Establishment of specific enzyme-linked immunosorbent assay (ELISA) for measuring Fsh and Lh levels in medaka (Oryzias latipes), using recombinant gonadotropins

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Method Article

Establishment of speci ﬁ c enzyme-linked

immunosorbent assay (ELISA) for measuring Fsh and Lh levels in medaka (Oryzias latipes), using recombinant gonadotropins

Susann Burow

^a

, Romain Fontaine

^a,1

, Kristine von Krogh

^a,1

, Ian Mayer

^b

, Rasoul Nourizadeh-Lillabadi

^a

,

Lian Hollander-Cohen

^c

, Yaron Cohen

^c

, Michal Shpilman

^c

, Berta Levavi-Sivan

^c

, Finn-Arne Weltzien

^a,

*

aDepartmentofBasicSciencesandAquaticMedicine,FacultyofVeterinaryMedicine,NorwegianUniversity ofLifeSciences,0454Oslo,Norway

bDepartmentofProductionAnimalClinicalSciences,FacultyofVeterinaryMedicine,NorwegianUniversityof LifeSciences,0454Oslo,Norway

cDepartmentofAnimalSciences,FacultyofAgriculture,FoodandEnvironment,TheHebrewUniversity, Rehovot76100,Israel

ABSTRACT

Thepaucityofinformationonunderstandingtheregulatorymechanismsthatareinvolvedinthecontrolof piscineFshandLhsynthesis,secretion,andfunction,promptedthepresentwork.Partoftheproblemisrelatedto themolecularheterogeneityand theunavailabilityof Fshand Lh assays forquantifyinggonadotropins, in particularassaysregardingthemeasurementofFsh,andsuchassaysareavailabletodayforonlyafewteleost species.The presentstudy reports thedevelopment andvalidation of competitive ELISAsforquantitative determinationofmedakaFshand Lhbyﬁrstproducingmedakarecombinant(md) gonadotropinsmdFshβ, mdLhβ,mdFshβα,andmdLhβαbyPichiapastoris,generatingspeciﬁcantibodiesagainsttheirrespectiveβsubunits, andtheirusewithinthedevelopmentofELISAs.

Theadvantagesofthisprotocolinclude:

^ThereproducibilityoftheELISAdemonstratedwasrelativelyhigh,asshownbyreasonablylowintra-(Fsh2.7%, Lh3%)andinterassayCVs(Fsh5.3%,Lh5.7%).

*Correspondingauthor.

E-mailaddresses:[email protected](S.Burow),[email protected](R.Fontaine),

[email protected](K.vonKrogh),[email protected](I.Mayer),[email protected] (R.Nourizadeh-Lillabadi),[email protected](L.Hollander-Cohen),[email protected](Y.Cohen), [email protected](M.Shpilman),[email protected](B.Levavi-Sivan),ﬁ[email protected] (F.-A.Weltzien).

1 Theseauthorscontributedequally.

https://doi.org/10.1016/j.mex.2019.06.011

creativecommons.org/licenses/by-nc-nd/4.0/).

ContentslistsavailableatScienceDirect

MethodsX

journal homepage:www.elsevier.com/locate/mex

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^The^high^degree^ofparallelismbetweenserialdilutionsoftherecombinantandnativepituitary-derivedFshand Lh,maybeasignofsimilarstructuresandimmunologicallysimilarity.

^Two^newcompetitiveELISAsforthequantiﬁcationofmedakaFshandLhwereestablishedfortheﬁrsttime.

ARTICLE INFO

Methodname:Specificenzyme-linkedimmunosorbentassay(ELISA)fordeterminingFshandLhlevelsinmedaka Keywords:Competitiveenzyme-linkedimmunosorbentassay,Follicle-stimulatinghormone,Gonadotropinquantification, Luteinizinghormone,Oryziaslatipes,Specificantibodies

Articlehistory:Received26December2018;Accepted12June2019;Availableonline17June2019

SpeciﬁcationsTable

SubjectArea: Biochemistry,GeneticsandMolecularBiology Morespeciﬁcsubjectarea: Quantiﬁcationofgonadotropinlevelsinmedaka

Methodname: Speciﬁcenzyme-linkedimmunosorbentassay(ELISA)fordeterminingFshandLhlevelsin medaka

Nameandreferenceof originalmethod:

Mañanós,E.L.,Swanson,P.,Stubblefield,J.,Zohar,Y.,PurificationofgonadotropinIIfroma teleostfish,thehybridstripedbass,anddevelopmentofaspecificenzyme-linked immunosorbentassay,Gen.Comp.Endocrinol.108(1997)209–222[1].

