RESEARCH ARTICLE
Ge m cell desquama ion-based es is
eg ession in a seasonal b eede , he Egyp ian
long-ea ed hedgehog, Hemiechinus au i us
Diaa Massoud
1☯
, Miguel Lao-Pe
´ ez
2☯
, Alicia Hu ado
2☯
, Walied Abdo
3
, Rogelio Palomino-
Mo ales
4
, F ancisco Da id Ca mona
2
, Miguel Bu gos
2
, Ra ael Jime
´nezID
2
*, F ancisco
J. Ba ionue o
2
1Depa men o Zoology, Facul y o Science, Fayoum Uni e si y, Fayoum, Egyp , 2Depa amen o de
Gene
´ ica e Ins i u o de Bio ecnologı
´a, Uni e sidad de G anada, G anada, Spain, 3Depa men o Pa hology,
Facul y o Ve e ina y Medicine, Ka El Sheikh Uni e si y, Ka El Sheikh, Egyp , 4Depa amen o de
Bioquı
´mica y Biologı
´a Molecula I, Uni e sidad de G anada, G anada, Spain
☯These au ho s con ibu ed equally o his wo k.
*[email p o ec ed]
Abs ac
Tes es o seasonally b eeding species expe ience a se e e unc ional eg ession be o e he
non-b eeding pe iod, which implies a subs an ial mass educ ion due o massi e ge m-cell
deple ion. Two al e na i e mechanisms o seasonal ge m-cell deple ion ha e been
desc ibed in mammals, apop osis and desquama ion (sloughing), bu hei p e alence has
no been de e mined ye due o educed numbe o species s udied. We pe o med a mo -
phological, ho monal, and molecula s udy o he mechanism o seasonal es icula eg es-
sion in males o he Egyp ian long ea ed-hedgehog (Hemiechinus au i us). Ou esul s show
ha li e, non-apop o ic, ge m cells a e massi ely deple ed by desquama ion du ing he es-
is eg ession p ocess. This is concomi an wi h bo h dec eased le els o se um es os e -
one and i egula dis ibu ion o he cell-adhesion molecules in he semini e ous epi helium.
The inac i e es es main ain some meio ic ac i i y as meiosis onse is no hal ed and spe -
ma ocy es die by apop osis a he pachy ene s age. Ou da a suppo he no ion ha apop o-
sis is no he majo es is eg ession e ec o in mammals. Ins ead, desquama ion appea s
o be a common mechanism in his class.
In oduc ion
In empe a e a eas o he Ea h, animals ep oduce when he en i onmen al condi ions a e
op imal o maximize g ow h a e and su i al o newbo ns. In mos species li ing a hese la i-
udes, pho ope iod is he cue con olling hei ep oduc i e hy hm. Howe e , o he ac o s,
such as ood a ailabili y, s ess, and wea he , can ei he modi y o e en o e come he in luence
o pho ope iod. En i onmen al cues ac by modula ing he exp ession o he ho mones
eleased by he hypo halamic–pi ui a y–gonadal (HPG) axis. The le els o se um gonado o-
pins a e lowe in he non-b eeding pe iod, a ac ha in males leads o a educ ion o ci cula -
ing es os e one, which is associa ed o spe ma ogenesis inhibi ion and, as a consequence, o
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0204851 Oc obe 4, 2018 1 / 16
a1111111111
a1111111111
a1111111111
a1111111111
a1111111111
OPEN ACCESS
Ci a ion: Massoud D, Lao-Pe
´ ez M, Hu ado A,
Abdo W, Palomino-Mo ales R, Ca mona FD, e al.
(2018) Ge m cell desquama ion-based es is
eg ession in a seasonal b eede , he Egyp ian
long-ea ed hedgehog, Hemiechinus au i us. PLoS
ONE 13(10): e0204851. h ps://doi.o g/10.1371/
jou nal.pone.0204851
Edi o : S e an Schla , Uni e si y Hospi al o
Mu¨ns e , GERMANY
Recei ed: Ma ch 8, 2018
Accep ed: June 5, 2018
Published: Oc obe 4, 2018
Copy igh : ©2018 Massoud 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.
Da a A ailabili y S a emen : All ele an da a a e
wi hin he pape .
Funding: This s udy has been suppo ed by he
Agencia de Inno acio
´n y Desa ollo de la “Jun a de
Andalucı
´a” h ough G oup PAI BIO-109 and he
Spanish “Sec e a ı
´a de Es ado de In es igacio
´n,
Desa ollo e Inno acio
´n” h ough he g an CGL-
2015-67108-P, which includes FEDER unds.
