Reproductive Dysfunction in Cultured Sablefish (Anoplopoma fimbria) José M. GUZMÁN ＊1 , J. Adam LUCKENBACH ＊1,2 , Frederick W. GOETZ ＊1 , William T. FAIRGRIEVE ＊1 , Mollie A. MIDDLETON ＊3 , and Penny SWANSON ＊1,2 Abstract: Sablefish Anoplopoma fimbria is a ground fish native to the North Pacific Ocean that is considered a promising new species for marine aquaculture in the US. However, efforts to establish sustainable production of sablefish have been constrained by the reproductive performance of females from the first-filial (F1) generation. Although some F1 females may mature after 5+years of age, some others fail to initiate puberty in captivity. Development of methods to unblock/induce early puberty are necessary to reduce costs associated with rearing F1 female broodstock and reduce generation times for selective breeding. Current research at the Northwest Fisheries Science Center (NOAA, Seattle, USA) integrates basic and applied biology to gain knowledge on the reproductive endocrine system of sablefish and develop approaches to unblock or reduce the age of puberty in F1 female sablefish. As part of our basic line of research, we compared the pituitary gonadotropin-ovary axis in wild-caught, maturing females and 8 year-old F1 females that had never shown signs of sexual maturation. Wild-maturing females had higher levels of pituitary gonadotropin subunit and ovarian gonadotropin receptor mRNAs and plasma sex steroids compared to F1 females, which were holding at the immature, perinucleolus ovarian stage. Anecdotal evidence from sablefish farms indicates that F1 female broodstock maintained in ~4 ℃ seawater mature in captivity. We hypothesize that culture conditions that use warmer water (10-15 ℃) suppress the pituitary gonadotropin-ovary axis in sablefish, and ultimately block the onset of puberty. As part of our applied line of research, we conducted a series of studies to determine the ability of exogenous hormones to stimulate the reproductive axis in prepubertal F1 females. Treatments with testosterone or estradiol 17-beta (E2) increased the expression of pituitary luteinizing hormone beta subunit 40- and 185-fold, respectively, relative to control. This finding suggests that pituitaries from immature females are responsive to exogenous hormones, and that sex steroids may be an important part of a hormone therapy to stimulate the reproductive axis of F1 females. In addition, using an in vitro ovarian tissue culture system, we demonstrated that fragments of prepubertal sablefish ovaries incubated with human-chorionic gonadotropin increased the secretion of E2. This indicates that ovaries of prepubertal females are equipped to synthesize and release sex steroids critical for vitellogenesis under the appropriate hormone stimulation, and that the failure to initiate puberty is likely due to a lack of adequate gonadotropin signaling. These data provide the foundation for the development of hormone treatments aimed at inducing puberty in prepubertal F1 female sablefish. Key words: sablefish, marine aquaculture, reproduction, gonadotropins 2015年 1 月30日受理（Received on January 30, 2015） ＊1 Environmental and Fisheries Sciences Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, Seattle, WA 98112, USA ＊2 Center for Reproductive Biology, Washington State University, Pullman, WA 98164, USA ＊3 School of Aquatic and Fishery Sciences, University of Washington, Seattle, WA Corresponding author: José M. GUZMÁN, E-mail: [email protected]水研センター研報，第40号，111－119，平成27年 Bull. Fish. Res. Agen. No. 40，111－119，2015 111
