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17α‑e hynyles adiol p e en s
he na u al male‑ o‑ emale sex
change in gil head seab eam
(Spa us au a a L.)
M. Pila Ga cía He nández1*, Isabel Cabas1, M. Ca men Rodenas1, Ma a A izcun2,
Elena Cha es‑Pozo2, Debo ah M. Powe 3 & Al onsa Ga cía Ayala1
Exposu e o 17α‑e hynyles adiol (EE2, 5 μg/g ood) impai s some ep oduc i e e en s in he
p o and ous gil head seab eam and a sho eco e y pe iod does no allow ull eco e y. In his s udy,
spe mia ing seab eam males in he second ep oduc i e cycle (RC) we e ed a die con aining 5 o
2.5 μg EE2/g ood o 28 days and hen a comme cial die wi hou EE2 o he emaining RC. Indi iduals
we e sampled a he end o he EE2 ea men and hen a he end o he RC and a he beginning
o he hi d RC, 146 and 333 days a e he cessa ion o ea men , espec i ely. Inc eased hepa ic
ansc ip le els o he gene coding o i ellogenin ( g) and plasma le els o V g indica ed bo h
concen a ions o EE2 caused endoc ine dis up ion. Modi ica ions in he his ological o ganiza ion o
he es is, ge m cell p oli e a ion, plasma le els o he sex s e oids and pi ui a y exp ession le els
o he genes coding o he gonado opin β‑subuni s, shβ and lhβ we e de ec ed. The plasma le els
o V g and mos o he ep oduc i e pa ame e s we e es o ed 146 days a e ea men s. Howe e ,
al hough 50% o he con ol ish unde wen sex e e sal as expec ed a he hi d RC, male‑ o emale
sex change was p e en ed by bo h EE2 concen a ions.
Nowadays he e is a b oad a ie y o chemicals discha ged om indus ial and u ban sou ces ha ha e been
desc ibed as endoc ine dis up ing chemicals (EDCs). EDCs exe ad e se e ec s by means o hei in e ac ions
wi h ho mone ecep o s o by in e e ing wi h he no mal syn hesis, me abolism, anspo , o sec e ion o
endogenous ho mones in wildli e and humans1. Among EDCs, 17α-e hynyles adiol (EE2) has ecei ed much
a en ion in ecen decades. EE2 is a syn he ic es ogen ha mimics E2 ac ions and is equen ly used in con acep-
i e pills and ho mone he apy and is widesp ead in aqua ic en i onmen s2–5. Exposu e o sub-le hal concen a-
ions o EE2 causes al e a ions in ep oduc i e capaci y6–14 and sex di e en ia ion15–17 in ish. The exp ession o
he gene coding o i ellogenin (V g, a p o ein no mally syn hesized by emales du ing oocy e ma u a ion) and/
o plasma le els o V g in male ish a e e e ence ma ke s widely used o es ogenic dis up ion6–8,12. Al e a ions
in ep oduc i e endpoin s such as plasma le els o s e oid ho mones7–9,12,13, gonad mo phology6–9,13,14 and he
exp ession le els o genes coding o es ogenic ecep o s o o s e oidogenic enzymes8–10,13 help o assess he
dis up i e e ec s o his compound which a y acco ding o dose, ime o exposu e, age and he s age in he
ep oduc i e cycle o he ea ed indi iduals7,8,17. Howe e , only a ew s udies ha e deal wi h he e ec o EE2
on he exp ession o genes coding o he β subuni o gonado opins, lu einizing ho mone (lhb) and ollicle
s imula ing ho mone ( shb)13,18,19, ha a e egula ed by E2 in some species20,21 and a e essen ial o egula ion o
game ogenesis and gonadal di e en ia ion in ish.
Less a en ion has been paid o he po en ial o eco e y om EE2 exposu e and mos s udies a e limi ed o
assessmen o sexual di e en ia ion and he ep oduc i e capaci y o de eloping zeb a ish, a ish model widely
used in esea ch. Re e sal o he e ec s o EE2 exposu e di e ed wi h he endpoin s udied, he leng h o he
exposu e and he du a ion o he eco e y pe iod17,22–24. In addi ion, EE2 exposu e has been epo ed o a ec
sex a ios25, o ha e ansgene a ional e ec s on ish su i al and ecundi y and o dis up popula ion dynamics
o small model ish17,26.