Compe ing in e es s: The au ho s ha e decla ed
ha no compe ing in e es s exis .
he loss o he ge mina i e epi helium and he subsequen educ ion in es icula size and mass
[1]. In e speci ic di e ences ha e been epo ed ega ding he condi ion in which he inac i e
es is is main ained a e seasonal eg ession. In some species, meio ic ac i i y is no
comple ely abolished du ing he non-b eeding season as meiosis onse con inues and p ima y
spe ma ocy es a e subsequen ly deple ed by apop osis [2,3]. In con as , in o he species meio-
sis onse is hal ed and he semini e ous ubules e ain only Se oli and spe ma ogonial cells [4,
5]. Massoud e al., [6] epo ed ha sou he n and no he n popula ions o he g ea e whi e-
oo hed sh ew, C ocidu a ussula, exhibi di e en seasonal b eeding pa e ns in he Ibe ian
Peninsula. In he no h, ep oduc ion occu s in summe and males unde go comple e es is
eg ession in win e . On he o he hand, in he sou h ep oduc ion akes place in win e and
no es is eg ession occu s du ing he non-b eeding season ( emales a e no ecep i e in sum-
me ). The au ho s p oposed ha his isa case o adap i e lack o seasonal in olu ion.
In addi ion o ge m cells, Leydig cells also show species-speci ic di e ences in he eg essed
es is [7].
The mechanisms o es is eg ession a e also a iable among species. Mos s udies ha e
poin ed o apop osis as he main cellula e en esponsible o es is eg ession [8,9]. Howe e ,
mo e ecen ly, ge m cell desquama ion (sloughing) has been p oposed as a new mechanism o
es is eg ession, as epo ed o bo h he Ibe ian mole Talpa occiden alis [10] and he la ge
hai y a madillo Chae oph ac us illosus [11]. Dadhich e al. [10] p oposed ha low le els o
in a- es icula es os e one de egula e he exp ession o he cell-adhesion molecules, leading
o he loss o Se oli-Se oli and Se oli-ge m cell junc ions, which esul s in he sloughing o
he meio ic and pos -meio ic ge m cells placed in he adluminal compa men o he semini -
e ous ubules. Mo eo e , in a e y ecen s udy, Gonza
´lez e al. [12] p oposed a no el mecha-
nism o es is eg ession in which he balance be ween apop osis and au ophagy egula es his
p ocess, as epo ed in he izcacha, Lagos umus maximus.
Thus, analysing he ci cannual es icula dynamics in new mammalian species would help
1) o elucida e which mechanisms o es is eg ession a e e olu iona y conse ed and 2) o es i-
ma e he in e -species a ia ion exis ing in he p ocess. The long ea ed-hedgehog, Hemiechinus
au i us, is one o he six A ican species o spiny hedgehogs belonging o he amily E inacei-
dae. I is dis ibu ed in he coas al semi-dese a eas o Libya and Egyp , in he Cyp io island,
he Middle Eas , and Cen al Asia. H.au i us is a e es ial noc u nal mammal ha lies in bu -
ows and p e e s mesic habi a s including ga dens, oli e g o es, and cul i a ed a eas [13]. This
species has a ci cannual cycle o i al ac i i y, showing i s maximum in summe (wi h a peak
in July), whe eas mos indi iduals hibe na e in win e o pe iods o up o 40 days. This cycle
is accompanied by annual body mass luc ua ions, being maximal in summe and minimal in
win e [14]. Consis en ly, H.au i us is a seasonal b eede . A ailable da a indica e ha , inde-
penden ly o he geog aphical loca ion, hedgehogs o his species b eed in la e sp ing-summe
and a e inac i e in la e au umn and win e [14–18].
He e we analysed he ci cannual es icula changes occu ing in he long-ea ed hedgehog
om no hwes e n Egyp du ing i s seasonal b eeding cycle. Tes icula his ology was s udied
and he spa ial gene-exp ession pa e ns o se e al soma ic and ge m cell ypes we e de e -
mined. The incidence o apop osis and he se um es os e one le els we e also quan i ied in
bo h sexually ac i e and inac i e hedgehogs.
