Title. Author(s)Shimizu, Munetaka; Fukada, Haruhisa; Hara, Akihiko; CitationAquaculture, 273(2-3): Issue Date Doc URL.

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1 Title Response of the slmon somtotropic xis to growth h Author(s)Shimizu, Munetk; Fukd, Hruhis; Hr, Akihiko; CittionAquculture, 273(2-3): Issue Dte Doc URL Type rticle (uthor version) File Informtion ShimizuAqu273.pdf Instructions for use Hokkido University Collection of Scholrly nd Ac

2 Title Response of the slmon somtotropic xis to growth hormone dministrtion under two different slinities Authors Munetk Shimizu 1, Hruhis Fukd 1*, Akihiko Hr 2 nd Wlton W. Dickhoff Affilitions 1 Northwest Fisheries Science Center, NOAA Fisheries, nd School of Aqutic nd Fishery Sciences, University of Wshington, Settle, WA, USA, 2 Reserch Fculty of Fisheries Sciences, Hokkido University, Hkodte, Hokkido, Jpn Present ddresses: Reserch Fculty of Fisheries Sciences, Hokkido University, Minto, Hkodte, Hokkido , Jpn; *Fculty of Agriculture, Kochi University, B200 Monoe, Nnkoku, Kochi , Jpn Correspondence: Munetk Shimizu, Reserch Fculty of Fisheries Sciences, Hokkido University, Minto, Hkodte, Hokkido , Jpn; Tel&Fx: ; e-mil: mune@fish.hokudi.co.jp Key words insulin-like growth fctor-i, growth hormone, sewter, slmon 23

3 Astrct We compred the response of plsm insulin-like growth fctor-i (IGF-I) to growth hormone (GH) dministrtion under two different slinities to test the hypothesis tht environmentl slinity lters the ctivity of the GH-IGF-I xis. In July, postsmolt coho slmon rered in fresh wter (FW) were trnsferred to either FW or hlf sewter (1/2 SW) (15 ppt) tnk. During the experiment, wter temperture ws mintined t 10 C for oth slinities; photoperiod ws djusted to tht of Settle (48 N), nd fish were not fed. Two dys fter trnsfer, fish were injected once with porcine GH (pgh) t dose of 2 or 8 µg/g ody weight. Liver nd lood smples were collected 1, 2 nd 3 dys fter injection. Liver GH receptor (GHR) mrna expression ws nlyzed y quntittive rel-time RT-PCR, nd plsm IGF-I, 41-kD IGF-inding protein (min crrier of IGF- I) nd pgh were quntified y rdioimmunossys. Trnsfer to 1/2 SW resulted in trnsient increses in sl levels of liver GHR mrna nd 41 kd IGF-inding protein (IGFBP) ut not IGF-I. The GH-injection incresed liver GHR mrna, plsm IGF-I nd 41-kD IGFBP in fish in oth FW nd 1/2 SW. However, the time course nd mgnitude of the response differed etween slinities. Fish in FW receiving 8 µg/g pgh hd the highest IGF-I levels (63.7 ± 6.8 ng/ml) one dy fter injection, wheres fish in 1/2 SW showed pek (88.8 ± 14.3 ng/ml) two dys fter injection of the sme dose. It is speculted tht the prolonged response to GH y fish in 1/2 SW my e due to slower disppernce of pgh from the circultion in fish in 1/2 SW. The trnsient increse in sl liver GHR mrna my lso contriute to greter response for fish in 1/2 SW. These results suggest tht slinity is cple of ltering the ctivity of the GH-IGF-I xis in slmon. 46