Resourceavailability: Theinformationaboutallreagents,hardwareandsoftwareisincludedinthepresentarticle.

Methoddetails Background

Thegonadotropinsfollicle-stimulatinghormone(Fsh)andluteinizinghormone(Lh)playessential roleswithinthebrain-pituitary-gonadal(BPG)axisbycontrollinggonaddevelopmentandmaturation inallvertebrates[2].Detailedknowledgeoffunctionandregulationofthesehormonesisstilllimited.

The accepted model in fish suggests that Fsh is important for early stage of gametogenesis, spermatogonialproliferation,andvitellogenesisinfemale,whereasLhismostlyinvolvedinprocesses leadingtofinalgametogenesis,oocytematurationandovulationinfemalesandspermiogenesisand spermiationinmales[3,4].Whenstudyinggonadotropinregulationandfunction,animportanttoolis thequantificationofhormonelevelsinbloodandpituitarybyenzyme-linkedimmunosorbentassay (ELISA)orradioimmunoassay(RIA).ELISAandRIAassaystodeterminegonadotropinlevelsinfish traditionally have been based on native gonadotropins from fish pituitaries and their specific antibodies.Thepurificationofnativegonadotropinshasbeenshownchallenging,andrecombinant gonadotropinsarenowbeingused tosubstitutenative hormones.Theyhavevariousadvantages comparedtonativelypurifiedhormonesbecausetheycanbecontinuallyproduced,andpotential cross-contaminationwithotherrelatedglycoproteinsisminimized[4].Basedontheabovementioned considerations,theobjectiveofthisstudywastodevelopandvalidatehomologouscompetitiveELISAs formedaka(Oryziaslatipes)FshandLhusingrecombinantgonadotropins,thusenablingquantification ofbiologicallymore relevantproteinlevels,which willconsiderablyadvancethevalueof future studiesofgonadotropinphysiologyinthisimportantfishmodel.

Animals

Japanesemedaka(Oryziaslatipes)ofthed-rRstrainwerekeptandbredinourﬁshfacilityinre- circulatingsystemsundera photoperiodofL14:D10andwatertemperatureof281C.Embryos wereincubatedinembryoculturemedium(E3;5mMNaCl,0.17mMKCl,0.33mMCaCl2,0.33mM

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MgSO4(allSigma-Aldrich,St.Louis,U.S.A.)),andkeptat26Cuntilhatchingandtransfertosystem tanks.Thefishwerefedthreemealsperdaywithacombinationofdryfeedandlivebrineshrimp nauplii larvae (Artemia salina). All fish were raised under the same conditions regarding to temperature,photoperiod,food,tanksize,and density.Handling,husbandryandallexperimental proceduresoffishwereincompliancewiththeguidelinesandrequirementsforthecareandwelfare ofresearchanimalsoftheNorwegianAnimalHealthAuthorityandoftheNorwegianUniversityofLife Sciences.Inaddition,theworkdescribedhasbeencarriedoutinaccordancewiththeEUDirective 2010/63/EU for animal experiments and Uniform Requirements for manuscripts submitted to Biomedicaljournals,andinformedconsentwasobtainedforexperimentationwithanimalsubjects.

