en.2007-0311

0013-7227/07/$15.00/0PrintedinU.S.A.

Endocrinology148(12):5822–5830

Copyright©2007byTheEndocrineSociety

doi:10.1210/en.2007-0311

SexualMaturationModulatesExpressionofNuclearReceptorTypesinLaser-CapturedSingleCellsoftheCichlid(Oreochromisniloticus)Pituitary

TakashiKitahashi,SatoshiOgawa,TomokoSoga,YasuoSakuma,andIshwarParhar

SchoolofMedicineandHealthSciences(T.K.,S.O.,T.S.,I.P.),MonashUniversity,46150BandarSunway,Malaysia;andDepartmentofPhysiology(T.K.,S.O.,T.S.,Y.S.,I.P.),NipponMedicalSchool,Sendagi,Tokyo113-8602,Japan

Theroleofsteroid/thyroidhormonesintheregulationofen-docrinecellsatthelevelofthepituitaryhasremainedun-clear.Therefore,usingsingle-cellquantitativereal-timePCR,weexaminedabsoluteamountsoftranscriptsfornuclearre-ceptors[estrogenreceptors(ERs) , ,and ;androgenre-ceptors(ARs)aandb;glucocorticoidreceptors(GRs)1,2a,and2b;andthyroidhormonereceptors(TRs) 1, 2,and ]inpituitarycellsofimmature(IM)andmature(M)maletilapia,Oreochromisniloticus.Inthetworeproductivestages,ACTHcellsexpressedonlyER ,whereasallotherpituitarycelltypesexpressedER ,andasubpopulationcoexpressedARa,ARb,GR1,GR2b,andTR butlackedER ,GR2a,TR 1,andTR 2.IMmaleshadhighpercentagesofLHcells(IM46.0%vs.M10.0%),GHcells(IM23.3%vs.M7.9%),andprolactincells(IM68.8%vs.M6.0%)withER ,andTSHcells(IM19.2%vs.M

0.0%)andMSHcells(IM25.6%vs.M0.0%)withER TR .AhighpercentageofFSHcellsinIMmalesexpressedER (IM46.9%vs.M18.8%),andFSHcellsinMmalesshowedsignifi-cantlyhighGR1transcripts(IM76.0 5.0vs.M195.0 10.7copiespercell;P<0.05),suggestingthatFSHcellsareregu-lateddifferentlyinthetworeproductivestages.CoexpressionofER inhighpercentagesofcellsoftheGHfamily(GH,IM43.8%vs.M14.3%;prolactin,IM8.3%vs.M59.7%;somato-lactin,IM22.2%vs.M42.2%)suggeststhattheexpressionofbothERsisimportantforfunctionality.Thus,differentialco-expressionofgenesfornuclearreceptorsinsubpopulationsofpituitarycelltypessuggestsmultiplesteroid/thyroidhor-moneregulatorypathwaysatthelevelofthepituitaryduringthetworeproductivestages.(Endocrinology148:5822–5830,2007)

TISWELLdocumentedthatsteroid/thyroidhormonesregulatethesynthesisandreleaseofpituitaryhormones[FSH,LH,TSH,GH,prolactin(PRL),somatolactin(SL),ACTH,andMSH]throughcomplexinteractionswithhypo-thalamicneurons.

Thereisevidencethatsteroid/thyroidhormonescanin-fluencethesynthesisandreleaseofpituitaryhormonesininvitrocultures(1–4).Furthermore,severalstudiesinteleostshaveshownthepresenceofbindingsitesforsteroidhor-monesinthepituitary(5),andthepresenceofglucocorticoidreceptor(GR)inFSHandLHcells(6),aswellasaromataseinthepituitary(7).Thesefactssuggestdirecteffectsofste-roid/thyroidhormonesandaromatizableandrogenatthelevelofthepituitary.