4 Introduction Smoltifiction in slmonids is pre-dpttion to ocen life ccompnied y series of morphologicl, iochemicl nd ehviorl chnges (Hor, 1988). One of the mjor chievements of smoltifiction is successful dpttion to sewter (SW). Slmon dpted to SW generlly rech lrger sizes thn those in fresh wter (FW). On the other hnd, if juvenile slmon re trnsferred premturely to SW, their growth is severely retrded nd they ecome stunts (Folmr et l., 1982). These circumstnces led scientists to hypothesize tht slinity ffects growth in slmonids, nd successful SW dpttion results in n improved growth. However, the effect of slinity on growth in slmonids is inconsistent, proly due to vrying experimentl conditions such s wter temperture, feeding rtion, developmentl stge, seson nd experimentl period (Smith nd Thorpe, 1976; McCormick et l., 1989; Morgn nd Iwm, 1991; Usher et l., 1991; Duston, 1994; Hndelnd et l., 1998). On the other hnd, the interction etween growth nd slinity hs een demonstrted in severl fish species (for review, Bœuf nd Pyn, 2001). In tilpi (Oreochromis mossmicus), evidence indictes tht SW rering improves growth of this species (Kuwye et l., 1993; Riley et l., 2002). In other mrine fishes, optiml growth is sometimes seen t n intermedite slinity (Bœuf nd Pyn, 2001). Improved growth t intermedite slinity my e explined y reduction of the metolic cost for osmoregultion, wheres ppetite nd/or the endocrine system my lso ply role (Bœuf nd Pyn, 2001). Growth hormone (GH) nd insulin-like growth fctor-i (IGF-I) re key hormones regulting growth of nimls. In the clssicl somtomedin hypothesis, GH from the pituitry glnd stimultes heptic production of IGF-I vi the GH receptor, nd IGF-I

5 from the liver medites mny of the ctions of GH (Dughdy nd Rotwein, 1989). Recent findings hve pointed out tht GH hs direct ctions independent of IGF-I, nd virtully ll tissues express IGF-I tht cts through utocrine nd prcrine mnners (Butler nd LeRoith, 2001). Regrdless of the site of production, the ctivity of IGF-I is modulted y fmily of six IGF inding proteins (IGFBPs). In the circultion, IGFBPs prolong the hlf-life of IGF-I nd deliver IGF-I to certin tissues (Rjrm et l., 1997). Incresing evidence indictes tht the fish GH-IGF-I xis lso plys n importnt role in osmoregultion s well s growth regultion (Skmoto et l., 1993; Dickhoff et l., 1997; McCormick, 2001). However, how the GH-IGF-I xis opertes these two processes simultneously is poorly understood nd the endocrine mechnism, if ny, y which slinity influences growth is not known. In study y Riley et l. (2002) on tilpi, the GH-IGF-I xis ws ctivted y tretment with 17α-methyltestostelone (MT) nd SW rering. The etter growth of tilpi treted with MT nd SW rering ws reflected y higher IGF-I levels (Riley et l., 2002). Circulting levels of IGF-I hve een considered to e n index of growth in fish (Beckmn et l., 1998; Uchid et l., 2003; Dyer et l., 2004). In coho slmon (Oncorhynchus kisutch), series of experiment hs shown tht plsm IGF-I levels re positively correlted with growth rtes of individuls in fresh wter nd sewter (Beckmn et l., 2004,), implying tht plsm IGF-I levels could e used to evlute growth potentil of slmon. In n ttempt to understnd how slinity might influence the "ctivity" of the GH-IGF-I xis nd in turn growth in slmon, we exmined response of the GH-IGF-I xis to GH dministrtion under two different slinities. 91

6 Mterils nd Methods Fish rering conditions One-yer-old postsmolt coho slmon were rered in FW t the Northwest Fisheries Science Center in Settle, WA, USA. They were mintined in recirculted FW (dechlorinted city wter tht is uffered with sodium icronte) in circulr fierglss tnks under nturl photoperiod djusted to tht of Settle, WA (48 N); flow rte ws 25 L/min; temperture rnged from 10.5 C to 13.0 C. Before fish were used for experiments, they were fed rtion of 1.25% ody weight/dy of commercil diet (Biodiet Grower; Bioproducts Inc., Wrrenton, OR, USA) Tretment of fish In erly July, 2002, fter 24 hr of fsting, 156 fish were trnsferred directly to one of eight tnks contining fresh wter or hlf sewter (15 ppt; 1/2 SW) mde from rtificil se slt (Aqurium Systems Inc., Mentor, OH, USA). The verge ody length nd weight of fish were 15.6 ± 0.2 cm (men ± SE) nd 41.8 ± 2.2 g, respectively. Throughout the experiment, slinity ws monitored dily; wter temperture ws kept t 10 C for oth slinities, nd fish were not fed. Blood nd liver smples from untreted fish were collected 1, 2 nd 5 dys fter trnsfer (dy 1, 2 nd 5, respectively) s descried elow. Other fish were injected intrperitonelly with porcine GH (Sigm, St. Louis, MO, USA) in sline t dose of 2 or 8 µg/g ody weight two dys fter trnsfer (dy 4). Shm injected fish received sline only. Blood nd liver smples were collected 1, 2 nd 3 dys fter the single injection (dy 3, 4 nd 5, respectively). 114

