General and Comparative Endocrinology

Transcription

1 Generl nd Comprtive Endocrinology 75 () 7 Contents lists ville t SciVerse ScienceDirect Generl nd Comprtive Endocrinology journl homepge: Differentil regultion of the expression of cytochrome P5 romtse, estrogen nd ndrogen receptor sutypes in the rin pituitry ovrin xis of the Jpnese eel (Anguill jponic) revels steroid dependent nd independent mechnisms Shn-Ru Jeng,, Jeremy Psquier, Wen-Shiun Yueh, Gun-Ru Chen c, Yn-Horn Lee d, Sylvie Dufour, Ching-Fong Chng e,f, Deprtment of Aquculture, Ntionl Kohsiung Mrine University, Kohsiung 8, iwn Reserch Unit BOREA, Biology of Aqutic Orgnisms nd Ecosystems, CNRS 78/IRD 7/UPMC, Muséum Ntionl d Histoire Nturelle, 7 rue Cuvier, 75 Pris Cedex 5, Frnce c Freshwter Aquculture Reserch Center, Fisheries Reserch Institute, Lukng 55, iwn d ungkng Biotechnology Reserch Center, Fisheries Reserch Institute, Pintung, iwn e Deprtment of Aquculture, Ntionl iwn Ocen University, Keelung, iwn f Center of Excellence for Mrine Bioenvironment nd Biotechnology, Ntionl iwn Ocen University, Keelung, iwn rticle info strct Article history: Received My Revised Octoer Accepted Novemer Aville online 5 Novemer Keywords: Aromtse Steroid receptor Estrdiol estosterone Eel eleost his study imed t investigting the role of sexul steroids in the regultion of the expression of the single romtse gene nd steroid receptor sutypes in the rin pituitry ovrin xis of the Jpnese eel. Unlike other teleosts, which possess duplicted genes for romtse, cyp nd cyp, expressed in the gonds nd in the rin, respectively, eel species possess single cyp. Phylogenetic nlysis indicted tht eel rin/gondl cyp rnches t the sis of oth teleost gondl cyp nd rin cyp cldes. Femle eels treted with ctfish pituitry homogente (CPH) to induce sexul mturtion showed n increse in the expression of cyp nd romtse enzymtic ctivity in the rin nd in the ovries. retments with sex steroids (estrdiol-7, E or testosterone, ) reveled tht the increse in cyp expression in the rin my result from E -specific induction. In contrst, the increse in cyp expression in the ovries of CPH-treted eels is result of steroid-independent control, proly from direct effect of gondotropins contined in the pituitry extrct. Anlysis of the expression of estrogen nd ndrogen receptor sutypes,,, r- nd r-, in eels treted with CPH or sex steroids reveled differentil regultions. In CPH-treted eels, the expression of nd r- ws significntly incresed in the rin, while the expression of r- nd r- ws incresed in the ovries. No chnge ws oserved in esr in ny orgn. Steroid tretments induced n upregultion y E of, ut not expression, in the rin, pituitry nd ovries, while no utoregultion y of its own receptors could e oserved. hese results revel oth steroid-dependent nd -independent mechnisms in the regultion of cyp nd steroid receptor sutype expression in the eel. Ó Pulished y Elsevier Inc.. Introduction Estrogens re involved in vrious spects of sexul differentition, vitellogenesis, gondl development, reproduction nd reproductive ehviors []. Cytochrome P5 romtse is the key enzyme tht ctlyzes the conversion of ndrogens to estrogens [5]. It hs een shown tht romtse is expressed in vrious Corresponding uthors t: Center of Excellence for Mrine Bioenvironment nd Biotechnology, Ntionl iwn Ocen University, Keelung, iwn (C.-F. Chng). Fx: E-mil ddresses: srjeng@mil.nkmu.edu.tw (S.-R. edu.tw (C.-F. Chng). tissues, especilly in rin nd gonds, in different verterte species [,5]. In mmmls, there is only single copy of the romtse gene, CYPA, except in pigs, which hve multiple copies of the CYPA gene []. he tissue-specific regultion of CYPA is chieved through distinct promoters nd lterntive splicing of 5 - untrnslted exons under the control of vrious fctors, including gondotropins, growth fctors nd steroids []. Erly studies reveled tht teleosts exhiit much higher rin romtse enzymtic ctivity (- to -fold) thn mmmls [,]. his high enzymtic ctivity is relted to the specific expression of the duplicted romtse gene cyp in the rin of teleosts. In most teleosts, two seprte genes, nmed cyp nd cyp, hve een identified. As other duplicted teleost genes, -8/$ - see front mtter Ó Pulished y Elsevier Inc. doi:./j.ygcen...5

2 S.-R. Jeng et l. / Generl nd Comprtive Endocrinology 75 () 7 they re supposed to e relted to the whole genome dupliction tht occurred erly in the teleost linege, efore the seprtion of the Elopomorph superorder group [,]. Cyp nd cyp re predominntly expressed in the gonds nd rin, respectively, nd encode the distinct romtse isoforms romtse A nd romtse B (goldfish, Crssius urtus: [7,5]; zerfish, Dnio rerio: [8,8]; Europen se ss, Dicentrrchus lrx: [5,]; rinow trout, Oncorhynchus mykiss: [,5]; rice field eel, Monopterus lus: [7]). In rinow trout, due to dditionl slmonid gene dupliction, two distinct cyp genes, cyp nd cyp, were found; the cyp gene hs een identified s n unusul duplicte of cyp lcking the first two exons of cyp []. In ddition, von Schlurg et l. [] showed tht there re t lest five cyp trnscripts with different 5 - untrnslted regions in the ovries nd testis of rinow trout. In contrst, our previous studies in the Jpnese eel, Anguill jponic, indicted tht the enzymtic properties of rin romtse in this species re similr to those of mmmlin romtse, with lower ffinity nd lower mximl rection rte thn rin romtse from other teleots [5]. In ddition, s suggested y Ijiri et l. [] for the Jpnese eel nd zchori et l. [] for the Europen eel, our dt supports the hypothesis tht eels (Anguill species) my hve retined single cyp gene [5], expressed oth in the gonds nd in the rin, similr to mmmls. Brin romtse enzymtic ctivity is significntly incresed during the spwning seson in vrious teleost species (goldfish: [5]; lck porgy, Acnthopgrus schlegeli: []; Europen se ss: []). Both E nd romtizle ndrogens were shown to upregulte rin cyp expression nd enzymtic ctivity (goldfish: []; lck porgy: []; zerfish: []). Gondl cyp trnscript levels nd enzymtic