Therecentdiscoveryofmorethanonesteroid/thyroidhormonereceptor(TR)subtype[estrogenreceptors(ERs) , ,and (8);androgenreceptors(ARs) and (9,10);GR1,GR2a,andGR2b(11);andTR 1,TR 2,andTR (12)]invertebratesraisesnewquestionsastowhethermultipletypesorsubtypesoftranscriptsfornuclearreceptorsarecoex-pressedinindividualpituitarycells.Identificationofnuclear

FirstPublishedOnlineSeptember6,2007

Abbreviations:AR,Androgenreceptor;DIG,digoxigenin;E2,17 -estradiol;ER,estrogenreceptor;GR,glucocorticoidreceptor;IM,im-mature;LCM,lasercapturemicrodissection;M,mature;POMC,pro-opiomelanocortin;PRL,prolactin;SL,somatolactin;T,testosterone;TR,thyroidhormonereceptor.

EndocrinologyispublishedmonthlybyTheEndocrineSociety(http://wendang.chazidian.com),theforemostprofessionalsocietyservingtheI

receptorexpressioninpituitaryendocrinecellswillhelptoelucidatethemechanismsofactionofsteroid/thyroidhor-monesatthepituitarylevel,whichpresentlyremainsunclearinvertebrates.

Toaddressthesequestions,weexaminedtheexpressionofgenesformultiplenuclearreceptortypesinindividualpituitarycellsduringsexualmaturationinthetilapia,Oreo-chromisniloticus.Weusedanoveltechniqueintegratinglasercapturemicrodissection(LCM)ofsingledigoxigenin(DIG)-labeledpituitarycellscoupledwithquantitativereal-timePCRthatallowspreciseharvestingofidentifiedcells,highpreservationofmRNAforanalysis,andhighsensitivityforthedetectionofmultipletargets(13).Furthermore,weusedimmunocytochemistrytoconfirmthepresenceofGRandaromataseinpituitarycells.

MaterialsandMethods

Animals

ExperimentalproceduresinthepresentstudywereperformedundertheguidelinesoftheAnimalCareCommitteeofNipponMedicalSchool,Tokyo.Thetilapiausedinthepresentstudyweremaintainedinfreshwaterat27 1Cwithanaturalphotoregime(14-and10-hlightanddarkcycles,respectively).

Tissuepreparationandinsituhybridizationforpituitaryhormones

Immature(IM)(standardlength 6.5 0.3cm;bodyweight 11.6 3.4g;gonadosomaticindex 0.25 0.04;n 25)andmature(M)(standardlength 13.7 0.4cm;bodyweight 89.4 6.9g;gona-dosomaticindex 1.23 0.18;n 20)maleswereanesthetized

Kitahashietal. NuclearReceptorsinPituitaryEndocrineCellsEndocrinology,December2007,148(12):5822–58305823

(Sigma,St.Louis,MO)beforetheywerekilledbydecapitation.GonadswerefixedinBouin’ssolution,processedthroughethanolseries,andembeddedinParaplastPlus(OxfordLabware,St.Louis,MO)forhis-tologicalexamination.Thebrainswithpituitariesattachedweredis-sectedandfixedin4%bufferedparaformaldehydefor6hatroomtemperature,cryoprotectedin20%sucrose,andembeddedinTissue-TekOCTcompound(SakuraFinetechnical,Tokyo,Japan).Pituitarysectionswerecutinsagittalplanes(6 m),mountedontoaminopropyltriethoxysilane-treatedglassslides(MatsunamiGlass,Tokyo,Japan),andstoredat 80Cuntiluseforlocalizationofpituitarycelltypes.ForDIGinsituhybridization,weusednucleotidesequencesofcDNAfortilapiapituitaryhormonesobtainedfromtheGenBank[FSH sub-unit,AF289173;LH subunit,AY294016;TSH subunit,AB120769;GH1,M97766;PRL188,M27010;SL,AB120767;proopiomelanocortin(POMC) ACTHandMSH,AF116240].BecauseGH1andGH2mRNAsencodeanidenticalpolypeptide(14),andPRL188andPRL177arecolo-calizedinthesamepituitarycellsinthetilapia(15),weusedGH1andPRL188cDNAstosynthesizeriboprobesforinsituhybridization.ThepGEM-TEasytranscriptionvectorconstructs(Promega,Madison,WI)thatcontainedthecDNAwerelinearizedwithSpeIorNcoIendonu-clease(NipponGene,Tokyo,Japan)andusedastemplates.SenseandantisenseriboprobesweresynthesizedwithT7orSP6RNApolymerase(Toyobo,Tokyo,Japan)andDIGRNA-labelingmix(RocheDiagnostics,Tokyo,Japan).DIGinsituhybridizationwasperformedonsagittalpituitarysections,asdescribedpreviously(16,17).