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Reproductive Dysfunction in Cultured Sablefish (Anoplopoma fimbria)
José M. GUZMÁN, J. Adam LUCKENBACH, Frederick W. GOETZ, William T. FAIRGRIEVE, Mollie A. MIDDLETON, and Penny SWANSON
Infishes,as inothervertebrates, reproduction isprimarilycontrolledby thehypothalamic-pituitary-gonadal axis.The hypothalamic neuroendocrinesystem regulates synthesis and release of thepituitary gonadotropins , fo l l ic le -st imulat inghormone (Fsh)and luteinizinghormone (Lh).Bothgonadotropinsareessentialintheendocrinecontrolof reproductionbyregulatingsteroidogenesisandgermcell development through interactionswiththeirrespectivereceptorsingonadaltissues(Levavi-Sivanet al., 2010;Zoharet al., 2010). Optimal functionof the reproductiveendocrinesystem is critical for fish to complete gonadaldevelopment,maturationand spawnsuccessfully.Undercultureconditions,however,manyfishspeciesexhibit somedegree of reproductivedysfunction.These canvary from inconsistent spawning to acomplete failure toundergopuberty (i.e., the timeduringwhich an individual becomes capable ofreproducingforthefirsttime) (Mylonaset al., 2010;Tarangeret al., 2010).Suchadelayinageofpubertyposes amajorproblem for selectivebreeding infinfishaquacultureandnecessitatesfurtherresearch. Hormone therapies arewidely used to controlreproduction in cultured fish species.During thepast twodecades, hormone therapies havebeendevelopedtoinducepubertyinstripedbass(Morone saxatilis) (Hollandet al., 2002),Europeanseabass(Dicentrarchus labrax) (Zanuyet al., 1999),Europeaneel (Anguilla anguilla) (Vidal et al., 2004), andgreymullet (Mugil cephalus) (Aizen et al., 2005).Therapies for inducingpuberty or to acceleratethis process were based on an understandingof how the endogenous reproductive system ishormonallyregulatedandwhere the insufficiency/failureoccursinthereproductivesystem(e.g.,brain,pituitaryglandand/orgonad)thatdelaysorblockspuberty onset.Therefore, characterization of thereproductivedysfunctioninagivenspecieshelpstotailorspecificstrategiestooptimizeitsreproductionincaptivity. Sablefish(Anoplopoma fimbria),knownasgindarainJapan,isagroundfishnativetotheNorthPacificOcean ranging fromBajaCalifornia toAlaska’sBering Sea and Japan.The primarymarket forsablefish is inJapan,wheredemandandpricesarehigh,butan increasingamountofthewildcatch is
staying in theUSmarket.Dueto itsrapidgrowthrate (1.5kgafter12-monthsofgrowout)andhighmarketvalue (22USD/lb. approx.marketprice),sablefishhasbeen identifiedasanexcellentmarineaquaculturespecies in theUS.However,efforts toestablishsustainableandefficientproductionhavebeenconstrainedbythereproductiveperformanceof females fromthe first filial (F1)generation (i.e.,producedandmaintained in captivity).AlthoughsomeF1 femalesmaymature at 5+years old inthe industrysetting,othersnever initiatepubertyin captivity (B.Campbell, SablefishCanada Inc.,personalcommunication).Thissituationcompromisesthedevelopment of selectivebreedingprogramsand increasescostsassociatedwithrearing femalesablefishbroodstock. Our approach to this issue integrated a seriesof basic and applied studies aimed at gaining abasicunderstandingof thereproductivephysiologyof sablefish anddeveloping amethod thatwouldeliminatetheblockorreducetheageofpubertyinF1femalesablefish.