Ma e ial and me hods
Animals and issue p epa a ion
Twel e adul males o long-ea ed hedgehog we e cap u ed ali e in Ma ouh go e no a e (31˚
21’ N, 27˚ 14’ E) in he no hwes e n pa o Egyp , 400 km om Cai o. Two s udy g oups
Tes is eg ession in Hemiechinus au i us
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0204851 Oc obe 4, 2018 2 / 16
we e es ablished: animals cap u ed in summe ( om May o Augus , N = 6) and hose cap-
u ed in win e (Janua y and Feb ua y, N = 4). We also collec ed wo indi iduals in he pe iod
o es is eg ession, a he end o Sep embe . Ju enile males (N = 2), iden i ied on he basis o
ex emely low es is mass (<200 mg), educed semini e ous ubule diame e (<70 μm), and
low body mass (<140 g), we e excluded om he s udy. Tes es and epididymides we e col-
lec ed, weighed, and ixed o e nigh in a 50× olume o Se a’s ixa i e (100% e hanol, 40%
o maldehyde, and glacial ace ic acid in a p opo ion o 6:3:1, espec i ely). This s udy was ca -
ied ou in s ic acco dance wi h he ecommenda ions o he Guide o he Ca e and Use o
Labo a o y Animals o he Na ional Ins i u es o Heal h. The cap u e and expe imen al p o o-
cols we e app o ed by he E hics Commi ee o Animal Expe imen a ion o he Zoology
Depa men , Facul y o Sciences, Fayoum Uni e si y. The long-ea ed Hedgehog is ca ego ized
by he Egyp ian Wildli e P o ec ion Sys em as (LR/lc), (Lowe isk/ leas conce n), hus, i is
no an endange ed o p o ec ed species. Animals we e cap u ed by a p o essional hun e wi h
pe mission om he Egyp ian Fede a ion o Wild Hun ing. To minimize animal su e ing,
hedgehogs we e cap u ed by hand in hei bu ows and immedia ely anspo ed o he labo a-
o y whe e hey we e eu hanised by C0
2
inhala ion.
His ology and immuno luo escence
Tes es we e embedded in pa a in, sec ioned (5 μm), moun ed on polylysine-coa ed slides (VWR,
Belgium), and s ained wi h hema oxylin and eosin acco ding o s anda d p ocedu es o mo pho-
logical analysis. Single and double immuno luo escences we e pe o med as p e iously desc ibed
[6]. Table 1 summa izes he an ibodies and wo king concen a ions used in his s udy.
Analysis o apop osis
Apop osis was assessed wi h he e minal deoxynucleo idyl ans e ase deoxy-UTP-nick end
labeling (TUNEL) assay, using he Fluo escen In Si u Cell Dea h De ec ion Ki (Roche e .
11684795910), acco ding o he manu ac u e ’s ins uc ions. In o de o quan i y he incidence
o apop osis, he o al numbe o TUNEL
+
cells we e coun ed in a o al o 100 semini e ous
ubule sec ions o each animal.
Mo phome ics and s a is ics
The diame e s o 30 ans e sely sec ioned semini e ous ubules o di e en es is sec ions
we e measu ed as p e iously desc ibed [6]. Semini e ous ubule diame e (exp essed in
Table 1. An ibodies used in his s udy.
Gene p oduc Hos species Wo king dilu ion Re e ences
Laminin Rabbi 1:100 Sigma L9393
Smoo h muscle alpha-ac in Mouse 1:100 Sigma A2547
Claudin 11 Rabbi 1:100 San a C uz Bio echnology, CA sc-25711
DMC1 Goa 1:100 San a C uz Bio echnology, CA sc-8973
PCNA Mouse 1:100 San a C uz Bio echnology, CA sc-56
P450scc Goa 1:100 San a C uz Bio echnology, CA sc-18043
SOX9 Rabbi 1:500 MERCK Millipo e AB5535
Be a-Ca enin Mouse 1:100 Sigma C7082
N-Cadhe in Ra 1:1 Hyb idoma bank
Connexin 43 Rabbi 1:10 San a C uz Bio echnology, CA sc-9059
h ps://doi.o g/10.1371/jou nal.pone.0204851. 001
Tes is eg ession in Hemiechinus au i us
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0204851 Oc obe 4, 2018 3 / 16
mic ons) as well as body and es is mass (exp essed in g ams and millig ams, espec i ely) a e
epo ed as mean ±s anda d de ia ion alues. Since hese g oups o da a i a no mal dis ibu-
ion, we used S uden ’s - es o compa e he espec i e means.