7 Smple collection Fish were nesthetized in 0.05% tricne methnesulfonte (MS-222; Argent Chemicl Lortories, Redmond, WA, USA). Blood ws withdrwn y cutting the cudl peduncle nd letting lood flow into heprinized glss tue. Plsm ws collected fter centrifugtion t 700g for 15 min nd stored t -80 C until use. Liver pieces were excised, immeditely frozen in liquid nitrogen nd stored t -80 C until use Smple nlysis Expression of growth hormone receptor mrna in the liver ws mesured y rel-time reverse trnscript polymerse chin rection (RT-PCR) s descried in Fukd et l. (2004). Expression levels were normlized with n cidic riosoml phospoprotein P0 (ARP). ARP is superior to 18S riosoml RNA s reference gene nd hs een dopted to RT-PCR for slmon IGF-I mrna in the liver (Pierce et l., 2004). For mesurement of IGF-I, plsm IGF-I ws extrcted with n cid-ethnol followed y cryoprecipittion s descried in Breier et l. (1991) nd quntified y RIA (Shimizu et l., 2000). Plsm 41-kD IGFBP levels were mesured y RIA s descried in Shimizu et l. (2003). Porcine GH levels were mesured y commercil RIA kit (Linco Reserch Inc., St. Chrles, MO). This RIA showed no cross rectivity with shm-operted slmon plsm (dt not shown) Sttisticl nlysis All mesured vlues were not normlly distriuted nd thus nturl-log trnsformed efore nlyses to otin norml distriution (D Agostino nd Person omnius

8 normlity test). Dt sets for ech dependent vrile (liver GHR mrna expression, nd plsm IGF-I, 41-kD IGFBP nd porcine GH levels) were first nlyzed y two- or three-wy nlysis of vrince (ANOVA) y including GH tretment, slinity nd time s fctors. When significnt effects were found, the dt were further nlyzed y one- or two-wy ANOVA for ech time point. Differences etween groups were identified y Fisher s protected lest-significnt difference (PLSD) test. Differences etween groups were considered to e significnt t P < Results Chnges in the sl levels of liver GHR mrna, plsm IGF-I nd 41-kD IGFBP in fish trnsferred to FW or 1/2 SW were compred (Fig. 1). There were overll effects of slinity nd time on the liver GHR mrna nd plsm 41-kD IGFBP levels, ut no interction ws found (two-wy ANOVA). Slinity nd time hd no significnt effect on plsm IGF-I. Heptic GHR mrna nd circulting 41-kD IGFBP were significntly higher in the 1/2 SW group one dy fter trnsfer (dy 1) (Fig. 1,c) nd the difference ecme insignificnt therefter. A similr trend of higher levels in 1/2 SW ws seen in plsm IGF-I, lthough this ws not sttisticlly significnt (P = ). There were overll min effects of GH tretment, slinity nd time on the liver GHR mrna expression (three-wy ANOVA). An increse in liver GHR expression y the low dose of GH injection ws evident in fish in oth FW nd 1/2 SW on dy 3 (one dy fter injection) (Fig. 2). On dy 4 (two dys fter injection), the effect of GH ws not seen in fish in FW ut ws seen in 1/2 SW (Fig. 2). GHR mrna levels ecme similr etween the shm nd treted groups on dy 5 (three dys fter injection). When