ProductionandpuriﬁcationofrecombinantgonadotropinsmdFshβ,mdLhβ,mdFshβα,andmdLhβα

UsingthemethylotrophicyeastP.pastorisexpressionsystemrecombinantproteinswereproduced generallyaccordingtoKasutoandLevavi-Sivan[5]and Yom-Dinetal.[6].Synthesis ofgenesfor medakafshb(AccessionNumberNM_001309017.1),lhb(AccessionNumberAB541982.1),fshba,and lhba(gpa;AccessionNumberNM_001122906)wasoutsourcedtoGenScript,NewJersey,U.S.A.(see sequencesinBurowetal.[7]).Geneexpressionconstructswereproduced(seeFig. 1inBurowetal.[7]) andgeneexpressioncassettesweregeneratedwithP.pastoriscodonoptimizedDNAsequence.The syntheticgeneswerejoinedtoformafusiongenethatencodesa"tethered"polypeptide(mdFshβα, mdLhβα)inwhichoneoftheβchainsformstheN-terminaldomainandtheαchainformstheC- terminal domain. A "linker" sequence containing six amino acids (three Gly-Ser pairs) [8] was positionedbetweentheβandαchainstoassistinchimerizationofthesubunits.TheGly-Serlinker sequenceisminimallyhydrophobicandcanthusmaximizethepossibilityforchimerizedsubunits folding into their native conformational structure enabling independent interaction with their receptors[5]. A six-His tailwas placed atthe end of theβ subunitenabling puriﬁcation of the recombinantprotein.SynthesizedDNAfragmentswereclonedintopPIC9KvectorusingEcoRIand NotIrestrictionssitesandconﬁrmedbysequencing(WeizmannInstitute,Rehovot,Israel).ThepPIC9K plasmidcontainedtheyeastαmatingfactor(αMF)secretionsignalthat directstherecombinant proteinintothesecretorypathway.TheconstructsweredigestedwithSalIandusedtotransformP.

pastorisstrainGS115(Invitrogen,Carlsbad,U.S.A.)byelectroporation(GenePulser,Bio-Rad,Hercules, U.S.A.).ThisresultedininsertionoftheconstructattheAOX1locusofP.pastoris,generatingaHis⁺ Mut^S(slowmethanolutilization)phenotype.TransformantswereselectedfortheHis⁺phenotypeon 2%agarcontainingregenerationdextrose-biotin medium(1Msorbitol,1.34%yeastnitrogenbase withoutaminoacids,410⁵%biotin,and0.005%ofL-glutamicacid,L-methionine,andL-leucine(all fromSigma-Aldrich),and0.005%ofL-lysine,andL-isoleucine(allfromBiologicalIndustries,Kibbutz Beit-Haemek,Israel)).AnadditionalselectionforantibioticgeneticinG418(Gibco-BRL,Carlsbad,U.S.

A.)resistancewasperformedon2%agarcontaining1%yeastextract(Becton,DickinsonandCompany, FranklinLakes,U.S.A.),2%peptone(Becton,DickinsonandCompany),2%dextrosemedium(Sigma- Aldrich)andtheantibioticG418(1mg/ml)(Gibco-BRL).TenHis⁺Mut^Sclonesfromeachconstruct werecultivatedwithshakingfor24h(growthphase)in1.5mlofbufferedminimalglycerol(BMG) (1.34%yeastnitrogenbasewithoutaminoacids,1%glycerol,410⁵%biotin,and100mMpotassium phosphatebuffer(allfromSigma-Aldrich),pH6)at28C,supplementedwithmethanol0.5%[v/v]

every24h.Afterwards,cellswereharvested(1500g,5min),resuspendedandcultivatedfor3days (inductionphase) in 1ml of bufferedminimal methanol (BMM) (BMG, supplemented with0.5%

methanolevery24h).Theproteinwasexpressedinashakerflaskandharvestedat72hafterinduction bymethanol.RecombinantmedakaFshβ(mdFshβ),medakaLhβ(mdLhβ),medakaFshβα(mdFshβα), andmedakaLhβα(mdLhβα)werepurifiedusingnickelnitrilotriaceticacidagarose(Ni-NTA)(Qiagen, Hilden,Germany)accordingtothemethoddescribedbyKasutoandLevavi-Sivan[5]andYom-Din etal.[6]withcertainmodifications.Briefly,thepHofthesupernatantwasadjustedto8.0with5N sodium hydroxide (Sigma-Aldrich). Beads (QIAexpressionist, Qiagen) were supplemented to the mediumandstirredonmagneticstirrerfor18hat4C.Thepurificationwasperformedbybatch purificationofthehis-taggedproteinsbywashingwith5mMimidazole(Sigma-Aldrich)thatprevents bindingofunspecificproteinstothebeads.TheboundproteinwaselutedwithPBSpH4.5containing 250mMimidazole.TheelutedfractionsofmdFshβ,mdLhβ,mdFshβα,andmdLhβαweredialyzedwith

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Slide-A-Lyzer Dialysis Cassette (Thermo Fisher Scientiﬁc, Waltham, U.S.A) according to the manufacturer’sprotocols against PBS pH 7.5, meaning unwanted compounds were removed by selectiveandpassivediffusionthroughasemi-permeablemembrane.Thepuriﬁedproteins,tagged withasix-Histail,weredetectedonaWesternblotasdescribedbelow.

