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RESEARCHOpenAccessMedakafishexhibitslongevitygendergap,anaturaldropinestrogenandtelomereshorteningduringaging:auniquemodelforstudyingsex-dependentlongevitySingaramGopalakrishnan,NapoKMCheung,BillWPYipandDorisWTAu*AbstractIntroduction:Femaleshavingalongertelomereandlifespanthanmaleshavebeendocumentedinmanyanimals.
Suchlinkagehoweverhasneverbeenreportedinfish.
Progressiveshorteningoftelomerelengthisanimportantagingmechanism.
Mountinginvitroevidencehasshownthattelomereshorteningbeyondacriticallengthtriggeredreplicativesenescenceorcelldeath.
Estrogenhasbeenpostulatedasakeyfactorcontributingtomaintenanceoftelomereandsex-dependentlongevityinanimals.
Thispostulationremainsunprovenduetothelackofasuitableanimalsystemfortesting.
Here,weintroduceateleostmodel,theJapanesemedakaOryziaslatipes,whichshowspromiseforresearchintothemolecularmechanism(s)controllingsexdifferenceinaging.
Results:Usingthemedaka,wedemonstrateforthefirsttimeinteleostthat(i)sexdifferences(female>male)intelomerelengthandlongevityalsoexistinfish,and(ii)anatural,'menopause'-likedeclineofplasmaestrogenwasevidentinfemalesduringaging.
Estrogenlevelssignificantlycorrelatedwithtelomeraseactivityaswellastelomerelengthinfemaleorgans(notinmales),suggestingestrogencouldmodulatetelomerelengthviatelomeraseactivationinasex-specificmanner.
Ahypotheticalinvivo'critical'terminalrestrictionfragment(TRF,representingtelomere)lengthofapproximately4kbwasdeducedinmedakaliverforpredictionoforganismalmortality,whichishighlycomparablewiththatforhumancells.
Anageconversionmodelwasalsoestablishedtoenableagetranslationbetweenmedaka(inmonths)andhuman(inyears).
Thesenoveltoolsareusefulforfutureresearchoncomparativebiologyofagingusingmedaka.
Conclusion:ThestrikingsimilarityinestrogenprofilebetweenagingfemaleO.
latipesandwomenenablesstudyingtheinfluenceof"postmenopausal"declineofestrogenontelomereandlongevitywithouttheneedofinvasiveovariectomy.
Medakafishisadvantageousforstudyingthedirecteffectofincreasedestrogenontelomerelengthandlongevitywithoutthebreastcancercomplicationsreportedinrodents.
ThefindingsstronglysupportthenotionthatO.
latipesisauniquenon-mammalianmodelforvalidationofestrogenicinfluenceontelomereandlongevityinvertebrates.
Thislaboratorymodelfishisofpotentialsignificancefordecipheringtheostensiblyconservedmechanism(s)ofsex-associatedlongevityinvertebrates.
Keywords:Lifespan,Aging,Telomeraseandtelomere,Estrogenprofile,SexdifferenceandmedakaO.
latipes*Correspondence:bhdwtau@cityu.
edu.
hkStateKeyLaboratoryinMarinePollution,DepartmentofBiologyandChemistry,CityUniversityofHongKong,83TatCheeAvenue,Kowloon,HongKongSAR2013Gopalakrishnanetal.
;licenseeBioMedCentralLtd.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/2.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
TheCreativeCommonsPublicDomainDedicationwaiver(http://creativecommons.
org/publicdomain/zero/1.
0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.
Gopalakrishnanetal.
FrontiersinZoology2013,10:78http://www.
frontiersinzoology.
com/content/10/1/78IntroductionLongevitygendergap(LGG)isthelongevitydifferencebetweenthetwosexes.
Manyanimalsexhibitalongerlifespaninfemalesthanmales(Table1).
Severaltheorieshavebeenproposedtoexplainthepossiblecause(s)ofLGG(females>males)[1].