ctivity in ovrin follicles re lso significntly incresed during vitellogenesis in teleosts (rinow trout: [5]; tilpi, Oreochromis niloticus: [8]; red serem, Pgrus mjor: [8]; Atlntic croker, Micropogonis undultus: []), ut the role of sex steroids in this upregultion is less cler thn for rin cyp. Severl steroid nucler receptor sutypes hve een identified in teleosts. At lest two mjor sutypes of estrogen receptors ( nd ) re found in teleosts including the Europen silver eel nd Jpnese eel [,5]. As first discovered in the Jpnese eel [,57], there re two ndrogen sutypes (r- nd r-) in teleosts, likely resulting from the whole genome dupliction in the teleost linege. Eels present unique life cycle with lockde of sexul mturtion t prepuertl stge (silver stge) s long s the ocenic reproductive migrtion is not chieved (for review: [5]). Deciphering specil endocrine mechnisms of eel reproduction is current chllenge with oth sic nd pplied perspectives. o investigte the potentil roles of sex steroids nd their receptors in the differentil regultion of the single romtse gene long the rin pituitry ovrin xis, we nlyzed the vritions in romtse trnscript levels, enzymtic ctivity, nd trnscript levels of estrogen nd ndrogen receptor sutypes during induced ovrin development nd fter sex steroid tretment in Jpnese eels.. Mterils nd methods.. Animls hree-yer-old femle Jpnese eels (t the prepuertl silver stge), A. jponic, were otined from n quculture frm in iwn nd trnsferred t the culture sttion of the Ntionl Kohsiung Mrine University, iwn. Experimentl fish were plced in outdoor.5 ton-tnks (one tnk/experimentl group), with running freshwter, under nturl light nd temperture (wter temperture rnge: 7 C). All procedures nd investigtions were pproved y the Ntionl Kohsiung Mrine University Institutionl Animl Cre nd Use Committee nd were performed in ccordnce with stndrd guiding principles... Gondotropic tretment Chronic tretments with teleost (crp or slmon) pituitry homogentes re usully pplied to induce ovrin development of femle Europen nd Jpnese eels [,,]. In the present study, we chose to use ctfish (Clris fuscus) pituitry homogentes (CPH) prepred in sline (.75% NCl) from cetone-dried pituitries purchsed from Kohsiung locl fishermen. CPH is commonly used for inducing finl mturtion nd spwning in vrious teleost species in quculture frms in iwn. Femle eels (n = 8, BW = 75. ±.7 g, BL = 75.7 ±. cm) were divided into two groups: control eels (for romtse enzymtic ctivity, n = 8; for cyp nd steroid receptor sutype trnscript levels, n = ) nd treted eels (for romtse enzymtic ctivity, n = 8; for cyp nd steroid receptor sutype trnscript levels, n = ). he treted fish were injected intrperitonelly (ip) weekly with the homogente of one ctfish pituitry ( mg dry weight) in.5 ml sline/fish for 7 weeks. he control fish were injected with sline lone. Fish were scrificed dys fter the lst injection... Sex steroid tretments Chronic tretments with sex steroids were performed ccording to the protocols previously descried []. E nd were purchsed from Sigm nd dissolved in coconut oil (Sigm Aldrich Corp., St. Louis, MO). Femle eels (BW = 7.5 ± 7 g, BL = 85. ±. cm) were divided into five groups (n = 8 eels per group). reted eels received ip injections of.75 mg or.75 mg of steroid ( or E )/kg BW. eels received injections of coconut oil lone. Fish were injected weekly for weeks nd were scrificed week fter the lst injection... Smpling procedure nd treted eels were nesthetized with 8 ppm -phenoxyethnol efore eing scrificed. he totl ody weight (BW), liver weight nd ovrin weight were mesured for the clcultion of the heptosomtic index (HSI = liver weight/bw %) nd gondosomtic index (GSI = gond weight/bw %). he forerin, midrin, pituitry nd ovries were collected nd stored t 8 C for enzymtic nd quntittive rel-time PCR nlysis. A piece of ovrin tissue ws lso stored in Bouin s solution for histologicl oservtion. Blood smples were llowed to clot t C, nd serum ws collected nd stored t C for steroid EIAs..5. Mesurement of romtse enzymtic ctivity Aromtse enzymtic ctivity of rin, pituitry nd ovries ws mesured using the tritited wter ssy s previously descried [,5]. he protein concentrtions of the crude superntnt frction were mesured with Bio-Rd protein ssy kit (Bio-Rd Co., Hercules, CA). Aromtse enzymtic ctivity ws expressed s fmol H O/h mg protein... Quntifiction of cyp,,, r- nd r- trnscripts y rel-time PCR nlysis Quntittive rel-time PCR (qpcr) nlysis for gene trnscripts ws conducted ccording to previously descried methods []. Prtil sequences of Jpnese eel cyp (7 p), (8 p), (87 p), r- (5 p), nd r- (7 p) cdnas were cloned nd

3 S.-R. Jeng et l. / Generl nd Comprtive Endocrinology 75 () 7 5 used s stndrds for qpcr. hese cdnas were cloned from the totl RNA of Jpnese eel rin (cyp), liver ( nd ) or testis (r- nd r-) y R-PCR using specific primers designed from the pulished sequences of Jpnese eel neurl romtse (GenBnk Accession No. AY7.; [5]), Europen eel (GenBnk Accession No. EU75.; []), Jpnese eel (Gen- Bnk Accession No. AB5.; [5]), r- (GenBnk Accession No. AB.; [57]), nd r- (GenBnk Accession No. AB5.; []). he sequences otined were % identicl to the pulished Jpnese eel sequences (cyp,, r-, nd r-) nd 7% identicl to the pulished Europen eel (GenBnk Accession No. HM558 for Jpnese eel ). Specific primers for cyp,,, r-, r- nd reference gene glycerldehyde--phosphte dehydrogense (gpdh) (Gen- Bnk Accession No. AB58.; [5]) were designed for qpcr (le ). Gene quntifiction of stndrds (plsmids with cdna sequence) nd smples were conducted simultneously y qpcr (GeneAmp 57 Sequence Detection System; Applied Biosystems, Foster City, CA) with SYBR green I s dsdna minor-groove inding dye. Melting curves indicted the mplifiction of single mplicon for ech gene. he slopes of the respective stndrd nd smple curves of the log (cdna concentrtions) vs. Ct (the clculted frctionl cycle numer t which the PCR-fluorescence product is detectle ove threshold) were. to.5, indicting n mplifiction