SevemlSpeciesorhleosts MicroscopicObservationorItoCellsPresentintheLiversor

日本海域研究，第42号，1-8ページ，２０１１ Ｍ力o"Ｋｍ流ＫＣ"ﾉIﾘﾉ",vol42,ｐ1－８，２０１１

MicroscopicObservationorItoCellsPresentintheLiversor SevemlSpeciesorhleosts

MitsukoASADAl,HajimcNAKABAYASHI2andYilichiSASAYAMA3＊

2010年９月１４日受付，Receivedl4September2010 2010年11月８日受理，Accepted8November2010

Abstract

Itoccllswithcytoplas血cprotrusionswereclearlystamedmtheliversof7speciesoftelcostsby Otsukaissilverstaining,whichisusuallyusedtodctectreticularflbersinneuronsandconnectivetis‐

sues・Amongthosespecies，mmedakaandblackscraper,hnmunostainingwasconductedusingan

anti-l3-tubulmantibody(TU27),whichrecogmzesaUisotypesofp-tubulmdistributedfomprotozoato manⅢnalsThisantibodyreactedtoltoceUsmbothspeciesSmcel]-tubulmisoneofthecompo‐

nentsofmicrotubulesoftheaxonlnneurons,thisresultsuggeststhatItocellsexpress6-mbulmsimi‐

larlytoneuronSInaddition,anotherantibody(TU20),whichrecognizesClasslII6-tubulmwasap‐

pliedThistypeofl3-tUbulmisexpressedonlyinneuronsTheantibodystainedsomecells,which appearedtobeItocells,mblackscraperbutnotinmedakaOnthebasisoftlleseresults,attllispomt，

atleast,mblackscraper,Itocellsmayhavesimilarfimctionstoneurons,althougllitiscertamthatthey

KeyWords:Itocells,teleostliver,l3-tubulm,ncuronalfimction

１．lntroduction

TheycontamalargeqUantityofvitaminAmthefht dropletsandparticipatemthemetabolismofvitanmnA (Wake，1971）Furthennore,Itocellssccretecollagen ln厚catqUantitiesmtheprocessesofliverregeneration

（Ramadori,1991)．Inhuman,ithasbeenthoughtthat

ｌｎｔｈｅｓｐａｃｅｏｆＤｉｓｓｅｏｆｍａｍｎａｌｉａｎliver，unique cellsexist,whicharediHerenthDmhepaticparenchymal cellsandKupfferceUs(ItoandNemoto,1956）These cellsarecalledItocellsafterDr・Ito,whoidentifiedthem Theyarealsodesclibedasfht-stormgcells，smcemany fatdropletsexistmthecytoplasm（ItoandShibasaki，

1968）Furthennorc,becauseofthedistmctivemor- phologyofthcsecells，theyarecalledstellateceUsas welLItocellshavepluralprotrusionsofcytoplasmand

'DivisionofBiologicalSciences，GraduateSchoolofNaturalScienceandTbchnologyぅKanazawaUniversityぅKakuma-machi，

Kanazawa,920-11g2Japan

2ResearchFellow）ResearchAdviseEDivisionofBiologicalMeasurememandApplications,InstituteofNatureandEnvironmemal

TechnologpLKanazawaUniversity)Kakuma戸machi,Kanazawa,920-1192Japan

3MarineLaboratoryぅDivisionofBiodiversityぅInstituteofNatureandEnvironmentalTechnologyぅKanazawaUniversity）

Kakuma-machi,Kanazawa,920-1192Japan

*Correspondenceauthor

１

、lkingthesefactsmtoconsideratio､，theoriginoflto cellshasbeenregardedtobemyogemccells，suchas smoothmusclecellsormuscularfiberblastcells(Ybkoi eM/､,1984）

Duringthepasttenyears,however,theoriginoflto

cellshasbeendebaｔｅｄＩｔｉｓｋｎｏｗｎｔｈａｔＩｔｏｃｃｌｌｓｅｘ‐

presstheneuralcelladhesionmoleculeandtheglialfi‐

brillaryacidicprotem(NakatanieM/､,1996;Neubauere／

α/､，1996）NishieM/､（1999)conductedcloningofa newGTP-bmdiｎｇｐｒｏｔｅｍＢｏｍｔｈｅｓｐｍａｌｃｏｒｄｏｆｒａｔｓ andfbundthatitisexpressedmItocellsaswellSyn‐

aptophysmisknownasamarkermoleculeofthenerv‐

ous-endocrinesystem(Edehanne/α1,1995)．Itocclls cxpresssynaptophysm（Cassmane/α/､，1999）Fur- thelmore,CassnaneM/､(1999)notedthatltocellshave synapticvesiclesandsuggestedthepossibilitythatthey mayhaveunknownfimctionsmneighboringcellslto cellshavereceptorsfbrthencrvegrowthfactor(TTime／