9 fish received high dose of GH, slinity enhnced the GH effect on the liver GHR expression one dy fter injection (Fig. 2). For plsm IGF-I, GH tretment, slinity nd time hd significnt min effects, nd n interctive effect ws lso seen (three-wy ANOVA). Plsm IGF-I levels were incresed y the low dose of GH tretment in oth FW nd 1/2 SW for two dys (Fig. 3). On dy 3 (one dy fter injection), plsm IGF-I levels in the FW group with the lowdose GH injection were higher thn those in 1/2 SW, nd decresed grdully over time (Fig. 3). When fish received the high dose of GH (8 µg/g), plsm IGF-I levels in the 1/2 SW group showed pek on dy 4 (two dys fter injection) nd were higher thn those in the FW group (Fig. 3). The response of 41-kD IGFBP to GH tretment ws essentilly the sme s tht of IGF-I (Fig. 4). A positive effect of pgh injection lsted for two dys. Slinity hd positive effect on the increse in 41-kD IGFBP with the high-dose GH on dy 4 (two dys fter trnsfer) (Fig. 4). The disppernce of exogenously injected pgh from the circultion ws monitored y homologous RIA (Fig. 5). The pgh levels decresed rpidly fter injection in oth FW nd 1/2 SW. However, the levels were lwys higher in fish in 1/2 SW thn those in FW, showing tht injected pgh ws retined longer in the circultion in 1/2 SW Discussion This study exmined the effect of slinity on the sl levels of the GH-IGF-I xis components nd their response to GH dministrtion in postsmolt coho slmon. A reltively mild slinity chnge (FW to 1/2 SW) ws chosen for the experiment s

10 trnsferring fish directly to full sewter (30-33 ppt) likely cuses stress response. A reltively short period of SW exposure (up to five dys) ws pplied to the present study. Folmr nd Dickhoff (1981) found tht when yerling coho slmon were trnsferred to sewter, plsm ions (sodium, chloride nd potssium) reched stedy stte within few dys. On the other hnd, Pierce et l. (2005) reported tht fsting of coho slmon induced decline of plsm IGF-I s erly s dy 4. For these resons, fish were cclimted for two dys prior to the GH dministrtion in order to void the influence of the rpid physiologicl chnge during the initil phse of sewter dpttion nd the negtive effect of food deprivtion on the GH-IGF-I xis. Under these experimentl conditions, we evluted the ctivity of the somtotropic xis y plsm IGF-I levels since severl studies hve reveled tht circulting IGF-I levels re positively correlted with growth rtes of fishes including slmon (Beckmn et l., 1998, 2004,; Uchid et l., 2003; Dyer et l., 2004). During sewter dpttion of slmonids, increses in secretion nd metolic clernce rte of GH, nd occupncy nd totl numer of liver GHR hve een oserved (Skmoto et l.,1991; Skmoto nd Hirno, 1991, 1993). Plsm GH levels re lso known to chnge during sewter dpttion of slmon (Björnsson et l., 1998). These chnges in GH nd its receptor support the concept tht GH is key hormone for sewter dpttion in slmonids. The ction of GH in osmoregultion my e, t lest prtly, medited y IGF-I. McCormick et l. (1991) demonstrted tht IGF-I promotes osmoregultory ility of slmon. Prticiption of the GH-IGF-I xis in osmorgultion hs een lso suggested in non-slmonid fishes (Mncer nd McCormick, 1998). Despite the importnce of the GH-IGF-I xis in osmoregultion, little is known out the

11 response of GHR mrna, plsm IGF-I nd its crrier protein (41-kD IGFBP) levels during sewter dpttion in slmon. In the present study, liver GHR mrna expression ws trnsiently incresed one dy fter 1/2 SW trnsfer. GHR expression tended to e higher in fish in 1/2 SW therefter lthough it ws not significntly different. This grees with the finding y Skmoto nd Hirno (1991) tht totl GH inding sites in the liver of rinow trout (Oncorhynchus mykiss) trnsferred to 80% SW tended to e high nd ecme significntly higher two weeks fter trnsfer. This suggests tht GH inding cpcity my e regulted t the trnscriptionl level. Averge plsm IGF-I levels were similr etween FW nd 1/2 SW despite tendency of higher IGF-I levels in 1/2 SW. There ppers to e no negtive impct of fsting for up to five dys on the sl IGF-I levels ecuse IGF-I levels were similr to those in fed fish (dt not shown). Mixed results in the response of growth regulting hormones to slinity re seen in nonslmonid species. Trnsfer of tilpi from SW to FW for five months resulted in decline in growth rte, increses in plsm GH nd IGF-I, nd decreses in pituitry GH nd liver IGF-I mrna levels (Riley et l., 2003). In lck se rem (Mylio mcrocephlus) heptic IGF-I incresed in fish dpted to 1/3 SW nd full SW fter eight months (Dene et l., 2002). In the four spine-sculpin (Cottus kzik), dpttion to full SW for 44 dys resulted in n elevtion of heptic IGF-I mrna, ut not pituitry GH mrna (Inoue et l., 2003). There is only one study exmining the effect of slinity on circulting IGFBPs (Shepherd et l., 2005). In their experiment, juvenile rinow trout were grdully cclimted to 66% SW over five dys. As result, the intensity of four IGFBP nds t 21, 32, 42 nd 50 kd on lignd lotting ws higher in fish t higher slinity. In the