TABLE1.SequencesofPCRprimersandfluorogenicprobesusedforRT-PCRandreal-timePCR

Primer

Sequence(5 –3 )

LCMofpituitarycells

Thecellharvestingprotocolwasthesameasthatdescribedelsewhere(13,17,18).Thedehydratedpituitarysectionwasoverlaidwithather-moplasticmembranemountedonanopticallytransparentcap(CapSureMacroLCMCaps;Arcturus,MountainView,http://wendang.chazidian.comingaPixCellIILasercaptureinstrument(Arcturus),DIG-identifiedpituitarycellsweremicrodissectedbyfocalmeltingofthemembranethroughlaseractiva-tion(laserpulsepower,25–65mW;laserpulseduration,1.5msec;laserspotsize,10 mdiameter).Becausethelaserbeamwaslargerthanthediameterofthecells,undesirabletissuewasalsocapturedalongtheperipheryofthecells.Heat-pulledborosilicateglassmicrocapillarypi-pette(1.5-mmouterdiameter,HarvardApparatusLtd.,Edenbridge,Kent;micropipettepuller;TypePE-2,Narishige,Tokyo,Japan)attachedtoamicromanipulator(Narishige)wasusedtoremoveundesirabletissuearoundtheperipheryofthesingle-lasercapturedcells.Byusingnegativepressure,singlecellswerethenharvestedfromtheLCMcapintothemicropipetteundervisualcontrol,thenexpelledintoasterile1.5-mlreactiontubecontaining50 lofthelysisbufferindividually,andstoredat 80CuntiltotalRNAisolation.Forunbiasedcellsampling,eightto10cellswereharvestedatrandom(approximatelytwocellsperalternatesection)alongtherostral-caudalextentofthewholepopulationofeachpituitarycelltypeineachanimal.

RT-PCRforpituitaryhormones

CCAGAAGGATCCCAACTTCAFSH -F

FSH -RCAGAGGGAAGAGGTGCAAAGLH -FGACACTGCATCACCAAGGACLH -RGGTTGCATGCTCTCAAAGGTTSH -FTCAACACCACCATCTGCATGGGTSH -RAGGGCCACGGGGTAGGTGAAGGH1-FAGTCCTGGGAGTTTCCCAGTGH1-RGGTAGGTCTCCACCTTGTGC

GCAACAGCTAACGGACAGAAPRL188-F

PRL188-RCTGAGCAGGTGAACCCATCTSL-FCAACTGTGCATAAAATATAAACTSL-RGAAATCAATGGCAGGATATGPOMC-FGCAACATGATGGAGTGCATCPOMC-RCAGCGGAAGTGCTTCATCTTReal-timePCRfornuclearreceptorsER -FGCCTGCGACAGAGACAAACCER -RCACGCTTCCTTCCATCCTGAER probeCCTGCCACACACAAGGGCGTCCER -FAAGCCACAGAGAGGGTGAGCER -RTATACCCCACAGGAGGGCTGER probeCCCCGGGACTTCTGCCTGCTATGER -FGCTATGACGGACGCTCTGGER -RCGAGTGTACTGCTGCCGGAARa-FTGACCAGGGTGGCAAAGGTARa-RGGCACTTGGGTAACTTGACATTTARaprobeATGCGAGCGACGGCACAAGGATAARb-FAAGCAGTGCCGCGAGTTTARb-RTCACAGGCTAGCGGGATAATGARbprobeCAGAATCCGAGAATGCAAACCTGTACGGGR1-FCCTCCTACCCCACCAGCTTTGR1-RTCCCGCTCTTTCCTTGTGTGGR1probeCAGGCAGGAAGGGAGCACCGCGR2-FGGGTTGTAACATCTGGCAGCTGR2-RAATGATGCACTCATTTCTCCCTGGR2bprobeTCGTGCTCTCCATCCTTCAACGGCTTR 1-FAGTGGAAGCAGAAGCGCAATR 1-RTGATGTTGGAGCGACTGGAGTR 2-FCCCATCGTCACACCAATGCTR 2-RTCACAAGGCAGCAGGAATTTGTR -FGAAATTCCTGAGTGCAGCGGTR -RCAGGTGCATTACCCGTGGA