OPEN
1Depa men o Cell Biology and His ology, Facul y o Biology, Uni e si y o Mu cia, Campus de Espina do,
30100Mu cia,Spain.2Oceanog aphicCen e o  Mu cia,Spanish Ins i u e o Oceanog aphy(IEO),Ca e e ade
laAzohías/n,Pue odeMaza ón,30860Mu cia,Spain.3Cen o de Ciências Do Ma , Uni e sidade Do Alga e,
CampusdeGambelas,8005-139Fa o,Po ugal. *email: [email p o ec ed]
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The gil head seab eam (Spa us au a a L.) is a p o and ous he maph odi ic ish species ha de elops as a
unc ional male o he i s 2yea s o li e and hen 40% o a gi en popula ion de elops as emales du ing he
hi d yea 27.Li le a en ion has been paid o he na u al sex change in his species27,28 despi e i s g ea comme cial
and economic in e es in he Medi e anean a ea29. Al hough he na u al sex change in he maph odi e ish has
been he subjec o nume ous s udies ( e iew in30–32) mos dealing wi h he male- o- emale change ha e been
ca ied ou in he p o and ous black po gy20,33,34.
Based on he s a us o he es is, he second ep oduc i e cycle (RC) o he gil head seab eam can be di ided
in o ou main s ages: spe ma ogenesis, spawning, pos -spawning and es icula in olu ion. Du ing spe ma o-
genesis, he es icula ubules con ain spe ma ogonia s em cells and cys s o spe ma ogonia, spe ma ocy es and
spe ma ids and a ying amoun s o spe ma ozoa exis in he lumen. A his s age, ci cula ing le els o es os-
e one (T) and 11-ke o es os e one (11-KT) a e high and 17β-es adiol (E2) is low. A spawning, he ubules o
he es is con ain mainly spe ma ogonia s em cells and cys s o p ima y spe ma ogonia and spe ma ozoa ill
he lumen; ci cula ing le els o T and 11-KT all and E2 le els ise. A pos -spawning, he ubules main ain he
cell a angemen , bu he lumen con ains no, o e y ew, spe ma ozoa and se um E2 le els all. A he es icula
in olu ion s age, ubules con ain mainly spe ma ogonia s em cells andha e no lumen and nume ous nec o ic
a eas su ounded by abundan in e s i ial issue ich in eosinophilic cells appea in he in e nal es icula a ea,
while se um le els o s e oids emain low and cons an 27,29.
P e iously we demons a ed ha he e ec o EE2- eeding on ep oduc i e pa ame e s o ma u e gil head
seab eam a ied wi h he concen a ion o EE2 and he s age a which i was applied du ing he RC. The lowes
concen a ion es ed, 5µg EE2/g ood7,8,35, is a highe han hose ou inely eco ded in aqua ic en i onmen s,
which a e usually below 50ng/L36, bu is simila o he highes concen a ion epo ed by Bicchi e al.37 in I al-
ian su ace wa e s collec ed downs eam o a was ewa e ea men plan (4381ng/L). Fu he mo e, when EE2
exposu e was ollowed by a eco e y pe iod o 25days when ish we e ed a s anda d die , ep oduc i e pa am-
e e s did no eco e 35.
The aim o he p esen s udy was o de e mine whe he in he p o and ous gil head seab eam: (1) exposu e
o lowe concen a ions o EE2 han p e iously used (2.5µg EE2/g ood) a ec ed ep oduc i e pa ame e s; (2)
i longe pe iods o eco e y a e cessa ion o EE2 ea men (146 and 333days) es o ed no mal ep oduc i e
pa ame e s; and (3) i exposu e o EE2 a ec ed he sex change in his he maph odi e ish. Fo his eason, ma u e
gil head seab eam males in spe ma ogenesis du ing he second RC we e ed a comme cial die con aining 2.5 o
5µg EE2/g ood o 28days and hen ed comme cial die wi hou EE2 un il he ollowing RC. Fish we e sampled
a e eco e y o 146-days on a s anda d comme cial die , du ing he es icula in olu ion s age when ho mone
le els a e no a ec ed by game ogenesis o spawning. Fu he samples we e collec ed a e 333days o eeding on
he s anda d comme cial die , a he beginning o he hi d RC when he sex change can be de ec ed.