12 present study, plsm 41-kD IGFBP levels incresed one dy fter 1/2 SW trnsfer while its increse lsted for just one dy. This conflicts with the finding y Shepherd et l. (2005). As discussed ove, differences in species nd experimentl conditions my contriute to the discrepncy. Overll, direct trnsfer of postsmolt coho slmon to 1/2 SW hd trnsient, positive effect on liver GHR mrna expression nd 41 kd IGFBP levels. Mny of the components in the GH-IGF-I xis re regulted y their own control system. For exmple, GH induces production of IGF-I nd IGFBP-3 in the liver, nd IGF-I is cple of inhiiting GH synthesis nd secretion y the pituitry (Le Roith et l., 2001). This regultory mechnism ppers to e opertive in teleosts (Dun, 1997; Björnsson et l., 2002). The present study exmined the effect of slinity on the potency of GH to stimulte the GH-IGF-I components (i.e. GHR mrna, plsm IGF-I nd 41- kd IGFBP) in slmon. Biologicl ctions of GH re medited y trnsmemrne GH receptor. GHR is composed of two suunits nd forms dimer with nother GHR upon inding GH (Argetsinger nd Cter-Su, 1996). It is not cler, however, if GHR expression is under control y GH. In mouse heptocyte culture, GH lone hd no effect on GHR mrna cellulr concentrtions, wheres synergistic effect with estrogen ws seen (Contrers nd Tlmntes, 1999). In fish, no study hs exmined the effect of GH on GHR mrna expression. The present study showed tht liver GHR mrna ws incresed one dy fter GH injection. When fish were held in FW, the GH effect diminished in two dys, wheres the effect ws still evident in fish in 1/2 SW. This difference is presumly ttriuted to reltively high levels of GHR mrna in the shm-

13 operted group in FW on dy 4 (two dys fter injection). However, it is cler tht GH induces its own receptor in the liver in slmon. Induction of circulting IGF-I y GH dministrtion is well-known response in slmonids (Moriym et l., 1994; Moriym, 1995). A new finding of this study is tht the time course nd mgnitude of the IGF-I response differed etween two slinities. Fish in FW showed mximum response on dy 3 (one dy fter injection) nd IGF-I levels grdully decresed therefter. In contrst, plsm IGF-I levels in fish in 1/2 SW peked on dy 4 (two dys fter injection). Moreover, when fish were injected t the high dose (8 µg/g), IGF-I levels were higher in fish in 1/2 SW thn those in FW on the sme dte. It is possile tht IGF-I levels in fish in FW reched pek in less thn 24 hr, lthough Moriym (1995) reported tht in trout in FW plsm IGF-I levels followed y GH injection continued to increse until 24 hr. The 41-kD IGFBP is one of three mjor circulting IGFBPs in slmon (Shimizu et l., 2003,). Although its identity is still not cler due to the lck of complete mino cid sequence dt, severl lines of evidence from physiologicl nd iochemicl studies suggest tht it is physiologic equivlent of mmmlin IGFBP-3 (Shimizu et l., 2003,; Beckmn et l., 2004,). IGFBP-3 prolongs the hlf-life of IGF-I nd therefore forms lrge pool of IGF-I in the circultion (Rjrm et l., 1997). A similr IGFBP with moleculr mss of kd hs een detected in other fish species nd shown to e induced y GH, s is mmmlin IGFBP-3 (Sihrth et l., 1995; Prk et l., 2000). The result from the present study is in good greement with the previous findings in fish. It is worth noting tht the response of the 41-kD IGFBP to GH is lmost identicl to tht of IGF-I. In ddition, simple regression nlysis confirmed tht their levels re positively,

