induced spawning in the sea cucumber apostichopus ......induced spawning in the sea cucumber...

Induced Spawning in the Sea Cucumber Apostichopus japonicus by Neuropeptide, Cubifrin

KeisukeYAMANO＊1,AtushiFUJIWARA＊2,MichiyasuYOSHIKUNI＊3

Abstract:Inhatcheriestheinductionofspawninginseacucumbershasbeentypicallycarriedoutbyregulationofrearingconditionssuchastemperatureandlightintensity.However,thismethodisrelativelyineffectiveandtherateofspawningisunpredictable.Inthisstudyweestablishedanefficientmethodfor inducingspawning intheJapaneseseacucumberApostichopus japonicusbyinjectinganeuropeptide,cubifrin.　Wepurifiedpeptides thatcan induceoocytematuration fromthebuccal tissuescontainingthenerve ring, using liquid chromatography and in vitro assaywith ovarian fragments incombination.Theeffectivedose of eachpeptidewas evaluatedwith chemically synthesizedpeptides.Consequently, themostpotentpeptidewas identifiedasNGIWY-amide.Wealso foundthatsyntheticderivativesreplacedthethirdaminoacid, isoleucine,withadifferentbasicaminoacidcouldbe10-100timesmorepotentthanthenaturalhormone.　When injected into thebodycavityof sexuallymatured individuals,NGIWY-amide, or itsderivative,inducedspawninginbothmalesandfemales.NGIWY-amidewasthennamedcubifrinaftertheJapaneseword“kubifuri”meaningwavinghead,whichisareproductivebehaviorofseacucumbers.Gametesheddingstartedabout60minand80minafter the injection inmalesandfemales,respectively,andwascompletedalmostsimultaneously inthetwosexesabout2hoursafter theadministration.The in vitro responsivenessofbiopsiedovarian fragmentswaswellcorrelatedwiththespawningsuccessinducedbyaninjection.Therefore,cubifrincanbeusedasanexcellentdetectorofmaturityaswellasaninducerofspawninginA. japonicus inahatcherysetting.

Key words:reproduction,hormone,aquaculture,echinoderm

　TheJapanesecommonseacucumberApostichopus japonicus is the most commercially-importantholothurianspecies inJapan.Untilrecentyearstheannualcatchhadbeenabout5,000-10,000tons (wetweight) but fishingpressurehasbeen increasingmainlybecauseofagrowingdemandforexport toChina.Therefore, thesustainableproductionof theseacucumberiscrucial.Forover30years,culturedjuveniles of this specieshavebeen released intofishinggrounds tosupplementnatural stocks.Thecurrentsituationconcerningseacucumberfisheriesmakesthisactivitymoreimportantthanever.

　Inhatcheries, the inductionof spawning in seacucumbers is typically carried outby regulationof rearingconditions suchas temperature,waterexchange,andlightintensity(Battagleneet al., 2002;Sui, 2004;WangandCheng, 2004;Liuet al., 2004;Shaoet al., 2006).InJapan,wild-caughtA. japonicusbroodstock are induced to spawn in thedark intanksof seawaterat temperatures thatare~ 5℃higher thannatural seawater (Ito, 1995).However,thesemethods are relatively ineffective and therateofspawning isunpredictable.Therefore,moreeffectivemethodof spawning inductionhasbeen

2015年 1 月30日受理（ReceivedonJanuary30, 2015）＊1 NationalResearchInstituteofAquaculture,FisheriesResearchAgency,Minamiise,Watarai,Mie516-0193,Japan E-mail:yamano@fra.affrc.go.jp＊2 NationalResearchInstituteofFisheriesScience,FisheriesResearchAgency,Fukuura,Kanazawa,Yokohama,Kanagawa236-8648,Japan＊3 FisheryResearchLaboratory,KyushuUniversity,Fukutsu,Fukuoka811-3304,Japan

水研センター研報，第40号，121－128，平成27年Bull.Fish.Res.Agen.No. 40，121－128，2015

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desired.　Starfisheshave longbeenusedasmodelanimalsfor the study of reproductive biology after thefinding of spawning-inducing activity in a nerveextract in themiddle of the 19th century (ChaetandMcConnaughy, 1959).At long last, thenervoussubstancewaspurifiedand identifiedasarelaxin-likeprotein (Mitaet al., 2009).Sincebothstarfishesandseacucumbersbelong to theEchinodermata,theirreproductivemechanismmightbedrivenbysimilar endogenous factors.Thus,we started thestudytodiscoveraspawning-inducingsubstanceinA. japonicus,followingthestrategyemployedinthestudyinstarfish.　Here,we report a nativeneuropeptidepotentin inducingoocytematurationandspawning inA. japonicus, andpropose a procedure of using thepeptideinahatcherysetting.

