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