efficiency of %. he trnscript vlues of ech gene were clirted with the internl reference gene (gpdh). No significnt chnges were oserved in gpdh trnscript levels fter steroid tretments..7. Immunoenzymtic ssys of serum E nd he concentrtions of E nd in serum were mesured y Cymn Chemicl s ACE enzyme immunossy kit (EIA) Kits (Estrdiol EIA Kit nd estosterone EIA Kit, Cymn Chemicl Compny, Ann Aror, MI)..8. Sttistics he dt from the enzymtic ssys, qpcr nd EIAs were expressed s mens ± SEM. All sttisticl nlyses were performed with SPSS. for Windows. he vlues were sujected to onewy ANOVA to test significnce, followed y t-test or Duncn multiple-rnge test... Phylogenetic nlysis he mino cid sequences of verterte cyp were first ligned using ClustlW [55]. he protein sustitution mtrix of the resulting lignment ws determined s J + I + G + F using the Pro- est softwre []. he phylogenetic nlysis of the cyp sequence lignment ws performed using mximum likelihood method using the RxML softwre [5] with ootstrp replictes.. Results.. Anlysis of romtse sequences Prtil cloning ( p) of cyp from Jpnese eel rin cdna (GenBnk Accession No. AY7.) resulting in sequence tht ws % identicl to tht of Jpnese eel ovrin cyp []. No other cyp sequence could e detected in the eel. Sequence lignment nd comprisons were mde using ville cyp sequences in vertertes nd chondrichtyn cyp s rooting group (Fig. ). he phylogenetic tree shows two min clusters corresponding to tetrpod cyp nd teleost cyp. he eel single gondl/rin cyp rnches t the se of the teleost cyp cluster. All the other teleost sequences re distriuted into two cldes corresponding to gondl cyp (cyp) nd rin cyp (cyp). hese two cldes reflect the dupliction of cyp in teleosts. he single eel cyp does not pper to e more relted to one teleost clde thn to nother... Effect of gondotropic tretment in femle... Biometric prmeters nd serum steroid levels Chronic tretment with CPH induced significnt development of the ovries, s shown y the lrge increse in GSI (. ±.% in the treted eels vs..7 ±.% in the control eels, p <.). Histologicl oservtion showed tht the oocytes of control eels were t the previtellogenic stge (no visile yolk gloules), while the oocytes of CPH-treted eels were t the dvnced vitellogenic stge (with numerous yolk gloules) (dt not shown). HSI ws significntly incresed in CPH-treted eels (.5 ±.% in treted eels vs.. ±.5% in the control eels, p <.5). he concentrtions of sex steroids in serum were significntly incresed in CPH-treted eels (E levels:.5 ±. ng/ml in the treted eels vs..5 ±. ng/ml in the control eels, p <.; levels:. ±.7 ng/ml in the treted eels vs..5 ±. ng/ml in the control eels, p <.5).... Aromtse enzymtic ctivity Aromtse ctivity could e mesured in the rin, pituitry nd ovries. he ctivity ws the highest in the pituitry. Aromtse enzymtic ctivity ws significntly incresed in the CPH-treted eels in the forerin (-fold, p <.), midrin (7-fold, p <.) nd ovries (-fold, p <.5), while no significnt chnge ws oserved in the pituitry s compred to the controls (Fig. ).... Cyp trnscripts As for enzymtic ctivity, cyp trnscript levels were higher in the pituitry thn in the rin nd ovries s indicted y comprison of Ct vlues (dt not shown). Cyp trnscript levels le Specific primers used for quntittive rel-time PCR nlyses (S, sense strnd; AS, ntisense strnd). Gene Sequences Amplicon size GenBnk Accession No. cyp S 5 -CCGCCACCCGGGC- 8 p AY7. cyp AS 5 -GACAAGCCGCACCAGACA- S 5 -CGCCCACGACCAGGGCAG- 8 p HM558. AS 5 -GCGCCACACCAGCCCGAA- S 5 -AGGACCAAGGGCACAAG- 5 p AB5. AS 5 -CGCGCCGGCGC- r- S 5 -CCCACCGGCCGAAAAAC- 8 p AB. r- AS 5 -CGCCCCGACCGAACGA- r- S 5 -GGCCCAGGAGCC- p AB5. r- AS 5 -GCCCGCGCGCAAAC- gpdh S 5 -GCCAGCCAGAACACAC- p AB58. gpdh AS 5 -GACACGGAAAGCCAACC-

4 S.-R. Jeng et l. / Generl nd Comprtive Endocrinology 75 () 7 Fig.. Consensus phylogenetic tree of the verterte cyp constructed sed on the mino-cid sequences of cyp using the mximum likelihood method with ootstrp replictes. he numer shown t ech rnch node indictes the ootstrp vlue (%); only vlues nd rnching ove 5% re shown. he sequences used for this phylogenetic reconstruction were tken from NCBI dt se (GenBnk Accession No. etween rckets): ov/r/cyprinus crpio (ACB7., ACB8.), ov/r/ictlurus puncttus (AAB., NP_87.), ov/r/kryptoleis mrmortus (ABC8., ABC8.), A/B/kifugu ruripes (NP_7., BAF5.), ov/r/goiocypris rrus (ADB5., ADB88.), ov/r/fundulus heteroclitus (AAR78., AAR7.), ov/r/oreochromis mossmicus (AAD., AAD.), ov/r/cromileptes ltivelis (AAV78., AAV8.), ov/r/goiodon histrio (AAV77., AAV8.), ov/r/lrimichthys croce (ACO5., ACO5.), ov/r/cynoglossus semilevis (ABL77., ABM.), A/B/Epinephelus coioides (AAR7., AAR7.), ov/r/hlichoeres tenuispinis (AAR78., AAR77.), ov/r/pseudolrus jponicus (ABB85., ABB8.), ov/r/rhdosrgus sr (ABC788., ABC78.), ov/r/anguill jponic (AAS78., AA.), ov/slmo trutt (AAR775.), ov/r-i/r- II/Oncorhynchus mykiss (85A, CAG85., CAG85.). o, ov/r/dnio rerio (AAB5788., AAV.), ov/r/dicentrrchus lrx (CAC78, AAM555), ov/r/ Rutilus rutilus (BAD7., BAD8), ov/r/epinephelus kr (AAS588., AAS587.), ov/r/hippoglossus hippoglossus (CAC., AAY.), ov/r/mugil cephlus (AAW77., AAW77.), ov/r/monopterus lus (ABX5., ABX5.), ov/r/oreochromis niloticus (AAB8, AAG858.), ov/r/crssius urtus (BAA758., BAA757.), ov/r/oryzis ltipes (BAA5., AAP8.), ov/r/lrimichthys croce (ACO5., ACO5.), ov/r/odontesthes onriensis (ABK87., AAQ88.), Homo spiens (NP_.), Equus cllus (AAC8.), Bos turus (CAA85.), Xenopus levis (BAF85.), eniopygi guttt (NP_75.), Squlus cnthis (ABB58.), Dsytis Sin (AAF7.), Rttus norvegicus (NP_5878.), Cynops pyrrhogster (BAD.), rchemys script (AAG7.), Bufo mrinus (ACN55.), Rn rugos (BAD5.), Gllus gllus (AAA878.), Coturnix jponic (AAN75.), Ovis ries (CAB5.), Cnis lupus fmiliris (CAH.), Sus scrof (NP_5.), Oryctolgus cuniculus (NP_.), Eulephris mculrius (BAE.), Alligtor mississippiensis (AAK8.). ov: ovrin form romtse, r: rin form romtse, te: testiculr form romtse. he tree ws rooted using the sequences of Squlus cnthis nd Dsytis sin cyp.