ProductionandvalidationofspeciﬁcantibodiesformdFshβandmdLhβ

PolyclonalantiseraagainstrecombinantmdFshβandmdLhβweregeneratedgenerallyfollowinga procedure described by Aizen et al. [9]. Two different rabbits for each protein received three intradermalinjections ofpurifiedprotein(mdFshβ; 1mg first injection, 0.5mg secondand third injection;mdLhβ;0.7mgfirstinjection,0.4mgsecondandthirdinjection)in0.9%NaClandemulsified inanequalvolumeofcompleteFreundsadjuvant(Sigma-Aldrich)at3-weekintervals.Twoweeks afterthefinalinjection, therabbitswerebled,and theserumwas aliquotedand lyophilized.To validate the produced antibodies, the recombinant proteins and medaka pituitary extract were visualizedusinganti-mdFshβ,oranti-mdLhβantisera.Toensurethattheplasmaoftherabbitbefore thefinalinjectionsdidnotreactwithmdFshβandmdLhβ,aWesternblotusingmedakapre-immune serumagainstmedakapituitaryextract,mdFshβ,andmdLhβwasconductedasdescribedbelow.

Westernblotanalysis

Fromculturesupernatants,reducedand non-reducedsampleswereresolvedbySDS-PAGEfor Westernblotanalysis.RecombinantmdFshβ,mdLhβ,mdFshβα,and mdLhβαwerevisualizedusing anti-His(diluted1:2000)(QIAexpressanti-Hisantibodies;Qiagen),generallyaccordingtoYom-Din etal.[6],andvalidatedbymolecularweight.Tovalidatetheproducedantibodies,therecombinant proteinsandmedakapituitaryextractwerevisualizedusinganti-mdFshβ,oranti-mdLhβ(bothdiluted 1:2000,1:100,000,1:600,000)antisera.

ToconﬁrmthattheplasmaoftherabbitbeforetheﬁnalinjectionsdidnotreactwithmdFshβand mdLhβ, a Western blot using medaka pre-immuneserum as a negative controlagainst medaka pituitary extract, mdFshβ, and mdLhβ was performed. Precisely, reduced samples from culture supernatantswereelectrophoresedon15%SDS-polyacrylamiderunninggelswitha5%stackinggel.

Thegelswereblottedontonitrocellulosemembranes(SchleicherandSchuell,Dassell,Germany)and blockedwith3% BSAin Tris BufferedSaline(TBS) buffer(20mMTris,150mM NaCl) toprevent nonspeciﬁcbindingoftheantibodiesduringsubsequentsteps.ThemembraneswereincubatedinPBS containing1%nonfatmilkwiththeantisera(dilutions1:2000,1:100,000,1:600,000)for1hatroom temperature(RT)andafterwardswithgoatantirabbithorseradishperoxidase(GAR-HRP)conjugate (dilution1:5000;JacksonImmunoResearchLaboratories)for1hatRT.Allmembraneswerewashed andtreatedwithenhancedchemiluminescencereagent(ChemiluminescencedetectionkitforHRP, BiologicalIndustries)torevealimmunoreactivebands.Forglycosylationanalysis,priortoWestern blotanalysis,N-glycosidaseF(PNGaseF)wasusedtoproducedeglycosylatedproteinsbyhydrolyzing all types of N-glycan chains. According to supplier recommendations (Roche Applied Science, Mannheim,Germany),100ng of reduced and denaturedmdFshβ,mdLhβ,mdFshβα,mdLhβα,and medakapituitaryextractwereincubatedfor2hat37CinthepresenceorabsenceofPNGaseF.

Fluorescenceinsituhybridization(FISH)

