Expression profile for metabolic and growth-related genes in domesticated and transgenic coho

12 Pages·2010·0.76 MB·English

by  Ken Overturf

Aquaculture307(2010)111–122
ContentslistsavailableatScienceDirect
Aquaculture
journal homepage: www.elsevier.com/locate/aqua-online
Expression profile for metabolic and growth-related genes in domesticated and
transgenic coho salmon (Oncorhynchus kisutch) modified for increased growth
hormone production
Ken Overturfa,⁎, Dionne Sakhranib, Robert H. Devlinb
aUSDA-ARS,HagermanFishCultureExperimentStation,3059-FNationalFishHatcheryRoad,Hagerman,ID83332,USA
bFisheriesandOceansCanada,4160MarineDrive,WestVancouver,BC,Canada,V7V1N6
a r t i c l e i n f o a b s t r a c t
Articlehistory: To gain a better understanding of the aspects underlying growth in salmonids, quantitative expression
Received24September2009 analysis was performed for a number of genes related to muscle growth, metabolism, immunology and
Receivedinrevisedform8June2010 energyregulationintheliverandmuscleofwild-typecohosalmon(Oncorhynchuskisutch),domesticated
Accepted9June2010
coho salmon selected for growth, growth hormone-transgenic coho salmon fed to satiation, and growth
hormone-transgenic coho salmon reared on restricted rations. In comparisons among these four
Keywords:
experimentalgroups,ourfindingsshowasignificantandcorrelativeup-regulationintheexpressionofa
Metabolism
number of muscle determination and development factors infull-fed (FF)transgenic and domestic coho
Growth
Geneexpression salmon.Expressionofseveralmetabolicgenesinvolvedwithaminoacidturnoverandutilizationalsowas
Growthhormone correlatedbetweenthesetwofaster-growinggroups.However,distinctdifferencesbetweendomesticated
Transgenic andFF-transgenicfishwerefoundforsomegenesinvolvedwithmusclecelldifferentiation,innateimmune
Cohosalmon system,metabolism,andaminoacidregulation.Wild-typeandration-restrictedgrowthhormone-transgenic
fishcorrelatedveryhighlyintheexpressionofmostgenestested,suggestingthatlimitingavailableenergy
reduces the effects of elevated growth hormone and down-regulates muscle and metabolic regulatory
pathwayssimilarlytothereducedpotentialforgrowthhormoneproductionseeninwild-typefish.
PublishedbyElsevierB.V.
1.Introduction growth hormone (GH) gene constructs can result in significant
improvementsinweightgainandgrowthrate(Duetal.,1992;Devlin
TheFoodandAgricultureOrganizationoftheUnitedNations(FAO) et al., 1994; Nam et al., 2001; Rahman et al., 1998). However,
predictsthatthegrowthofaquaculturewillcontinuetoriseandsurpass domesticationoffish(selectedforfastgrowth)seemstointeractwith
capture fisheries in the near future (FAO, 2009). Recently, it was transgenicmanipulation.Inrainbowtrout,expressionofGHgenesor
reported that aquaculture now provides more than half of the fish treatmentwithexogenousGHproteinhadareducedeffectinahighly
consumedglobally(Nayloretal.,2009).Infact,theFAO(2009)suggests domesticatedstrainvs.wild-type,whereasasynergisticeffectwasseen
that within the next 20years, aquaculture production will need to forcohosalmon(Devlinetal.,2001).Researchhasbeeninitiated to
increase by at least 40million tons/year in order to meet demand. characterize the expression at coincidental pathways involved with
Increasing concerns regarding the effects of aquaculture on the growthregulationbetweenthesetwogeneticsystems.
environment and use of unsustainable practices for rearing fish are In order to accurately determine the similarities and differences
forcingtheindustrytooptimizeproduction.Forcommerciallyvaluable between selected domesticated fish, wild-type, and transgenic fish
species, selective breeding programs have been initiated to develop modified withenhancedgrowth hormoneproduction,samples were
faster, more efficiently growing strains of fish. Another method for taken from the liver and muscle of these groups and analyzed for
improving growth in fish has been the development of transgenic changesinexpressionofspecificgenes.Apreliminarystudyinvolved
strainsdesignedtoenhanceweightgainand/orgrowthrate.Studies the use of microarrays to study expression differences in liver and
have shown that the genetic transformation of wild-type fish with muscle, and found that domesticated fish and growth hormone-
transgenicstrainsmodulatetheexpressionofcertaingenesinasimilar
and strongly correlative manner (Devlin et al., 2009a). To further
investigatethesimilaritiesanddifferencesbetweenthesegroups,andto
gainabetterunderstandingofenergypartitioning,musclegrowth,and
⁎ Correspondingauthor.Tel.:[phone];fax:[phone]. metabolism, the expressions of several specific genes involved in
E-mailaddress:[email protected](K.Overturf). development,regulation,metabolism,anddegradationinmusclewere
0044-8486/$–seefrontmatter.PublishedbyElsevierB.V.
doi:10.1016/j.aquaculture.2010.06.010
112 K.Overturfetal./Aquaculture307(2010)111–122
analyzed. In addition, genes involved in immunological, growth, ykinase, pyruvate dehydrogenase, glutamate dehydrogenase, and
metabolism and cellular protection functions in the liver have been aspartateaminotransferase.
examined. Fox01(forkheadbox01)isamemberofafamilyofforkheadbox
Inmuscle,genesinvolvedwithdifferentiationandpropagationwere transcription factors in muscle and can induce the muscle-specific
studied,includingtheconservededgeexpressedprotein(cee)andPax7. ubiquitinligasesatrogin-1andMuRF-1whichareknowntoparticipate
Cee originally was identified in a genome-wide search for genes inmuscledegradation(Sandrietal.,2004;Leckeretal.,2004;Langetal.,
associatedwithmyotubeformationusingpufferfish(Macqueenetal., 2006).Intheliverofmammals,fox01actstoregulatelipidandglucose
2007). In salmonids, cee has been found in the superficial layers of metabolism(Valentietal.,2008;KamgateandDong,2008).Fructose1,6
developingtissuesandisbelievedtoplayaroleinnormalgrowthand bisphosphatase is found mainly in the liver, where it functions in
development(Femandesetal.,2008).Pax7isexpressedinquiescent gluconeogenesis by converting smaller substrate molecules into
satellite cells. When satellite cells are activated, Pax7 is found co- glucose. Prior studies have demonstrated changes in the expression
expressedwithMyoD.ItisbelievedthatPax7isinvolvedinmaintaining for this gene in the muscle ofrainbow troutunder different feeding
muscle cell proliferation and preventing precocious differentiation conditions(JohansenandOverturf,2006).Further,REDD-1expression
(Zammitetal.,2006).Infish,ithasbeenshownthatdietarycomponents intheliveralsowasmeasured.REDD-1isinvolvedinregulatingmTOR
andavailableenergycanregulatemusclemyogenesisthroughPax7and (mammalian target of rapamycin) activity and protein synthesis in
othermyogenicfactors(Chapalamaduguetal.,2009). mammalsandinturnisregulatedtosomedegreebyubiquitinligases
Differencesinmuscledevelopmenthavebeenexaminedusingthe (Katiyaretal.,2009).RecentstudieshaveshowntheTORpathwaytobe
muscle-specific genes myogenin, MyoD2, Myf5, Mef2A, and Mef2C. importantinregulatingproteinaccretionandgrowthinskeletalmuscle
Myogenin,MyoDandMyf5aremuscleregulatoryfactors(MRFs)that in fish (Seiliez et al., 2008b). Another gene found to be linked with
aremembersofafamilyofbasichelix–loop–helix(bHLH)transcrip- metabolismistheperoxisomalproliferator-activatedreceptorgamma
tionfactorsknowntoregulatethetranscriptionofothermusclegenes (PPARγ) which has been linked with metabolic regulation of energy
(Royetal.,2002).MyoDandMyf5areknowntofunctioninapartially from incoming nutrients, particularly those involved with fatty acid
redundant manner during the specification and proliferation of metabolism(Kennedyetal.,2006;Leaveretal.,2008).