Resul s
EE2 did no a ec body weigh bu dis up ed he ansc ip le els o he g gene and he
plasma le els o V g. Exposu e o 2.5 o 5μg EE2/g ood o 28days did no a ec he ish body weigh a
he end o ea men o a e 146days o eco e y using he s anda d die . A e 333days eco e y he e we e
no signi ican di e ences be ween he weigh o male con ol ish and he males ha had been ed ei he 2.5 o
5μg EE2/g ood. The emale con ol ish we e signi ican ly hea ie han he con ol o EE2- ea ed males (Fig.1).
Exposu e o 2.5 o 5μg EE2/g ood o 28days had dis up i e e ec s, since i caused a signi ican inc ease in
he hepa ic exp ession le els o g (Fig.2a) and plasma le els o V g compa ed o he con ol ish (Fig.2b). The
Figu e1. E ec s o EE2 die a y in ake on ish body weigh a e 28days o ea men wi h 0 (con ol), 2.5 and
5µg/g ood, and a e 28days o ea men wi h 0 (con ol), 2.5 and 5µg/g ood ollowed by 146 and 333days o
eeding a s anda d die ( eco e y). Da a ep esen he means ± S.E.M. o duplica e samples. M, male specimens;
F, emale specimens. *Deno e s a is ically signi ican di e ences be ween ea ed and con ol g oups and le e s
indica e di e ences in he con ol g oup a dis inc sampling imes, as de e mined by one-way ANOVA and LSD
pos hoc es s.
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V g plasma le els we e es o ed o con ol le els in all ea ed ish a e a eco e y pe iod o 146days (Fig.2b).
In e es ingly, a day 333 when he sex change was accomplished in 50% o he con ol ish, he con ol emales
had inc eased plasma V g compa ed o he con ol and EE2 ea ed ish ha main ained he male a e (Fig.2b).
The e ec o a 28‑days exposu e o EE2 on he mic oscopic o ganiza ion o he gonads a ‑
ied acco ding o concen a ion and specimens. A he end o a 28-day pe iod o EE2- eeding, all he
specimens in he con ol g oup displayed he mo phological ea u es (Fig.3a) and cell p oli e a ion pa e n
as e ealed by an i-PCNA immunolabelling (Fig.3b,c) ypical o he la e spe ma ogenesis s age in wo-yea
old specimens27. In he es is ubules a ge minal epi helium consis ing o spe ma ogonia s em cells, p ima y
spe ma ogonia (some o hem p oli e a ing), nume ous cys s o p oli e a i e spe ma ogonia and spe ma ocy es
and non-p oli e a i e spe ma ids we e e iden and abundan spe ma ozoa illed he lumen (Fig.3). Fish ed
wi h 2.5μg EE2/g ood showed a iable le els o gonad issue dis up ion (Fig.3d–i). Thus, among he six speci-
mens s udied, hal o hem had a es icula mo phology and p oli e a i e pa e n simila o hose o con ol ish
(Fig.3d–e). Howe e , one specimen displayed a dec eased numbe o cys s and he an i-PCNA immunolabel-
ling e ealed a educed numbe o p oli e a i e spe ma ogonia and spe ma ocy es (Fig.3 –g) compa ed o he
con ol ish. The emaining wo specimens ed 2.5μg EE2/g ood had es is esembling hose o ish ed wi h a
die con aining 5μg EE2/g ood (Fig.3h–i) ha a e desc ibed below. The ish ed wi h 5μg EE2/g ood had a
ge minal epi helium lacking meio ic cells, wi h sca ce p oli e a i e spe ma ogonia,Se oli and in e s i ial cells,
and abundan spe ma ozoa illing he lumen o he ubules (Fig.3j–l).