14 highly correlted (dt not shown). These results further support our ssumption tht the 41-kD IGFBP is the min crrier of circulting IGF-I in slmon. The present study showed tht the response of circulting IGF-I to GH dministrtion differed etween two slinities. Wht cused the difference? An ovious possiility is difference in the clernce of exogenous GH. Mesurement of pgh y specific RIA enled us to monitor pgh levels fter injection nd reveled tht pgh ws retined longer in the circultion in fish in 1/2 SW. This my e due to difference in the glomerulr filtrtion rte t the kidney under different slinities. The kidney is one of the min sites of GH clernce in mmmls, where 20-50% of circulting GH is clered (Feld nd Hirscherg, 1996). In slmonids, the glomerulr filtrtion rte t the kidney is known to e reduced in hyperosmotic environment (Brown et l., 1978). GH-inding protein (GHBP) might contriute to the slower disppernce of pgh from the circultion since GHBP hs een shown to increse in SW in trout (Sohm et l., 1998). Another possile reson for the difference in the GH effect on IGF-I my e GHR. The trnsient increse in GHR mrna levels in 1/2 SW might induce greter response. The reson for the significntly higher IGF-I levels in fish tht received 2 µg/g GH in FW is not cler. It might to due to n inhiition of GH effect y GHBP incresed in 1/2 SW. In mmmls, GHBP is cple of inhiiting GH interction with GHR (Brnrd nd Wters, 1997). Levels of GHBP in fish in 1/2 SW might hve een high enough to prevent the low dose of pgh, ut not for the high dose of GH, from inding GHR. However, we hve no dt on GHBP levels. The iologicl significnce of the prolonged induction of IGF-I in SW oserved in the present study is difficult to interpret; It is not known whether induced IGF-I would

15 e utilized for growth or osmoregultion. Collie et l. (1989) found tht two-dy predpttion of trout in 1/3 SW prior to trnsfer to 80% SW enhnced the plsm ion lowering effect of ovine GH. Although IGF-I levels were not mesured in their study due to the lck of the immunossy t tht time, IGF-I ws most likely induced in the circultion nd might medite the ion lowering ction of GH. It is thus possile tht in the present study the induced IGF-I might hve n osmoregultory ction rther thn growth promoting ction. To distinguish these two ctions, longer cclimtion period in SW (with feeding) should e helpful in future work. In summry, the present study suggests tht slinity is cple of ltering the ctivity of the GH-IGF-I xis. The prolonged response of plsm IGF-I in fish in 1/2 SW my e due to slower clernce of pgh. The trnsient increse in the sl liver GHR mrna levels in fish in 1/2 SW my lso contriute to the greter response Acknowledgments We thnk Dr. Brin R. Beckmn, NOAA Fisheries Service, Settle, WA, for his invlule comments on the experimentl design. We lso thnk Brd Gderry of NOAA Fisheries Service for the mintennce of fish nd help in setting up the trnsfer experiment. This project ws supported y Ntionl Reserch Inititive Competitive Grnt no from the USDA Coopertive Stte Reserch, Eduction, nd Extension Service, nd Bonneville Power Administrtion (Projects nd ) References

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19 Hndelnd, S.O., Berge, Å., Björnsson, B.Th., Stefnsson, S.O., Effects of temperture nd slinity on osmoregultion nd growth of Atlntic slmon (Slmo slr L.) smolts in sewter. Aquculture 168, Hor, W.S., The physiology of smolting slmonids. In: Hor, W.S, Rndll, D. (Eds), Fish Physiology. Acdemic Press, Orlndo, FL, pp Inoue, K., Iwtni, H., Tkei, Y., Growth hormone nd insulin-like growth fctor I of Euryhline fish Cottus kzik: cdna cloning nd expression fter sewter cclimtion. Gen. Comp. Endocrinol. 131, Kuwye, T.T., Okimoto, D.K., Shimod, S.K., Howerton, R.D., Lin, H.-R., Png, P.K.T., Gru, E.G., Effect of 17α-methyltestosterone on the growth of the euryhline tilpi, Oreochromis mssmicus, in fresh wter nd in se wter. Aquculture 113, Le Roith, D., Bondy, C., Ykr, S., Liu, J.L., Butler, A., The somtomedin hypothesis: Endocr. Rev. 22, Mncer, J.M., McCormick, S.D., Osmoregultory ctions of the GH/IGF xis in non-slmonid teleosts. Comp. Biochem. Physiol. 121B, McCormick, S.D., Endocrine control of osmoregultion in teleost fish. Amer. Zool. 41, McCormick, S.D., Sunders, R.L., McIntyre, A.D., The effect of slnity nd rtion level on growth rte nd conversion efficiency of Atlntic slmon (Slmo slr) smolts. Aquculture 82,