Materials and methods
Study I: Assessment of the pituitary gonadotropin-ovary axis in wild maturing and F1 non-maturing female sablefish: To understand the endocrinedi f ferences that under l ie the reproduct iveimpairment of female sablef ish propagatedin captivity, we first compared the pituitarygonadotropin-ovaryaxisof twostocksof sablefishwith different reproductive status:wild-caughtmaturingfemalesand8year-oldF1femalesthathadnevershownsignsofsexualmaturation. Wild female sablef ish were caught usingsportfishinggearnear themouthof theQuinaultRiver (Washington,USA) inOctober2010.ThefishweretransportedtotheManchesterResearchStation(PortOrchard,Washington,USA)andmaintainedintankssuppliedwithflow-through,sand-filteredandUV-treated seawater.DuringFebruary2011, thepeakspawningperiodalong theWashingtoncoast(Masonet al., 1983), thewild femalesshowedsignsof sexualmaturationbyultrasound.F1 sablefish(broodyear 2003)were reared at theManchesterResearchStation innet-pensuntil fall 2010when
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theywere transported to theNorthwestFisheriesScienceCenter (NWFSC,Seattle,Washington,USA)where theywererearedonrecirculatedseawater.These females were maintained under similarrearing conditions as thewild-caught fish at theManchesterResearchStation. In contrast to thewildfemales,theF1femalesdidnotexhibitsignsofovarianmaturationwhenassessedbyultrasound. Wild and F1 females were sampled on 28February 2011 and 8March 2011, respectively.Three females fromeachbroodstockgroupweredeeplyanesthetizedwithMS-222andbodyweight(BW)and fork length (FL) recorded (wild, 6413.0 ± 712.9gBWand81.3 ± 3.1cmFL;F1, 4160.0 ± 303.3gBWand69.8 ± 2.7cmFL).Plasmawasobtainedbycentrifugationofwholebloodandstoredat －20 ℃forsexsteroidanalyses.Thepituitaryglandandasmallpieceofovary(~80mg)werealsocollected fromeach fish, frozen in liquidnitrogenandstoredat－80 ℃untilRNAextraction.Genesofinterestincludedthepituitarygonadotropinsubunits(fshb, lhb, cga)andovariangonadotropinreceptors(fshrand lhcgr).Forhistology,amiddleportionoftheovarywaspreservedinBouin’sfixativefor48hprior to storage in70%ethanolandprocessingasdescribedelsewhere(Campbellet al., 2006).Study II: Effect of sex steroids on pituitary gonadotropin gene expression in vivo:Todeterminepotential effects of sex steroids on pituitarygonadotropinsandwhethersexsteroidswillbeanimportantpartofhormonetherapiestoreducetheageofpuberty insablefish,weevaluatedtheeffectof testosterone (T) and estradiol-17beta (E2) onpituitarygonadotropinbetasubunit (fshband lhb)mRNAlevelsinprepubertalF1femalesin vivo. Twelve2-yearoldprepubertalF1femalesablefish(1187.4 ± 49.4 gBW and 469.9 ± 5.3 cmFL)maintained at theNWFSCon 12 ℃recirculatedseawaterweredistributed into threegroups (n=4sablefish/group) and implantedwith cholesterol-basedpellets (0.2x0.5mm)containingnohormone(control)orcontainingTatdosesof0.75or3.75mg.Twenty-eight days after implantation, fishwereeuthanizedandtheirpituitariesremovedandsnapfrozen for laterRNA isolation. Inaparallel study, 6 females fromthesamecohortof fishreceived6intramuscular injections of 2mgE2/kgdissolved
inethanol: 0.9%NaCl(1:7)orvehiclealone(control),everyotherday.Twodaysafter the last injection,fish were euthanized and pituitaries removedto determine gonadotropin beta subunit geneexpression.DosesofTandE2wereselected frompreviousstudiesinotherfishspecies(Guzmánet al., 2008;Hollandet al., 2002;Vidalet al., 2004)Study III: Effect of gonadotropins on ovarian secretion of estradiol in vitro : To determinewhether immatureovaries ofF1 female sablefishare responsive to gonadotropin stimulation,we evaluated the effect of two gonadotropinpreparations,recombinantcohosalmonFshanalog(sFsha)andhumanchorionicgonadotropin(hCG),onE2productionin vitro. For this, a 3-year old prepubertal F1 femalesablefish(2910gBW, 67.6cmFL)maintainedattheNWFSCwaseuthanizedand