Materials and Methods

Preparation of neural extract for purification of a spawning-inducing substance: Buccal tissuescontaining the ring nervewere homogenized inthe same volume of ice-chilled 4N acetic acidcontaining 0.4mM ㌼-aminoethyl benzensulfonylfluoride, 10µg/ml leupeptin and4µMpepstatinA.Thehomogenateswere centrifuged at 15,200xg for15min.at4℃.Theresultingsupernatantswere thenultracentrifugedat45,000xg for1hrat 4℃.These supernatants ofultracentrifugationwerekeptasacrudeneuralextract inaliquotsat-80℃.ThecrudeextractwasseparatedbyserialultrafiltrationwithAmiconUltraYM30andYM10 (Millipore).Thegonadotropicactivitydeterminedbythein vitromaturationassaywasmainlyrecoveredinthefractionoflessthan10Kdaltons.AfterYM10filtration, thefiltratewas lyophilizedandpreservedforasubsequentpurificationprocess.Liquid chromatography:The lyophilizedpreparationwasdissolved inMilliQwater.Aftercentrifugation,the supernatantwas applied to aDevelosil C8-UG-5 column (20 x 50mm) and elutedwith 80 % acetonitrile containing 0.1 % trifluoro aceticacid (TFA) for desalting.The desalted effluentwas lyophilized. The lyophilized extract wasfractionatedby reversed-phasehighperformance

l iqu id chromatography (RP-HPLC) us ing aDevelosil RPAQUEOUS-AR-5 column (10 x 250mm)with a linear gradient of acetonitrile from10 to 40 %with 0.1 %TFA.Each fractionwaslyophilized, thendissolved in 0.5mlMilliQwatertodetect thegonadotropicactivityby the in vitromaturation assay.The fractions containing thegonadotropicactivitywereseparatedbyaDevelosilRPAQUEOUS-AR-5 column (10 x 250mm)witha lineargradient of acetonitrile from10 to 30 %containing20mMammoniumacetate (pH6.0).Thefractionswerelyophilizedandassayedasmentionedabove.The active fractionswere separatedbyaDevelosilRPAQUEOUS-AR3column (2x250mm)with a linear gradient of acetonitrile from15 to22 %with0.1 %TFA.Thegonadotropicactivitywas detected in fractions 30 and 33 of the lastchromatographystep.De t e rm i n a t i o n o f c h em i c a l s t r u c t u r e o f gonadotrophic substances:Thefractions30and33wereanalyzedforaccuratemassvaluesandaminoacid sequencesby liquid chromatograph-tandemmass spectrometers (Quattro Premier,Waters;nanoFrontierLD,HitachiHigh-Technologies)andaproteinsequencer(491cLC,AppliedBiosystems).Preparation of peptides:NGIWY-amide (cubifrin)and itsderivativeswerechemicallysynthesized.Astock solutionof 1mMdissolved inMilliQwaterwasstoredat-80℃.Priortouse,itwasdilutedwithfilteredseawatertothedesiredconcentrations.In vitro maturation assay:Aportionofovarywasexcised throughashort incision in thebodywall.The excised ovarian tissue was cut into smallfragmentsabout3mmlong.Forthepurificationofamaturation-inducingsubstance,ovarian fragmentswere incubatedwith each fraction for 1.5 hr at20℃.Thegonadotropicactivitywasdeterminedbygerminal vesiclebreakdown (GVBD) and alsobythe incidenceofovulationofoocytes fromovarianfragments.　For the detection of the responsiveness topeptides,ovarian fragmentswere incubatedeitherwith thepeptidesolutionat100, 10, 1nM,or100pM in filteredseawaterorwith filteredseawateralone.Thetestwasduplicated foreach individual.The incidenceofGVBDwerescoredonascaleof0–3 inwhich3 indicatesGVBD inover two-thirds

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ofoocytes in the fragment; 2, inoverone-thirdofoocytes; 1,inuptoone-thirdofoocytes; 0.5,inafewpercentageof oocytes; 0,no responseof oocytes.Thesumofscoresofall treatedovarian fragmentsfromoneindividual(8fragmentsforeachindividual)wasregardedastheoocytematurationscoreofthatindividual.After theassay, oocytes in thecontrolovarian fragmentsweremechanically separatedbygentlepipettingand thediametersof 20well-developedoocytesweremeasured.Injection of cubifrin and observation of reproductive behavior : Cubifrin solution (10 µM) or filteredseawater was injected into the body cavity oftheseacucumbers; the injectionvolumewas0.1%ofbodyweight(v/w).Eachseacucumberwasthenseparatelyplacedonthebottomofa21-l tank,anditsbehaviorwascontinuouslymonitoreduntil thecompletionofspawning,or for2hr ifnospawningoccurred.We recorded the times atwhich theundersideof thewatersurfacewasreached,whenheadwavingcommenced,andwhenspawningwasstarted and completed.After the completion ofspawning, thenumberof spawnedeggs in 10mlof tankwaterwas counted to estimate the totalnumberofspawnedeggsforeachfemale.