To further evaluate the antibody specificity of mdLhβ and mdFshβ, fluorescence in situ hybridization(FISH)and immunofluorescence (IF)wereconducted.FISHwas performedonfree- floatingparasagittalbrain-pituitarysections,generallyaccordingtoFontaineetal.[10].Briefly,after beingsacrificedwithicewater,brainandpituitaryfrom12unsexed6month-oldadultfishwere dissectedandfixatedovernightwith4%PFAat4C.Tissueswerethengraduallydehydratedwitha seriesofincreasingconcentrationsofethanolandstoredin100%methanoluntilused.Tissueswere rehydrated,embeddedin3%agarose,para-sagittallysectioned(60

m

^m^sections)^using^a^vibratome

(VT1000SLeica,Wetzlar,Germany),and then treatedwithproteinaseK(1

m

^g/ml;^P6556,^Sigma-

Aldrich) for 30min. fshb riboprobe was cloned using AGAGCAGAGGAAGCAACACT and

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GGGGCACAGTTTCTTTATTTCAGasprimers,andsynthesizedusingPGEM-Tvector(Promega,Madison, WI),whereasweusedthelhbriboprobepreviouslydescribed[11].fshbandlhbsenseandantisense riboprobeswereconjugatedwithdigoxigenin(DIG;11277073910;Roche,Basel,Switzerland)using SP6 or T7 RNA polymerase (Promega). Tissues were hybridized with either sense or antisense riboprobesfor18hat65Candthenincubatedwithsheepanti-DIGconjugatedwithperoxidase(POD;

1:500; 11207733910; Roche) over night. Signalwas revealedusing TAMRA-conjugated tyramide constructedinourlab.

Immunoﬂuorescence(IF)

IFstainingwasconductedonfree-ﬂoatingsectionsaccordingtoFontaineetal.[10]usinganti-Lhβ andanti-Fshβ(describedearlier).Foranti-Lhβ,thetissueslabelledforlhbmRNAbyFISHwereused (seeabove).Foranti-Fshβ,IFcouldnotbecarriedoutafterinsitulabellingduetoanantigenretrieval treatmentthatwasnecessarybeforetheIF,destroyingthelabelingoftheFISH.Thus,IFwasperformed onconsecutiveparasagittalsectionsoftheoneusedforthefshbFISH.Tissuesectionsweretreated with2NHydrochloricacid(HCl)for1hat37Candthenincubatedwithprimaryantibody(anti-Lhβ; 1:2000, anti-Fshβ; 1:1000) overnight at 4C. A secondary goat anti-rabbit antibodycoupled to AlexaFluor488(A-11034,ThermoFisherScientiﬁc,Waltham,U.S.A.)ataconcentrationof1:1000was usedfor4hincubation.Controlexperimentwithouttheprimaryantibodywasincluded.Tissuesfor anti-Lhβ were then treated for nuclei staining with DAPI (4⁰, 6-Diamidino-2-phenylindole dihydrochloride;32670Sigma)byincubationatRTfor20minatatiterof1:1000andrinsing.

Imaging

Stained tissues were mountedbetween slide and coverslip with the mounting mediumfor fluorescenceVectashield H-1000(Vector, California,U.S.A.). Imageswereobtained usinga Zeiss LSM710 confocalmicroscopewith25(LCI Plan-Neofluar25x/0.8NA)objective.Channelswere acquiredsequentiallyinordertoavoidsignalcrossoverbetweenthedifferentfilters.Imageswere processed using the ZEN software (Carl Zeiss AG, Oberkochen, Germany). Z-projections from confocal stacks of images were acquired using Image J software (http://rsbweb.nih.gov/ij/).

Composites were assembled using Adobe Photoshop and Illustrator CS6 (Adobe Systems, San Francisco,California).

Gonadalhistology

Histologicalanalysisoftesteswasperformedtoassessthematurationalstagesofﬁshsampledfor developmentalseriesbyidentifyingthegermcellstages(seeTable3inBurowetal.[7]).Testesfrom 36males, grouped according to standard body length (SL), were dissected and transferred to phosphate buffered saline (PBS; Sigma-Aldrich) prior to overnight ﬁxation in ice-cold 4%

glutaraldehyde phosphate bufferedsolutionat 4C. Note that fish fromthesmaller size groups hadtesticulartissueofsuchlowvolumesthat,infearoflosingitlaterinthefixationprocess,itwasnot dissectedoutofthebodycavity.Rather,allotherorganswereremovedfromtheabdomen,andbody andgonadsfixatedasawhole.Glutaraldehydefixativesolutionwaspreparedfreshbymixing28ml 0.2MNaH2PO4-H2Owith72ml0.2MNa2HPO4-2H2O(bothfromMerckMillipore,Billerica,MA,USA), adjustingthemixturetopH7.2,andthenadding68mlmq-H2Oand32mlof25%glutaraldehyde (MerckMillipore).TissuesweredehydratedinaseriesofincreasingconcentrationsofEtOH(70–100%) atRT,eachsteplastingatleast30min.Thelaststep(100%)wasrepeatedtriceandreplacedwith approx.5mlofpreparationsolution(100mlTechnovit7100added1gofHardenerI(HeraeusKulzer, Hanau,Germany))andkeptatslowshakingovernight.Allfixationstepswereperformedinsmallglass bottles.Afterinfiltration,tissuesampleswereembeddedincoldHistoformS(HeraeusKulzer)added approx.1mlpreparationsolutionw/50