No mo phological al e a ions we e obse ed in he es is o EE2‑ ed specimens a e 146 days
eco e y on a s anda d die . A e 28days o ea men and 146days o eco e y he es is o ish ed 2.5
(Fig.4d) o 5μg (Fig.4c) EE2/g ood had a simila mo phology o he con ol ish wi h a his ological o ganiza-
Figu e2. (a) T ansc ip le els o g gene in ish ed wi h EE2 a concen a ions o 0 (con ol), 2.5 and 5µg/g
ood o 28days. (b) Plasma V g le els o ish ed 0 (con ol), 2.5 and 5µg/g ood o 28days ( ea men ) and
o ish ed 0 (con ol), 2.5 and 5µg/g ood o 28days and a e wa ds a s anda d die o 146 and 333days
( eco e y). M, male specimens; F, emale specimens. *Deno e s a is ically signi ican di e ences be ween ea ed
and con ol g oups and le e s indica e di e ences in he con ol g oup a dis inc sampling imes, as de e mined
by one-way ANOVA and LSD pos hoc es s.
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Figu e3. Tes is o gil head seab eam ed wi h 0 (con ol, a–c), 2.5 (d–i) o 5 (j–l) µg o EE2/g o ood o
28days showing ubules wi h spe ma ogonia s em cells (a ow), p ima y spe ma ogonia (solid a owhead) and
cys s o spe ma ogonia (SG), spe ma ocy es (SC) and spe ma ids (SD wi h plen y o spe ma ozoa (SZ) in he
lumen. No e ha besides some in e s i ial cells (as e isk) and Se oli cells (open a owhead), diminished ( ,g) o
absen (h,i) cys s o p oli e a ing ge m cells we e ound in some indi iduals ed wi h 2.5 (g,i) and in hose ed
5µg o EE2/g o ood (j–l). H–E (a,d, ,h,j); PCNA-immunolabelling (b,c,e,g,i,k,l).
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ion ypical o he es icula in olu ion s age27 (Fig.4a,b). The ubule lumen was e y na ow o lacking and
he ge minal epi helium consis ed mainly o spe ma ogonia s em cells, p ima y spe ma ogonia and a e cys s
o spe ma ogonia and conspicuous nec o ic a eas we e ound in he inne es icula a ea (Fig.4a–d). PCNA-
immunolabelling e ealed low p oli e a ion o in e s i ial (Fig.4 ,h), Se oli cells (Fig.4e,g,h) and spe ma ogonia
(Fig.4g) in he con ol (Fig.4e, ) and EE2 ea ed ish a e 146days eco e y (Fig.4g,h).
Exposu e o EE2 o 28 days du ing he spe ma ogenesis o he second RC p e en ed he sex
change. A 333days a e he cessa ion o EE2 ea men s hal o he con ol ish had es is a he spe ma o-
genesis s age (supplemen a y da a). In he o he 50% o he con ol ish he es icula issue was a mino pa
o he gonad and con ained collapsed ubules, consis ing o da k cells and spa se spe ma ogonia s em cells; he
o a ian a ea was dominan and con ained abundan i ellogenic oocy es (Fig.5a,b). Howe e , he gonads o all
he specimens ha had been ed ei he 2.5 (Fig.5c) o 5μg (supplemen a y da a) EE2/g ood we e simila o
hose o he con ol males a his s age.
Sex s e oid plasma le els we e a iable in EE2‑ ed ish and in eco e ed ish ed s anda d
die s o 146 days. Feeding wi h EE2 a concen a ions o ei he 2.5 o 5μg EE2/g ood caused a signi ican
inc ease in plasma le els o E2 (Fig.6a) and a signi ican dec ease in plasma le els o P (Fig.6b), 11-KT (Fig.7c)
and T (Fig.6d) leading o a a io o 11-KT/E2 lowe han he con ol ish (Fig.6e). Con ol le els o sex s e oids in
EE2 ea ed ish we e es o ed a e 146days o eeding a s anda d die (Fig.6b,d), wi h he excep ion o E2 which
emained signi ican ly ele a ed in ish ha had been ed wi h 5μg EE2/g ood bu no in ish ed wi h 2.5μg
EE2/g ood (Fig.6a). A e 333days eco e y on a s anda d die he sex s e oid plasma le els o male EE2- ed ish
did no di e om he male con ol. Howe e , he con ol ish ha de eloped as emales had signi ican ly lowe
le els o 11-KT and a lowe 11-KT/E2 a io han bo h con ol and ea ed males (Fig.6a,e).Fish ed 2.5μg EE2/g
ood had inc eased plasma T le els compa ed o con ol emales.