Se um le els o es os e one
F esh blood samples ob ained om he H.au i us males included in his s udy we e s o ed a
4˚C o e nigh and cen i uged nex mo ning a 6,000 pm o 20 min a 4˚C. The supe na an
(se um) was hen s o ed a -80˚C un il u he use. Tes os e one le els we e measu ed using
he chemiluminescen enzyme immunoassay me hod in an Immuli e 2000 analyze (Siemens
Heal hca e).
Resul s
Tes es o Hemiechinus au i us emain s e ile du ing he win e
Long-ea ed hedgehogs cap u ed in summe we e signi ican ly la ge han hose om win e
(summe body mass: 225 ±24 g; win e body mass: 192 ±10 g; wo- ailed - es , P = 0.019;
Fig 1A). In he summe g oup, he mean es is mass was h ee imes highe han ha in he
win e g oup (summe es is mass: 887 ±88 g; win e es is mass: 277 ±40 g; wo- ailed - es ,
P<0.001; Fig 1B). A he his ological le el, es es in he summe g oup p esen ed all ea u es
o an ac i e and e ile es is, wi h semini e ous ubules in all s ages o he spe ma ogenic cycle
and spe miogenesis comple ed (Fig 2A). Acco dingly, we ound abundan spe m in he epi-
didymides (Fig 2B). In con as , win e es es showed ma ked educ ion in semini e ous
ubules (summe ubula diame e : 195 ±18 μm; win e ubula diame e : 80 ±11 μm; wo-
ailed - es , P <0.001; Figs 1C and 2C). In hese ubules, no s age o he spe ma ogenic cycle
could be iden i ied and mos o hem we e illed wi h p ima y spe ma ocy es, lacking second-
a y spe ma ocy es, spe ma ids, and spe m (Fig 2C). Consis en ly, he diame e o he epididy-
mal ubule was educed and emp y (Fig 2D).
Spe ma ogenesis is in e up ed a he meio ic pachy ene s age in he
eg essed es es o Hemiechinus au i us
Nex , we s udied he exp ession o soma ic-cell-speci ic molecula ma ke s by means o immu-
no luo escence in bo h summe (he ea e e e ed o as ac i e) and win e (he ea e e e ed
Fig 1. Compa isons o h ee mo phome ic pa ame e s be ween summe and win e g oups o H.au i us:(A) body mass, (B) es is mass and (C)
semini e ous ubule diame e .
h ps://doi.o g/10.1371/jou nal.pone.0204851.g001
Tes is eg ession in Hemiechinus au i us
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0204851 Oc obe 4, 2018 4 / 16
o as inac i e) es es o H.au i us. SOX9 is a ansc ip ion ac o necessa y o adul Se oli cell
su i al and unc ion [19]. In he ac i e es es, SOX9
+
cells we e e enly dis ibu ed a basal posi-
ions o he semini e ous ubules (Fig 3Aa). In he inac i e es es, SOX9
+
cells we e also local-
ized a he pe iphe y o es is ubules; howe e , he dis ance be ween neighbou ing SOX9
+
cells
was no ably educed when compa ed o ha o ac i e ubules, con i ming ha Se oli cells in
he eg essed es es unde go a se e e sh inkage (Fig 3Ae). We also analysed α-smoo h muscle
ac in (ACTA2), which in he es is is a ma ke o pe i ubula myoid cells and a e ial muscle
ib es [20], and laminin (LAM), a p incipal componen o he basemen memb ane [21]. In he
ac i e es es, we ound a s ong and mode a e immuno eac i i y o ACTA2 and LAM, espec-
i ely, su ounding he semini e ous ubules (Fig 3Ab and 3Ac). In he inac i e es es, he s ain-
ing o bo h ACTA2 and LAM, was weake and i egula in shape, again, as a consequence o
Fig 2. Hema oxylin-eosin-s ained his ological sec ions o es es (A and C) and epididymides (B and D) om H.au i us males belonging o he summe (A and B)
and win e (C and D) s udy g oups. In he summe g oup, he his ology o he semini e ous ubules was he expec ed o sexually ac i e males and he epididymides
con ained spe m. In con as , es is ubules o animals collec ed in win e we e educed in size. They we e illed wi h ound cells esembling spe ma ocy es and no
ma u e spe m was isible. In his season, he epididymal ubules a e educed in diame e and de oid o spe m. Scale ba shown in D ep esen s 100 μm o all pic u es.
h ps://doi.o g/10.1371/jou nal.pone.0204851.g002
Tes is eg ession in Hemiechinus au i us
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0204851 Oc obe 4, 2018 5 / 16

Tes is eg ession in Hemiechinus au i us
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0204851 Oc obe 4, 2018 6 / 16
he in olu ion o he semini e ous ubules (Fig 3A and 3Ag). Finally, we also s udied he
exp ession pa e n he choles e ol side-chain clea age enzyme, P450scc, a p o ein in ol ed in
he syn hesis o es os e one which is p oduced by Leydig cells. In bo h ac i e and inac i e es-
es, P450scc exp ession was clea ly isible in he in e s i ial cells, indica ing ha Leydig cells o
eg essed es es main ain he s e oidogenic unc ion in some ex en (Fig 3Ad and 3Ah).