myoblasts (Braun et al., 1992; Rudnicki et al., 1993). Mef2A and Degradation factors studied in both the liver and muscle in the
Mef2C belong to a separate group of transcription factors (myocyte presentstudyincludecathepsinL,calpain1,20Sproteasome,andthe
enhancerfactor2family)andfunctiontogetherwithMRFstoregulate longandshortformsofcalpastatin(castLandcastS).Alsoincludedwas
muscledevelopmentandgrowth.ItisbelievedthatMEF2proteinsare the analysis of two muscle ubiquinase genes, MuRF1 and atrogin-1.
mainly involved in regulating muscle differentiation, but not Regulationofthedegradationsystemhasbeenreportedascrucialin
specification. The MEF2 proteins alone are not sufficient to induce regulating protein and muscle accretion, particularly in starved fish
myogenesis, but interact with MRF family proteins by forming (Martinetal.,2001;Houlihanetal.,1988).Therearefourmajorsystems
heteroproteincomplexesthatcontrolthetranscriptionofnumerous involvedinproteindegradation,includingcalpains,cathepsins,protea-
muscle structural genes, including myosin heavy and light chains, someandapoptosis.However,thefirstthreehavebeenshowntobethe
desmin, and troponins, as well as those encoding the bHLH MRFs primarysystemsinvolvedwithmaintainingandregulatingmetabolic
themselves(BlackandOlson,1998).Myostatinisasecretedproteinof andgrowthhomeostasis.Calpainsareafamilyofcalcium-dependent,
thetransforminggrowthfactorβ(TGFβ)familythatinmammalshas non-lysosomalcysteineproteases.Theyhavebeenshowntobeactivein
beendemonstratedtoactasanantagonisttomusclegrowth(Sharma, processes such as cell mobility, cell cycle progression, cell fusion in
etal.,1999;Thomasetal.,2000).Severalmyostatinsequenceshave myoblasts,celldegradation,andmuscleproteinturnoverandgrowth
been characterized in salmonids with different expression patterns (HuangandForsberg,1998).Previousresearchhascharacterizedthe
(Rescanetal.,2001;RobertsandGoetz,2001).Twoofthesegenes, isoformsandcatalyticsubunitsforμandmcalpainsinrainbowtrout
Tmyostatin1andTmyostatin2,wereassessedonmuscleisolatedfrom (Golletal.,2003;Salemetal.,2005a).Cathepsinsareafamilyofcysteine
fishsamplesinthisstudy. and aspartic proteases that are distinguished by their structure and
Recentresearchhasstartedtomoreintensivelyanalyzeconnec- substratespecificity.TheyfunctionatlowpHandarealmostexclusively
tionsbetweenfactorsinvolvedwithgrowthormetabolism(Stelling found in lysosomes. In mammalian systems, cathepsins have been
etal.,2002;Picahetal.,2009).Inbalancingenergypartitioningand shown to play a vital role in cellular turnover and bone resorption
nutrientutilization,metabolismneedstobetightlylinkedforefficient (Yasudaetal.,2005).Insalmonids,cathepsinhasbeenidentifiedasa
growth. Previously, it had been determined that enhanced growth proteinwhoseabundanceisincreasedintheliverduringshort-term
might be achieved through selection for individuals with increased starvation(Martinetal.,2001).Multiplestudiesalsohaverecognized
appetite or who demonstrate a greater efficiency in partitioning thatchangesintheregulationandactivityofproteasomeproteolysisare
energy from nutrients (Hill et al., 2000; Silverstein et al., 2000; important for protein turnover in fish at different life stages and on
Johnsonetal.,2003).Tounderstandtheeffectsofselectivebreeding differentdietsorfeedingregimes(Seiliezetal.,2008a;Martinetal.,
andtransgenesisontheseprocesses,expressionsofthegenesIGF-I, 2002;Doblyetal.,2004;OverturfandGaylord,2009).Inthisstudy,the
IGF-II, fox01, and fructose 1,6 bisphosphatase (F1,6 Bp) have been expressionofcalpain1(μcalpain),castSandcastLwasmeasuredasan
examined in muscle and liver. IGF previously has been studied in indicationofcalpainactivity,cathepsinLforlysosomaldegradation,and
fishesbyresearchersinterestedingrowthinteractionsinvolvingthe the proteosomal subunit proteasome 20S for evaluation of ubiquitin
somatotropic axis (Pierce et al., 2001; Gomez-Requeni et al., 2003, proteasomal degradation activity. MurF1 and atrogin-1 are ubiquitin
Raven et al., 2008). Although IGF-I is known for its direct role in ligasesthathavebeenlinkedtomanyareasincludingmetabolism,but
increasinganimalsizethroughthesomatotropicaxis,italsohasbeen theirmajorrolehasbeendepictedasfactorsinvolvedwithmyofibril
showntostimulategrowthbysuppressingtheexpressionofatrogin-1 breakdownandmuscleatrophy(Glass,2003;Leckeretal.,2004;Hirner
andMuRF1(Sachecketal.,2004).IGF-IIbelongstothesamefamilyas etal.,2008).
IGF-Iandalsoisinvolvedintheregulationofmetabolism,growth,and There has been some evidence of immunological changes in
differentiation(Planasetal.,2000).Alsoanalyzedforexpressionwere transgenically modified fish (Guo et al., 2003; Wang et al., 2006).
theIGF-Ireceptor1a,whoseexpressionhasbeencorrelatedwithIGF ReducedresistancetoVibrioanguillarumwasfoundforGH-transgenic
levels (Greene and Chen, 1999; Gabillard et al., 2003), and IGF-I cohosalmonatcertainlifestages(Jhinganetal.,2003).Inthepresent
bindingprotein1,oneofatleastsixIGF-bindingproteinsknownin study, potential differences in the innate immune system between
salmonids(Kamangaretal.,2006).Theexpressionofotherrelevant transgenic and non-transgenic coho salmon were evaluated by
metabolicgenesanalyzedincludedpyruvatekinase,pyruvatecarbox- measuringtheexpressionofcomplementfactorC3,TNFα,CXCL-8,and
K.Overturfetal./Aquaculture307(2010)111–122 113
NFκβ. Complement factor C3 is a component of the innate immune additiontopublisheddatadescribedbyDevlinetal.(2001,2009b)and
systemandhasmultiplefunctions,includingdownstreamactivationof Tymchuketal(2006),suggestthatthisdomesticatedstraingrowsfaster
thecomplementcascadeandbindingtopathogensurfacestofacilitate than native wild coho salmon populations. Hemizygous growth
phagocytosis(MedzhitoxandJaneway,2000).TNFα(tumornecrosis hormone-transgenic salmon were generated in January 2006 by
factorα)isacytokinethatfunctionsinsystemicinflammationandcan matings between GH-transgenic strain M77, F generation, (Devlin
6
regulateotherimmunecells.TNFαalsohasbeenshowntoinhibittumor et al., 1994, 2004) and Chehalis River wild-type salmon. Utilizing
developmentandviralreplicationandtoinduceapoptosisandcellular previous knowledge of these fish, including growth histories and
inflammation.Morerecently,TNFαalsohasbeenfoundtobeinvolved spawningtimes(Tymchuketal.,2006;Ravenetal.,2006;Devlinetal.,
in metabolic regulation, including glucose signaling, regulation of 2001),size-matchedindividualsweregeneratedforthisstudy.Tenfish
plasma triglycerides and body fat in mammals (Ventre et al., 1997; fromeachgroupwereusedforanalysis.Weightsandtotallengthswere
SaltielandKahn,2001).CXCL8(interleukin8)isachemokineproduced takenforeachfish,andspecificgrowthrateswerecalculated(%growth/
bymacrophagesthatalsofunctionduringaninflammatoryresponse. day).Fishwererearedin10°Cfreshwellwaterandfedartificialsalmon
CXCL-8issecretedbyseveralcelltypesanditsprimaryfunctionisto diets (Skretting Canada) to satiety three times per day for the
recruit neutrophils for phagocytosis of antigen (Gangur et al, 2002). domesticated,wild-typeandtransgenicfull-fedgroups.Ateachfeeding,
NFκβisarapid-actingtranscriptionfactorthatrespondstostimuli,such ration-restrictedGH-transgenicsalmon(Tran-RR)werepair-fedtothe
as stress, cytokines, free radicals and antigenic pathogens. NFκβ rationlevelconsumedbythewild-typestrain.