Amongthem,sexdifferencesintelomerelength(longerinfemales)andestrogen(higherinfemale)havebeengiventhemostattention,particularlyinmammalianstudies[2-5].
TelomeresareDNAcappingstructuresthatprotectchromosomeendsfromrecombinationandfusion,maintaininggenomicstability[6].
Telomeresshortenwithageduetoineffi-ciencyinDNAreplication(a.
k.
a.
end-replicationproblem)[7].
Shortenedtelomerelength(TL)belowthresholdlevelinducescellularsenescenceorcelldeath[8].
Progressiveerosionoftelomerelengthisanimportantagingprocess,whichiswellrecognizedbyextensiveinvitroandinvivostudiesonmammals[4,9].
Theenzymetelomerasepro-motestelomericrepairandreducestelomereerosionbyaddingconservedrepeatsofTTAGGGtochromosomalends[10].
Invitromammalianstudiesdemonstratedthatestrogencanstimulatetelomeraseactivityviaestrogen-receptor-mediatedtranscriptionandpost-translationalactivationofTERT(telomerasereversetranscriptase;thecatalyticunitoftelomerase)[11,12].
Estrogenissynthesizedinallvertebratesandsomeinvertebrates[13,14],thereforeestrogen-mediatedtelomeraseacti-vationandtelomeremaintenancearelikelyconservedinanimals.
Despiteestrogenbeingdescribedasakeyfactorcon-tributingtotheobservedsexdifferencesintelomerelength(female>male)andlongevityinanimals,thishy-pothesishasneverbeenvalidatedusingasuitablemodelsystem.
Thecommonanimalmodelsemployedforagingstudies,CaenorhabditiselegansandDrosophilamela-nogaster,havepostmitoticcellspredominantlyinthesomatictissuesofadult,makingitunfeasibletoinves-tigatetelomere-associatedreplicativesenescence[46].
Theconventionalrodentmodelsarenotdesirablefortelomere-andestrogen-relatedagingstudies.
Thisisbecausetelomeresofinbredrodentsareextraordinarilylong[47],makingitdifficulttostudytheeffectsoftelo-mereerosiononagingandLGGineithershort-termex-perimentsorwithinasinglegeneration.
Moreover,theincreasedriskofbreastandovariancancerdevelopmentuponestrogenadministrationinrodents[48,49]compli-catesthestudyofthedirecteffectsofestrogenontelo-merelengthandlongevityinvivo.
Thesmallsizedfish,Japanesemedaka(Oryziaslatipes),sharessimilarestrogenbiologywithmammals[50].
Theyundergogradualsenescence,increasingmortalityratewithage[51-53]andprogressivetelomereshorteninginmostorgansasitages[54].
Ourpreliminarystudyfurtherre-vealsfemalemedakalivelongerthanthemales.
However,sexdifferenceintelomerehasneverbeenreportedinO.
latipesoranyteleost[45].
UsingthemedakaO.
latipesasamodel,thepresentstudywasdesignedtoanswerthefundamentalquestion"Dosexdifferences(female>male)inbothtelomerelengthandlongevityexistinfish"Sup-portedbythepositiveoutcomes,wefurtherexaminedage-associatedchangesinsexhormones,telomeraseactiv-ityandtelomere(lengthandattritionrate)intheliver(organcommoninvertebrates)andgill(organuniqueinfish),withanattempttoinferthemechanisticrelationshipamongestrogen(sex),telomereandlongevityinO.
latipes.
Atheoretical'critical'telomerelengthwasderivedinme-dakaliverforpredictionoforganismalsenescenceandmortality.
OurfindingsalsohighlightastrikingsimilaritybetweenfemaleO.
latipesandwomenregardingtheirage-associatedchangeinestrogenprofileandtelomeredynamic.
ThemedakaO.
latipesisthereforeuniqueforstudyingthemechanismsofestrogenic(sex)influenceonlongevityinvertebrates,particularlyhuman.
Tofacilitatefutureresearchinthisdirection,weputforwardama-thematicalmodelthatpermitsageconversionbetweenmedakaandhuman.
