense ansc ip s. Simila dis ibu ion o iso o ms was de ec ed o each sample g oup sepa a ely (da a no shown). In o al 161 362 ansc ip s we e iden i ied in he mouse es is. The GO e m en ichmen o he di e en ially exp essed iso o ms gene a ed by di e en ial p omo e usage we e analyzed using GO illa wi h di e ences decla ed a high signi icance le el (p,10 26 ). Genes wi h di e en ial p omo e usage we e associa ed wi h molecula unc ion GO e ms ca aly ic ac i i y and binding (p o ein, ion, nucleo ide) in all compa isons. On he whole, gene al biological p ocesses we e en iched wi hin genes ela ed o Figu e 4. Venn diag am and en iched cellula complex GO e ms o up egula ed genes in biological p ocess GO e m game e gene a ion. Only lowe le el GO e ms a e shown. doi:10.1371/jou nal.pone.0061558.g004 T ansc ip ome o he Mouse Tes is PLOS ONE | www.plosone.o g 8 Ap il 2013 | Volume 8 | Issue 4 | e61558 di e en ial p omo e usage (Figu e 6A). Genes associa ed wi h en iched biological p ocesses a each ime poin a e p esen ed in able S3. Co esponding en ichmen pa e n was also de ec ed in cellula componen GO e ms (Figu e 6B). Cy oplasm, nucleus, cy oplasmic pa and p o ein complex we e en iched in all compa isons. Genes associa ed wi h mi ochond ia we e en iched be ween PND 14/17 and 17/21. Mic o ubule and cell p ojec ion ela ed genes we e en iched be ween PND 17/21 and cy oskele on genes be ween PND 17/21 and 21/28. Finally, genes localized o cen osome we e en iched be ween PND 21 and 28 (Figu e 6B). Cen osome associa ed genes we e in ol ed in cen osome duplica ion, spindle o ganiza ion, mi osis, p o ein localiza ion, egula ion o mic o ubule cy oskele on o ganiza ion and cilium assembly. In o al 81 genes we e iden i ied as di e en ially exp essed speci ically a iso o m le el (Table S4). Six o he iden i ied genes ( o al n = 12) a ea ly spe ma ogenesis we e associa ed wi h p o ein binding (Table S4). Mos o he DE iso o ms (n = 23, no assigned Z p706, Usp9x and I na 1) be ween PND 14 and 17 we e in ol ed in cellula p ocesses and se en o hese genes we e associa ed wi h p o ein domain speci ic binding, nine wi h nucleo ide binding and wo wi h elome ic DNA binding (Table S4). DE iso o ms be ween PND 17/21 and 21/28 did no show any signi ican en iched p ocesses o unc ions. Al hough he exp ession pa e n o iso o ms gene ally co ela ed wi h he gene le el exp ession pa e ns, a high numbe o iso o ms iden i ied in his analysis and di e en ial exp ession pa e ns o some speci ic iso o ms p o ide e idence o he complexi y o gene exp ession in male ge m cells. Exp ession o Non-coding RNAs Fo iden i ica ion o long non-coding RNAs (lncRNAs) he colo space eads we e mapped agains he NONCODER 3 lncRNA da ase using CLC Genomics Wo kbench ( .5.5.1). In o al close o 30 000 ncRNAs we e iden i ied as exp essed in he analysed sample g oups. The ncRNAs wi h highes exp ession in all sample g oups we e mainly ibosomal RNAs wi h some p o ein anspo (SRP_7SL), ansc ip ion elonga ion ela ed (7SK) and mi ochond ial RNAs (AK131596) (Table S5). The di e en ial exp ession analysis o lncRNA highligh ed speci ic g oups o up- and down egula ed genes a each ime poin du ing he i s wa e o spe ma ogenesis. In o al o 108 down egula ed (Figu e 7A) and 947 up egula ed lncRNAs (Figu e 7B) we e iden i ied wi h high co ela ion be ween samples o he same g oup (Figu e 7C). Highes numbe o up egula ed DE lncRNAs was iden i ied a c i ical phases o spe ma ogenesis du ing he appea ance o spe ma ocy es (PND 7/14) and spe ma id di e en ia ion (PND 21/28) (Figu e 7B). The highes numbe o down egula ed genes was de ec ed du ing ea ly spe ma ogenesis (PND 7/14), and also be ween PND 17 and 21 du ing he ansi ion om he meio ic o he pos -meio ic p og am (Figu e 7A). The down egula ion old change (FC) was low, al hough some s a is ically signi ican (p,0.01, FC .2) DE lncRNAs we e iden i ied (Figu e 7A). Up egula ed lncRNAs Figu e 5. Gene and iso o m coun s and p omo e and ansc ip ion s a si e usage in sample g oups. A. Exp essed gene, known and no el iso o m coun s a di e en ime poin s du ing he i s wa e o spe ma ogenesis. FPKM .1. B. Di e en ial p omo e usage in all sample g oup compa isons. C. Di e en ial ansc ip ion s a si e usage be ween all sample g oups. The compa isons in ch onological o de du ing he i s wa eo spe ma ogenesis a e indica ed in black (B and C). doi:10.1371/jou nal.pone.0061558.g005 Table 3. Desc ip ion o iden i ied gene iso o ms classes. Desc ip ion T ansc ip s % Exonic o e lap wi h e e ence on he opposi e s and 937 0.6 Gene ic exonic o e lap wi h a e e ence ansc ip 692 0.4 Po en ially no el iso o m: a leas one splice junc ion is sha ed wi h a e e ence ansc ip 45580 28.2 Comple e ma ch 74122 45.9 No e e ence ansc ip o e lap 40030 24.8 To al numbe o ansc ip s 161362 100 doi:10.1371/jou nal.pone.0061558. 003 T ansc ip ome o he Mouse Tes is PLOS ONE | www.plosone.o g 9 Ap il 2013 | Volume 8 | Issue 4 | e61558