20 McCormick, S.D., Skmoto, T., Hsegw, S., Hirno, T., Osmoregultory ctions of insulin-like growth fctor-i in rinow trout (Oncorhynchus mykiss). J. Endocrinol. 130, Morgn, J.D., Iwm, G.K., Effects of slinity on growth, metolism, nd ion regultion in juvenile rinow trout nd steelhed trout (Oncorhynchus mykiss) nd fll chinook slmon (Oncorhynchus tshwytsch). Cn. J. Fish. Aqut. Sci. 48, Moriym, S., Incresed plsm insulin-like growth fctor-i (IGF-I) following orl nd intrperitonel dministrtion of growth hormone to rinow trout, Oncorhynchus mykiss. Growth Regul. 5, Moriym, S., Swnson, P., Nishii, M., Tkhshi, A., Kwuchi, H., Dickhoff, W.W., Plisetsky, E.M., Development of homologous rdioimmunossy for coho slmon insulin-like growth fctor-i. Gen. Comp. Endocrinol. 96, Prk, R., Shepherd, B.S., Nishiok, R.S., Gru, E.G., Bern, H.A., Effects of homologous pituitry hormone tretment on serum insulin-like growth-fctorinding proteins (IGFBPs) in hypophysectomized tilpi, Oreochromis mossmicus, with specil reference to novel 20-kD IGFBP. Gen. Comp. Endocrinol. 117, Pierce, A.L., Dickey, J.T., Lrsen, D.A., Fukd, H., Swnson, P., Dickhoff, W.W A quntittive rel-time RT-PCR ssy for slmon IGF-I mrna, nd its ppliction in the study of GH regultion of IGF-I gene expression in primry culture of slmon heptocytes.

21 Pierce, A.L., Shimizu, M., Beckmn, B.R., Bker, D.M., Dickhoff, W.W., Time course of the GH/IGF xis response to fsting nd incresed rtion in chinook slmon (Oncorhynchus tshwytsch). Gen. Comp. Endocrinol. 140, Rjrm, S., Bylink, D.J., Mohn, S., Insulin-like growth fctor-inding proteins in serum nd other iologicl fluids: regultion nd functions. Endocr. Rev. 18, Riley, L.G., Richmn, N.H., III, Hirno, T., Gordon Gru, E., Activtion of the growth hormone/insulin-like growth fctor xis y tretment with 17αmethyltestosterone nd sewter rering in the tilpi, Oreochromis mossmicus. Gen. Comp. Endocrinol. 127, Riley, L.G., Hirno, T., Gru, E.G., Effects of trnsfer from sewter to fresh wter on the growth hormone/insulin-like growth fctor-i xis nd prolctin in the Tilpi, Oreochromis mossmicus. Comp. Biochem. Physiol. 136B, Skmoto, T., Hirno, T., Growth hormone receptors in the liver nd osmoregultory orgns of rinow trout: chrcteriztion nd dynmics during dpttion to sewter. J. Endocrinol. 130, Skmoto, T., Hirno, T., Expression of insulin-like growth fctor I gene in osmoregultory orgns during sewter dpttion of the slmonid fish: possile mode of osmoregultory ction of growth hormone. Proc. Ntl. Acd. Sci. USA 90, Skmoto, T., Iwt, M., Hirno, T., Kinetic studies of growth hormone nd prolctin during dpttion of coho slmon, Oncorhynchus kisutch, to different slinities. Gen. Comp. Endocrinol. 82,