AAGGAAGCTAAGCCTGAGGATATTGGTCAAGCTR probeAllprobeswerelabeledwith5 FAMreporterdyeand3 TAMRA

quencherdye.

clonedandidentifiedpartialcDNAsequencesoftilapiaER (GenBankaccessionno.AB110981),GR1(GenBankaccessionno.AB245405),andGR2b(GenBankaccessionno.AB245406),whereassequencesofothertilapianuclearreceptorswereobtainedfromtheGenBank(accessionnos.:ER ,U75604;ER ,U75605;ARa,AB045211;ARb,B045212;TR 1,AF302248;TR 2,AF302249;andTR ,AF302247).Onetenthofthesinglecell’scDNAswaspooledfrom25samplesofeachcelltype,andthenone12thofthepooledcDNAwassubjectedtoPCRusinggene-specificprimers(Table1).Aspositivecontrols,clonedcDNAfornuclearrecep-torsweresubjectedtoPCR.No-RTsampleswerealsosubjectedtoPCRasnegativecontrols.ThePCRproductswereelectrophoresedona4%agarosegel,stainedwithethidiumbromide,andvisualizedbyillumi-nationwithUVlight.

RT-PCRforpituitaryhormonesandnuclearreceptorsinpituitarycells

TheconditionsforRT-PCRweresimilartothosedescribedelsewhere(13,17,18).Briefly,theharvestedsinglecellfromthepituitarywasdigestedwith1 gproteinaseK(GentraSystems,Minneapolis,MN)for1hat53C.Thecelllysatewasincubatedfor1hat37Cwith1Uribonuclease-freeDNaseI(Promega)toeliminategenomicDNA,andheatdenaturedat95Cfor10mintoseparatethemRNAfromtheDIG-labeledriboprobe.TotalRNAwasextractedfromthecelllysatebyISOGEN(NipponGene),andreversetranscribedtocDNAinareactionmixture(20 l)with100pmolrandomhexamer(Takara,Tokyo,Japan)and10UPrimeRNaseInhibitor(Eppendorf,Hamburg,Germany)using40USuperScriptIIIReverseTranscriptase(Invitrogen,Carlsbad,CA).Toconfirmthepresenceofpituitaryhormonetranscripts,thesinglecell’scDNAwassubjectedtoRT-PCRusinggene-specificprimersforpituitaryhormones(Table1)andone20thofasinglecell’scDNAsolution.ThePCRproductswereelectrophoresedona2%agarosegel,stainedwithethidiumbromide,andvisualizedbyilluminationwithUVlight.OnlythosecellsthatwerepositiveforeachpituitaryhormonemRNAwereusedforfurtheranalysis(n 3–9cellsperanimal;26–73Real-timePCRofnuclearreceptorsinpituitarycells

WeusedcDNAsamplesofGH,PRL,SL,MSH,andACTHcellsthathadbeenpreparedinourpreviousstudy(17).Moreover,weharvestedFSH,LH,andTSHcellsfrompituitarysectionsthatwerepreparedatthesametimeandstoredat 80C,aswerequiredmorecDNAsamples.To

5824Endocrinology,December2007,148(12):5822–5830Kitahashietal. NuclearReceptorsinPituitaryEndocrineCells