5 S.-R. Jeng et l. / Generl nd Comprtive Endocrinology 75 () 7 7 P5rom Activity (fmole/h. mg protein) () () Reltive expression levels (cyp/gpdh) cyp cyp cyp cyp Reltive expression levels ( /gpdh) () Reltive expression levels ( /gpdh) () Fig.. Effect of ctfish pituitry homogentes on forerin, midrin, pituitry nd ovry () cyp trnscript levels nd () romtse enzymtic ctivity in femle Jpnese eels, s mesured y rel-time quntittive PCR. Brs represent men ± - SEM. Significnt differences etween the two groups re denoted y the following: p <.5, p <. nd p <.. Fig.. Effect of ctfish pituitry homogentes on forerin, midrin, pituitry nd ovry () nd () trnscript levels in femle Jpnese eels, s mesured y rel-time quntittive PCR. Brs represent men ± SEM. Significnt differences etween the two groups re denoted y the following: p <.5 nd p <.. were significntly incresed in CPH-treted eels in the forerin (-fold, p <.), midrin (-fold increse, p <.5) nd ovries (-fold increse, p <.5), wheres pituitry cyp trnscripts remined t similr level in the CPH-treted eels s compred to the control eels (Fig. ).... Estrogen receptors ( nd ) trnscripts Esr- trnscript levels were significntly incresed in CPH-treted eels in the forerin (-fold, p <.), midrin (-fold, p <.5) nd pituitry (-fold, p <.5) when compred with the control eels, wheres trnscripts in the ovries were not significntly different (Fig. ). Esr- trnscript levels were not significntly different in ny tissue in the CPH-treted eels s compred with the control eels (Fig. )...5. Androgen receptors (r- nd r-) trnscripts Ar- trnscript levels were significntly incresed in the CPHtreted eels in the midrin (-fold, p <.5) nd ovries (-fold, p <.5), while significnt decrese ws oserved in the pituitry (-fold decrese, p <.) nd no chnge ws oserved in the forerin s compred to the control eels (Fig. ). Ar- trnscript levels were significntly incresed in the ovries of CPH-treted eels (7-fold, p <.5), while no significnt chnges were oserved in the forerin, midrin nd pituitry s compred to the control eels (Fig. )... Effect of sex steroid tretments... Biometric prmeters nd serum steroid levels No significnt chnges in GSI were oserved fter tretment with E or (.77 ±.% in the controls,. ±.% in the low E -,. ±.% in the low -,.55 ±.7% in the high E -,.5 ±.% in the high -treted eels, NS). Histologicl oservtion showed tht oocytes remined t the previtellogenic stge (no visile yolk gloules) in steroid-treted eels s oserved in the controls (dt not shown). he HSI ws not significntly chnged fter low E - (. ±.%, NS) or low -tretment (. ±.5%, NS), ut significntly incresed fter high E - (.57 ±.%, p <.5) or high - tretment (. ±.%, p <.5) s compred to the controls (.8 ±.%). E serum levels were significntly incresed fter low E - (8. ±. ng/ml, p <.) nd high E -tretment (77. ±.7 ng/ml, p <.) s compred to controls (. ±. ng/ml). serum levels were significntly incresed fter low - (.8 ±. ng/ml, p <.) nd high -tretment (5.5 ±. ng/ml, p <.) s compred to the controls (. ±. ng/ml). E serum levels remined low even fter high -tretment (. ±. ng/ml, NS s compred to the controls).... Cyp trnscripts Cyp trnscript levels were significntly incresed y E tretment in dose-dependent mnner oth in the forerin nd midrin. In the forerin, 5-fold increse (p <.) ws induced y low dose of E, nd 8-fold increse (p <.) ws induced y high dose of E s compred to the controls. In the midrin, -fold increse (p <.5) ws induced y low dose of E, nd n -fold increse (p <.) ws induced y high dose of E s compred to the controls. In contrst, pituitry nd ovry cyp trnscript levels remined unchnged fter E tretment. Brin, pituitry nd ovry cyp trnscripts were not significntly chnged y ny dose of tretment (Fig. 5).... Estrogen receptor ( nd ) trnscripts Esr- trnscript levels were significntly incresed in the forerin, pituitry nd ovries (-, 7- nd -fold, respectively;

6 8 S.-R. Jeng et l. / Generl nd Comprtive Endocrinology 75 () 7 Reltive expression levels (r- /gpdh) () p <.5) fter high dose of E tretment, while low dose of E hd no significnt effect (Fig. ). t low or high dose did not induce ny chnges in trnscript levels in ny tissue, except for slight increse (-fold increse, p <.5) in the forerin in eels treted with low dose of (Fig. ). Neither E nor induced ny chnges in trnscript levels in the rin, pituitry nd ovries (Fig. 7).... Androgen receptors (r- nd r-) trnscripts retments with E or did not induce ny chnges in r- (Fig. 8) or r- (Fig. ) trnscript levels in the rin, pituitry nd ovries.. Discussion r- r- r- r- r-.. A single cyp gene is expressed in the rin nd gonds in the eel Duplicted genes for cyp hve een found in teleosts, likely resulting from the whole genome dupliction event tht occurred t the sis of their linege []. eleost cyp is expressed in the gonds, while cyp is expressed in the rin. In contrst, single cyp gene is found in eel species, nd we showed tht it is expressed in oth the rin nd the gonds, similr to tetrpods. o further understnd the unique chrcteristics of eel cyp, we performed phylogenetic nlysis of cyp sequences. Eel cyp rnches t the se of the teleost cyp cluster in greement with the sl phylogenetic position of Elopomorphs mong teleost species. Eel cyp does not pper to e more relted to teleost cyp thn to teleost cyp cldes. his suggests tht single eel cyp my hve conserved ncestrl properties of ctinopterygin cyp. Reltive expression levels (r- /gpdh) () r- r- r- Fig.. Effect of ctfish pituitry homogentes on forerin, midrin, pituitry nd ovry () r- nd () r- trnscript levels in femle Jpnese eels, s mesured y rel-time quntittive PCR. Brs represent men ± SEM. Significnt differences etween the two groups re denoted y the following: p <.5 nd p <.. Reltive expression levels (cyp/gpdh) 5 5 E c E cyp We my consider two lterntive scenrios for the single cyp gene in the eel. In the first scenrio, whole genome dupliction would hve occurred erly in the teleost linege, efore the seprtion of the Elopomorph group from the other teleosts, nd eel single cyp would reflect specific loss of one duplicted cyp gene in Elopomorh/Anguill linege. In the lterntive scenrio, whole genome dupliction would hve occurred fter the seprtion of the Elopomorph linege from the other teleosts groups nd the single cyp in the eel would simply reflect the ncestrl sitution of unique gene. Incresing dt re indicting the presence of vrious duplicted genes in the eel s well s in other teleosts, in fvor of the first scenrio. his is the cse for instnce for duplicted ndrogen