We also s udied he exp ession o ge m cell-speci ic ma ke s. DMC1 is p esen in lep o-
ene- o-ea ly pachy ene p ima y spe ma ocy es [22] and he p oli e a ing-cell nuclea an igen
(PCNA) is exp essed in spe ma ogonia as well as in zygo ene and pachy ene, bu no lep o ene
spe ma ocy es [23]. Ac i e es es exhibi ed bo h DMC1 and PCNA posi i e cells (Fig 3Ba–
3Bc), which we e loca ed a he pe iphe y o he semini e ous ubules showing an exp ession
pa e n ha a ied depending on he spe ma ogenic cycle s age, as desc ibed p e iously [6,
24]. DMC1
+
cells we e only obse ed in ubules o he spe ma ogenic s ages VII-IX (s a s in
Fig 3Ba), whe eas PCNA was exp essed in p oli e a ing spe ma ogonia as well as in a a iable
numbe o spe ma ocy es depending on he s age o he spe ma ogenic cycle (Fig 3Bb). Bo h
p o eins co-localized in zygo ene and ea ly pachy ene spe ma ocy es o semini e ous ubules
a s ages VII-IX (s a s in Fig 3Bc), bu no in semini e ous ubules con aining la e spe ma o-
cy es (as e isks in Fig 3Bc). The inac i e es is also showed a dynamic pa e n o exp ession o
bo h ma ke s, al hough immuno eac i e cells did no exhibi a ing-like o ganiza ion and clus-
e ed oge he (Fig 3Bd–3B ). A a highe magni ica ion, we could obse e ha he cen es o
he es is ubules we e comple ely illed wi h ei he DMC1
+
PCNA
+
(zygo ene and ea ly pachy-
ene) o DMC1
-
PCNA
+
(la e pachy ene) spe ma ocy es (Fig 3C). Al oge he , ou esul s
show ha in he inac i e es es o H.au i us meiosis onse is no hal ed bu meio ic a es
occu s a he pachy ene s age.
P ima y spe ma ocy es unde go apop osis in he inac i e es is o
Hemiechinus au i us
Since apop osis is a p ocess no mally occu ing in he eg essed o es es o seasonal b eeding
mammals [25], we pe o med TUNEL assay in bo h, ac i e and inac i e es es o H.au i us.
Ac i e es es only con ained some ew posi i e cells in a educed numbe o semini e ous
ubules, whe eas he numbe o apop o ic cells was no ably highe in inac i e es es (ac i e:
17 ±4 apop o ic cells pe 100 ubula sec ions; inac i e 334 ±54 apop o ic cells pe 100 ubula
sec ions; wo- ailed - es , P = 0.0094; Fig 4A and 4B). To iden i y he cell ypes dying in he
inac i e es es, we pe o med TUNEL assay oge he wi h immuno luo escence o cell ype-
speci ic ma ke s. We obse ed ha SOX9
+
cells we e ne e TUNEL
+
(Fig 4Ca–4Cc), sugges -
ing ha Se oli cells we e no a ec ed. In con as , all TUNEL
+
cells showed immuno eac i i y
o PCNA (yellow cells in Fig 4C ), showing ha an apop osis-media ed massi e loss o ge m
cells occu s in he inac i e es es o H.au i us.