Figu e4. Tes is o gil head seab eam ed wi h 0 (con ol, a,b,e, ), 2.5 (d,g) o 5 (c,h) µg o EE2/g o ood o
28days ollowed by a eco e y pe iod o 146days o eeding wi h a s anda d die showing collapsed ubules
consis ing o mainly spe ma ogonia s em cells (a ow) and p ima y spe ma ogonia (solid a owhead), expanded
in e s i ial issue (IT) and nec o ic a eas (Na). No e he sca ce p oli e a i e in e s i ial (as e isk, ,h), Se oli
(open a owhead, e,h) and ge m (solid a owhead, g) cells. H–E (a–d); PCNA-immunolabelling (e–h).

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Gonado opin gene ansc ip s we e al e ed a e 28 days o EE2 ea men and du ing eco ‑
e y. The pi ui a y exp ession le els o sl we e no a ec ed by EE2 ea men a any dose (Fig.7a). Howe e ,
hey we e inc eased 333days a e cessa ion o ea men in ish ed 2.5μg EE2/g ood (Fig.7a). Pi ui a y exp es-
sion le els o shb we e signi ican ly down- egula ed a e 28days o 5μg EE2/g ood-in ake and es o ed a e
146days o eeding wi h a s anda d die . Howe e , in males 333days a e cessa ion o eeding he 5μg EE2/g
ood he e was a signi ican down- egula ion o shb ansc ip abundance and le els esembled ha o con ol
emales (Fig.7b). Compa ed o he con ol ish he pi ui a y exp ession le el o lhb was signi ican ly up- egula ed
in ish ed 2.5μg EE2/g ood o 28days (Fig.7c) and also in ish a 146days a e cessa ion o eeding 5μg EE2/g
ood (Fig.7c). A e a 333days eco e y pe iod, lhb exp ession le els in EE2- ed ish we e signi ican ly lowe han
in con ol emales (Fig.7c) and we e simila o hose in con ol males.
Spe m quali y was simila in all males 333 days a e he cessa ion o EE2 ea men . The
concen a ion and iabili y o spe m om ish ed s anda d die s o 333days a e exposu e o 2.5 o 5μg EE2/g
ood du ing he spe ma ogenesis s age o he second RC we e simila o con ol males a he spe ma ogenesis o
he hi d RC (Table1).
Discussion
Exposu e o 5µg EE2/g ood o 25 o 28days had a dis up i e e ec in male gil head seab eam, since i inc eased
he hepa ic exp ession o g and dis up ed spe ma ogenesis and s e oidogenesis. The obse ed e ec s a ied wi h
he EE2 concen a ion, ime o exposu e, age and ep oduc i e s age o he indi iduals7,8,35. Mo eo e , a 25-day
eco e y pe iod wi h a s anda d die a e EE2 ea men did no es o e he al e ed ep oduc i e pa ame e s in
ma u e gil head seab eam35.
In he p esen s udy, ma u e gil head seab eam males a spe ma ogenesis o he second RC we e used o
in es iga e he dis up i e e ec o EE2 using a simila dose o ou p e ious s udy and a lowe dose (2.5µg EE2/g
ood in die ). The e ec was assessed by analyzing some pa ame e s p e iously s udied (his ological o ganiza-
ion o he es is, he plasma le els o he sex s e oids E2, T and 11-KT and he spe m quali y) and new selec ed
ep oduc i e endpoin s, such as he plasma le els o P and he pi ui a y exp ession le els o he gene coding
o SL, sl, a ho mone ela ed o ep oduc i e ac i i y38–41 and o sex di e en ia ion42, and o he genes coding
o he β-subuni s o he gonado opic ho mones, lhb and shb. Exp ession le els o g and/o plasma le els o
V g we e de e mined o assess he dis up i e e ec s o EE2 ea men s. Fu he mo e, he eco e y o 146 and
333days a e EE2 exposu e was in es iga ed o explo e whe he he ea men caused dis up ion o he na u al
sex change o his species. Fish ha eco e ed om EE2 ea men o 146days we e a he end o he RC, when
he b ain-pi ui a y-gonad axis is no condi ioned by he cou se o game ogenesis o spawning and many ishes
change sex30. Fish ha eco e ed om EE2 ea men o 333days we e unde going he game ogenesis o he hi d
RC, when app oxima ely 40% o he gil head seab eam no mally de elop as emales27, which was con i med in
he p esen s udy, o he con ol ish only.