theovariesremoved.Ovarian fragments (~60mgeach)were culturedinCortland’s solutionalone (control) orcontainingsFsha (providedbyDr.W.R.Moyle,RobertWoodJohnsonMedicalSchool,NewJersey,USA)atdosesof50and500ng/mlorhCG (Sigma)atdosesof1, 10and100 IU/ml.After24h incubationat11 ℃,theculturemediumwascollectedandstoredat-20 ℃ forE2analysis.Dosesof sFshaandhCGwereselectedbasedonpreviousstudies inotherspecies(Luckenbachet al., 2011;Sorberaet al., 2001).RNA isolation, cDNA synthesis and quantitative PCR: Total RNA from sablefish pituitaries andovarian tissue was isolated with Tri-Reagent(MolecularResearchCenter)usingaTissueLyserII (Qiagen).Analiquotof totalRNAwasdilutedto~250ngRNA/µl innuclease-freewaterandDNasetreatedusingtheDNAFreekit’s‘‘rigorous’’protocol(Ambion).RNAyields andqualitywere assessedbyNanoDrop (ND-1000Spectrophotometer)andgelelectrophoresis.Forreverse transcription, 1µgoftotalRNAofeachsamplewasreverse transcribedin a 20-µl reaction with the Superscript II kit(Invitrogen).QuantitativePCRswere conductedaspreviouslydescribed (Luckenbachet al., 2011).Reactionsconsistedof1xPowerSYBRGreenPCRmastermix (AppliedBiosystems), 150nMofgene-specificprimers(seeGuzmánetal., 2013forprimersequences)and0.5ngofpituitaryorovariancDNAtemplate.AssayswererunonanABI7700Sequence
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Detectorusingstandardcyclingconditions.Sex steroids:PlasmalevelsofE2weremeasuredbyradioimmunoassayand levelsofTwerequantifiedby enzyme-linked immunosorbent assay usingprotocolspreviouslyvalidatedforsablefish(Guzmánet al., 2013).Statistical analyses : Statistical analyses wereperformedusingPrism5 software forMacOSX(GraphPadSoftware)with theminimum level ofsignificancesettoP<0.05.Differenceswereexaminedusing t-test or one-wayANOVA followed by aTukeymultiplecomparisons test.Whennecessary,datawerelogorLntransformedinordertocomply
Study I: Assessment of the pituitary gonadotropin-ovary axis in wild maturing and F1 non-maturing female sablefish Endocrineparameters indicated amore activereproductiveaxisinwildversusF1femalesablefish(Fig. 1).At thepituitary level, lhbandcga mRNA
Fig. 1. Relative gene expression of pituitary gonadotropin subunits (A)andovariangonadotropin receptors (B), plasma sex steroid levels (C), andrepresentativeovarianhistologicalsections(D)ofwildmaturingandF1femalesablefish.Geneexpression levelsweredeterminedbyqPCRandnormalizedto eef1a.Levels ofE2weredeterminedbyRIA,whereas levels ofTweredeterminedbyELISA.Dataareexpressedasthemean±SEM(n=3).Asterisksindicatesignificantdifferences(t-test,p<0.05).Maturingfollicleswiththenucleusmigrating to theperipherypredominated in the ovaries ofwildmaturingfemales,whereasonlyfolliclesattheperinucleolusstagewerefoundinovariesofF1females(D).Scalebars: 100μm.ND,nondetectable.
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levelsweresignificantlyhigher inwildfishthan inF1 fish.Levels of fshbwerealsoelevated inwildfishrelativetoF1fish(7.1-fold),althoughdifferenceswerenot statistically significantdue to thehighvarianceacrossindividuals.Intheovary,transcriptsfor both gonadotropin receptors, fshr and lhcgr,weresignificantlyhigherinwildthaninF1females.PlasmalevelsofE2werealsosignificantlyhigherinwildthan inF1females,whereasonlywildfemaleshaddetectablelevelsofTinplasma. The ovary ofwild females consisted of post-vitellogenicpreovulatory folliclescharacterizedbythenucleusmigrating to theperiphery andyolkcoalescence,aswellasanotherclutchof folliclesat
Study II: Effect of sex steroids on pituitary gonadotropin gene expression in vivo Theeffectof treatmentwithTorE2on in vivopituitarygonadotropinbetasubunit (fshband lhb)mRNAlevelsisshowninFig.2.Transcriptsforfshbwerenot significantly affectedby treatmentwithTorE2. Incontrast, transcripts for lhb increasedsignificantly in fish treatedwith thehighestdoseofTormultiple injectionsofE2 (40-and185-fold,respectively,relativetocontrol).