receptors (r- nd r-) first evidenced in the eel [57,], or for duplicted dopmine D receptors s investigted y our group [7]. Furthermore, this hs een recently highlighted y the study of hox genes in the Jpnese eel reveling whole genome dupliction []. his supports the hypothesis tht the single cyp gene in the eel would result from specific cyp gene loss in the Elopomorph/Anguill linege. he presence of single cyp gene in the eel my ccount for its low rin romtse ctivity, similr to mmmls, ut differently from the other teleosts [5]... Upregultion of cyp expression in the rin nd ovries during induced mturtion in the eel he cyp trnscripts coding for the single rin/gondl romtse in the eel were significntly incresed in the CPH-induced cyp cyp cyp Fig. 5. Effect of sex steroid tretments on forerin, midrin, pituitry nd ovry cyp trnscript levels in femle Jpnese eels, s mesured y rel-time quntittive PCR. hree-yer-old cultivted femle eels received weekly injections of estrdiol-7- (E ) or testosterone () (low dose:.75 mg or high dose:.75 mg in coconut oil/kg BW) for weeks. eels were injected with coconut oil lone. Brs represent men ± SEM. Different letters represent significnt differences mong the groups. (p <.5 or p <.). 5 5 Reltive expression levels (cyp/gpdh)

7 S.-R. Jeng et l. / Generl nd Comprtive Endocrinology 75 () 7 esr β Reltive expression levels ( /gpdh) Reltive expression levels ( /gpdh) Reltive expression levels (β/gpdh) β β Reltive expression levels (β/gpdh) β E E E E Fig.. Effect of sex steroid tretments on forerin, midrin, pituitry nd ovry trnscript levels in femle Jpnese eels, s mesured y rel-time quntittive PCR. hree-yer-old cultivted femle eels received weekly injections of estrdiol-7- (E ) or testosterone () (low dose:.75 mg or high dose:.75 mg in coconut oil/kg BW) for weeks. eels were injected with coconut oil lone. Brs represent men ± SEM. Different letters represent significnt differences mong the groups. (p <.5). Fig. 7. Effect of sex steroid tretments on forerin, midrin, pituitry nd ovry trnscript levels in femle Jpnese eels, s mesured y rel-time quntittive PCR. hree-yer-old cultivted femle eels received weekly injections of estrdiol- 7- (E ) or testosterone () (low dose:.75 mg or high dose:.75 mg in coconut oil/ kg BW) for weeks. eels were injected with coconut oil lone. Brs represent men ± SEM. Different letters represent significnt differences mong the groups. (p <.5). mtured eels in oth the rin nd ovries. Similr increses were mesured for romtse enzymtic ctivity in the rin nd ovries of CPH-induced mtured eels. hese results re consistent with other studies in teleosts concerning oth types of romtse genes. For instnce, the rin cyp trnscripts levels nd romtse enzymtic ctivity were significntly incresed during the spwning seson in goldfish [7], lck porgy [], se ss [] nd lue gourmi (richogster trichopterus) []. Similrly, the ovrin cyp trnscript levels nd romtse enzymtic ctivity were significntly elevted during vitellogenesis in rinow trout [5], Nile tilpi [8] nd red serem [8]. In contrst to the increses in cyp trnscripts levels in the rin nd ovries, no significnt chnges were oserved in the pituitry in CPH-treted femle eels s compred to control femle eels. he expression of cyp trnscripts lso incresed significntly in the rin, ut not in the pituitry of grey mullet (Mugil cephlus) when the fish reched the puerty stge []. In contrst, Kzeto nd rnt [7] indicted tht rin nd pituitry cyp expression levels incresed drmticlly prior to spwning in chnnel ctfish (Ictlurus puncttus)... Estrdiol-specific role in the upregultion of cyp expression in the rin Circulting E nd levels were elevted in the CPH-induced mtured femle eels, in greement with previous studies in vrious eel species ( A. jponic: []; Anguill nguill: []; Anguill dieffenchii: [5]). Both E nd could therefore e potentilly involved in feedck regultions of gene expression during sexul mturtion. For instnce, our previous studies demonstrted n E -dependent up-regultion of mgnrh (mmmlin type gondotropin relesing hormone) nd testosterone-dependent downregultion of cgnrh-ii (chicken type GnRH) in the rin of mtured femle Europen eels [8]. We investigted the potentil role of these sex steroids in the increse in cyp expression y performing steroid tretments. We oserved dose-dependent stimultory effect of E on cyp expression in the rin, ut not in the ovries. his result revels differentil regultion y steroids of eel cyp trnscripts in the rin nd in the ovries, with n estrdiol-specific upregultion in the rin. he effect of E in the rin ws specific, s no chnge ws induced y ndrogen. In other teleosts, oth romtizle ndrogens nd estrogens re known to upregulte rin cyp trnscripts (goldfish: [7]; zerfish: [8,7]). Androgens re considered to ct through estrogen receptors fter locl romtiztion, ut not vi ndrogen receptors (for review: []). In zerfish, Mouriec et l. [] recently found tht rin cyp ws upregulted y nd lso y dihydroxytestosterone (DH), n ndrogen generlly considered s non-romtizle in mmmls. hey suggested tht the effect of DH on the rin cyp gene ws due to the conversion of DH into metolite with estrogenic ctivity in zerfish. In our study, hd no significnt effect on rin cyp expression in contrst to the upregultory effect of E. he low level of rin romtse ctivity in eel, unlike in zerfish nd other teleosts, likely ccounts for the

8 7 S.-R. Jeng et l. / Generl nd Comprtive Endocrinology 75 () 7 r- r- Reltive expression levels (r- /gpdh) r- r- Reltive expression levels (r- /gpdh) Reltive expression levels (r- /gpdh) r- r Reltive expression levels (r- /gpdh) r- r- E E E E Fig. 8. Effect of sex steroid tretments on forerin, midrin, pituitry nd ovry r- trnscript levels in femle Jpnese eels, s mesured y rel-time quntittive PCR. hree-yer-old cultivted femle eels received weekly injections of estrdiol- 7- (E ) or testosterone () (low dose:.75 mg or high dose:.75 mg in coconut oil/kg BW) for weeks. eels were injected with coconut oil lone. Brs represent men ± SEM. Different letters represent significnt differences mong the groups. (p <.5). Fig.. Effect of sex steroid tretments on forerin, midrin, pituitry nd ovry r- trnscript levels in femle Jpnese eels, s mesured y rel-time quntittive PCR. hree-yer-old cultivted femle eels received weekly injections of estrdiol- 7- (E ) or testosterone () (low dose:.75 mg or high dose:.75 mg in coconut oil/kg BW) for weeks. eels were injected with coconut oil lone. Brs represent men ± SEM. Different letters