α1,2000）Bmdmgwiththegrowthfactorcauses apoptosisofltocellsspecificallｙＴＴｉｍｅＭ/､（2000）

havesuggestedthatItocellsareaselectivetargetofthc nervoussystem・A1thougllnestinoriginallyconsistsof intennediatefilamentsexpressedspecificallymneurons，

ｔｈｅｓａｍｅｆｌｌａｍｅｎｔｓｈａｖｅａｌｓｏｂｅｅｎｆｂｕｎｄｉｎｌｔｏｃｅｌｌｓ

Ｏｎｔｈｅｏｌｈｅｒｈａｎｄ，phylogenetically;Itocellshave alreadybeenfbundmlampreyliver(Lα叩e〃/cIpo"ｊｃａ）

(Ｗａｋｅｅｊα/､,1987）WakeeraLpointedoutthatthese cellshavecontractilityonthebasisofelectronnmcro‐

scopicobscrvatlons、Ingoldfishhver(Cｍａｓｓ伽α"、‐

伽)andstoneflounderliver(肋"伽bjco/０，伽),des‐

mosomesdevelopmallcellscomposmgthelivcrs，and areremarkablyabundantaroundItocells(FUjitae'αﾉ.，

1980mlnumaeMﾉ.,1982)．Observationsofthelivers ofl9speciesofEeshwaterandseawaterfishesrevcalcd thatltocellsremarkablydevelopdesmosomes（Sakano andFUjital982）Thehepatocytesofcod(Gα伽加”

/i"α'，zacmcepﾉ､伽）contamedalargenumbcroffat

droplets(FUjitaerα/､,1986）Itocellshavelongprotru‐

sionsofcytoplasmandadheretoeacｈｏｔｈｃｒｂｙｔｈｅｄｅｓ‐

mosomespresentatthetipoftheprotrusionsFUjitM α/､（1986）suggestedtllat,ｍｃｏｄ,thismorphological characteristicamongltocellsconstitutesaskeletalsys‐

temthathasthefhnctionofmamtainingthefbrmofthe

liverbecauseoftheaccumulationoffat・InthePacific

halibut(』肋e"s伽sevelmα""j),ItocellswithfIneand longprotrusionsofcytoplasmwerestainedbygoldcmo- 1idestanng(YOshikawae/α1,2006）

Asmentionedabove,mltocellsofteleosts,longand elongatedprotrusionsofcytoplasmareemphasized，m additiontothedevelopmentofdesmosomesThese factssuggestthat,mteleosts,ItoceUshavethefimction ofmamtainingthefbnnofthelivcr，

Becauseteleostsoccupyaplylogeneticallyfimda- mentalpositionamongvertebrates,theyareannerest- 1ngtargctmthesmdyoftheoriginofltocells・How- ever,therehasbeennoresearchaboutthiｓｐｏｉｎｔｓｏｆｈｒ・

Inthepresentsmdy）ｍ７ｓｐeciesofteleosts,weapphed silverstainingtodetecttheltocells・Furthennore，ｗｅ conductedimmunostahnngwithtwokindsof anti-p-mbulmantibodiestoexaminewhetherItocells haveneuｒａｌｃｌｅｍｅｍｓｏｒｎｏｔＴｈｉｓｉｓｔｈｅｆｉｒｓｔreportm whichantibodiesof6-mbulmwereusedtodetectlto

cells．

ⅡMaterialsandmethods

Freshwatermedaka（ｏｒ)』z伽肋ipes）ｗａｓobtained fiomacomnercialsource、Sixspeciesofseawater fishes,damselfish(Ｃｈｍ伽〃ＭＪ"),geenfish(G舵ノル p""伽"),fine-pattemedpuffbr(TＭ１/i`gz`poecﾉﾉo"o伽)，

bamboo-leafwrasse（Pse"ぬ肋7ｚ４Ｆノ〃o"ｊｃ妬)，pud- dingwifewrasse(肋/jchoe形spoecj/qp妙蛎),andblack scraper（Ｚｈａｍ"αＣＯ"zJsmodbs伽),werecollectedBom themarinestationofourunivcrsitybTheliverofcach speciesoffIshwasanatomizedafterhavmgcompletely