Fig. 2.Relativegeneexpressionofpituitaryfollicle-stimulatinghormone beta subunit (A) and luteinizing hormone betasubunit (B) inprepubertalF1 femalesablefishtreatedwithT(leftgraphs)orE2 (rightgraphs).ForTtreatments, femalesreceiveda single implant containinghormoneatdosesof 0 (control), 0.75 and3.75mg for 28days.ForE2 treatments,femalesreceived6intramuscularinjectionsof2mghormone/kgdissolvedinethanol: 0.9%NaCl(1:7)orvehiclealone(control,CNT),everyotherday.GeneexpressionlevelsweredeterminedbyqPCRandnormalizedtoeef1a.ForTtreatments,barswithdifferentsuperscriptlettersaresignificantlydifferent(ANOVA,p<0.05).FortheE2treatment,anasteriskindicatesasignificantdifferencerelativetocontrol(t-test,p<0.05).
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Study III: Effect of gonadotropins on ovarian secretion of estradiol in vitro TheeffectofsFshaorhCGonovariansecretionofE2in vitroisshowninFig. 3.IncubationwithsFshahadasubtleeffectonthesecretionofE2withonlythehighestdoseofsFshahavingasignificanteffectcompared tocontrol. Incontrast, treatment withhCGregardlessofdosesignificantlystimulatedE2secretion(>4-fold).
Discussion
Currently, theemergingaquaculture industry forsablefish ismostlydependent on spawningwild-caught individuals.Theexpansionandoptimizationof this industry requires a closer understandingof thereproductivephysiologyof this speciesandthedevelopmentofprotocols tocontrol theageofpubertyinculturedbroodstock. In Study I,we demonstrated that transcriptlevels of pituitary lhb and ovarian lhcgr werehigherinwild-caughtmaturingfemalesthaninnon-maturingF1females.ThisisinaccordancewiththepredominantroleofLhintheregulationofgonadalmaturation,ovulationandspawninginfishes(Gothilfet al., 1997;Swansonet al., 2003;Yaronet al., 2003).
Interestingly,ovarian fshrmRNA levelswerealsohigher inmaturing femalesandasimilar tendencywasobserved for thepituitary fshbmRNA levels.Little isknownabout specific rolesofFshduringgametogenesisinfishes,althoughitisacceptedthatFshisimportantforearlygonadaldevelopmentandvitellogenesis (Lubzenset al., 2010;Luckenbachet al., 2011).Theelevated levelsofpituitary fshbandovarian fshrthatweobserved inwild femalesmayindicate thatapopulationofperinucleolaroocyteswithintheovarywerepreparingforthetransitiontovitellogenicgrowth,orthatFshalsoplaysarole infinaloocytematuration.Sincetheovaryofthewildmaturefemaleshadamixedpopulationofoocytesitisnotpossible todistinguishthesetwopossibilities.Plasma levels of E2 andTwere also higher inwild females thanF1 females as expected fromtheobserveddifferences inpituitarygonadotropinsubunit andovarian receptormRNA levels.Thisdemonstrates classicalgonadotropin-mediated sexsteroidsecretion(Lubzenset al., 2010)bytheovariesofwildfemalesandindicatesalackofgonadotropinsignalinginF1females. AlthoughF1 females fromStudyIwere8yearsold, theyremainedarrested inaprepubertalstage.Interestingly, it hasbeen recently demonstratedthatsomeF1femalescan initiatevitellogenesis (i.e.,onset of puberty) if they aremaintained in coldwater (~4 ℃) after 5years of age (B.Campbell,personal communication). It isgenerally acceptedthat every fish species has an optimal range ofwater temperatures forvitellogeninsynthesis,andboth higher and lower temperatures affect thenormal progression of vitellogenesis (Guzmán et al., 2008;KimandTakemura, 2003).Consideringthatsablefishisadeep-waterspeciesandtheonsetofvitellogenesismaynaturallyoccuratvery lowtemperatures, it ispossiblethatthetemperatureatwhich fishweremaintained (fluctuatedbetween8and15 ℃throughout theyear)hadadeleteriouseffectonreproductiveaxis function,andultimatelyontheonsetofvitellogenesisinF1females. Although someF1 femalesmaintained in coldwater can achieve puberty after 5+ years, thedevelopmentofmethodstoreduceandsynchronizetheageofpuberty isessential forreliableandcosteffective sablefish breeding programs.Hormone