represent significnt differences mong the groups. (p <.5). lck of n effect of on cyp expression. he low centrl romtse ctivity in the eel, unique feture mong teleosts, my hve fvored vrious estrogen-specific or inversely ndrogen-specific regultory processes, s lso highlighted y our previous neuroendocrine studies (i.e., [,7,]). Estrogen response elements hve een found on the rin cyp promoter, ut not on the gondl cyp promoter in ll investigted teleosts (goldfish: [7], zerfish: [58], grey mullet: []). his ERE is supposed to medite the upregultory effect of E on rin cyp in these teleosts. In mmmls, no typicl ERE ws found in the promoter of the single CYPA. Yilmz et l. [5] demonstrted tht in the mouse, E upregultes rin CYPA expression vi the rin-specific promoter I.f y enhncing the inding of n estrogen receptor-/jun complex to distinct ctivtor protein (AP-) motifs in hypothlmic cells. Further studies in the eel will im t deciphering if the E -dependent stimultion of the expression of the single romtse gene cyp in the rin is medited vi n ERE similr to teleost cyp or vi mmmlin-like pthwy... Steroid-independent upregultion of cyp expression in the ovries In contrst to the E upregultory effect on cyp expression in the rin, neither E nor hd ny effect on cyp expression in eel ovries. Although previous study in lck porgy hd demonstrted higher gondl romtse enzymtic ctivity fter long-term tretment with E [], most of the studies on teleosts showed tht ovrin cyp expression ws not estrogen-inducile (goldfish: [7]; zerfish: [8]). As mentioned ove, in teleosts, no ERE could e detected on the promoter of gondl cyp. hese findings suggest steroid-independent regultory mechnism for the increse in cyp expression in the ovries of CPHinduced mtured eels. In mmmls, Yong et l. [] indicted tht FSH nd LH strongly stimulted humn CYPA mrna expression in mture humn grnulos cells. In slmonids, Montserrt et l. [] lso showed tht coho slmon FSH directly stimulted ovrin cyp expression levels nd romtse enzymtic ctivity in vitellogenic ovrin follicles of rown trout (Slmo trutt) in vitro. We my therefore hypothesize tht the increse in ovrin cyp expression nd enzymtic ctivity in CPH-treted eels my result from direct stimultion y gondotropins, without excluding possile other indirect effects..5. Differentil regultion of steroid receptor sutype expression during induced mturtion nd under the effects of sex steroids We oserved differentil regultion of the expression of eel estrdiol receptor sutypes during induced mturtion. he expression levels of incresed in the rin nd pituitry in CPHtreted eels, with no significnt chnge in the ovries. No chnge in the expression of ws oserved in ny orgn. retment with high dose of E ws lso le to increse, ut not expression. his upregultion ws E -specific, s no significnt effect ws oserved with tretment on or expression. his

9 S.-R. Jeng et l. / Generl nd Comprtive Endocrinology 75 () 7 7 suggests tht the selective increse in expression in CPHtreted mtured eels my e medited y endogenous E. hese results indicte differentil utoregultion of E receptor sutypes y E in the eel, with n up-regultion of, ut not expression. In contrst, in goldfish, Nelson et l. [] oserved tht the ovrin trnscripts of, nd were ll significntly upregulted y E. he presence of n ERE in the promoter of hs een first demonstrted in the rinow trout []. More recently, in zerfish, n imperfect ERE hs een found in the promoter of long with n ERE hlf site with no ERE consensus in the promoter of [,]. Concerning ndrogen receptor sutypes, we found in CPH treted eels, n increse in r- expression levels in the midrin, no chnge in the forerin nd decrese in the pituitry. However, neither E nor tretment induced ny significnt up- or downregultion of r- nd r- in the eel rin nd pituitry. his suggests tht up nd down regultion of r- in CPH-treted eels my not result from uto-regultion y ndrogens nor from cross regultion y estrogens, ut from other indirect mturtion-relted mechnisms. Recently, single r ws identified in cyprinid fish, Spinirus denticultus, nd no significnt vrition of its mrna levels ws found throughout the ovrin recrudescence, in rin, pituitry nd ovries []. he ovrin r- nd r- trnscript levels were remrkly incresed in CPH-treted eels. he importnce of ARs in eel mturtion hs een suggested ecuse exogenous ndrogen, ut not estrogen, in comintion with pituitry extrcts cn enhnce eel mturtion []. In in vitro studies, -ketotestosterone (-K), ut not E, induced significnt increse in the dimeters of previtellogenic oocytes nd oocyte nuclei in dose-dependent mnner in New Zelnd shortfinned eel []. More recently, dt showed tht r- trnscript levels were high from the lte oil droplet stge to the lte vitellogenic stge, while r- trnscript levels were high from the lte oil droplet stge to the midvitellogenic stge in the Jpnese eel [5]. In contrst to the cler-cut effect of CPH tretment on ovrin r- nd r- expression levels, E or tretment did not hve ny significnt effect; this suggests tht the stimultory effect of CPH would not e medited y endogenous E nd in femle eels. Further studies re required to decipher whether this effect on ovrin AR my e the result from direct ction of pituitry hormones, s hypothesized ove, for the increse in ovrin romtse. In conclusion, the present study showed tht the expression of the single romtse gene cyp present in the eel is upregulted in oth the rin nd the ovries during mturtion induced y CPH tretment. However, different regultory mechnisms my e involved, with n estrdiol-specific induction of cyp expression in the rin, ut not in the ovries, where cyp would e sumitted to steroid-independent control. he differentil regultion of the expression of sex steroid sutypes, r- nd r- ws evidenced during induced mturtion, while no chnges were oserved for. An upregultion of, ut not expression, y E in the rin nd ovries ws evidenced. In contrst, no pprent utoregultion y of the r- nd r- receptors ws found. hese results revel oth steroid-dependent nd -independent mechnisms in the regultion of romtse nd steroid receptor expression in the eel. Acknowledgment his study ws supported y the ilterl Frnce ANR/iwn NSC project PUBEREEL: NSC8--B---MY. References [] F. Ascl, R. Zrdoy, D. Posd, Protest: selection of est-fit models of protein evolution, Bioinformtics (5) 5. [] A. Amores, A. Force, Y.L. Yn, L. Joly, C. Amemiy, A. Fritz, R.K. Ho, J. Lngelnd, V. Prince, Y.L. Wng, M. Westerfield, M. Ekker, J.K. Postlethwit, Zerfish hox clusters nd verterte genome evolution, Science 8 (8) 7 7. [] E. Andersson, B. Borg, J.G. Lmert, Aromtse ctivity in rin nd pituitry of immture nd mture Atlntic slmon (Slmo slr L.) prr, Gen. Comp. Endocrinol. 