functions as a first responder to harmful cellular stimuli, and over-
activation can lead to a number of different inflammatory diseases
2.2.SamplingandRNAisolation
(Gilmore,2006).Otherfactorsstudied,suchasglutathioneperoxidase,
whichhaveaknownroleinprotectingcellsfromoxidativedamage,also
Fishbecamesize-andstage-matchedinOctober2006.Thefishwere
were tested for evaluating potential stress effects from the different
notfedonthedaywhensamplesweretakenandbeforeeuthanization
treatments(OttoandMoon,2009).Theglutathionesystemhasbeen
(100mg/LofMS222plus200mg/Lsodiumbicarbonate).Tissuesfrom
shown to be up-regulated in GH-transgenic salmon (Leggatt et al.,
tenfishfromeachgroupwereremovedrapidlyfromindividualfishby
2007),butwhethersucheffectsareinfluencedatthegeneregulation
team dissection and placed into RNAlater (Ambion, Austin, TX)
levelisnotwellknown.
followingthemanufacturer'sinstructions.RNAisolationswerecarried
Prior research has suggested similarities in gene expression at a
outusingTRIzolaccordingtothemanufacturer'sprotocol(Invitrogen,
global level between domesticated fish that have been selected for
Rockville,MD).Tissueswerehomogenizedbyaddinga5mmstainless
growthforseveralgenerationsandfishthathaveundergonetransgenic
steelbeadtotheRNA-TRIzolmixandthenshakingtwicefor2minat
modificationforincreasedproductionofgrowthhormone(Devlinetal.,
40HzusingaQiagenMM301shaker(Valencia,CA).RNAfromeachfish
2009a).Toexamineindetailspecificpathwaysinvolved,real-timePCR
was DNAse-I treated (Promega, Madison, WI). Quantity and quality
analysis was used to assess the expression of genes involved in
checksoftheRNAweredonebyspectrophotometry.
metabolism,muscledevelopmentandgrowth,andimmunefunction.
Expressionwasassessedinwild-type,domesticatedandGH-transgenic
cohothathavebeeneitherfully-fedorfeed-rationedtodiscernsomeof 2.3.Quantitativereal-timeRT-PCRanddataanalysis
the more specific metabolic and other physiological differences
generated by either domestication or GH transgenesis. Further, QuantitativePCR(QPCR)analyseswereperformedon10fishfor
assessment of expression of the different gene panels used in this each experimental treatment (wild-type, domesticated, GH-trans-
studywillimproveourknowledgeofhowincreasedgrowthhormone genicfull-fed,andGH-transgenicfeed-restricted).Primersandprobes
productionistranslatedintoanimal growth anditseffectonmuscle weredesignedeitherwithPrimerExpress3softwareorbyApplied
accretion.Additionally,withtheuseofappropriatecontrols,effectssuch BiosystemAssaybyDesign(ABI,FosterCity,CA).Todetectthelevelof
asdomestication,GH-transgenicmanipulationandavailableenergycan gene expression at each time-point, real-time quantitative RT-PCR
bedeterminedsothatanenhancedunderstandingofthepathwaysand was carried out using an ABI Prism 7900HT Sequence Detection
mechanisms involved with metabolism and growth can be better System and the TaqMan One-Step RT-PCR Master Mix Reagents kit
understoodandusedtoimproveselectionorenhancefuturetransgenic fromABI,accordingtotheprotocolprovidedbyABI(FosterCity,CA).
models. Thefinalconcentrationofeachreactionwas:MasterMix,1x(contains
AmpliTaqGoldenzyme, dNTPsincludingdUTP,a passive reference,
2.Materialsandmethods andbuffercomponents);MultiScribereversetranscriptase,0.25U/μL;
RNaseinhibitormix,0.4U/μL;forwardprimer600nM;reverseprimer
2.1.Animalcareandhusbandry 600nM;probe,250nM;totalRNA,75ngwithwateraddedforatotal
reactionvolumeof15μL.Probesandprimersequencesarelistedin
Strainsofcohosalmon(Onchorhynchuskisutch)weregrownwithin Table1.Cyclingconditionsforgenestestedwereasfollows:30minat
theTransgenicFishFacilityattheFisheriesandOceansCanadaCentre 48°C,10minat95°C,then40cyclesofPCRconsistingof15sat95°C
forAquacultureandEnvironmentalResearch(CAER)inWestVancou- followedby1minat60°C.Foreachgene,assayswereruninduplicate
ver,Canada.Thisbio-containedfacilityisdesignedtopreventtheescape onRNAsamplesisolatedfromindividualfish.
offishtothenaturalenvironment.Fishwererearedunderanapproved Relativecopynumberfortheexpressionofeachgenetestedwas
AnimalUseProtocolinaccordancewiththeCanadianCouncilonAnimal determined by serially diluting a random experimental sample 10-
Care Guidelines. Experimental fish were selected from families foldandincludingthisineachsetofreal-timeassaysrun.Thedataare
generatedbyartificialfertilizationofeggs(10single-pairmatingsper reported as a ratio of relative mRNA copy number of each gene to
genotype).Gametesfromwild-strainsalmonwereobtainedfromthe absolutemRNAcopynumberofβ-actininliver(Kreuzeretal.,1999)
Chehalis River in southwestern British Columbia in fall 2004, and or 18S ribosome in muscle (multiplied by a constant for ease of
gametes from domesticated coho salmon mass-selected for growth interpretation)andexpressedasmeans±standarderrors.Microsoft
wereobtainedinNovember2005fromaBritishColumbiaaquaculture Excelwasusedtoproducegraphicalrepresentationsofthedata.One-
producer. Briefly, this growth-enhanced domesticated strain was way ANOVA and t-test analysis of the data were performed using
originallyderivedfromKitimatRiverandRobersonCreekpopulations SYSTAT ver. 9 (SPSS, Chicago, IL). One-way ANOVA with post-hoc
andwasselectedforenhancedgrowthforsixgenerations.Substantial testingbyStudent–Newman–Keulswasusedforcomparisonanalysis
anecdotalinformationfromaquacultureandlaboratoryobservations,in offishgrowth.
114 K.Overturfetal./Aquaculture307(2010)111–122
Table1
Genbankaccessionnumbersandsequencesofprimersandprobesusedforreal-timePCRanalysis.