treatments are widely used in aquaculture toimprove the reproductiveperformanceof captivefishes, including activation of the reproductiveendocrine system and promotion of gonadaldevelopmentinimmaturestages(Hollandet al., 2002;Zanuyet al., 1999).WeconductedStudiesIIandIIItodeterminetheabilityofexogenoushormonestostimulate thereproductiveaxis inprepubertalF1sablefish. In Study IIwe demonstrated that treatmentwithT orE2 significantly elevated transcriptsforpituitary lhb,butdidnot affect levels of fshbmRNA.Effects of sex steroids on levels of thepituitarygonadotropinsubunitmRNAshavebeendemonstratedinanumberoffishes,althoughresultsvaryamongspecies.Forexample, treatmentwithTelevatedpituitarytranscriptsof lhb inEuropeanseabassduringtheperiodof“sexualresting,”whileT andE2 reduced pituitary fshbmRNA levels(Mateos et al., 2002). In immatureEuropean eel(Anguilla anguilla), treatmentswithE2butnotTelevatedtranscripts for lhb,whereasnoeffectwasobservedon fshbmRNAlevels (Arouaet al., 2007).Lastly,treatmentswithTandE2increasedpituitarytranscriptlevelsforfshbandlhbinzebrafish(Danio rerio) (LinandGe, 2009).Ourresultssuggest thatpituitaries fromprepubertal female sablefish areresponsive to exogenoushormones, and that sexsteroidsmaybean importantpart of ahormonetherapy to stimulate the reproductive axis ofF1females. In linewith this, treatmentswith sexsteroids were necessary to induce puberty inEuropeanseabass(Zanuyet al., 1999),stripedbass(Hollandet al., 2002)andEuropeaneel (Vidalet al., 2004). InStudy III,wedemonstrated that treatmentwith hCG, an Lh-like gonadotropin, stimulatesovarian secretionofE2 in vitro.Treatmentswithgonadotropinpreparations,which actdirectly onthegonad,haveproveneffective inthestimulationof gonadalmaturation, ovulation and spawningin anumber of fish species (Zohar andMylonas, 2001).Most importantly, inspecies fromthegenusAnguilla, captive fish often remain arrested in aprepubertal stage in captivity.However,multipleinjectionswithpituitaryextractscantriggerovarianvitellogenesis,maturationandovulation in females
(OhtaandTanaka, 1997),whilemultiple injectionsof hCG can induce testicular development andspermiation inmales (Dou et al., 2007).Ourdatasuggest that,as inanguillideels, immatureovariesofF1femalesablefishareequippedwiththeabilityto synthesize and release sex steroids (criticalfor vitellogenesis) under appropriate hormonestimulation,and that treatmentwithgonadotropinpreparationsmay effectively stimulate ovarianfunctioninthisspecies. Inconclusion,wild-maturingfemaleshadelevatedgonadotropinsignaling (pituitaryandovariangeneexpressionandplasmasteroid levels)compared to8year-oldF1 females,whichwereholdingat theimmature,perinucleolusovarian stage.AnecdotalevidenceindicatesthatF1femalesmaintainedin~4 ℃seawatermaymatureincaptivityafter5yearsofage.Itmightbepossiblethatcultureconditionsthatusewarmerwater(10-15 ℃)suppressthepituitarygonadotropin-ovaryaxis insablefish,andultimatelyblock the onset of puberty.Our results indicatethat hormone treatments, such as sex steroidsandgonadotropinpreparations, can stimulate thereproductiveendocrineaxis inprepubertal femalesandthereforeshouldbeconsideredascandidatestoreducetheageofpubertyinthisspecies.
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