anesthetizedthefishmasolutionwithasuitableconcen‐

trationoftricamemethanesulfbnateAstheentiIeliver

ofeachspecieswasquitelarge，ｉｔｗａｓｃｕｔｔｏａｓｉｚｅｏｆ

ａｒｏｕｎdlcm3andfixed・

InhematowIjnandcosmstahnng,livcrswerefixed

２

identifiedmevelyspeciesexaminedTherefbre，gen‐

eralstainingisnotsuitablefbrdetectmgthesecellsOn theotherhand，silverstanmngrevealedthepresenceof ItoceUsmtheliversofcveryspecies．ＰlateFiguresl

and2exhibitthosecellsmmedakaandbamboo-leaf

wrasse，respectivelyItocellsofthesefishesclosely resembledthosefbundmlampreyliverstamedusmgthe mprovedvonKupBfer1sgoldcmoridemethod(Ｗａｋｅｅｒ ａＬ,1987）FUjitae/ａＬ（1986)observedItocellmcod liverusmgascanmngelectronmcrOscopeLThe 3-demensionalmorphologyofthecellismcomplete agreementwiththaｔｏｆｌｔｏｃｅｌｌｓｓｔａｍｅｄｍｔｈｅｐｒｅｓｅｎｔ study・haddition，thecharacteristicsofltocellsob‐

servedwithsilverstainingwerelngoodagreementwith thoseofPacifichalibutstainedusingthegoldcmolide method(YbshikawaeM/､,2006）hevelyspeciesex‐

ammedmthepresentstudy;thenucleusofltocellshada triangularshape，whereasthenucleusofhepaticparen- chymalcellsiseithersphericalorovalmshapeThe cytoplasmicprotrusionofltocellswaslO-201unlong Thenumberofprotrusionswasusually2-4,asobserved mthepuddmgwifbwrasseofPlateFigure３．

Ontheotherhand，Itoccllsreactedpositivelywith theTU27antibodymtheliversofmedakaandblack scraper(P1ateFigures4and5,respectively）Thepro‐

teml3-mbulmisusuallyexpressedasacomponentofa

ncrombulemtheaxonsofnervecells・Therefbre,this

mzestheC1assIIIp-tubulmspecificallyexpressedm nervecellsHowever,thereactionofthisa[Iibodywas

restrictedAfewcellswhｉｃｈｓｅｅｍｅｄｔｏｂｅＩｔｏｃｅｌｌ

ｓｈｏｗｅｄａｐｏｓitivereactionmblackscraper(PlateFigure ６)．Inmedaka,noceUsreactedpositivelyTheposi‐

tivecellmPlateFigure6mblackscraperdoesnotap‐

peartohavecytoplasmicprotrusions,astypicalltocells do,althougdlthesectionmi8htnothavecontamedparts oftheprotrusionsbyaccident・Howeverlthenumberof ｗｉｔｈａ４％parafbnnaldehydesolutionfbr3Shrona

shaker,dehydratedwithanalcoholselies,andembedded mparaffinfbllowmgaroutmemethod、Paraffinblocks werecutmtoserialsectionswith81lmthicknessmall stauungmethods・

WeadoptedOtsuka,ssilverstaining（1962)．For thisstamng,liverswerefixedwithan80％ethylalcohol solutioncontaining０５％glacialaceticacidaswellas

0.5％fbrma]ｉｎｆｂｒ４８ｈｒｏｎａｓｈａｋｅｒ・Sectionswere

hydratedwithMilliporewaterandmcubatedwitha２０％

silvermtratesolutionfbr4hrat37゜Cunderconditionsof

shielding丘omlight・Afterthat,thesectionswereagain

incubatedwithasilvernltratebuB[ersolutionfbrl8hr

underthesameconditions、Usmgasodiumsulfiteand hydroquinonemixsolution,thesectionsweredeoxidized Agoldchloridesolutionwasusedfbrplatmgthesections Aftertreatmentｗｉｔｈ２％oxalicacid,asodmnthiosulfnte

solutionwasusedtofixthereactionofthesections

lnimnunostaining，２species，medakaandblack scraper,wereusedastherepresentativesofBeshwater

fishandseawaterfishTheirliverswerefixedwith４％

parafbnnaldehydefbr3S‐ＳｈｒｏｎａｓｈａｋｅｌｘＷｅｕｓｅｄ２ ｋｉｎｄｓｏｆａｎtibodyasaprimaryantibodyOneisan anti-6-tubulmmonoclonalantibody(clonenameTU27，