EvidencesprovidedhereinstronglyTable1Theexistenceoflongevitygendergap(femaleslivinglongerthanmales)indifferentanimaltaxaTaxaReferencesVertebratesMammalsHuman[2,3,15-17]Chimpanzees[18,19]Gorillas,Orangutans,gibbons,spidermonkeysandsifakas[18,20]Rat[15-17,21]Soaysheep,pilotwhalesandkillerwhales[22,23]ReptilesLizard[24-26]AmphibiansFrogsandnewts[27-30]FishesSpinydogfish;mosquitofishandscaldfish[31-33]InvertebratesArthropodsandannelidsFruitflies,medflies,butterflies,mosquitoes,seedbeetles,ants,bees,tarantulas,tearedspiders,copepodsandthegiantkidney-wormsinthemanedwolf[34-44]Footnote:Analternativeformoflongevitygendergap,i.
e.
maleslivinglongerthanfemales,isalsoexhibitedinafewanimaltaxa,especiallytheAves[45].
Gopalakrishnanetal.
FrontiersinZoology2013,10:78Page2of11http://www.
frontiersinzoology.
com/content/10/1/78supportthatmedakafishisdesirableforresearchincomparativebiologyofaging,unravelingtheevidentlyconservedmechanism(s)ofsex-dependentlongevityinvertebrates.
ResultsSex-dependentlifespanofmedakaMaleandfemalemedakarearedunderstablelaboratoryconditiondisplayedsignificantlydifferentsurvivalpro-files(Figure1;logranktest:χ2=6.
51,df=1,N=3117,p=0.
011).
Majordiscrepancyinsurvivalprobabilitybetweenthetwosexeswasobservedat4–15monthsold(Figure1).
Themedianlifespanofthemalesandfemaleswas13.
7(95%CI:13.
3–14.
1)monthsand14.
6(95%CI:14.
3–15.
2)months,respectively.
Sex-dependenttelomerelengthintheliverandgillofmedakaduringagingTelomerelength(TL)representedbythemeanterminalrestrictionfragmentsize(dubbed'TRF'hereafter)inbothgillandliverwasnotonlyage-dependent,butalsosex-specific(Figure2;two-wayANOVAinterac-tioneffect;Gill:F3,70=8.
43,p0.
05),exceptinthegillofmales(ρ=0.
62,FDR-adjustedpmale)wasthemostdistinct;whereaswhenE2leveldeclinedinseniorfemales,e.
g.
>12monthsold,suchsexdifferencewascorrespondinglyattenua-ted.
Ithasbeenwellprovenusingavarietyofhumancells[58]thatestrogenenhancestelomeremainten-ancebystimulatingtelomeraseactivitythroughERE-dependentupregulationofTERTexpression[11,12].
Ongoingstudyinourlabindicatesthepresenceoffunc-tionalEREsinO.
latipesTERTpromoter,suggestingthisestrogen-andtelomerase-dependentpathwayalsoexistsinO.
latipes.
Ontheotherhand,givenaveryweaklinkbetweentestosterone/11-keto-testosteroneandtelomeraseacti-vityinbothmaleandfemaleO.
latipes(Table2),theregulatoryeffectofthetwomasculinehormonesontelo-meremaintenanceislikelyindependentoftelomerase.
ThisseemscontradictorytopreviousinvitrostudiesshowingthatandrogencouldmodulatehTERTexpres-sionandtelomeraseactivityinprostateandhematopoieticcells[59-61].
Theinvivoregulatoryeffectofandrogenontelomeraseandtelomeremaintenanceinvertebrateswar-rantsfurtherinvestigation.
Figure3Changeoftelomeraseactivityinmale(blue)andfemale(pink)O.
latipesduringaging(upper:gill;lower:liver).
Relativetelomeraseactivityisdenotedbythebarlengthandthecoloredmarkontheoppositesideoftheorigin.
Errorbarsarestandarderrorofmean(SEM).