Results and Discussion

Purification of an oocyte maturation-inducing substance : The neural extract was separatedby three serial HPLCs (Fig. 1). On the thirdchromatography, the gonadotropic activitywasseparated intotwo independent fractions,Fractions30and33 (arrowheads inFig. 1c).Both fractionswereanalyzedbymassspectrometryandproteinsequencing.Theaminoacid sequenceofFraction30wasNGIWY, andFraction 33’s sequencewasQGLFSGV.While thecalculatedmonoisotopicmassvaluesofthesesequenceswere651.320and706.365,analyses bymass spectrometry indicatedmasssignals of 650.35and705.42, respectively.Furtherde novosequencinganalysesof thesemasssignalssuggested that thesehad the samesequencesasthose given through protein sequencing exceptfor anamidation at each C-terminal end of thepeptides.With theseanalyseswedetermined thatthe sequences of the components in Fractions

30 and 33 were NGIWY-amide and QGLFSGV-amide, respectively.Unexpectedly, both of thesepeptidescompletelydiffer fromarelaxin,which isanoocytematuration-inducingsubstanceinstarfish.NGIWY-amide has been reported as a peptideassociatedwith contractions ofmuscle, intestineand tentacles inA. japonicus (Inoue et al., 1999),

Fig. 1. Separation of the gonadotropic activitybyRP-HPLC.Dotted lines in the chromatogramsindicate changes of programmed concentrationsof acetonitrile in the eluents.Hormonal activityin each fraction is shownwith the help of barshangingfromthetopsideofthechromatograms(a,b).Arrowheads in (c) indicate fractions30and33containingthebioactivity.ReproducedfromKatoet al. (2009)withpermission.

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whereasQGLFSGV-amide has not been reportedever.In vitro maturation-inducing activity of peptides:Twopeptides,NGIWY-amideandQGLFSGV-amide,werechemicallysynthesizedandGVBD-inducingactivitywas estimated with in vitro maturation assay.NGIWY-amidewasextremelypotentevenat1nMor less (Fig. 2).QGLFSGV-amidewaslesspotent ininducingGVBD,witheffectiveconcentrationsbeing1µMormore.Thus,NGIWY-amide is stronglysuspectedtobeinvolvedintheregulationofoocytematuration. Incontrast,QGLFSGV-amidedoesnotappear tobe theprimaryendocrine regulator ofoocytematuration.　Theactivities of the twoderivatives,NGLWY-amideandNGIWY-COOH,werecomparedwiththatofthenaturalNGIWY-amide(Fig. 3).NGLWY-amidewaseffectiveat10pMorless,ahundredtimesmorepotent than the natural peptide,NGIWY-amide.However,NGIWY-COOHbarely inducedGVBDat100nM.Accordingly,C-terminal amidationof the

peptideisindispensableforthebioactivity.Effect of NGIWY-amide and NGLWY-amide in inducing spawning:NGIWY-amide andNGLWY-amidewere injected into sexuallymaturedmalesand females to examine the effect on inducingspawning behavior. Injections ofNGIWY-amideinducedspawning inmalesand femalesat10nM.NGLWY-amidemoreeffectively inducedspawninginmalesat1nMandinfemalesat100pM.NGLWY-amidewasthereforeatleasttentimesmorepotentthan NGIWY-amide, as in the case of in vitro experiments.Seacucumbers injectedwithNGIWY-amide orNGLWY-amide exhibited reproductivebehaviors that were independent of sex andtypically included: (1) climbingup the sidewallof the tank to theundersideof thewatersurface; (2) throwing back and swaying of the head; (3)sheddinggametes from thegonopore in theheadregion.Gamete release occurred 45-60min after

Fig. 2.ConcentrationdependenceofNGIWY-amideandQGLFSGV-amide to induceGVBD inovarianfragments.Ovarianfragmentswere incubatedwithNGIWY-amide (whitebars) andQGLFSGV-amide(graybars)atvariousconcentrations.Barsrepresentpercentages ofGVBD (means±SDof duplicatedeterminations of four separate experiments).Barswith different labels differ significantly (P< 0.05＊, P < 0.01＊＊). The grouping symbol atthe top side of the graph indicates a significantdifferencebetweenNGIWY-amideandQGLFSGV-amideateachconcentration.SignificantdifferencesweredeterminedusingWilcoxon’s ranksumtest.ReproducedfromKatoet al. (2009)withpermission.