Gene GenBank/TIGRaccessionno. Primer-probesequence(5′–3′)
18SrRNA AF243428 18SF:CCACGCGAGATTGAGCAAT
18SR:GCAGCCCCGGACATCTAA
18SPR:6FAM-ACAGGTCTGTGATGCC-NFQ
Atrogin1 BX082508 ATRF:CCACTCAGGACATCCAGAGATTTAC
ATRR:CAGCTGAGACCTGGCTATGAG
ATRPR:6FAM-CTATGTGGCAAAACTC-NFQ
Aspartateaminotransferase TC9251 AATF:CATGCCTGTGCCCATAACC
AATR:CACCAGGTCAGCGATCTCTTT
AATPR:6FAM-CCACTGCTCAGGCTTG-NFQ
β-Actin AF254414 BactF:CCCTCTTCCAGCCCTCCTT
BactR:AGTTGTAGGTGGTCTCGTGGATA
BactPR:6FAM-CCGCAAGACTCCATACCGA-NFQ
Calpain1 AY573919 Calp1F:CCAGGGCCAGGACTTTAAGT
Calp1R:AGGCGCACGGGAACAG
Calp1PR:6FAM-CCGCAGCCTGTTTGAG-NFQ
CalpastatinL(castL) AY937407 CastLF:GCTCCAGCTGTCCATGCT
CastLR:GCATCCAAGGCAAAGTCATCTGA
CastLPR:6FAM-CCCCAGCTCCTCCC-NFQ
CalpastatinS(castS) AY937409sd CastSR:AGCTGTCCAATCCTGTGAGAAAA
CastSR:TGAAGCAACATCACTGCAACATTG
CastSPR:6FAM-CTAGGGCAACACCTCCATTT-NFQ
CathepsinL AF358668 CathLF:CAGTGCTGCCAACGAGACT
CathLR:GCCTTCATCATAGCATGCTCCTT
CathLPR:6FAM-CTTTGTGGACATCCCC-NFQ
ComplementC3 AF271079 C3F:CATTGGCCTGTCCAAAACACAT
C3R:GCAGCTTCAGATCAAGGAAGAAGTT
C3PR:6FAM-CAGACACACAGATTCC-NFQ
Conservededgeexpressed EF036472 ceeF:CGCAGCGAGGTGGACAT
ceeR:TGTTCTTTAGACAGAGGAACTGTAGGA
ceePR:6FAM-CCTGCGCCACAAAC-NFQ
CXCL8 AJ279069 IL8F:AGCCAGCCTTGTCGTTGTG
IL8R:AGTTTACCAATTCGTCTGCTTTCC
IL8PR:6FAM-TCCTGGCCCCTCCTGACCATTACTGAG-TAMRA
Fox01 CX247152 Fox01F:CGCTGGTGGCAGTGGT
Fox01R:TCCAGGTCAGTGGGAAAGC
Fox01PR:6FAM-CCTAGCGAGCAGATTC-NFQ
Fructose1,6-bisphosphatase AY113693 FR16BPF:CGTTATGTCGGCTCCATGGT
FR16BPR:TGCCTCCGTACACCAGAGT
FR16BPPR:6FAM-CCTGTGCACATCAGGC-NFQ
Glutamatedehydrogenase TC9373 GDHF:GAGCAGAAGATGAAACGAGTGAAG
GDHR:CTCTCCGTTGTCCCTCTTGATG
GDHPR:6FAM-AAGCCCTGCAACCAC-NFQ
Glutathioneperoxidase AF281338 GPF:CCTCCTCCTCCTCACAAGCT
GPR:CAGGAGAGCACGGTACATGATTTAA
GPPR:6FAM-ATGGGTGCTGTGGTCACA-NFQ
IGF-I M95183 IGF1F:GTGTGTGGAGAGAGAGGCTTTTATT
IGF1R:CGTCCACAATACCACGGTTATGT
IGF1PR:6FAM-ATGGCCCCAGTTCACG-NFQ
IGF-II M95184 IGFIIF:GAGGCGTGGCAGAGGAA
IGFIIR:GCCTGCCTCCGGAACTT
IGFIIPR:6FAM-TCCCGGCCATCCTC-NFQ
IGF-Ireceptor1a AY100459 IGF1r1aF:GAAGGAGAGCCCCCCTGAGA
IGF1r1aR:CAGTGAAGGGCTGCAGGTT
IGF1r1aPR:6FAM-CTTGCTCCGGTCCTCC-NFQ
IGF-Ibindingprotein1 DQ190460 IGFbpF:CACAGCTCCCTAGCCTACTTC
IGFbpR:CTCTCCTGAGCACCCTCATTTT
IGFbpPR:6FAM-CTGGGACTCAACACCC-NFQ
MEF2A CA374878 MEF2AF:GGCCAGGCAGCTCTCA
MEF2AR:TTGGAGCCCTGAGGTAGGT
MEF2APR:6FAM-CACCCACCAAAGAGC-NFQ
MEF2C CA380324 MEF2CF:CCCTAGGCAACCACAACCT
MEF2CR:ACTGGGAGGTCTATGTGTGACA
MEF2CPR:6FAM-CCGTCCCATGACCCC-NFQ
Myf5 AY751283 Myf5F:CACAAGCTATGGCAACAACTACAG
Myf5R:GGCACCAGCACCTCTCT
Myf5PR:6FAM-TCCAGAGCTCACATTCT-NFQ
MuRF1 BX319947 MuRF1F:CGCAATCCCTACCGCTTCTC
MuRF1R:GGTCCAGGACCACCTCGAA
MuRF1PR:6FAM-TCCGCTGCCCCACCTG-NFQ
NFκβ CA356763 NFKBF:TGACAAAGGCATCTGCATCACA
NFKBR:CATGGAGGATGCCCAGGTT
NFKBPR:6FAM-CTGATGCTGGAGTCTTT-NFQ
Pax7 CB493668 PAX7F:CTATGTGGCAAAACTC
PAX7R:GTCCCAGCATGACTTCTCCAT
PAX7PR:6FAM-CAGCCTGGAGTCCTC-NFQ
K.Overturfetal./Aquaculture307(2010)111–122 115
Table1(continued)
Gene GenBank/TIGRaccessionno. Primer-probesequence(5′–3′)
PPARγ AJ292963 PPARgF:AGGCCATCCTCTCTGGAAAGA
PPARgR:CGTCAGAGACTTCATGTCATGGAT
PPARgPR:6FAM-CCCACGGAAACTCAC-NFQ
Proteasome20Δ AF115539 ProtΔF:GAGGGTCAGGATCCACCTATATCTA
ProtΔR:GCGAAGACACTGGTCCTTTGT
ProtΔPR:6FAM-ACTCCAACTACAAACCC-NFQ
Pyruvatecarboxylase TC105924 PCF:GCCCTGTAGACTGCCTCAA
PCR:CTCTTCAGCAATCTTGGGTCCAT
PCPR:6FAM-CACACGCCTGTTCCTC-NFQ
Pyruvatedehydrogenase TC28238 PDH2F:GTAGTGAGGTCCCAATGTCATACTT
PDH2R:TGGGCACAGTATCTGAGTCTTCA
PDH2PR:6FAM-CTGCCACATCTCTCCC-NFQ
Pyruvatekinase AY113695 PKF:ACAGCGTGGGCGATACC
PKR:GCTGGAGCTGTCATAGTACTCACT
PKPR:6FAM-TCAGCCCAGCTCCTG-NFQ
TMyoD2 Z46924 MyoDF:GCCGTCACCGACCAACT
MyoDR:CACTGTGTTCATAGCACTTGGTAGA
MyoDPR:6FAM:CCGTCCCATGACCCC-NFQ
Tmyogenin Z46912 MyogeninF:CATGGACCGGCGGAAAG
MyogeninR:GGCCTCGAATGCCTCGT
MyogeninPR:6FAM-CTTCTTCAGCCTCCTCTT-NFQ
Tmyostatin1 AF273035 TMSTAT1F:CCGCCTTCACATATGCCAA
TMSTAT1R:CAGAACCTGCGTCAGATGCA
TMSTATPR:6FAM-CATATTACATTTGGGATTCAA-NFQ
Tmyostatin2 AF273036 TMSTAT2F:AGTCCGCCTTCACGCAAA
TMSTAT2R:ACCGAAAGCAACCATAAAACTCA
TMSTAT2PR:6FAM-CGTATTCACTTTTGGATTTT-NFQ
3.Results wasnosignificantdifferenceinSGRbetweenwild-typeandTran-RR
fishforeitherweightorlength.