Barsannotatedwithdifferentalphabetsaresignificantlydifferent.
Asterisk(*)indicatessignificantsexdifferenceatthatparticularage(FDR-adjustedpmale)and(ii)sexdifferenceintelomerelength(female>male).
Thenatural,'menopause'-likedropofestrogeninfe-malesduringagingrendersmedakaauniquemodelforstudyingtheeffectof'postmenopausal'declineofestro-genontelomereandlongevitywithouttheneedofinva-siveovariectomyasrequiredinrodents.
Moreover,thedirecteffectofincreasedestrogenontelomereandlon-gevitycouldbeinvestigatedinfishwithoutthecompli-cationofbreastcancerasreportedinrodents.
AlltheseevidenceshighlightthestrongpotentialofO.
latipesasaFigure5ApredictionmodelestablishedtoforecastsurvivalprobabilityfromhepatictelomerelengthinO.
latipes.
ThesurvivalprobabilityisgrosslyproportionaltothehepaticTRFlength(R2=0.
893).
'Zero'survivalprobabilityofmaleandfemaleO.
latipesisequivalenttoa'critical'lengthofca.
3.
8kbp.
Crossesareoverlaiderrorbars(95%CI)forsurvivalprobability(vertical)andhepatictelomerelength(horizontal).
Gopalakrishnanetal.
FrontiersinZoology2013,10:78Page7of11http://www.
frontiersinzoology.
com/content/10/1/78uniquenon-mammalianmodeltoadvanceresearchonestrogen-(sex-),telomere-associatedagingandlongevityinvertebrates.
Theinvivocriticallengthofmedakaliverforpredictionoforganismalmortalityandtheagecon-versionmodelformedakaandhumanareusefultoolsforfutureresearchoncomparativebiologyofagingusingmedaka.
Overall,evidencesprovidedhereinhighlightthattheteleostO.
latipesissignificantfordecipheringtheos-tensiblyconservedmechanism(s)ofsex-associatedlonge-vityinvertebrates.
MaterialandmethodsMedakacultureOrange-red,outbredlineofJapanesemedaka(Oryziaslatipes)wasoriginatedfromtheDukeUniversity,Mo-lecularAquaticToxicologyLaboratory.
In2008,fishweretransferredandmaintainedasacolonyintheCityUniversityofHongKong.
Fertilizedeggswerecollecteddailyandreareduntilhatchasdescribedin[67].
Fishthathatchedinthesamemonthweregroupedasonecohort.
Uponsexualmaturation(approx.
3month-old),malesandfemalesfromthesamecohortwererandomlypairedandtransferredtoaquariumtanks(39.
5L*23.
5W*27.
5Hcm)atadensityof15pairspertank.
Thefishwerekeptinthesamesetoftanksthroughouttheirwholelife.
Allfishwerekeptunderstaticconditionof26±1°C,7.
2±0.
2mgO2L-1and14:10hrslight-darkcycle.
Halfofthetankwaterisreplacedwithcharcoal-filteredtapwatereveryday.
ThefishwerefeedtwicedailywithOtohimeβ1(NisshinCo,Japan)andsupplementedfreshlyhatchedbrineshrimp(Artemianauplia)(OceanStarInternationalLuckyBrand,Utah,USA)3daysperweek.
Fishhealthwascloselymonitored.
Unhealthyfish,usuallycharacterizedbylackofappetite,inactivity,lossofgolden-redcolor,hemorrhages,and/orexternalout-growth,werepromptlyisolatedintoindividualglasscon-tainers(diameter:14.
5cm;height:5cm)containing500mlcharcoal-filteredtapwater.
Everyquarantinedfishwasrearedunderidenticalconditionasdescribedaboveandwouldnotreturntooriginatedtankunlesssymptomsdisappeared('recovered')foratleast2weeks.
Recordsofstockingdensityanddailymortalityofmalesandfemalesinindividualtank(henceindividualcohort)werecuratedinSQLite3relationaldatabasemanage-mentsystem(www.
sqlite.
org).