Bo h doses o EE2 had a dis up i e e ec on gil head seab eam ep oduc ion as e ealed by inc eased hepa ic
g gene exp ession and plasma V g and inc eased plasma E2 and dec eased plasma T, 11-KT and P compa ed
o he con ol. Modi ica ions in plasma E2, T and 11-KT in esponse o EE2 exposu e in ish12,43,44 including he
gil head seab eam, depends on he ep oduc i e s age a which i occu s7,8. The e ec o EE2 on he es is his ol-
ogy depended on he dose and highe doses had a mo e p onounced e ec in common wi h p e ious epo s in
he gil head seab eam8,35 and o he eleos ish13,45. Al hough in ou p e ious s udy we epo ed ha 5µg EE2/g
Figu e5. Gonads o gil head seab eam ed wi h 0 (con ol, a,b) and 2.5µg o EE2/g o ood o 28days ollowed
by a eco e y pe iod o 333days o eeding wi h a s anda d die (c). The con ol ish is a emale and has an
o a y wi h seconda y oocy es (a) and sh unk es is wi h collapsed ubules consis ing o da k cells and spa se
spe ma ogonia s em cells (b). The ea ed ish showed es is a spe ma ogenesis (c) wi h ubules consis ing o
wi h spe ma ogonia s em cells (a ow), p ima y spe ma ogonia (solid a owhead) and cys s o spe ma ogonia
(SG), spe ma ocy es (SC) and spe ma ids (SD) and plen y o spe ma ozoa (SZ) in he lumen. H–E.
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ood caused a educ ion in he spe m concen a ion in gil head seab eam a he spe ma ogenesis s age35. I was
no possible o ob ain spe m samples om he ish ed 5µg EE2/g ood in he p esen s udy, al hough simila
obse a ions ha e been made in peje ey ma u e males exposed o 45ng EE2/L du ing he spawning season13.
Less a en ion has been paid o he e ec o EE2 exposu e on pi ui a y ho mones13,18,19. The doses used in
his s udy had a a iable e ec on pi ui a y exp ession le els o shb (dec eased wi h 5µg EE2/g ood) and lhb
(inc eased by 2.5µg EE2/g ood). Simila obse a ions ha e also been made in sub-adul emale coho salmon
(Onco hynchus kisu ch) wi h EE2 exposu e (12ng EE2/L o 6weeks) causing a high inc ease in he exp ession
o lhb and a mode a e dec ease in he exp ession o shb18. In adul Gobio cyp is a us o bo h sexes, mRNA le els
o shb we e no a ec ed and lhb gene ansc ip s we e supp essed by 1–125ng EE2/L19. In con as , in peje ey
(Odon es hes bona iensis) exposu e o ma u e males o 45ng/L o EE2 did no a ec pi ui a y exp ession le els
o lhb o shb13.
In gil head seab eam, mos e ec s o EE2 on he ep oduc ion- ela ed endpoin s s udied we e los 146days
a e he cessa ion o ea men s, including he V g plasma le els used as ma ke o endoc ine dis up ion. How-
e e , gil head seab eam we e no ully eco e ed om EE2 exposu e, as some al e a ions we e obse ed a he
Figu e6. Plasma le els o 17β-es adiol (E2) (a), p oges e one (P) (b), 11-ke o es os e one (11-KT) (c)
and es os e one (T) (d) o ish ed 0 (con ol), 2.5 and 5µg/g ood o 28days ( ea men ) and o ish ed 0
(con ol), 2.5 and 5µg/g ood o 28days and a e wa ds a s anda d die o 146 and 333days ( eco e y). Da a
ep esen he means ± S.E.M. o duplica e samples. M, male specimens; F, emale specimens. *Deno e s a is ically
signi ican di e ences be ween ea ed and con ol g oups and le e s indica e di e ences in he con ol g oup
a dis inc sampling imes, as de e mined by one-way ANOVA and LSD pos hoc es s (a–d) o K uskal Wallis
ollowed by Mann Whi ney es s (e).