Fig. 3.ConcentrationdependenceofNGIWY-amideand its derivatives to induceGVBD in ovarianfragments. Ovarian fragments were incubatedwithsyntheticanaloguesatvariousconcentrations.NGIWY-amide (white bars ) , NGLWY-amide(graybars) andNGIWY-COOH (blackbars)wereexamined.Bars represent percentages ofGVBD(means±SD of triplicate determinations of sixdifferent experiments), and barswith differentlabelsdiffersignificantlyineachpeptide(P< 0.05＊,P< 0.01＊＊).Thegrouping symbol at top sideofthegraph indicatesasignificantdifferenceamongtwo peptides at each concentration. SignificantdifferencesbetweenmeansweredeterminedusingSteel-Dwass’spair-wise comparisons.ReproducedfromKatoet al. (2009)withpermission.

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thepeptide injections inmales and70-90min infemales, andwascompletedalmostsimultaneouslyinthetwosexesabout2hraftertheadministration(Fig. 4).This series of actions from climbing tospawningobservedintheexperimentseemedtobea typicalbehaviorof spawningbyseacucumbers(McEuen, 1988;Battaglene, 2002).NGIWY-amideandNGLWY-amidewerenamedcubifrinandcubifrin-L,respectively, after the Japaneseword “kubifuri”meaningwaving head,which is a reproductivebehaviorofseacucumbers.A s s o c i a t i o n b e t w e e n o o c y t e s i z e a n d responsiveness to cubifrin: The competence ofovarian fragments to undergo in vitro oocytematuration and the induction of spawning bycubifrin-L stimulationwere examined in relationtooocytesize.Allovarian fragmentswithoocytes>155µm indiameter (animalnos. 17–25 inFig. 5)responded to cubifrin-L in vitro, as indicatedbytheiroocytematurationscore,whichwasameasureoftheirabilityforovulationandGVBD.Incontrast,all fragments but one (animal no. 11 inFig. 5)

with oocytes

Keisuke YAMANO, Atushi FUJIWARA, Michiyasu YOSHIKUNI

Fig. 5.Associationbetweenoocytesizeandresponsiveness tocubifrin.ResponsivenesstoNGLWY-amidewasdeterminedbyspawningsuccess,asindicatedbythenumberofspawnedeggsper100gbodyweight(a)andbyin vitromaturationassay,evaluatedfromtheoocytematurationscore (b).SeetheMaterialsandmethodsforadescriptionoftheoocytematurationscore.Oocytesizes(meandiameter±SD,n= 20)ofeachfemaleareshownin(c).

effectiveandsimplemethodforpracticalapplicationduring the artificial culture ofA. japonicus.Wepropose theprocedure of spawning induction asfollows:(1)preparationofseacucumbersduringthespawningseason; (2)sexdeterminationandgrossevaluationofmaturityfromtheappearanceofbiopsiedgonad; (3)evaluationofdefinitematuritywithbiopsiedovarybyin vitrotest; (4)Rearingmatureindividualsbelow15℃ to suppressunanticipatednatural spawninguntil spawning induction; (5)spawning inductionby the injectionof10µMcubifrinsolution (0.1%ofbodyweight (v/w)); (6) standard fertilizationandcultivationoffertilizedeggs.

Acknowledgements

　Thisworkwas supportedbygrants from theProgramforPromotionofBasicResearchActivitiesforInnovativeBiosciences(PROBRAIN).

References

BattagleneS.,SeymourJ.,RomafafiaC.,andLaneI., 2002:Spawning inductionof three tropical seacucumbers,Holothuriascabra,H. fuscogilvaandActinopyga mauritiana.Aquaculture, 207, 29-47.

Chaet A. B. , and McConnaughy R. A. , 1959:Physiologic activity of nerve extracts.Biol. Bull., 117, 407-408.

InoueM.,BirenheideR.,KoizumiO.,KobayakawaY., Muneoka Y., and Motokawa T., 1999:

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Localizationof theneuropeptideNGIWY-amidein the holothurians nervous system and itseffectsonmuscularcontraction.Proc. R. Soc. Lond. B, 266, 993-1000.