Fig 3. Immuno luo escence o se e al cell- ype-speci ic molecula ma ke s on his ological sec ions o es es om H.
au i us.A) Analysis o he soma ic cell ma ke s SOX9 (a, e), ACTA2 (b, ), LAM (c, g) and P450scc (d, h) in ac i e (a-d) and
inac i e (e-h) es es. Se oli cells (SOX9), pe i ubula myoid cells (ACTA2), basal lamina (LAM) and Leydig cells (P45scc) a e
iden i ied wi h hese p o ein ma ke s. B) Double immuno luo escence o he ge m cell ma ke s DMC1 and PCNA in ac i e (a-c)
and inac i e (d- ) es es. In ac i e es es, DMC1
+
cells (a) we e only obse ed in lep o ene- o-ea ly pachy ene spe ma ocy es o he
semini e ous ubules a he spe ma ogenic s ages VII-IX (s a s). PCNA exp ession (b) was de ec ed in mi o ic spe ma ogonia as
well as in zygo ene and pachy ene spe ma ocy es. Bo h p o eins co-exp ess in zygo ene and ea ly pachy ene spe ma ocy es o
ubules a s ages VII-IX (c; s a s), bu no in ubules con aining la e pachy ene spe ma ocy es (as e isks). In inac i e es es, bo h
ma ke s also showed a dynamic pa e n o exp ession simila o ha o ac i e es es, bu immuno eac i e cells did no p esen a
ing-like o ganiza ion and appea ed clus e ed oge he (d- ). C) Highe magni ica ion o an inac i e es is showing ha he
luminal egion o he eg essed ubules was illed wi h ei he DMC1
+
PCNA
+
(ea ly pachy ene) o DMC1
-
PCNA
+
(la e
pachy ene) spe ma ocy es (dashed lines ou line he ubula pe ime e ). Scale ba shown in Ah ep esen s 50 μm o A, scale ba in
B ep esen s 100 μm in B and scale ba in Cd ep esen s 50 μm in C.
h ps://doi.o g/10.1371/jou nal.pone.0204851.g003
Tes is eg ession in Hemiechinus au i us
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0204851 Oc obe 4, 2018 7 / 16
Tes is eg ession in Hemiechinus au i us
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0204851 Oc obe 4, 2018 8 / 16
Ge m cell desquama ion accoun s o he massi e ge m cell loss occu ing
du ing he es icula eg ession o Hemiechinus au i us
Two indi iduals cap u ed a he beginning o Au umn (end o Sep embe ) p esen ed es es in
which he p ocess o eg ession was aking place (he ea e e e ed o as inac i a ing). In hese
es es, he mean diame e o he semini e ous ubules was in be ween hose o he summe and
he win e g oup (120 ±12 μm). The ge mina i e epi helium looked diso ganised in many
ubules and p ima y and seconda y spe ma ocy e we e anomalously loca ed in he lumen (Fig
5Aa), showing ha hey we e sloughed om hei usual loca ion in mo e basal egions. We
also de ec ed his ype o ge m cells wi hin he lumen o he epididymal ubule (Fig 5Ab). We
pe o med TUNEL assay o check whe he apop osis was esponsible o he massi e loss o
ge m cells ha akes place in he eg essing es es o he long-ea ed hedgehog. Dying cells we e
obse ed wi hin es is co ds o inac i a ing es es, al hough ew o hem we e loca ed a , o
nea by, he lumen (Fig 5B), hus indica ing ha desquama ion mainly a ec s li ing cells.
These esul s sugges ha , al hough apop osis may con ibu e in some ex en o he massi e
ge m cell deple ion a ec ing inac i a ing es es, addi ional mechanisms mus be in ol ed in
he p ocess. Two ecen s udies ha e shown ha in he es es o wo seasonal b eede s, he Ibe-
ian mole T.occiden alis [10] and he la ge hai y a madillo C. illosus [11], he loss o Se oli-
ge m cell junc ions leads o a apid desquama ion o ge m cells du ing he es is eg ession
pe iod. To de e mine whe he he exp ession o p o eins o ming Se oli-Se oli and Se oli-
ge m cell junc ions was al e ed in he eg essing es es o H.au i us, we pe o med immuno-
luo escence o se e al impo an adhesion molecules on his ological sec ions o ac i e, inac i-
a ing, and inac i e es es. N-cadhe in (NCAD) and β-ca enin (β-CAT) a e s uc u al
componen s o he adhe ens junc ions as well as o he ec oplasmic specializa ion exis ing
be ween Se oli-Se oli and Se oli-ge m cells [26]. Immuno eac i i y o hese wo p o eins in
ac i e es es was ela i ely s onge in he basal compa men han in he adluminal egion,
which showed a ain s aining (Fig 5Ca and 5Cd). Claudin 11 (CLDN11) is a ansmemb ane
p o ein o he igh junc ions o ming he blood- es is ba ie es ablished be ween adjacen
Se oli cells [27], and Connexin 43 (CNX43) is a componen o he gap junc ions ound in Se -
oli-Se oli and Se oli-ge m cell con ac a eas [28]. In ac i e es es, hese wo p o eins we e
ound in a spe ma ogenic cycle-dependen pa e n, loca ed mainly in he basal compa men
o he ge minal epi helium (Fig 5Cg and 5Cj). In con as , in bo h inac i a ing and inac i e
es es, a homogeneous diso ganized s aining was obse ed o he ou molecules h oughou
he en i e ge mina i e epi helium (Fig 5Cb, 5Cc, 5Ce, 5C , 5Ch, 5Ci, 5Ck and 5Cl). These
esul s sugges ha he adhesion be ween Se oli cells and be ween Se oli and ge m cells is
comp omised in he eg essing es es, hus explaining he ge m cell sloughing aking place
du ing his pe iod.