7 (88). [] S. Arou, F.A. Weltzien, N. Le Belle, S. Dufour, Development of rel-time R-PCR ssys for eel gondotropins nd their ppliction to the comprison of in vivo nd in vitro effects of sex steroids, Gen. Comp. Endocrinol. 5 (7). [5] M. Blázquez, F. Piferrer, Cloning, sequence nlysis, tissue distriution, nd sex-specific expression of the neurl form of P5 romtse in juvenile se ss (Dicentrrchus lrx), Mol. Cell Endocrinol. () 8. [] G.V. Cllrd, Z. Petro, K.J. Ryn, Estrogen synthesis in vitro nd in vivo in the rin of mrine teleost (Myoxocephlus), Gen. Comp. Endocrinol. (8) 55. [7] G.V. Cllrd, A.V. choudkov, M. Kishid, E. Wood, Differentil tissue distriution, developmentl progrmming, estrogen regultion nd promoter chrcteristics of cyp genes in teleost fish, J. Steroid Biochem. Mol. Biol. 7 () 5. [8] X.. Chng,. Koyshi, H. Kjiur, M. Nkmur, Y. Nghm, Isoltion nd chrcteriztion of the cdna encoding the tilpi (Oreochromis niloticus) cytochrome P5 romtse (P5rom): chnges in P5rom mrna, protein nd enzyme ctivity in ovrin follicles during oogenesis, J. Mol. Endocrinol. 8 (7) 57. [] C.F. Chng, B.Y. Lin, Estrdiol-7 et stimultes romtse ctivity nd reversile sex chnge in protndrous lck porgy, Acnthopgrus schlegeli, J. Exp. Zool. 8 (8) 5 7. [] H. Chi, K. Iwtsuki, H.K. Kzuo, A. Hr, K. Ymuchi, Chnges in serum steroid hormones nd vitellogenin levels in cultured femle Europen eels Anguill nguill during rtificilly induced ovrin development, J. World Aqucult. Soc. () [] L. Dll Vlle, L. Lunrdi, L. Colomo, P. Belvedere, Europen se ss (Dicentrrchus lrx L.) cytochrome P5rom: cdna cloning, expression nd genomic orgniztion, J. Steroid Biochem. Mol. Biol. 8 () 5. [] L. Dll Vlle, V. offolo, S. Vinello, I. Girono,. Aoki, P. Belvedere, L. Colomo, Genomic orgniztion of the CYP genes in the rinow trout (Oncorhynchus mykiss Wlum), J. Steroid Biochem. Mol. Biol. (5) 55. [] N. Diotel, Y.L. Pge, K. Mouriec, S.K. ong, E. Pellegrini, C. Villnt, I. Anglde, F. Brion, F. Pkdel, B.C. Chung, O. Kh, Aromtse in the rin of teleost fish: expression, regultion nd puttive functions, Front Neuroendocrinol. () 7. [] S. Dufour, N. Le Belle, S. Bloche, Y.A. Fontine, Positive feedck control y the gonds on gondotropin (GtH) nd gondolierin (GnRH) level in experimentlly mtured femle silver eel Anguill Anguill, Fish Physiol. Biochem. 7 (8) 57. [5] S. Dufour, E. Burzw-Gérrd, N. Le Belle, M. Sihi, B. Vidl, Reproductive endocrinology of the Europen eel, Anguill nguill. In: K. Aid, K. sukmotom, K. Ymuchi (Eds.), Eel Biology, Springer, okyo,, 7 8. [] M. Ezgouri, S. Yom-Din, D. Golderg, K. Jckson, B. Levvi-Sivn, G. Degni, Expression of the two cytochrome P5 romtse genes in the mle nd femle lue gourmi (richogster trichopterus) during the reproductive cycle, Gen. Comp. Endocrinol. 5 (8) 8. [7] D. Gelins, G.A. Pitoc, G.V. Cllrd, Isoltion of goldfish rin cytochrome P5 romtse cdna: mrna expression during the sesonl cycle nd fter steroid tretment, Mol. Cell. Endocrinol. 8 (8) 8. [8] K. Gen, K. Okuzw, N. Kumkur, S. Ymguchi, H. Kgw, Correltion etween messenger RNA expression of cytochrome p5 romtse nd its enzyme ctivity during oocyte development in the red serem (Pgrus mjor), Biol. Reprod. 5 () 8. [] A. Gonzlez, F. Piferrer, Aromtse ctivity in the Europen se ss (Dicentrrchus lrx L.) rin. Distriution nd chnges in reltion to ge, sex, nd the nnul reproductive cycle, Gen. Comp. Endocrinol. (). [] L.G. Grddy, A.A. Kowlski, F.A. Simmen, S.L. Dvis, W.W. Bumgrtner, R.C. Simmen, Multiple isoforms of porcine romtse re encoded y three distinct genes, J. Steroid Biochem. Mol. Biol. 7 () 57. [] B. Guo, X. Gn, S. He, Hox genes of the Jpnese eel Anguill jponic nd Hox cluster evolution in teleosts, J. Exp. Zool. B Mol. Dev. Evol. () 5 7. [] S. Ijiri, Y. Kzeto, P.M. Lokmn, S. Adchi, K. Ymuchi, Chrcteriztion of cdna Encoding P-5 romtse (CYP) from Jpnese eel ovry nd its expression in ovrin follicles during induced ovrin development, Gen. Comp. Endocrinol. (). [] S. Ijiri, Y. Kzeto, N. ked, H. Chi, S. Adchi, K. Ymuchi, Chnges in serum steroid hormones nd steroidogenic ility of ovrin follicles during rtificil mturtion of cultivted Jpnese eel, Anguill jponic, Aquculture 5 (5). []. Ikeuchi,. odo,. Koyshi, Y. Nghm, CDNA cloning of novel ndrogen receptor sutype, J. Biol. Chem. 7 () [5] S.R. Jeng, S. Dufour, C.F. Chng, Differentil expression of neurl nd gondl romtse enzymtic ctivities in reltion to gondl development in Jpnese eel, (Anguill jponic), J. Exp. Zool. A (5) 8 8. [] S.R. Jeng, W.S. Yueh, G.R. Chen, Y.H. Lee, S. Dufour, C.F. Chng, Differentil expression nd regultion of gondotropins nd their receptors in the Jpnese eel, (Anguill jponic), Gen. Comp. Endocrinol. 5 (7) 7. [7] Y. Kzeto, J.M. rnt, Moleculr iology of chnnel ctfish rin cytochrome P5 romtse (CYPA): cloning, preovultory induction of gene expression,

10 7 S.-R. Jeng et l. / Generl nd Comprtive Endocrinology 75 () 7 hormonl gene regultion nd nlysis of promoter region, J. Mol. Endocrinol. 5 (5) [8] M. Kishid, G.V. Cllrd, Distinct cytochrome P5 romtse isoforms in zerfish (Dnio rerio) rin nd ovry re differentilly progrmmed nd estrogen regulted during erly development, Endocrinology () [] I.G. Lnge, A. Hrtel, H.H.D. Meyer, Evolution of oestrogen functions in vertertes, J. Steroid Biochem. Mol. Biol. 8 (). [] C.S. Lssiter, B. Kelley, E. Linney, Genomic structure nd emryonic expression of estrogen receptor et (ERet) in zerfish (Dnio rerio), Gene () 5. [] Y. Le Dren, G. Lzennec, L. Kern, D. Sligut, F. Pkdel, Y. Vlotire, Chrcteriztion of n estrogen-responsive element implicted in regultion of the rinow trout estrogen receptor gene, J. Mol. Endocrinol. 5 (5) 7 7. [] Y.H. Lee, F.Y. Lee, W.S. Yueh, P. con, J.L. Du, C.N. Chng, S.R. Jeng, H. nk, C.F. Chng, Profiles of gondl development, sex steroids, romtse ctivity, nd gondotropin II in the controlled sex chnge of protndrous lck porgy, Acnthopgrus schlegeli Bleeker, Gen. Comp. Endocrinol. (). [] X. Liu, H. Su, P. Zhu, Y. Zhng, J. Hung, H. Lin, Moleculr cloning, chrcteriztion nd expression pttern of ndrogen receptor in Spinirus denticultus, Gen. Comp. Endocrinol (). [] P.M. Lokmn, K.A. George, S.L. Divers, M. Algie, G. Young, -Ketotestosterone nd IGF-I increse the size of previtellogenic oocytes from shortfinned eel, Anguill ustrlis, in vitro, Reproduction (7) [5] P.M. Lokmn, R.. Wss, H.C. Suter, S.G. Scott, K.F. Judge, G. Young, Chnges in steroid hormone profiles nd ovrin histology during slmon pituitryinduced vitellogenesis nd ovultion in femle New Zelnd longfinned eels, Anguill dieffenchii gry, J. Exp. Zool. 8 (). [] A. Menuet, Y. Le Pge, O. orres, L. Kern, O. Kh, F. Pkdel, Anlysis of the estrogen regultion of the zerfish estrogen receptor (ER) revels distinct effects of ERlph, ERet nd ERet, J. Mol. Endocrinol. () [7] A. Menuet, E. Pellegrini, F. Brion, M.M. Gueguen, I. Anglde, F. Pkdel, O. Kh, Expression nd estrogen-dependent regultion of the zerfish rin romtse gene, J. Comp. Neurol. 