ConvanceCoLtd,USA)whichrecognizesaUisotypes of6-tubulmdistributed丘omprotozoatomanⅡnals・

Theotherisamonoclonalantibody(clonenameTU20，

AcrisAntibodiesCo､Ltd,Gemlany)whichrecognlzes onlyclassIII6-tUbulmexpressedmtheneuronsoffish andmanⅡnalsTheTU27antibodywasdilutedto ＺｌＬｇ/ｍｌｗｉｔｈＯＳ％blockingsolutionTheTU20anti‐

bodywasdilutedto2011g/mlwiththesameblockmg solution・Inbothimmunostahnngs，areactionwiththe pnmaryantibodywasconductedovemightatroomtem‐

pcrature・ｈｔｈｅｃａｓｅｏｆｉｎⅡnunostainmgusmgthe TU20antibody〉toactlvatethesurfaceofsections，the sectionwasprocessedwithprotemaseK(20Ⅱg/ml)。i‐

lutedwithaPBSsolutioncontainin９０．１％ＴＷｅｅｎ２０ｆｂｒ ｌＯｎｕｎｂｅｆｂｒｅｈａｎｄｌｍｇｗiththeprimaryantibody

Ⅲ．Resultsanddiscussion

InhematoⅣlm-eoslnstaining，Ｉｔｏｃｅｌｌｓｃｏｕｌｄｎｏｔｂｅ^●

３

ItocellsthatreactedwiththeTU20wastoolowwhen

comparedwitllthatｏｆｃｅｌｌｓｒｅａｃｔｍｇｗｉｔｈｔｈｅＴＵ２７ａnti- body・Thelcfbre,notallltocells,but,rather,omycells mspecificstages,nglltreactwiththeTU20antibody ItocellsmayexpresstheClassIIIP-mbulmomyundcr somerestrictedconditionsTbexaminetlleexpression ofClassIII6-tUbulmmdetail,mteleosts,weshould mvestigateUversatvanousstagesofthereproductive cycleofgonads，ofnourishmentofthebodyうｏｒｏｆｒｅ‐

generationfomilljurytoliver．

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ｑｇｌイ24,719-730．

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UmersuchungenanManthenerschenZellenvonFischen ルノＭ、/ilZCﾉﾘﾉｂＭ,58,33-50

Ramadori,Ｇ，1991:Thestellatecell(no-cell,fat-storingcell，

lipocyte,perisinusoidalcell)oftheliveIP71℃AowM"んｊｖｅＢ，

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ん"、α/q/１４"αｍ剛132,429-462

Wake，Ｋ､，Motomatsu，Ｋ・andSenoo，Ｈ､，1987：Stellaecells

ｊｃａ・Ｃｅ〃刀SF"ehseα７℃",249,289-299

YOkoi,Ｙ,Namihisa,Ｔ､,Kuroda,Ｈ､,Komatsu,Ｉ,Miyazaki,Ａ､，

Watanabe，ＳａｎｄＵｓｕｉ,Ｋ・'1987：Immunocytochemicalde- tectionofdesmininfat-storingcells(Itocells）Ｈ２ｐａｍﾉｂｇ〕ﾉ，

4,709-714．

Ybshikawa,Ｋ,Imai,Ｋ,Seki,Ｔ,Higashi-Kuwata,Ｎ,Kqjima，

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Ｃｂ叩α、"1′eBjoche"応〃α"リノロノ!〕人F/oﾉbg〕/IPm･'4,145,

280-28６．

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ExplanationsofPlateFigures

Fig.１Arrowsshowltocellswithcytoplasmicprotrusionsinmedaka,Silverstaining

Fig2Arrowsshowltocellswithcytoplasmicprotrusionsinbamboo-leefwrasse,Silverstaining，

Fig3Arrowsshowltocellswithcytoplasmicprotrusionsinpuddingwifewrasse､Silverstaining．

Fig4Anarrowshowsthemutualconnectionofthecytoplasmicprotrusionsofltocellsinimmunostaining withtheTU27antibodyinmedaka．

Fig5Anarrowshowsltocellsconnectedwitheachotherwithcytoplasmicprotrusionsinimmunostaining withtheTU27antibodyinblackscraper．

Fig.６Anarrowshowsanimmunopositiveceｌｌｗｈｉｃｈｓｅｅｍｓｔｏｂｅａｎｌｔｏｃｅｌｌｉｎｉｍｍunostainingwiththe TU20antibodyinbIackscrape［

６