3.1.Growthratesandrelativesizesoffish
Size-matched fish from the domesticated, wild-type, full-fed 3.2.Geneexpression
(Tran-FF)andrestrictedration(Tran-RR)growthhormone-transgen-
iccohosalmonwereusedforanalysisinthisstudy.Obtainingsizeand 3.2.1.Musclegeneexpression
weight-matchedindividualswaspossibleduetoexistingknowledge Theexpressionoftwenty-onegeneswasevaluatedinthemuscle
regardingtheirspawningtimesandgrowthhistory(Tymchuketal., oftheexperimentaltransgenicandnon-transgeniccohosalmon.The
2006;Ravenetal.,2006;Devlinetal.,2001).Nosignificantdifference genes are divided into the following groups; muscle propagation,
inweightwasobservedbetweenthefourgroups.Wild-typeandTran- muscledevelopmentandregulation,muscledegradation,andmuscle
RRfishweresignificantlygreaterintotallengththandomesticand metabolism.
Tran-FF fish, although the difference was slight (Fig. 1). Specific Muscle propagation factors analyzed included Pax7 and cee. The
growthrates(SGR)forbothweightandlengthweregreatestforTran- expressionofthePax7genewassignificantlyhigherinthemuscleof
FFfish,significantlymoresothanfordomesticatedfish,whichwere domesticatedfishcomparedtowild-type,Tran-FFandTran-RRsalmon.
significantlygreaterthanthoseforwild-typeandTran-RRfish.There Therewasnosignificantdifferencebetweenthelatterthreegroups.The
expressionoftheceegenesignificantlyincreasedinthemusclesofTran-
FFfishcomparedtotheotherexperimentaltreatments,amongwhich
nosignificantdifferencewasdetected(Fig.2).
Fig.1.Weight,totallength,andSGR(specificgrowthrate,%dailyforlength(L)and Fig.2.Expressionofmuscledifferentiationandpropagationfactorscee(conservededge
weight(w)dataforwild-type,andfull-fed(Tran-FF)orrestricted-rationed(Tran-RR) expressedprotein)andPax7inthemuscleofdomesticated,wild-type,andfull-fed(Tran-FF)
growthhormone-transgeniccohosalmon.BarswithinacategoryontheX-axisthatdo orrestricted-rationed(Tran-RR)growthhormonetransgeniccohosalmon.Foreachgene,
notshareidenticallowercaselettersaresignificantlydifferentwhencomparedamong barsthatdonotshareidenticallowercaselettersaresignificantlydifferentwhencompared
groupsatPb0.05. amongfishgroupsatPb0.05.
116 K.Overturfetal./Aquaculture307(2010)111–122
Muscledegradationgenesanalyzedforexpressionconsistedofthe
short and long forms of calpastatin (cast S and cast L), calpain 1,
cathepsinL,andtheproteosomalsubunit20Sdelta.Expressionsofthe
ubiquitinligasesMuRF-1andatrogin-1alsowereevaluated.Expres-
sionoftheproteasomedelta20Ssubunitwassignificantlyhigherin
the muscle of Tran-FF fish than in domesticated fish which was
significantlygreaterthanthatforwild-typeandTran-RRfish,among
whichtherewasnosignificantdifference.Calpain1wassignificantly
up-regulated in the muscle of domesticated fish relative to other
experimentalgroupswhichshowednosignificantdifferencesamong
one another. The calpain inhibitors cast S and cast L were similarly
expressedinallfourgroups,withwild-typeandTran-RRfishsimilarly
showingthelowestexpression.Expressionwashighestindomesti-
catedfish,significantlyhigherinthecaseofcastS,butnotcastL.For
both cast S and cast L, expression was significantly greater for the
Fig.3.Expressionofmyostatingenesinthemuscleofdomesticated,wild-type,andfull-fed domesticandTran-FFfishcomparedtowild-typeandTran-RRfish.
(Tran-FF)orrestricted-rationed(Tran-RR)growthhormonetransgeniccohosalmon.For CathepsinLexpressiondifferedsignificantlybetweenallthegroups,
eachgene,barsthatdonotshareidenticallowercaselettersaresignificantlydifferentwhen
comparedamongfishgroupsatPb0.05. withwild-typecohosalmonhavingthehighestexpression,followed
by Tran-RR then domesticated and Tran-FF fish. For the ubiquitin
ligases, MuRF1 expression was similar for domesticated, wild-type
and Tran-RR fish, but expression was strongly (N12-fold) and
Muscle developmental and regulation factors evaluated included
significantlyreducedinTran-FFfish.Atrogin-1geneexpressionwas
myostatin1andmyostatin2,myoD2,Myf5,mef2A,mef2C,andmyogenin.
significantly higher in domesticated and Tran-FF fish, with no
Therelativeexpression of myostatin 1 and 2 wasthesame, with the
differencebetweenthesegroups.Therewasnosignificantdifference
highest expression found in Tran-FF salmon. For myostatin 1, no
significantdifferenceswerefoundbetweentheotherthreeexperimental between the lower- expressing wild-type and Tran-RR fish groups
(Fig.5).
groups. However, for myostatin 2, Tran-FF and domesticated coho
salmonexpressionlevelswerenotsignificantlydifferent,butbothwere Genes associated with metabolism and growth whose expression
significantly higher thanthose for wild-type or Tran-RR fish (Fig. 3). wereexaminedinmuscleconsistedofIGF-I,IGF-Ibp1,IGF-II,fox01and
ExpressionofmyoD2wassignificantlydifferentbetweenallfourgroups, fructose 1,6 bisphosphatase. IGF-I expression was elevated in fast-
with Tran-FF fish showing the highest expression level, followed by growingTran-FFanddomesticgenotypesrelativetowild-typeandTran-
domesticatedfish,thenTran-RR,andfollowedbywild-typecohosalmon RRfish,withthehighestexpressioninTran-FFfishanddomesticated
coho salmon significantly lower (N3-fold). There was no significant
which had the lowest expression level. Relative Mef2A transcription
levels were similar to those found for myoD2 in that significant differencebetweenwild-typeandTran-RRfish,whichbothexpressed
IGF-I at levels significantly less than for domesticated coho salmon.
differenceswerefoundbetweenallthegroups,withTran-FFshowing
ExpressionofthebindingproteinIGF-Ibp1washighestinTran-RRfish,
thehighestlevelofexpressionfollowedbydomesticated,Tran-RR,and
wild-typecohosalmon.Myogeninexpressionwassignificantlyhigherin althoughnotsignificantlygreaterthanfordomesticatedfish.However,
FF-transgenicfish,followedbydomesticatedcohosalmonandTran-RR for both Tran-RR and domesticated fish, expression levels were
between which there was no significant difference, and both were significantly greater when compared to Tran-FF fish. There was no
significantlyhigherinexpressionthanwild-typefish.Expressionofmyf5 significant difference in expression between domesticated and wild-
wassimilarandsignificantlyhigherindomesticatedandTran-FFsalmon typefishforthisreceptor.NosignificantdifferenceswerefoundforIGF-II
compared to Tran-RR and wild-type fish. Mef2C expression was not expressionbetweenallfourgroups.Expressionoffox01wassignificantly
significantly different between domesticated, wild-type and Tran-RR higherinbothdomesticandTran-FFwhencomparedtowild-typeand
salmon;however,theexpressionofmef2Cwassignificantlyhigherin Tran-RRfish.Nosignificantdifferenceinexpressionwasfoundbetween
domesticatedthaninTran-FFfish(Fig.4). domestic and Tran-FF or between wild-type and Tran-RR fish.