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pi ui a y le el 146 and 333days a e he cessa ion o exposu e o 5µg EE2/g ood. In addi ion, all ish had es is
a spe ma ogenesis 333-days a e cessa ion o EE2 exposu e i espec i e o he dose (2.5 o 5µg EE2/g ood),
al hough in hal o he con ol ish he his ological changes we e consis en wi h he sex change ha na u ally
occu s in his species27. Thus, he exposu e o EE2 o adul gil head seab eam o a sho pe iod du ing he
second RC p oduced long-las ing dis up i e e ec s. S udies on zeb a ish exposed o EE2 om e iliza ion o
sexual ma u a ion desc ibed pa ial e e sal o he eminizing e ec s24 and he ep oduc i e consequences22 eigh
and i e mon hs a e cessa ion o exposu e. In he same species a 3-mon h depu a ion ollowing EE2 exposu e
h oughou de elopmen was no enough o es o e con ol le els o V g syn hesis and gonad mo phololy23,
Figu e7. T ansc ip le els o sl (a), shb (b) and lhb (c) gene in ish ed wi h EE2 a concen a ions o 0 (con ol),
2.5 and 5µg/g ood du ing 28days ( ea men ) and o ish ed 0 (con ol), 2.5 and 5µg/g ood o 28days and
a e wa ds a s anda d die o 146 and 333days ( eco e y). M, male specimens; F, emale specimens. *Deno e
s a is ically signi ican di e ences be ween ea ed and con ol g oups and le e s indica e di e ences in he
con ol g oup a dis inc sampling imes, as de e mined by one-way ANOVA and LSD pos hoc es s.
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al hough a o al escue om unde de elopmen and eminiza ion was desc ibed 40days a e he cessa ion o
EE2 exposu e unde simila condi ions17.
On he o he hand, he e ec s o EDCs in he sex a io o gonocho ic labo a o y ish models may be ansi o y
o pe manen and iming o de e mining ish sex a e exposu e is impo an 25. Hence, s udies on a a ie y o
species and condi ions a e needed o ully unde s and he e e sibili y o he endoc ine dis up i e e ec s o EE2.
Resea ch on he maph odi ic ma ine ish as he gil head seab eam would be e y aluable, especially conside ing
i s g ea economic in e es in he Medi e anean a ea.
No iceably, sex change ailed o occu in ish ea ed wi h 2.5 o 5µg EE2/g ood, whils as expec ed 50% o
con ol specimens became emales27. The p og ession o na u al sex change in ol es concomi an changes in
E2 syn hesis in many species, wi h le els inc easing in p o and ous46,47 and d opping in p o ogynous48 ish. In
he p o and ous sobai y (Spa iden ex has a) he a ainmen o a minimum h eshold le el o E2 ha enables he
o a ian po ion o he gonad o es ablish empo a y dominance and acili a e u he es ablishmen o a pe -
manen emale s a us seems o be necessa y o sex change o occu 49. On he o he hand, i has been p oposed
a de iciency in gonadal p oduc ion o E2 is he signal esul ing in he ailu e o sex change in he p o and ous
black po gy50. In he p esen s udy, an inc ease in he plasma le els o E2 was no de ec ed in con ol ish, which
we e simila a spe ma ogenesis and a es icula in olu ion s ages o he second RC. In addi ion, ea men s wi h
bo h doses o EE2 ele a ed plasma le els o E2 bu only ish ea ed wi h 5µg EE2/g ood main ained hese high
le els a e a 146-day eco e y. Howe e , all he EE2 ea ed ish i espec i e o dose emained male in he ollow-
ing RC. Thus, he inc ease o E2 plasma le els does no seem o be essen ial o s imula e sex change in gil head
seab eam. P o and ous ansi ion in ol es, besides he ele a ion o E2 le els, he cease o 11-KT syn hesis, and
a subsequen p oli e a ion o o a ian and degene a ion o es is, espec i ely30. In gil head seab eam, a d op in
plasma le els o 11-KT was obse ed in con ol ish a he es icula in olu ion s age compa ed o con ol ish a
spe ma ogenesis. Howe e , al hough bo h EE2 ea men s caused a d op in 11-KT plasma le els, which eco e ed
a 146days so ish had 11-KT plasma le els simila o he con ol ish a he es icula in olu ion s age, sex change
did no occu in any o he EE2- ea ed ish. Thus, he occu ence o low 11-KT plasma le els does no seem o
be de e minan o sex change in he gil head seab eam. On he o he hand, ou da a co obo a es he ou come
o p e ious s udies ha ha e ailed o di ec ly link T and P o sex change. Hence, unlike o he eleos ish46–50,
s e oid le els do no seem o be key igge s o sex change in he gil head sea b eam.