Theserecordswereex-tractedforgeneratingsurvivalprofilesandstatistics.
FishsamplingHealthyfishatfivedifferentagesweresampled:4-('young'),8-('mature'),12-('senior'),15-('old')and22-('veryold')months.
Ateachtimepoint,90fishes(45malesand45females)weredissected.
Fishwereanesthe-tizedinice-coldaquariumwaterfor30s,removedandmeasuredforbodylengthandweight.
ThefishwaskeptsedatedbygentlywrappingwithpapertowelfullysoakedFigure6AnageconversionmodelforO.
latipes(inmonths)andhuman(inyears):Agemedaka=0.
0954*Agehuman+0.
0171*Agehuman2.
Threemajorhallmarks:sexualmaturation,medianlifeexpectancyandup-to-daterecordofmaximumlifespanwereusedforregression.
Redcirclesrepresentdatafromthepresentstudy:sexualmaturation(O.
latipes:ca.
3month-old;human:12-15year-old),medianlifeexpectancy(O.
latipes:♂=13.
7|♀=14.
6months;human:world'saverage♂=65.
7|♀=70.
1years)andup-to-daterecordofmaximumlifespan(O.
latipes:♂=32.
0|♀=39.
5months;human:♂JiroemonKimura=116.
1|♀JeanneCalment=122.
4years).
Accordingtotheregressioncurve(R2=0.
996),theoccurrenceofestrogendeclineinthefemalemedaka(between8to12month-old)inourculturecouldbeinterpolatedtohumanageof46.
0to60.
4year-old,whichisakinto"menopause"inwomen.
Previousagingdatafrom[66](bluecross+)and[54](greentriangle)wereoverlaidforreference.
Gopalakrishnanetal.
FrontiersinZoology2013,10:78Page8of11http://www.
frontiersinzoology.
com/content/10/1/78withice-coldaquariumwater.
Acutwasmadetotailat1–2mmrostraltothecaudalfin.
BloodwasdrawnfromthecutusingP10micropipette(Eppendorf,Hamburg,Germany)attachedwithheparinizedpipettetipsandinstantlydilutedwith8μLdouble-distilledwatertopreventclotting.
Thefishwasthenimmediatelyde-capitated.
Gillandliverwereisolatedonicebedanddividedintotwoequalportionsforparalleltelomeraseactivityandtelomericlengthmeasurements.
Bloodandtissuesamplesfromthreefishofthesamesexwerepooledasonereplicate(i.
e.
finalsamplenum-ber=15persexpertime-point).
Pooledsamplesweresnap-frozeninliquidnitrogenandstoredat-80°Cuntilfurtherprocessing.
AnimalhandlingproceduresasmentionedabovewereacceptedbytheAnimalEthicsCommittee,CityUniversityofHongKong.
TelomerelengthmeasurementbySouthernblotanalysisGenomicDNAwasextractedfromtheliversandgillsofadultmaleandfemalemedakausingtheDNeasyBloodandTissueKit(Qiagen,Hilden,Germany)accordingtothemanufacturer'sinstructions.
Foreachassay,3μggenomicDNAwasdigestedtocompletionwithRsaIandHinfI(NewEnglandBiolabs,Massachusetts,USA)at37°Covernight.
ThedigestedDNAwasresolvedbyelec-trophoresisona1%agarosegel,runinparallelwithaλHindIII/EcoRImolecularmarker(Fermentas,Burlington,Canada)andsubsequentlytransferredtoHybond-XLmembrane(GEHealthcare,LittleChalfont,UnitedKingdom)forovernightthroughcapillarytransfer.
ThemembranewassaturatedwithExpressHybHy-bridizationSolution(Clontech,California,USA)at42°Cfor30mins,andhybridizedwith100pmolofDIG-labeledoligonucleotideprobes(TTAGGG)5(Invitrogen,California,USA)inExpressHybHybridizationSolution(ClontechLaboratories,California,USA).