in10 lreactionvolumesconsistingof1 TaqManUniversalPCRMasterMix(AppliedBiosystems,FosterCity,CA),300nmofeachforwardandreversenuclearreceptorprimer,200nmTaqManprobe(Table1),andone12thofasinglecell’scDNAsolutionorstandardcDNAusingaABIPRISM7700SequenceDetectionSystem(AppliedBiosystems).Amplificationwasperformedat50Cfor2min,95Cfor10min,45cyclesof95Cfor15sec,and60Cfor1min.TheabsoluteamountsoftranscriptsweredeterminedusingseveralconcentrationsofstandardcDNA(10,30,102,103,104,and105copies)thatwerereversetranscribedfromseriallydilutedstandardRNAs.Thecopynumbersoftranscriptsperreactionweremultipliedby12toobtainthecopynumbersoftran-scriptspercell.Foreachpituitarycelltype,givencopiesoftranscriptspercellwerecombinedtogiveexperimentalgroupmeans.Allvaluesareexpressedasmean sem,andstatisticalcomparisonsweremadebetweenIMandMmalesusingtheStudent’sttest.Plessthan0.01andlessthan0.05wereconsideredstatisticallysignificant.

primaryspermatogonia,secondaryspermatogonia,sper-matocyte,spermatid,andspermatozoa),includinganabun-danceofspermatozoa(Fig.1).

Localizationofpituitaryendocrinecells

DIGinsituhybridizationshowedlocalizationofpituitarycellsinIMandMmalesin:rostralparsdistalis(PRL,TSH,andACTHcells);proximalparsdistalis(FSH,LH,andGHcells);andparsintermedia(SLandMSHcells)(Fig.2).Thedistributionpatternofpituitaryendocrinecellswassimilartothatinourearlierreport(17).

Nuclearreceptorsinpituitarycells

Immunocytochemistry

Threeadultmaletilapiawereanesthetizedbyimmersingina0.01%solutionof3-aminobenzoicacidethylester,andkilledbydecapitation.Thebrainswithattachedpituitariesweredissectedout,fixedinBouin’ssolutionovernightat4C,processedthroughagradedseriesofethanol,andembeddedinParaplastPlus.Serialsectionswerecutinsagittalplanes(15 m)andprocessedfornuclearreceptorimmunocytochem-istryasdescribedbefore(19).

Deparaffinizedsectionswereincubatedwithprimaryantisera[anti-tilapiaGR1diluted1:2000;RACR21(20),raisedagainstsyntheticpeptidecorrespondingtoahydrophilicportionofhormonebindingdomainoftilapiaGR1,whichprobablyrecognizesbothGR1andGR2intilapia(kindlyprovidedbyDr.MasatomoTagawa,KyotoUniversity,Kyoto,Japan),andanti-tilapiaaromatasediluted1:6000wasraisedagainstsyntheticpeptidecorrespondingtotheC-terminalregionoftilapiaaro-matase(21)(kindlyprovidedbyDr.MasaruNakamura,UniversityoftheRyukyu,Okinawa,Japan)]for48hat4C.Thespecificitiesoftheseantiserahavebeenconfirmedandreportedpreviously(20,22,23).Thesectionswereincubatedwithbiotinylatedanti-IgGfor30min,“ABC”complexfor60min(Vectastain“ABC”Elitekit;VectorLaboratories,Burlingame,CA),andthentreatedwith0.05%3,3 diaminobenzidinetetrahydrochloride(Sigma)with0.03%H2O2for15min.Immunoreac-tivitywasseenasbrowngranularreactionproductincertaincellsandfibersinthepituitary.

TherewasnogenomicDNAcontaminationinthetotalRNAextractedfromharvestedsinglecellsofthepituitary(Fig.3).TheampliconsizesofpituitaryhormoneandnuclearreceptorcDNAswereoftheexpectedsizes(Fig.3;seeMa-terialsandMethodsforGenBankaccessionnumbers).BecauseGR2aandGR2baresplicevariants,theprimersusedforRT-PCRrecognizedbothtranscripts,andtheexpectedam-pliconsizesofGR2aandGR2bwere95and122bp,respec-tively.RT-PCRrevealedthatpituitarycelltypeshadER

,

Results

GonadalstatusofIMandMfish

GonadalsectionsstainedwithcresylvioletshowedthatIMmalespossessedonlyspermatogoniainthetestis,whereasMmalespossessedallstagesofgerminalcells(spermatogonia,

FIG.1.PhotomicrographsofgonadalsectionsofIMandMmales.A,IMfish.B,Mfish.Scalebars,20 m.Lc,Leydigcell;psg,primary