m

^l^HardenerÎI^(Heraeus^Kulzer)ândîncubatedât³⁷^C.^Cured

samplesweremounteduntoHistoblocsusingTechnovit3040(bothfromHeraeusKulzer),before sagittalsections(3

m

^m)^were^prepared^using^a^Leica^RM2245^microtome^(LeicaBiosystems,Wetzlar, Germany).Sectionswerecollectedfromtheperipheryuntilthemiddle ofthetestestissueevery

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30

m

^m ând ^placed ûnto ^microscope ^slides. ^Dried ^sections ^were ^stained ^with ^Toluidine ^Blue Ô

(Sigma-Aldrich)andmountedwithCoverquick4000(VWR International,Radnor, PA,USA)before histologicalanalysis.Germcellsweredeterminedaccordingto[12],withintheﬁvemaingermcell stages(spermatogoniatypaA(SPA),spermatogoniatypeB(SPB),spermatocytes(SC),spermatids(ST) andspermatozoa(SZ)),nofurtherdistinctionsweremade(seeTable2inBurowetal.[7]).

DevelopmentandvalidationofspeciﬁcELISAforFshandLh,andproﬁleofpituitarylevelsofFshandLhin malemedaka

TherecombinantgonadotropinswereusedtodevelopspeciﬁcandhomologouscompetitiveELISAs fordeterminationof mdFshandmdLh in thepituitary,generallyaccording to Mañanósetal.[1].

CompetitiveELISAs were developedusingspeciﬁcβ-subunitpolyclonalrabbitprimaryantibodies against mdFshβormdLhβ(describedearlier),recombinantβ-subunitsmdFshβormdLhβ(describedpreviously) tocoattheELISAmicroplates,andrecombinantmdFshβαormdLhβα(describedearlier)forthestandard curves.Brieﬂy,ELISAmicrotiterplates(Nunc-Immuno^TMPlates;Nunc,Denmark)werecoatedwith 100

m

^l/well^of¹⁰^ng/ml⁽¹^ng/well)^mdFshβor5ng/ml(0.5ng/well)mdLhβ.Thefollowingday,plates werewashedwith200

m

^l/well^phosphate^buffered^saline^with^Tween²⁰^(PBST)^buffer⁽¹⁰^mM^Na²^PO⁴^,

2mMKH₂PO₄(pH7.4),140mMNaCl,3mMKCl,and0.05%Tween20(allfromSigma-Aldrich)).Toreduce backgroundbyblockingtheunspeciﬁcbindingsites,plateswereblockedfor1hwith200

m

^l/well^of^PBST

buffercontaining1%bovineserumalbumin(BSA,Sigma).Singlepituitariesweredissectedandkepton iceuntilhomogenization(6m/s, 330susingceramicbeads,cat.no 116933050,MPBiomedicals, California,U.S.A.)usingFastPrep-24(MPBiomedicals)diluted1:2.7with0.1%BSAinPBST,insufﬁcient volumetoallowtechnicalduplicates(125

m

^l/well).^The^homogenate^wascentrifugedat10,000gfor 5min.Samplesandstandardswereﬁrstpre-incubatedovernightatRTwiththeprimaryantibodies (125

m

^l/well)⁽ﬁnaldilution1:10,000formdFshβand1:50,000formdLhβin0.1%BSAinPBST)in96-well microtestplates(Sarstedt,Nümbrecht,Germany).Afterpre-incubation,eachsamplewasdistributed intothewells(100

m

^l/well)ôf^the^coated^microtiter^platesândîncubated^for³^hât^RT.Âfterincubation, theplateswerewashedwithPBST.Formedantigen–antibodycomplexesweredetectedbyadditionof 100

m

^l/well^of^GAR-HRP^(Bio-Rad)^diluted^1:5000ⁱⁿ^PBST-0.1%^BSA^buffer^for²^h^at^RT.^The^plates^were