Gonado opins seem o be in ol ed in p o and ous sex change, al hough hei ole is no well unde s ood.
In clown ish induced o change sex by emo ing emales, exp ession le els o bo h shb and lhb inc eased as he
sex change p og essed51. Howe e , in he black po gy he ole o gonado opins is con o e sial. Lowe exp es-
sion le els o shb and lhb a e de ec ed du ing na u al sex change om male o emales20. In con as , inc eased
exp ession le els o lhb ha e been desc ibed in he ish unde going male- o- emale and emale- o-male induced
sex change52. Fu he mo e, a ise in plasma le els o LH has been eco ded in male ish compa ed o ish changing
sex ollowing he second spawning pe iod and be o e ec udescence o he hi d RC in his species (June–July50,
May–Augus 53, Ap il–June54).Likewise, in he p esen s udy he pi ui a y exp ession le els o shb we e lowe in
con ol gil head seab eam a he es icula in olu ion s age han a spe ma ogenesis o bo h he second and he
hi d RCs. Howe e , EE2 a ec ed his di e en ial exp ession only in ish ed 5µg EE2/g ood. Since ish o bo h
expe imen al g oups main ained he male s a us o he ollowing RC, he al e a ions o exp ession le els o shb
does no seem o be he cause o he ailu e o sex change in EE2-exposed gil head seab eam. On he o he hand,
on his s udy, con ol ish showed simila ly low exp ession le els o lhb in pi ui a y a spe ma ogenesis and es-
icula in olu ion s ages o he second RC and simila ly high pi ui a y exp ession le els o lhb in con ol males
and emales a he spe ma ogenesis o he hi d RC. As a esul o EE2 exposu e, pi ui a y exp ession le els o
lhb we e inc eased upon ea men in ish ed 2.5µg EE2/g ood and upon eeding and eco e ing o 146days
in ish ed 5µg EE2/g ood, compa ed o con ol ish. This inc eased LH signal could main ain he male a e and
a oid he de elopmen o emaleness, as p oposed o black po gy55. In his species, LH signal seems o p ese e
male a e55, h oughou he main enance o dm 1 exp ession54,55, whils emaleness would be swi ched on by
he eg ession o es icula de elopmen as a esul o he diminished dm 1 exp ession le els in es is54,55. In
gil head seab eam i has been epo ed ha exp ession le els o dm 1 dec ease du ing he es icula in olu ion
phase p io o sex change27. Mo eo e , exposi ion o 5µg EE2/g ood in die du ing spe ma ogenesis esul ed in
a dec ease in exp ession le els o dm 1 in he es is o ma u e gil head seab eam ha pe sis ed a e 25days o
eco e y wi h a s anda d die 35.
Hence, he exposu e o gil head seab eam o EE2 in he die (2.5 o 5µg EE2/g ood) o a sho pe iod o ime
(28days) a he spe ma ogenesis s age o he second RC caused al e a ions ha could be asce ained a long ime
a e (146days) in E2 plasma le els and e en longe (333days) in exp ession le els o genes coding o pi ui a y
ho mones in ol ed in he egula ion o ep oduc i e e en s. Howe e , hese dis u bances could no be di ec ly
Table1. E ec o he die a y in ake o 2.5 o 5 µg o EE2 g−1 o ood o 28 days ollowed by 333 days
eco e y wi h a s anda d die on he concen a ion (× 109 cell/mL) and iabili y o spe m. Da a ep esen he
means ± S.E.M. o 6 males om each expe imen al g oup. No signi ican di e ences we e iden i ied (ANOVA
es ).
T ea men Spe m concen a ion (× 109 cell/mL) Viabili y (%)
Con ol 6.21 ± 1.50 96.32 ± 0.73
2.5 µg o EE2 g−1 o ood 6.77 ± 0.84 97.40 ± 0.30
5 µg o EE2 g−1 o ood 6.73 ± 0.59 97.67 ± 0.31