ItoS., 1995:Studyonthetechnologicaldevelopmentof the mass production for sea cucumberjuvenile,Stichopus japonicus (inJapanese).SagaPrefecturalSeaFarmingCenter.

KatoS.,TsurumaruS.,TagaM.,YamaneT.,ShibataY., Ohno K., Fujiwara A., Yamano K., andYoshikuniM., 2009:Neuronalpeptides induceoocytematurationandgametespawningofseacucumber,Apostichopus japonicus. Dev. Biol., 326, 169-176.

LiuX.,ZhuG.,ZhaoQ.,WangL.,andGuB., 2004:Studies on hatchery techniques of the seacucumber,Apostichopus japonicus. In:LovatelliA et al (eds) Advance in sea cucumberaquaculture andmanagement, vol463. FAOfisheriestechnicalpaper,Rome,pp287–295.

McEuenF., 1988:Spawningbehaviorsofnortheastp a c i f i c s e a cucumber s (Ho l o thu r i d ae :Echinodermata).Mar. Biol., 98, 565–585.

MitaM.,YoshikuniM.,OhnoK., ShibataY.,PaulPrasanthB., Pitchayawasin S., IsobeM., andNagahama Y., 2009: A relaxin-like peptidepurified from radial nerves induces oocytematuration and ovulation in the starfish,Asterina pectinifera. PNAS, 106, 9507-9512.

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Annotated Bibliography

KatoS.,TsurumaruS.,TagaM.,YamaneT.,ShibataY., Ohno K., Fujiwara A., Yamano K., andYoshikuniM., 2009:Neuronalpeptides induceoocytematurationandgametespawningofseacucumber,Apostichopus japonicus.Dev. Biol., 326, 169-176.

　Extracts prepared from tissues containingbuccal ring nerve or longitudinal radial nerveof sea cucumber induce oocytematuration andovulation from ovarian tissues.Wepurified twosmallpeptides,apentapeptideandaheptapeptide,from thebuccal tissues of Japanese common seacucumber,Apostichopus japonicus.Bothpeptidesinducedoocytematurationandgamete spawning.Thepentapeptidewas identifiedasNGIWY-amide.This peptide induced in vitro germinal vesiclebreakdownandovulationoffully-grownoocytesatless than1pMand in vivospawningat10nM.Asyntheticderivativeof thepentapeptide,NGLWY-amide,was10–100 timesmorepotentcomparedtothenaturalNGIWY-amide.Theheptapeptidewaslesspotent, inducingovulationat 1µM.NGIWY-amideandNGLWY-amide inducedacharacteristicspawning behaviorwhen injected into sexuallymatured individuals. Mature eggs artificiallyspawnedwere fertilized, anddevelopednormallyandmetamorphosed into young sea cucumbers.ThedetailsoftheproductionandthemechanismofactionofNGIWY-amidearestillunclear,butthehighbiopotencyof thepeptidewillaidunderstandingoftheneuronalandhormonalcontrolofreproductionofseacucumber.

FujiwaraA.,YamanoK.,OhnoK., andYoshikuniM., 2010:Spawning inducedbycubifrin in theJapanesecommonseacucumberApostichopus japonicus. Fish. Sci., 76, 795–801.

　Theneuropeptide cubifrin-I and its derivativecubifrin-L have recently been demonstrated aspotent substances that induce oocytematurationin vitro and spawn in the Japanese commonsea cucumberApostichopus japonicus.Here, thereproductive behavior provoked by injection ofcubifrin-L intothebodycavityofA. japonicuswasexaminedwithaviewto thepracticalapplication

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of the peptide for induction of spawning in thehatchery.Ovarian fragmentswithoocytes largerthan155µm indiameter responded to cubifrin-Lin vitro.The in vitro responsiveness of ovarianfragmentswaswell correlatedwith thespawningsuccess induced in vivobya cubifrin-L injection.Mature sea cucumbers injectedwith cubifrin-Ldisplayedsequential reproductivebehaviors,whichcomprisedclimbingthesidewallofthetanktowardthewatersurface,wavingofthehead,andsheddingof gametes. Gamete shedding started about 60min and 80min after the injection inmales andfemales, respectively, andwas completed almostsimultaneouslyinthetwosexesabout2hoursaftertheadministration.Repeated injectionsofcubifrin-Lat intervalsofabout10dayssuccessfully inducedmultiplespawningsinmalesandfemales.Thisstudydemonstratedthatcubifrin-L isaneffective inducerofspawninginJapaneseseacucumbercultivation.

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