TheDIG-labelingwasachievedbytheuseofDIGOligonucleo-tideTailingKit,2ndgeneration(RocheAppliedScience,Penzberg,Germany)accordingtothemanufacturer'sin-structions.
Afterhybridization,themembranewaswashedtwotimesin2XSSCbufferwith0.
1%SDSat25°C(5minseach)andtwicefor15minsin0.
1XSSCwith0.
1%SDSat42°C.
Thewashedmembranewasblockedin3%solutionofnon-fatmilkpowder(inphosphatebufferedsaline,PBS;pH7.
4)for30minsatroomtem-perature,thenincubatedwith1:10000anti-DIG,AP-conjugatedantibodies(Roche).
ThemembranewasthenwashedthreetimesinPBSatRTandincubatedwithCDP-star(Roche)indarknessfor5mins.
Chemilumines-cencewasimagedbyluminiscentimageanalyzer(FujifilmLAS4000,Tokyo,Japan)andsavedlosslessasTIFF.
Telo-merelengthwasquantifiedinImageJasterminalrest-rictionfragment(TRF)lengthfollowingtheprocedureof[68].
Telomeraseactivityassaysbythereal-timequantitativetelomericrepeatamplificationprotocol(RTQ-TRAP)Frozentissuessampleswerethawedonicebedandlysedinice-coldCHAPS-containinglysisbuffer(10mMTris–HCl,pH7.
5,1mMMgCl2,1mMEGTApH8.
0,0.
5%CHAPS,10%glycerol,0.
1mMPMSF,5mMβ-mercaptoethanol)(Sigma-Aldrich,Missouri,USA).
Thely-sateswerecentrifugedat16000*g,4°Cfor30mins.
Thesupernatantswerecarefullytransferredtonewsterilizedmicrofugetubes.
ProteinconcentrationwasdeterminedusingtheBradfordproteinassay(Bio-Rad,California,USA),withreferencetostandardcurveconstructedfromserialdilutionsofproteinstandard(bovineserumalbu-min,BSA;Sigma-Aldrich).
Telomeraseactivitiesinliverandgillsamplesofme-dakawereassessedbytheRTQ-TRAPassayfollowingtheoptimizedprotocolby[69].
Briefly,40ngofproteinextractwasaddedtoa25μLreactionmixtureofTRAPbuffer[20mMTris-HCl(pH8.
3),63mMKCl,3.
5mMMgCl2,1mMEGTA(pH8.
0),0.
1mg/mLBSA,0.
005%Tween20],100μMdNTPs,1:25000SYBRGreenIdye,10nMROXreferencedye,1.
25UHotStarTaqpolyme-rase(Qiagen),0.
1μgtelomerasesubstrateprimer(a.
k.
a.
'TS';5′-AATCCGTCGAGCAGAGTT-3′;HPLCgrade;Invitrogen),and0.
065μganchoredreturnprimer(a.
k.
a.
'ACX';5′-GCGCGG(CTTACC)3CTAACC-3′;HPLCgrade;Invitrogen).
Thereactionmixturewasfirstin-cubatedat25°Cfor30minstoallowthetelomeraseintheproteinextracttoelongatetheTSprimerbyaddingTTAGGG-repeats.
Next,theTRAPreactionwashaltedandtheTaqpolymerasewasactivatedbyheatingat95°Cfor15min.
Theactivationwasfol-lowedby40cyclesof95°Cfor30s,60°Cfor30s,and72°Cfor60s.
Meltingcurveanalysiswasauto-maticallycarriedoutaftercompletionofthe40thcycletoverifytheamplificationspecificity.
ThermalcyclingwasconductedusingtheABI7500FastRealTimePCRSystem(AppliedBiosystems,California,USA).
Sampleswererunintriplicate.
TheCqvalueswerede-terminedfromsemi-logamplificationplots(login-creaseinfluorescencesignalagainstcyclenumber).
Therelativetelomeraseactivitywascalculatedbythe2-ΔCqmethod[69].