Expression of F1,6 Bp showed no significant difference between
Fig.4.Expressionofmuscleregulatoryfactorsinthemuscleofdomesticated,wild-type, Fig.5.Expressionofdegradationfactorsinthemuscleofdomesticated,wild-type,and
andfull-fed(Tran-FF)orrestricted-rationed(Tran-RR)growthhormonetransgenic full-fed(Tran-FF)orrestricted-rationed(Tran-RR)growthhormonetransgeniccoho
cohosalmon.Foreachgene,barsthatdonotshareidenticallowercaselettersare salmon. For each gene, bars that do not share identical lowercase letters are
significantlydifferentwhencomparedamongfishgroupsatPb0.05. significantlydifferentwhencomparedamongfishgroupsatPb0.05.
K.Overturfetal./Aquaculture307(2010)111–122 117
Fig.6.Expressionofmetabolic-relatedfactorsinthemuscleofdomesticated,wild-type, Fig.8.Expressionofglutathioneperoxidaseintheliverofdomesticated,wild-type,and
andfull-fed(Tran-FF)orrestricted-rationed(Tran-RR)growthhormonetransgenic full-fed(Tran-FF)orrestricted-rationed(Tran-RR)growthhormonetransgeniccoho
cohosalmon.Foreachgene,barsthatdonotshareidenticallowercaselettersare salmon.Barsthatdonotshareidenticallowercaselettersaresignificantlydifferent
significantlydifferentwhencomparedamongfishgroupsatPb0.05.(IGF1arec-IGF-I1a whencomparedamongfishgroupsatPb0.05.
receptor,F1,6Bp-fructose1,6bisphosphatase).
Glutathione peroxidase expression was examined to determine
domesticatedandTran-FFfish.However,domesticatedfishdidshow whether there were any changes involving oxidative damage
significantlyhigherexpressionthanwild-typeandTran-RRfish(Fig.6). protection.ExpressionofthisgenewassignificantlyhigherinTran-
FFanddomesticatedcomparedtoTran-RRandwild-typefish,withno
significantdifferencebetweentheselattertwogroups(Fig.8).
3.2.2.Livergeneexpression Growth factor genes examined in the liver included IGF-I, IGF-I
The expression of sixteen genes was evaluated in the liver of receptor1a(IGF-Irec1a),IGF-IIandfox01.Theexpressionoffox01was
transgenicandnon-transgeniccohosalmon.Thegeneswereselected significantlyhigherintheliverofdomesticatedcohosalmonthanfor
foranalysisbasedontheirinvolvementwithimmunologicalresponse, the other three groups, among which no significant difference in
cellulardamageresponse,growthfactors,and/orenergymetabolism. expressionwasdetermined.IGF-Iexpressionwashighestindomes-
Theimmunologicalgenesanalyzedforexpressionwerecomplement ticated fish, with no significant difference between these fish and
factor C3, tumor necrosis factor α (TNFα), CXCL-8 (IL-8) and NFκβ. Tran-FFfish.TherewasnosignificantdifferencebetweenTran-FFand
ComplementfactorC3wassignificantlyup-regulatedintheliverofFF Tran-RRgroups,withexpressionforbothofthesegroupssignificantly
transgenic fish when compared to the other treatments; however, higherthanforwild-typecohosalmon.ForIGF-Irec1a,wild-typefish
amongtheotherthreetreatmentstherewasnosignificantdifference. exhibitedhighestexpression,whichwhilebeingmorethan30%over
TNFαexpressionshowednodifferencebetweenwild-type,Tran-FF,and that for Tran-RR, was not significantly greater. Both wild-type and
Tran-RR,andtheexpressionforallthesegroupswassignificantlyhigher Tran-RR expression of IGF-Ibp showed significantly higher levels of
than for that of the domesticated fish. CXCL-8 expression was expressionthandomesticatedfish.Tran-FFfishexpressionofIGF-Ibp
significantlydifferentbetweenallgroups,withtheexpressioninTran- was significantly lower than the expression observed for the other
FF highest, followed by Tran-RR, wild-type, and domesticated coho three groups. Expression of IGF-II was found highest in the liver of
salmon.NFκβexpressionwashighestindomesticatedfish,andwhileit domesticatedfish,followedbyTran-FF,whereitwassignificantlyless.
wassignificantlyhigherthantheexpressionforTran-FFandTran-RR Wild-typeandTran-RRfishhadsignificantlylowerexpressionofIGF-
fish,itwasnotsignificantlyhigherwhencomparedtowild-typecoho. II compared to domesticated and Tran-FF fish, but there was no
TherewasnosignificantdifferenceintheexpressionofNFκβbetween significantdifferenceinexpressionbetweenthesetwogroups(Fig.9).
wild-type,Tran-FFandTran-RRfish(Fig.7). Expressionsofsevenenergymetabolism-relatedgenesalsowere
evaluatedintheliversofthesegroupsofcohosalmon.Thesegenes
included pyruvate dehydrogenase (PyrD), pyruvate carboxylase
Fig.7.Expressionofimmunologicalfactorsintheliverofdomesticated,wild-type,and
full-fed (Tran-FF) or restricted-rationed (Tran-RR) growth hormone transgenic coho Fig.9.Expressionofgenesrelatedtogrowthintheliverofdomesticated,wild-type,and
salmon.Foreachgene,barsthatdonotshareidenticallowercaselettersaresignificantly full-fed(Tran-FF)orrestricted-rationed(Tran-RR)growthhormonetransgeniccoho
differentwhencomparedamongfishgroupsatPb0.05.(C3—complimentarycomponent salmon. For each gene, bars that do not share identical lowercase letters are
C3,TNFα—tumornecrosisfactorα,CXCL-8). significantlydifferentwhencomparedamongfishgroupsatPb0.05.
118 K.Overturfetal./Aquaculture307(2010)111–122
(PyrC), pyruvate kinase (PK), glutamate dehydrogenase (GlutD), 1994,2001).Resultshaveshownthatwhenwild-typesalmonidsare
aspartateaminotransferase(AspAm),PPARγ,andfructose1,6bispho- transgenicallymodifiedtoexpressgreaterlevelsofgrowthhormone,
sphatase (F1,6Bp) . Expression of PyrD was highest in Tran-FF fish. relativebodysizesatspecificagescanincreaseupto37-fold(Devlin
ExpressionofPyrDdidnotdifferbetweendomesticatedandTran-FF et al., 1994). However, transfer of GH genes into domesticated fish
fish,butbothexpressedPyrDatlevelssignificantlyhigherthanthose that have been prior-selected for growth (Tymchuk et al., 2006)
found for wild-type and Tran-RR fish; there was no statistical showedareducedgrowthresponsetoGH(Devlinetal.,2001).Similar
difference in expression between wild-type and Tran-RR fish. PyrC attenuation of GH effects also were seen in domesticated Atlantic
wasexpressedindomesticatedcohoatsignificantlyhigherlevelsthan salmon and rainbow trout treated with GH protein (Devlin et al.,
in Tran-RR,which expressedthis geneat levelssignificantly higher 2001; Neregard et al., 2008). These data are consistent with the
thanthosefoundforwild-typeandTran-FFcohosalmon.Therewas hypothesis that selection for faster-growing fish is mediated by
no significant difference detected for PyrC between wild-type and increased stimulation of the growth hormone signaling pathway.