FIG.2.PhotomicrographsofDIG-labeledendocrinecellsinmidsag-ittalsectionsofthepituitary.A,Adiagramshowingsectioningplaneofthepituitary.B,AnillustrationshowinglocalizationofpituitarycelltypesinasagittalsectionatthelevelofplaneIinA.C,FSHcells.D,LHcells.E,TSHcells.Inset,TSHcellsathighermagnification.F,GHcells.G,PRLcells.H,SLcells.I,POMC(MSHandACTH).Inset,ACTHcellsathighmagnification.JandK,DIG-labeledFSHcellsbefore(J)andafter(K)LCM.Arrowsindicatetheharvestedcells.Scalebars,200 minC–I,40 minInsetE,50 minInsetI,and10 minJandK.NotethatallphotomicrographsexceptforFweretakenfrommidsagittalsectionsatthelevelofplane(I)inA.Fwasphoto-

Kitahashietal. NuclearReceptorsinPituitaryEndocrineCellsEndocrinology,December2007,148(12):5822–58305825

MthanIMmales(IM76.0 5.0vs.M195.0 10.7copiespercell;P 0.05)(Fig.4A).AbsolutecopiesofER ,ER ,ARa,andGR2bwerestatisticallynonsignificantbetweenthetworeproductivestages.

LHcells.LHcellshadER ,ER ,ARa,ARb,andGR1tran-scripts(Fig.3).ThetotalpercentageofLHcellsexpressingsingleormultiplenuclearreceptorwashigherinIMmales(IM100%,50of50cellsvs.M47.5%,19of40cells)(Table2).AhigherpercentageofLHcellsinIMmalesexpressedER (IM46.0%vs.M10.0%)andER ARa(IM20.0%vs.M0.0%)whencomparedwiththoseinMmales.AbsolutecopiesofER ,ER ,ARa,ARb,andGR1transcriptswerestatisticallynonsignificantbetweenthetworeproductivestages(Fig.4B).TSHcells.TSHcellshadER ,ER ,ARa,andTR transcripts(Fig.3).ThetotalpercentageofTSHcellsexpressingsingleormultiplenuclearreceptorswashigherinIMthanMmales(IM96.2%,25of26cellsvs.M39.4%,13of33cells)(Table2).ThepercentageofTSHcellsexpressingTR aloneorincombinationwithotherreceptorswashigherinIMthanMmales(IM88.5%vs.M3.0%).AbsolutecopiesoftranscriptsforER ,ER ,ARa,andTR werestatisticallynonsignificantbetweenthetworeproductivestages(Fig.4C).

GHfamily(GH/PRL/SL)

http://wendang.chazidian.compositegelshowingexistenceofmRNAsforpituitaryhor-monesandnuclearreceptorsinFSH,LH,TSH,GH,PRL,SL,MSH,andACTHcellstakenfromIMmales.Foreachcelltype,cDNAsfrom25cellswerecombinedandsubjectedtoPCR.NotethatPCRprimersforGR2bcanamplifybothsplicevariants.ThePOMCtranscriptencodesaprecursorpeptideofMSHandACTH.Thesizesofthebands,inbasepairs,aregivenintheright-handmargin.M,Marker,stable100-bpDNALadder(SIGMAGENOSYS,Sapporo,Japan);PC,positivecontrol;RT-,withoutreversetranscriptase.

ER ,ARa,ARb,GR1,GR2b,andTR butlackedER ,GR2a,TR 1,andTR 2transcripts(Fig.3).Asubpopulationofpituitarycelltypescoexpressedmultiplenuclearreceptors(seebelowfordetails).

Quantitativeanalysisofnuclearreceptorsinpituitarycells

GHcells.GHcellshadER ,ER ,andGR1transcripts(Fig.3).ThetotalpercentageofGHcellsexpressingsingleormultiplenuclearreceptorswashigherinIMthanMmales(IM87.7%,64of73cellsvs.M34.9%,22of63cells)(Table2).AlargepercentageofcellsinIMmalesexpressedER (IM23.3%vs.M7.9%)andER (IM43.8%vs.M14.3%).TheabsolutecopiesofER andER transcriptsweresta-tisticallynonsignificantbetweenthetworeproductivestages(Fig.5A).