Se um es os e one le els a e educed in win e long-ea ed hedgehogs
Reduc ion o he le els o se um es os e one in males du ing he non- ep oduc i e season is a
common ea u e o all seasonal b eeding mammals s udied o da e. In he case o H.au i us,
he mean le els o se um es os e one we e signi ican ly educed in he indi iduals o he
Fig 4. S udy o apop osis (TUNEL assay) in he es es o ac i e and inac i e males o H.au i us.A) The abundance
o apop o ic cells (g een) was clea ly lowe in ac i e (a) han in inac i e es es (b). B) Quan i ica ion o he incidence o
apop osis in ac i e and inac i e es es. C) (a-c) Double TUNEL-SOX9 immuno luo escence in inac i e es es. No e ha
ed (SOX9) and g een (TUNEL) signals ne e colocalize, showing ha Se oli cells we e no dying. (d- ) Double
TUNEL-PCNA s aining. All TUNEL
+
cells also exp essed PCNA (yellow cells in ), indica ing ha apop o ic cells a e
spe ma ocy es. Scale ba shown in Ab ep esen s 100 μm o A and scale ba in C ep esen s 50 μm in C.
h ps://doi.o g/10.1371/jou nal.pone.0204851.g004
Tes is eg ession in Hemiechinus au i us
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0204851 Oc obe 4, 2018 9 / 16
29. Luaces JP, Rossi LF, Me ico V, Zucco i M, Redi CA, Sola i AJ, e al. Spe ma ogenesis is seasonal in
he la ge hai y a madillo, Chae oph ac us illosus (Dasypodidae, Xena h a, Mammalia). Rep oduc ion,
e ili y, and de elopmen . 2013; 25:547–57. h ps://doi.o g/10.1071/RD12127 PMID: 22951275.
30. Hame G, No ak I, Kouzne so a A, Hoog C. Dis up ion o pai ing and synapsis o ch omosomes causes
s age-speci ic apop osis o male meio ic cells. The iogenology. 2008; 69:333–9. h ps://doi.o g/10.
1016/j. he iogenology.2007.09.029 PMID: 17997150.
31. McClusky LM. Coo dina ion o spe ma ogenic p ocesses in he es is: lessons om cys ic spe ma ogen-
esis. Cell and issue esea ch. 2012; 349:703–15. h ps://doi.o g/10.1007/s00441-011-1288-1 PMID:
22314845.
32. Shukla KK, Mahdi AA, Rajende S. Apop osis, spe ma ogenesis and male in e ili y. F on Biosci (Eli e
Ed). 2012; 4:746–54. PMID: 22201910.
33. T ipa hi R, Mish a DP, Shaha C. Male ge m cell de elopmen : u ning on he apop o ic pa hways. Jou nal
o ep oduc i e immunology. 2009; 83:31–5. h ps://doi.o g/10.1016/j.j i.2009.05.009 PMID: 19889463.
34. S benc M, Faza inc G, Ba dek SV, Pogacnik A. Apop osis and p oli e a ion du ing seasonal es is
eg ession in he b own ha e (Lepus eu opaeus L.). Ana omia, his ologia, emb yologia. 2003; 32:48–
53. PMID: 12733273.
35. Blo ne S, Hings O, Meye HH. In e se ela ionship be ween es icula p oli e a ion and apop osis in
mammalian seasonal b eede s. The iogenology. 1995; 44:321–8. PMID: 16727732.
36. Fu u a I, Po kka-Heiskanen T, Sca b ough K, Tapanainen J, Tu ek FW, Hsueh AJ. Pho ope iod egu-
la es es is cell apop osis in Djunga ian hams e s. Biology o ep oduc ion. 1994; 51:1315–21. PMID:
7888511.