washedagainwithPBST.Thevisualizationofthepresenceofenzymecomplexeswasperformedby additionof100

m

^l/well^of^3,3⁰^,5,5⁰-tetramethylbenzidine(TMB)peroxidasesubstrate(KPL,Zotal,Israel) diluted1:4.ThereactionwascarriedoutincompletedarknessatRTandstoppedafterapproximately 15minwithTMB stopsolution1MH2SO4 (50

m

^l/well). Âbsorbance^was^read ât⁴⁵⁰^nm, ûsingâ

MicroplateSpectrophotometer(Epoch2,Biotek,Winooski,U.S.A.).

TheELISAwasvalidatedformedakaLhandFshdeterminationsinpituitaryextractofmedaka.

Displacementcurvesforpituitarysampleswereachievedbyserialdilutionsofthesamplein0.1%

BSAinPBSTandcomparisonwiththestandardcurveusingrecombinantmdFshβαormdLhβα.Forthe parallelismanalysis,pituitarieswerecollectedfromsexuallymaturemaleﬁshandhomogenized usingdifferentconditions(10ceramicbeads,6m/s;20ceramicbeads,4m/s;20ceramicbeads,6m/

s;all330s).Thehomogenatewascentrifugedat10,000gfor5minandtheresultingsupernatant wasusedasthepituitaryextract.ThesensitivityoftheassayisdefinedasthelowestdoseofFshorLh capableofreducingtheopticaldensitymorethanthemeanplustwostandarddeviationsofthezero doseofFshorLh[B0-2SD];itwascalculatedbyaddingthemeanoftheblanktotwotimesthe standarddeviationoftheblank.Intra-assaycoefficientofvariation(CV)wasdeterminedbyassaying sixreplicatesofoneofthestandardconcentrations(1.56ng/ml)onthesameassayplate.Inter-assay CVwascalculatedbyassayingthesamesamplefivetimesindifferentplates.

ProﬁleofpituitarylevelsofFshandLhinmalemedakacomparingjuvenilesversusadults

AprofileofFshandLhinmalemedakapituitarieswasconductedusingtheELISAmethoddescribed above.ToachieveaprofileforFsh,pituitariesfrom24juvenilemaleswithSLbetween12mmand 16.5mm,andof24adultmalesbetween21mmand25.5mmweredissected.FortheprofileofLh pituitariesfrom12juvenilemaleswithSLbetween12mmand16mm,andof12adultmalesbetween 22.5mmand26.5mmweredissected(forbothFshandLh1pituitaryin40

m

^l^0.1%^BSAⁱⁿ^PBST^per

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biologicalreplicatewasused).Thedistinctionbetweenjuvenileandadultwasbasedonunpublished resultsrelatingmedakatesticularmaturationstagetoSL[7],andshowingthatmaleswithSLbelow 16mmwerecompletelyimmatureandmaleswithSLabove20mmtobefullymature.

Statisticalanalysis

DataarepresentedasmeanSEM.Alldataweretestedfornormaldistribution(Shapiro–Wilk normalitytest).Forsamplegroups,whichdidnotfollowanormaldistribution,thedatawereﬁrstlog- transformed.ForELISAdatacalculations,sigmoidcurveswerelinearizedusinglogittransformation.

CorrelationswerecalculatedbyGraph-PadPrismsoftware(version7;GraphPad,SanDiego,U.S.A.).

Signiﬁcancelevelwassetto0.05.

Funding

Thisresearchwas supportedﬁnanciallybytheNorwegianUniversityof LifeSciencesand the ResearchCouncil ofNorway(grantnumber 248828 BioTek2021,and 231767FriPro).Thefunding sourceshadnoinvolvementinthedesignofthestudy,inthecollection,analysisandinterpretationof data,inthewritingofthereport,andinthedecisiontosubmitthearticleforpublication.

Acknowledgement

TheauthorswouldliketothankLourdesGenoveTanforexcellentﬁshhusbandry.

References

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[2]F.A.Weltzien,E.Andersson,Ø.Andersen,K.Shalchian-Tabrizi,B.Norberg,Thebrain-pituitary-gonadaxisinmale teleosts,withspecialemphasisonﬂatﬁsh(Pleuronectiformes),Comp.Biochem.Physiol.A:Mol.Integr.Physiol.137(3) (2004)447–477.

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