PRLcells.PRLcellshadER andER transcripts(Fig.3).PRLcellsexpressedER ,ER ,orER (IM79.2%,38of48cells;M83.6%,56of67cells)(Table2).AhigherpercentageofPRLcellsexpressedER aloneinIMmales(IM68.8%vs.M6.0%)andER inMmales(IM8.3%vs.M59.7%).AbsolutecopiesofER transcriptsinPRLcellsweresignif-icantlyhigherinMmales(IM189.6 45.4vs.M1784.1 369.9copiespercell)(P 0.01;Fig.5B).

SLcells.SLcellshadER andER transcripts(Fig.3).SLcellsexpressedER ,ER ,orER (IM54.2%,39of72cells;M84.4%,38of45cells)(Table2),butahigherpercentageofcellsinMmalesexpressedER whencomparedwithIMmales(IM22.2%vs.M42.2%).AbsolutecopiesofER tran-scripts(IM184.8 21.2vs.M2911.7 643.9copiespercell)andER transcripts(IM296.4 76.8vs.M997.3 312.0copiespercell)inSLcellsweresignificantlyhigherinMmales(P 0.01;Fig.5C).

POMCfamily(MSH/ACTH)

Real-timePCRshowedthat4.7–100%ofpituitarycellsexpressedsingleormultiplenuclearreceptorgenes(Table2).Alargevariationincopiesofnuclearreceptortranscriptswasobservedbetweenindividualcells(Figs.4,A–C,and5,A–E,leftpanels).ER and/orER transcriptswerepredominantinmostpituitarycelltypes.AbsolutecopiesofARaandGR2btranscriptsinFSHcells,ARaandARbinLHcells,GR1inGHcells,andTR inMSHcellswereunquantifiableinMmales(Figs.4,A–C,and5,A–E,rightpanels).

Glycoproteinhormones(FSH/LH/TSH)

FSHcells.FSHcellshadER ,ER ,ARa,GR1,andGR2btranscripts(Fig.3).AsignificantpercentageofFSHcellsexpressedsingleormultiplenuclearreceptors(IM79.6%,39of49cells;M56.3%,18of32cells)(Table2).ThepercentageofFSHcellspossessingonlyER transcriptswasrelativelyhigherinIMthanMmales(IM46.9%vs.M18.8%).Expres-sionofGR1wasseenonlyinMmales(Table2).OfFSHcellsinIMandMmales,28.6and6.2%,respectively,expressedmultiplenuclearreceptors.ER ARacombinationwasseenin14.3%ofFSHcellsonlyinIMmales.AbsolutecopiesMSHcells.MSHcellshadER ,ER ,andTR transcripts(Fig.3).ThetotalpercentageofMSHcellsexpressingsingleormultiplenuclearreceptorswashigherinIMthanMmales

5826Endocrinology,December2007,148(12):5822–5830Kitahashietal. NuclearReceptorsinPituitaryEndocrineCells

TABLE2.Percentageofpituitarycellswithsingleormultiplenuclearreceptortranscripts

FSHIM

M

IM

LH

M

IM

TSH

M

IM

GH

M

IM

PRL

M

IM

SL

M

IMMSH

M

ACTHIM

M

ER ER ARaARbGR1GR2bTR

ER ARaER GR1ER TR ER ARaER ARbER GR1ER GR2bER TR ER

ER ARaER ARbER GR1ER TR OthercomboRpositiveRnegative

0.046.92.00.02.00.014.36.14.14.1

12.518.80.018.80.03.10.00.00.03.1

0.046.00.00.00.00.00.020.06.04.012.02.06.00.04.0100.00.0

7.510.02.52.55.05.02.50.00.00.010.00.00.02.50.047.552.5

3.90.00.015.26.16.1

15.123.31.4

12.77.90.0

2.168.817.96.020.811.126.715.65.12.620.07.5

10.04.7

19.20.019.20.0

0.03.0

1.4

0.03.0

0.0

25.625.6

0.00.0

3.93.93.915.426.996.23.8

0.00.03.00.03.039.460.6

43.82.787.712.3

14.30.0

8.359.722.242.2

12.80.00.05.0

23.1

34.965.1

79.220.8

83.616.4

54.245.8

84.415.6

94.95.1

0.032.567.5

10.090.0

4.795.3

79.620.456.343.7

RpositiveandRnegativerepresentthetotalpercentageofcellswithandwithoutnuclearreceptortranscripts,respectively.PleasenotethatER ,TR 1,TR 2,andcombinations(combo)ofER ARb,ER GR2b,andER ER GR2bwerenotpresent.