85 (5). [8] M. Montero, N. Le Belle, J.A. King, R.P. Millr, S. Dufour, Differentil regultion of the two forms of gondotropin-relesing hormone (MGnRH nd cgnrh-ii) y sex steroids in the Europen femle silver eel (Anguill nguill), Neuroendocrinology (5) [] N. Montserrt, A. Gonzlez, E. Mendez, F. Piferrer, J.V. Plns, Effects of follicle stimulting hormone on estrdiol-7 et production nd P-5 romtse (CYP) ctivity nd mrna expression in rown trout vitellogenic ovrin follicles in vitro, Gen. Comp. Endocrinol. 7 (). [] K. Mouriec, M.M. Gueguen, C. Mnuel, F. Percevult, M.L. hieulnt, F. Pkdel, O. Kh, Androgens upregulte cyp (romtse B) gene expression in the rin of zerfish (Dnio rerio) through estrogen receptors, Biol. Reprod. 8 () [] E.R. Nelson, W.B. Wiehler, W.C. Cole, H.R. Hii, Homologous regultion of estrogen receptor sutypes in goldfish (Crssius urtus), Mol. Reprod. Dev. 7 (7) 5. [] J.N. Nocilldo, A. Elizur, A. Avitn, F. Crrick, B. Levvi-Sivn, Cytochrome P5 romtse in grey mullet: cdna nd promoter isoltion; rin; pituitry nd ovrin expression during puerty, Mol. Cell Endocrinol. (7) [] B.S. Nunez, S.L. Appleum, issue- nd sex-specific regultion of CYPA expression in the Atlntic croker (Micropogonis undultus), Gen. Comp. Endocrinol. () 5. [] A.P. Plstr, D. Schnel, M.C. Nieveen, H.P. Spink, G.E. vn den hillrt, emporl expression of heptic estrogen receptor, vitellogenin nd vitellogenin in Europen silver eels, Gen. Comp. Endocrinol. (). [5] M. Psmnik, G.V. Cllrd, Chnges in rin romtse nd 5 lph-reductse ctivities correlte significntly with sesonl reproductive cycles in goldfish (Crssius urtus), Endocrinology (88) 5. [] M. Psmnik, B.A. Schlinger, G.V. Cllrd, In vivo steroid regultion of romtse nd 5 lph-reductse in goldfish rin nd pituitry, Gen. Comp. Endocrinol. 7 (88) [7] C. Psqulini, F.A. Weltzien, B. Vidl, S. Bloche, C. Rouget, N. Gilles, D. Servent, P. Vernier, S. Dufour, wo distinct dopmine D receptor genes in the Europen eel: moleculr chrcteriztion, tissue-specific trnscription, nd regultion y sex steroids, Endocrinology 5 () 77. [8] S.J. Swyer, K.A. Gerstner, G.V. Cllrd, Rel-time PCR nlysis of cytochrome P5 romtse expression in zerfish: gene specific tissue distriution, sex differences, developmentl progrmming, nd estrogen regultion, Gen. Comp. Endocrinol. 7 () 8 7. [] S. Sestin, S.E. Bulun, A highly complex orgniztion of the regultory region of the humn CYP (romtse) gene reveled y the Humn Genome Project, J. Clin. Endocrinol. Met. (). [5] E.R. Simpson, C. Clyne, G. Ruin, W.C. Boon, K. Roertson, K. Britt, C. Speed, M. Jones, Aromtse A rief overview, Annu. Rev. Physiol. () 7. [5] A. Stmtkis, M. Ott, Efficient computtion of the phylogenetic likelihood function on multi-gene lignments nd multi-core rchitectures, Philos. rns. R. Soc. Lond. B. Biol. Sci. (8) [5] Y. kei, K. Inoue, K. Ando,. Ihr,. Ktfuchi, M. Kshiwgi, S. Hirose, Enhnced expression nd relese of C-type ntriuretic peptide in freshwter eels, Am. J. Physiol. Regul. Integr. Comp. Physiol. 8 () R77 R75. [5] M. nk,.m. elecky, S. Fukd, S. Adchi, S. Chen, Y. Nghm, Cloning nd sequence nlysis of the cdna encoding P-5 romtse (P5rom) from rinow trout (Oncorhynchus mykiss) ovry; reltionship etween the mount of P5rom mrna nd the production of oestrdiol-7 et in the ovry, J. Mol. Endocrinol. 8 () 5. [5] A. choudkov, G.V. Cllrd, Identifiction of multiple CYP genes encoding different cytochrome P5 romtse isozymes in rin nd ovry, Endocrinology (8) 7 8. [55] J.D. hompson, D.G. Higgins,.J. Gison, CLUSAL W: improving the sensitivity of progressive multiple sequence lignment through sequence weighting, position-specific gp penlties nd weight mtrix choice, Nucleic Acids Res. () 7 8. [5]. odo, S. Adchi, K. Ymuchi, Moleculr cloning nd chrcteriztion of Jpnese eel estrogen receptor cdna, Mol. Cell Endocrinol. () 7 5. [57]. odo,. Ikeuchi,. Koyshi, Y. Nghm, Fish ndrogen receptor: cdna cloning, steroid ctivtion of trnscription in trnsfected mmmlin cells, nd tissue mrna levels, Biochem. Biophys. Res. Commun. 5 () [58] S.K. ong, B.C. Chung, Anlysis of zerfish cyp promoters, J. Steroid Biochem. Mol. Biol. 8 () 8 8. [5] R. osk,. odo, Y. Kzeto, P.M. Lokmn, S. Ijiri, S. Adchi, K. Ymuchi, Expression of ndrogen receptor mrna in the ovry of Jpnese eel, Anguill jponic, during rtificilly induced ovrin development, Gen. Comp. Endocrinol. 8 (). [] I. zchori, G. Degni, A. Hurvitz, B. Mov, Cloning nd developmentl expression of the cytochrome P5 romtse gene (CYP) in the Europen eel (Anguill nguill), Gen. Comp. Endocrinol. 8 () 7 8. [] K.R. von Schlurg, M. Ysuike, W.S. Dvidson, B.F. Koop, Regultion, expression nd chrcteriztion of romtse (cyp) trnscripts in ovry nd testis of rinow trout (Oncorhynchus mykiss), Comp. Biochem. Physiol. B Biochem. Mol. Biol. 55 () 8 5. [] Y.S. Wng, S.W. Lou, Structurl nd expression nlysis of heptic vitellogenin gene during ovrin mturtion in Anguill jponic, J. Steroid Biochem. Mol. Biol. (). [] F.A. Weltzien, C. Psqulini, M.E. Séert, B. Vidl, N. LeBelle, O. Kh, P. Vernier, S. Dufour, Androgen-dependent stimultion of rin dopminergic systems in the femle Europen eel (Anguill nguill), Endocrinology 7 () 7. [] K. Ymmoto, K. Ymuchi, Sexul mturtion of Jpnese eel nd production of eel lrve in the qurium, Nture 5 (7). [5] M.B. Yilmz, A. Wolfe, Y.H. Cheng, C. Glidewell-Kenney, J.L. Jmeson, S.E. Bulun, Aromtse promoter I.f is regulted y estrogen receptor lph (ESR) in mouse hypothlmic neuronl cell lines, Biol. Reprod. 8 () 5 5. [] E.L. Yong, S.G. Hillier, M. urner, Differentil regultion of cholesterol sidechin clevge (P5scc) nd romtse (P5rom) enzyme mrna expression y gondotrophins nd cyclic AMP in humn grnulos cells, J. Mol. Endocrinol. (). [7] Y. Zhng, W. Zhng, H. Yng, W. Zhou, C. Hu, L. Zhng, wo cytochrome P5 romtse genes in the hermphrodite rice field eel Monopterus lus: mrna expression during ovrin development nd sex chnge, J. Endocrinol. (8) 7.