37. Mo ales E, Pas o LM, Fe e C, Zuas i A, Palla es J, Ho n R, e al. P oli e a ion and apop osis in he
semini e ous epi helium o pho oinhibi ed Sy ian hams e s (Mesoc ice us au a us). In e na ional jou nal
o and ology. 2002; 25:281–7. PMID: 12270025.
38. Seco-Ro i a V, Bel an-F u os E, Fe e C, Saez FJ, Mad id JF, Pas o LM. The dea h o se oli cells
and he capaci y o phagocy ize elonga ed spe ma ids du ing es icula eg ession due o sho pho ope-
iod in Sy ian hams e (Mesoc ice us au a us). Biology o ep oduc ion. 2014; 90:107. h ps://doi.o g/10.
1095/biol ep od.113.112649 PMID: 24719257.
39. Blo ne S, Schon J, Roelan s H. Apop osis is no he cause o seasonal es icula in olu ion in oe dee .
Cell and issue esea ch. 2007; 327:615–24. h ps://doi.o g/10.1007/s00441-006-0328-8 PMID: 17093922.
40. Yazawa T, Yamamo o T, Abe S. P olac in induces apop osis in he penul ima e spe ma ogonial s age
o he es es in Japanese ed-bellied new (Cynops py hogas e ). Endoc inology. 2000; 141:2027–32.
h ps://doi.o g/10.1210/endo.141.6.7492 PMID: 10830286.
41. Ande sen Be g K, Wige R, Dahl E, To p T, Fa s ad W, K ogenaes A, e al. Seasonal changes in spe -
ma ogenic ac i i y and in plasma le els o FSH, LH and es os e one, and he e ec o immuniza ion
agains inhibin in he male sil e ox (Vulpes ulpes). In e na ional jou nal o and ology. 2001; 24:284–
94. PMID: 11554986.
42. Zhang L, Han XK, Qi YY, Liu Y, Chen QS. Seasonal e ec s on apop osis and p oli e a ion o ge m cells
in he es es o he Chinese so -shelled u le, Pelodiscus sinensis. The iogenology. 2008; 69:1148–58.
h ps://doi.o g/10.1016/j. he iogenology.2008.01.028 PMID: 18377973.
43. Islam MN, Tsukaha a N, Sugi a S. Apop osis-media ed seasonal es icula eg ession in he Japanese
Jungle C ow (Co us mac o hynchos). The iogenology. 2012; 77:1854–65. h ps://doi.o g/10.1016/j.
he iogenology.2012.01.002 PMID: 22401836.
44. Young KA, Zi kin BR, Nelson RJ. Sho pho ope iods e oke es icula apop osis in whi e- oo ed mice
(Pe omyscus leucopus). Endoc inology. 1999; 140:3133–9. h ps://doi.o g/10.1210/endo.140.7.6870
PMID: 10385406.
45. W igh S. The oles o mu a ion, inb eeding, c ossb eeding, and selec ion in e olu ion. P oceedings o
he Six h In e na ional Cong ess on Gene ics: I haca, New Yo k; 1932.
46. Kau G, Thompson LA, Du ou JM. Se oli cells—immunological sen inels o spe ma ogenesis. Semi-
na s in cell & de elopmen al biology. 2014; 30:36–44. h ps://doi.o g/10.1016/j.semcdb.2014.02.011
PMID: 24603046; PubMed Cen al PMCID: PMC4043859.
47. S an on PG. Regula ion o he blood- es is ba ie . Semina s in cell & de elopmen al biology. 2016;
59:166–73. h ps://doi.o g/10.1016/j.semcdb.2016.06.018 PMID: 27353840.
48. Pelle ie RM. Cyclic modula ion o Se oli cell junc ional complexes in a seasonal b eede : he mink
(Mus ela ison). The Ame ican jou nal o ana omy. 1988; 183:68–102. h ps://doi.o g/10.1002/aja.
1001830105 PMID: 3189199.
49. Ta ulli GA, Meachem SJ, Schla S, S an on PG. Regula ion o es icula igh junc ions by gonado o-
phins in he adul Djunga ian hams e in i o. Rep oduc ion. 2008; 135:867–77. h ps://doi.o g/10.1530/
REP-07-0572 PMID: 18502899.
Tes is eg ession in Hemiechinus au i us
PLOS ONE | h ps://doi.o g/10.1371/jou nal.pone.0204851 Oc obe 4, 2018 16 / 16