Tran-FFfish.PKwasexpressedhighestandapproximatelyequallyin Frompriorexperiments,itappearsthattheanswerisyesandno.That
thetwotransgenicgroups.WhileexpressionofPKwassignificantly is,thelevelofgrowthhormoneishigherinfaster-growinganimals,
higherintheTran-FFfishthaninthedomesticatedfish,therewasnot butcertainphysiologicalandmoleculardifferenceshavebeennoticed
a significant difference in expression between the Tran-RR and the betweenthetransgenicandselecteddomesticatedfish(Devlinetal.,
domesticated coho salmon. Wild-type fish expressed PK at a 2001).Theresearchdescribedhereinshowedseveralcorrelationsin
significantlylowerlevelthaninothergroups.Theenzymeglutamate the expression of different genes for the fish that grow the fastest,
dehydrogenase was similarly expressed in domesticated, wild-type, domesticselectedandTran-FFcoho,versusthesmallerfish,wild-type
andTran-RRfish,onlyshowingsignificantreductioninexpressionin and Tran-RR coho salmon. However, the expression of some genes
theliverofTran-FFfish.Aspartateaminotransferasewasexpressedat wasquitedivergentbetweenfull-fedtransgenicfishanddomesticat-
similarhigherlevelsindomesticatedandTran-FFfishasthaninwild- edcohosalmon.
typeandTran-RRcohofish,wherenosignificantdifferencewasnoted
betweeneitherdomesticatedandTran-FFfishorwild-typeandTran- 4.1.Muscle
RR fish. Similarly, expression of PPARγ was highest in Tran-FF and
domesticatedfish,withnosignificantdifferencebetweenthesetwo Analysisofmusclegeneexpressionrevealedbothsimilaritiesand
groups. However, in the Tran-RR group PPARγ was expressed at differencesregardingexpressionofgeneswhoseproductsplayarole
significantlylowerlevelsthanindomesticatedandTran-FFfish.Wild- inthemusclephysiologyofwild-type,domesticatedandtransgenic
typefishexpressedPPARγatasignificantlylowerlevelthantheother coho salmon. The muscle development factors cee and Pax7 both
threegroups.Unlikeinthemuscle,wherefructose1,6bisphosphatase showedsignificantdifferencesamongTran-FFanddomesticatedfish.
wasexpressedhighestintheTran-FFfish,intheliverthisenzymewas Tran-FF coho salmon exhibited up to a 2.5-fold increase in the
expressedatthelowestlevelinTran-FF,butnotinamannerthatwas expression of cee in muscle compared to the other experimental
statisticallydifferentthanindomesticatedandwild-typefish.Inthis groups. The cee gene originally was isolated from genome screens
case,Tran-RRwasfoundtoexpresstheF1,6bpgeneatasignificantly seekinggenesassociatedwithmyotubeformation(Fernandesetal.,
higher level than in the other experimental groups. REDD-1 2005). In fugu, cee was up to 15-fold up-regulated in fast-twitch
expression was highest in Tran-FF coho salmon, where it was muscleandrelativelyunchangedinothertissues.Insalmonids,ceeis
significantly higher than in wild-type and Tran-FF fish, among expressedindevelopingorgansandtissues(Macqueenetal.,2007).
whichnodifferencewasdetected.Domesticatedcohosalmonwere TheincreasedexpressionofceeinTran-FFsalmonmuscleduringthis
foundtoexpressREDD-1atasignificantlylowerlevelthanthethree studysuggeststhatrelativetotheothertreatments,whentransgenic
othergroups(Fig.10). fish have sufficient nutrients available, they potentially have the
abilitytocontinuallygrowmuscleinahyperplasticfashion,consistent
4.Discussion withthegreaternumberofsmallfibersseeninGH-transgenicorGH-
treatedfish(Fauconneauetal.,1996;Hilletal.,2000).Unlikecee,Pax7
A number of fish species have been genetically engineered to was significantly up-regulated in the muscle of domesticated fish
enhancetheexpressionofspecificgrowthfactors,primarilygrowth compared to wild-type and transgenic coho salmon. Pax genes are
hormones(Duetal.,1992;RahmanandMcLean1997;Devlinetal., tissue-specifictranscriptionfactorsthatareimportantduringdevel-
opment and have been associated with myogenesis and the
developmental specification of satellite cells in the skeletal muscle
inthepresenceofmyoD(Zammitetal.,2006).Pax7functionsthrough
the regulation of the bHLH factor Myf5, whose expression was
significantlygreaterforbothdomesticatedandTran-FFcomparedto
wild-typeandTran-RRfish.Thesefindingssuggestthattheenhanced
muscle growth identified in the faster-growing fish is being
modulated from multiple signals, beginning during differentiation
and most likely continuing through developmental regulation and
degradationofmuscletissue.Hence,whileceeexpressionishigherin
Tran-FFfish,Pax7expressionwasincreasedindomesticatedfish.In
othervertebrates,reducedPax7/MyoDtranscriptratiosareknownto
beatranscriptionalsignaturereflectingtheenhancementofsatellite
cellmyogenesis(HoltermanandRudnicki,2005;Olguinetal.,2007).
InthisstudytransgenicfishoverallhadreducedratiosofPax7/MyoD,
Fig.10.Expressionofmetabolic-relatedgenesintheliverofdomesticated,wild-type, average 0.021 for transgenic fish vs 0.071 for non-transgenic fish.
andfull-fed(Tran-FF)orrestricted-rationed(Tran-RR)growthhormonetransgenic However,downstreamMRFswereallincreasedfordomesticatedand
cohosalmon.Foreachgene,barsthatdonotshareidenticallowercaselettersare Tran-FF,withMyoD2andmyogeninsignificantlyincreasedforTran-FF
significantlydifferentwhencomparedamongfishgroupsatPb0.05.(PyrD—pyruvate fish. This finding would suggest that enhanced levels of growth
dehydrogenase,PyrC—pyruvatecarboxylase,PK—pyruvatekinase,GlutD—glutamate
dehydrogenase, AspAm — aspartate aminotransferase, PPARγ — peroxisomal prolif- hormone in salmonids can enhance satellite cell activation and
erator-activatedreceptorγ,F1,6Bp—fructose1,6bisphosphatase). differentiation. Since some of these changes were not observed in
K.Overturfetal./Aquaculture307(2010)111–122 119
Tran-RR, these responses are likely contingent upon availability of secretedproteinsinvolvedingrowthregulation(KaneandGottesman,
energy. 1990;IshidohandKominami,1995).AlthoughcathepsinLisrecognized
Regardingtheothermuscleregulatorygenesevaluated,myogenin asalysosomalprotease,italsoissecretedandthereforeispotentin
andmyoD2showedhighestexpressionlevelsinTran-FF,followedby degradingextracellularandstructuralproteins(Mason,1989).Inwild-
domesticated, then Tran-RR, with wild-type coho salmon as the typecohosalmon,cathepsincouldbeplayingaroleinincreasedprotein
lowest-expressinggroupforthesetwogenes.Theseresultsalsowere breakdown and turnover linked to the relatively inefficient use of
found for the expression of the muscle regulatory factor, myf5. availableenergyinthesefish.Expressionoftheubiquitinproteasome
ExperimentsinfisheshaveobservedincreasedexpressionofmyoDin pathway gene, delta subunit of proteasome 20, was expressed
faster-growinganimals(AlvesdeAlmeidaetal.,2008;Wilkesetal., significantly higher in the faster-growing domesticated and Tran-FF
2001).Itispossibleinfishthatalevelofhyperplasiaisalwayspresent fish.Thispathwayisbelievedtofunctionasthemainmodeofmuscle
astheygrow,andthatthislevelisfurtheraffectedbysizeandrateof degradation(Hochstrasser,1996;Ciechanover,2005)andturnoverin
growthoftheindividual. mostvertebratespeciesanddemonstratesthatwithenhancedprotein
Myocyteenhancerfactor(MEF)familygenes,whichtypicallywork accretiontherealsooccursincreasedmuscleturnover.