MSHcellsexpressingTR genealoneorincombinationwithER orER werefoundonlyinIMmales.AbsolutecopiesofER transcriptsweresignificantlyhighinIMmales(IM402.4 73.7vs.M213.4 32.4copiespercell;P 0.05)(Fig.5D).

ACTHcells.AsmallpercentageofACTHcellsinbothre-productivestageshadER transcripts(IM10.0%,5of50cellsvs.M4.7%,2of43cells)(Table2).AbsolutecopiesofER transcriptwere345.0 82.0copiespercellinIMmalesand80.2copiespercellinMmales(Fig.5E).

GRandaromataseimmunoreactivityinthepituitary

Glycoproteinhormonefamily

GRimmunoreactivitywasseenintheproximalparsdis-talis,whereFSH,LH,andGHcellsarelocated,andaro-mataseimmunoreactivitywasseeninthefiberterminalsoftheproximalparsdistalisandparsintermedia(Fig.6).Buffercontrolsdidnotshowanyimmunoreactivityinthepituitarysections(datanotshown).

Discussion

Thepresentstudyshowscoexpressionofmultiplenuclearreceptorsinindividualpituitarycells,whichconfirmsdirectactionofsteroid/thyroidhormonesatthelevelofthepitu-itary.Inthetworeproductivestages,ACTHcellsexpressedonlyER ,whereasallotherpituitarycelltypesexpressedER ,andcoexpressedARa,ARb,GR1,GR2b,andTR inasubpopulation(Fig.7).AllofthepituitarycelltypeslackedER ,GR2a,TR 1,andTR 2.IntheGHfamily,ahighpercentageofER transcript-containingcellsalsocontainedER transcripts,indicatingthepossibilityofERheterodimer-Inmammals,immunocytochemicalstudieshaveshownthepresenceofERsandARsinFSHcells(24,25),andtheregulationofFSHcellsby17 -estradiol(E2)andtestosterone(T)atthepituitarycelllevel(26).TheexistenceofERandARtranscriptsinFSHcells,showninthepresentstudy,andtheirresponsetoE2andTinvitroculturesystem(27)suggestevolutionallyconservedregulatorymechanismsinFSHcellsfromfishtomammals.Therelativelyinsignificantpercent-ageofFSHcellsthatpossessARtranscriptsandtheverylowcopiesofARtranscriptsinthemsuggestthatthestimulatoryeffectofTonculturedFSHcellsinmaletilapia(27)isme-diatedbyaromatizationofTtoE2.Thisideaisfurthersup-portedbythepresenceofaromataseimmunoreactivityintheFSHcellzone(7)(presentstudy)andtheexistenceofhalf-siteofestrogenresponseelementinthe5 flankingregionoftilapiaFSH gene(28).TheroleofARinFSHcells,ifany,isprobablylimitedtoIMfishbecausenoARtranscriptsweredetectedinMmales.

ThepresenceofGRtranscriptsandGRimmunoreactivityinFSHcellsinteleost(6)andmammals(29)indicatesdirectactionofcortisolonFSHcells.AsabsolutecopiesofGRtranscriptsincreasedinFSHcellsduringmaturation,theinactivationofreproductivefunctionsbycortisol(30)mayoccurbyinhibitingFSHcellactivity.

Furthermore,theincreasedlevelsofGRandER tran-scriptsinMmalessuggestthatFSHcellshaveanimportantroleintheMstage,aswellasintheIMstage(31),whichsupportsourpreviousfindingsofincreasedGnRHreceptorsinFSHcellsinMmales(17).

OurdemonstrationofERandARtranscriptsinLHcells