SexhormoneanalysisBloodsamplesweredilutedwith300μLdouble-distilledwaterandmixedwith2mLofdiethylether(Sigma-Aldrich).
Themixturewasvortexedfor10secondsandcentrifugedat2000*gfor10mins.
Theupper,organicphasewastransferredtoglasscentrifugetubecarefully.
Theextractionprocedurewasrepeated3timestoensurecompleterecoveryofthesexhormones.
Diethyletherwasthenevaporatedundergentlestreamofnitrogengas(HongKongOxygenLtd,HongKong).
ConcentrationsofGopalakrishnanetal.
FrontiersinZoology2013,10:78Page9of11http://www.
frontiersinzoology.
com/content/10/1/78estradiol,testosteronesand11-ketotestosteronewerequantifiedusingcommerciallyavailableenzymeimmuno-assaykit(Cat.
582251,Cat.
582701,Cat.
582751,respect-ively;CaymanChemical,Michigan,USA)followingthemanufacturer'srecommendedprocedures.
PlasmaproteinconcentrationofthewholebloodwasmeasuredbytheuseofBradfordproteinassay(Bio-Rad)andwasusedtonormalizetheconcentrationofsexhormones.
StatisticalanalysesAllstatisticalanalyseswereperformedintheRenviron-ment2.
15.
1.
Survivalprobabilityofmaleandfemaleme-dakaacrossagewasestimatedusingtheKaplan-Meiermethodadjustedforleft-truncation(<3month-old)andright-censoring(up-to-datesurvivorshipdataatthetimeofthiswriting).
Sexdifferenceinlongevitywasindicatedbycontrastingthesurvivalcurvesofthetwosexesusinglogranktest.
Theage-dependentTRFlengthwasmodeledasexponentialdecay:TRFt–Plateau=(TRF0–Plateau)·e–λt,whereTRFtisthemodeledmeanTRFlengthataget,TRF0istheextrapolatedinitialmeanTRFlength,Plateauisthehorizontalasymptote,andλistheratecon-stant.
Modelparameterswerefittedusingself-startingnonlinearleastsquaresasymptoticregressionmodel(R/nlsinconjunctionwithSSasympfunction).
Sex-dependencyofthetelomeraseactivityandtelomericlengthweretestedusingStudent'st-test.
Age-dependentvariationinplasmaconcentrationofthethreesexhor-mones'levelwasillustratedbytheuseofone-wayanalysisofvariance(ANOVA)followedbyTukey'shonestlysignifi-cantdifference(HSD)test.
Therelationshipbetweentelo-merelengthandtelomeraseactivityandthecirculatorylevelsofsexhormoneswereillustratedbySpearmancor-relationanalyses.
Wheneverapplicable,thenormalityandhomoscedascityassumptionsforallparametricinferenceswereverifiedbytheShapiro-WilktestandLevene'stestonmediancenter,respectively.
p-valuesfrommultiplecomparisonswereadjustedtocontrolforfalsediscov-eryrate(FDR)byuseoftheBenjaminiandHochbergprocedure[70].
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
Authors'contributionsExperimentaldesign:DWTA.
Financialsupport:DWTA.
Performedtheexperiments:GS.
Dataanalysisandinterpretation:NKMC,DWTA,GS,andBWPY.
Manuscriptpreparation:GS,NKMCandDWTA.
Allauthorsreadandapprovedthefinalmanuscript.
AcknowledgementsTheworkdescribedinthispaperwassupportedbygrantsfromtheResearchGrantsCounciloftheHongKongSpecialAdministrativeRegion,China(ProjectNo.
9041468;CityU160009),theUniversityGrantsCommittee,AreaofExcellenceGrant(AoE/P-04/04)andtheStateKeyLaboratoryinMarinePollution.
TheauthorsgreatlyappreciatedJunBo,RoyR.
Ye,AndyC.
K.
CheungandHong-LinRenfortheirassistanceonfishsampling,andJosephL.
Humbleforproofreadingthemanuscript.
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