togetherwithMRFstoregulatemuscledifferentiationandgrowthbut Fox01 is a negative regulator of myogenesis and its induction of
notspecification,showedvariableresults.Mef2Ahasbeenshowntobe proteasome-dependentdegradationofaspecificsubsetofcomponents
crucialformaintainingappropriatemitochondrialcontentandarchi- inthemTORsignalingnetworkofmammaliancellsduringdifferenti-
tecturalintegrityinpostnatalheartmuscleinmice(Nayaetal.,2002). ationimpairsIGF-IIexpression(Wuetal.,2008).Theactivityoffox01is
Mef2AshowedsimilarexpressionpatternsasthatseenfortheMRFs, knowntobecontrolledbyitsphosphorylationstatus,whichwasnot
highestinTran-FFanddomesticatedfishandlowerinTran-RRandwild- determinedinthisstudy;howeveritsregulatoryactionsoccurmainly
type fish. Regulation of differentiation and muscle development is viamodulationofgenetranscription.Fox01inducesexpressionofboth
regulatedlargelythroughheteroproteincomplexesofMRFsandMEFs, atrogin-1 and MuRF1. Interestingly, MuRF1 expression was strongly
whichthencontrolthetranscriptionofmusclestructuralgenes.Little reducedinthemuscleofTran-FFfishcomparedtotheothertreatments.
change was detected for the expression of mef2C, suggesting that Pastresearchhasshownup-regulationofMuRF1andatrogin-1during
regulationistakingplacethroughamixtureofMEFsandMRFsandthat muscleatrophy(Gomesetal.,2001)anddown-regulationduringage-
mef2Amay bemore directly involved.In all cases,itwasnoted that related loss of muscle (Edstrom et al., 2006). Feed deprivation in
expressionwashighestinTran-FFfish,followedbydomesticatedfish, salmonids and rats up-regulates expression of ubiquitin ligases;
furtherdemonstratinghowincreasedgrowthhormonelevelenhances however, treatment with IGF-I prevents this induced up-regulation
muscledevelopment.Inmostcases,itwasfoundthatinTran-RRfish, (Clevelandetal.,2009;Dehouxetal.,2004).Alternativestudieshave
expressionofmusclefactorswasnotablyhigherthanthosefoundfor demonstratedthattheleveloftheseubiquitinligasesisincreasedduring
wild-type fish. This observation indicates that even with reduced rapid muscle development due to increased protein breakdown
available energy, growth hormone is essential for the allocation of observedduringcatabolicconditions(Langetal.,2007).Inthepresent
available energy for muscle development and suggests that higher experiment,therapidly-growingcohosalmonappearedtobebreaking
growthhormonelevelscanleadtomoreefficientfeedutilization,ashas downandturningovermusclemoreextensivelyduringgrowth,thereby
beenpreviouslysuggested(Ravenetal.,2006;Oakesetal.,2007). showing an increased level of only certain degradation factors and
The relative expression of myostatin 1 and 2 genes was not ubiquitin ligases. Conversely, the expression of these genes was
significantly different, which differs from previous results where basicallyidenticalbetweenwild-typeandTran-RRfish,demonstrating
MSTN2geneexpressionwasreducedintransgeniccomparedtonon- thatreducedcapacityofgrowthhormoneeffectsisregulatedbyboth
transgenic salmon (Roberts et al., 2004). Prior research has demon- geneticsandavailableenergy.
stratedthattheageandcurrentgrowthstateofthefishwhenmuscle
samples are analyzed have a significant effect on the expression of 4.2.Liver
myostatin and other genes (Johansen and Overturf, 2005; Levesque
et al., 2007). In this study, expression of both myostatin genes was Therewereanumberofgeneexpressionchangesintheliverthat
highestinTran-FFfish.Furthermore,priorresearchhasdemonstrated seemtobetiedspecificallytothetransgenicmodificationofthefish.
thattheexpressionofthemyostatingenesishigherinlarger,faster- Specifically,whentheexpressionofimmunologicalfactorsintheliver
growingfish(JohansenandOverturf,2005).Whenenergyisavailable, wasexamined,wefoundthatCXCL-8wassignificantlyup-regulated
myostatinexpressionappearstobeincreasedinfaster-growingfish,but intransgenicfish,andwasexpressedlowestindomesticatedfish.Ina
apparentlyisnotnegativelyactingtoregulatemusclegrowth.Thus,it similar manner, TNFα was expressed lowest in domesticated fish,
wouldappearthatenergyavailabilityinfluencesmuscledevelopment while inversely NFκβ wasexpressed highest in non-transgenic fish.
morethanrepressionbymyostatin. Prior research has suggested that growth hormone-transgenic fish
Expressionofgenesinvolvedinregulatingproteindegradationin mighthaveaslightlydepressedimmunesystem(Jhinganetal.,2003),
musclesamplesshowedsimilarresponsesforboththelongandshort yet here our findings imply that certain subsystems of the innate
formsofcalpastatin,agenewhichactsasanegativeregulatorofcalpain. immune system actually might be up-regulated. Similar studies in
Bothformsofcalpastatinweresignificantlyup-regulatedinthemuscles growthhormone-transgeniccommoncarpsuggestedthatenhanced
ofdomesticatedandTran-FFfishcomparedtowildandTran-RRfish. growth hormone production stimulated non-specific immune func-
However,eventhoughdomesticatedfishdisplayedthehighestlevelof tions(Wangetal.,2006).However,steady-stateincreasedimmune
castLandSexpression,theexpressionofcalpain1wassignificantlyup- expressionmaynotpredicthowwelltheanimalswillrespondupon
regulatedinthemuscleofdomesticatedfishwhencomparedtothatin infectionwithcertainpathogens.However,previousanalysisofthe
theotherthreegroups.Itispossiblethatcalpastatinlevelswerejust expression of these genes in rainbow trout did find a correlation
beginningtoincreasetocompensateforthehighcalpainexpression; between expressionand resistance to bacterial and viral pathogens
samplingandevaluationatmultipletime-pointswouldbenecessaryto (Overturf and LaPatra, 2006). Glutathione peroxidase expression is
clarifyanymodulationoftheregulationofthesegenes.Althoughcalpain anotherindicatorofcellularhealthinanimals(Michielsetal.,1994).
activity has been demonstrated to influence fillet quality, it is not Here again, it appears that increased growth rate, likely correlated
believedtofunctionsubstantiallyinactivemuscleturnoverinsalmonids withenhancedgrowthhormonelevels,isinvolvedwithglutathione
(Salemetal.,2005b).ForcathepsinL,expressionwassignificantlyhigher peroxidase production (Leggatt et al., 2007). In GH-knockout mice,
inwild-typefish,followedbyTran-RR,domesticated,andfinallyTran-FF glutathione peroxidase production has been linked with muscle
fish.CathepsinLplaysamajorroleintheturnoverofintracellularand satellitecellproliferationandincreasedlifespan(Mulleretal.,2007).
120 K.Overturfetal./Aquaculture307(2010)111–122
Therefore, up-regulation of growth hormone production might be Acknowledgements
beneficialtocellsoftransgenicandgrowth-selectedsalmonids.
Theexpressionofcertaingrowthregulatoryfactorsintheliverwas TheauthorsthankUSDA-ARSbiologicaltechnicianKarenFrankat
similartoresultsofearlierstudieswiththesefishregardingIGF-Iand theHagermanFishCultureExperimentStationinHagerman,Idahofor
II (Devlin et al., 2009a,b). IGF-I expression levels were slightly but her assistance in sample analysis and the fish culture staff at the
significantly increased in Tran-RR fish compared to wild-type. This Transgenic Fish Facilityat Fisheriesand OceansCanada'sCentrefor
risecouldbethroughpotentialenergyenhancementfromthegrowth AquacultureandEnvironmentalResearch(CAER).
hormone transgenesis providing increased utilization of dietary
energy (Oakes et al., 2007; Higgs et al., 2009). Domesticated fish
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