22 Skmoto, T., McCormick, S.D., Hirno, T., Osmoregultory ctions of growth hormone nd its mode of ction in slmonids: A review. Fish Physiol. Biochem. 11, Shepherd, B.S., Drennon, K., Johnson, J., Nichols, J.W., Plyle, R.C., Singer, T.D., Vijyn, M.M., Slinity cclimtion ffects the somtotropic xis in rinow trout. Am. J. Physiol. 288, R Shimizu, M., Swnson, P., Fukd, H., Hr, A., Dickhoff, W.W., Comprison of extrction methods nd ssy vlidtion for slmon insulin-like growth fctor-i using commercilly ville components. Gen. Comp. Endocrinol. 119, Shimizu, M., Swnson, P., Hr, A., Dickhoff, W.W., Purifiction of 41-kD insulin-like growth fctor inding protein from serum of chinook slmon, Oncorhynchus tshwytsch. Gen. Comp. Endocrinol. 132, Shimizu, M., Hr, A., Dickhoff, W.W., Development of n RIA for slmon 41 kd IGF inding protein. J. Endocrinol. 178, Sihrth, K., Nishiok, R.S., Mdsen, S.S., Bern, H.A., Regultion of IGF-inding proteins y growth hormone in the striped ss, Morone sxtilis. Mol. Mr. Biol. Biotech. 4, Smith, M.A.K., Thorpe, A., Nitrogen metolism nd trophic input in reltion to growth in freshwter nd sewter Slmo girdneri. Biol. Bull. 150, Sohm, F., Mnfroid, I., Pezet, A., Rentier-Delrue, F., Rnd-Wever, M., Kelly, P.A., Bœuf, G., Postel-Viny, M.C., de Luze, A., Edery, M., Identifiction nd modultion of growth hormone-inding protein in rinow trout (Oncorhynchus

23 mykiss) plsm during sewter dpttion. Gen. Comp. Endocrinol. 111, Uchid, K., Kjimur, S., Riley, L.G., Hirno, T., Aid, K., Gru, E.G., Effects of fsting on growth hormone/insulin-like growth fctor I xis in the tilpi, Oreochromis mossmicus. Comp. Biochem. Physiol. 134A, Usher, M.L., Tlot, C., Eddy, F.B., Effects of trnsfer to sewter on growth nd feeding in Atlntic slmon smolts (Slmo slr L.). Aquculture 94,

24 Figure legends Fig. 1 Chnges in liver GHR mrna, plsm IGF-I nd 41-kD IGFBP levels fter trnsfer to 1/2 SW. Postsmolt coho slmon rered in FW were directly trnsferred to either FW or 1/2 SW. GHR mrna ws quntified y rel-time RT-PCR using cidic riosoml phospoprotein P0 (ARP) s n internl control. Plsm levels of IGF-I nd 41-kD IGFBP were mesured y rdioimmunossys. Vlues re men ± SEM (n = 6). For sttisticl nlysis, vlues were nturl-log trnsformed. Asterisks indicte significnt difference etween FW nd 1/2 SW for given time point (Fisher s PLSD, P < 0.05) Fig. 2 Effect of GH dministrtion on liver GHR mrna levels in postsmolts in FW nd 1/2 SW. Fish were cclimted in FW or 1/2 SW for two dys nd injected once with 2 µg/g ody weight porcine GH () or 8 µg/g (). Shm fish received sline only. GHR mrna ws quntified y rel-time RT-PCR using cidic riosoml phospoprotein P0 (ARP) s n internl control. Vlues re men ± SEM (n = 6). For sttisticl nlysis, vlues were nturl-log trnsformed. Symols shring the sme letters re not significntly different from ech other for given time point (Fisher s PLSD, P < 0.05) Fig. 3 Effect of GH dministrtion on plsm IGF-I levels in postsmolts in FW nd 1/2 SW. Fish were cclimted in FW or 1/2 SW for two dys nd injected once with 2 µg/g ody weight porcine GH () or 8 µg/g (). Shm fish received sline only. Plsm levels of

25 IGF-I were mesured y rdioimmunossy. Vlues re men ± SEM (n = 6). For sttisticl nlysis, vlues were nturl-log trnsformed. Symols shring the sme letters re not significntly different from ech other for given time point (Fisher s PLSD, P < 0.05) Fig. 4 Effect of GH dministrtion on plsm 41-kD IGFBP levels in postsmolts in FW nd 1/2 SW. Fish were cclimted in FW or 1/2 SW for two dys nd injected once with 2 µg/g ody weight porcine GH () or 8 µg/g (). Shm fish received sline only. Plsm levels of 41-kD IGFBP were mesured y rdioimmunossy. Vlues re men ± SEM (n = 6). For sttisticl nlysis, vlues were nturl-log trnsformed. Symols shring the sme letters re not significntly different from ech other for given time point (Fisher s PLSD, P < 0.05) Fig. 5 Disppernce of injected porcine GH from the circultion of postsmolts in FW nd 1/2 SW Fish were cclimted in FW or 1/2 SW for two dys nd injected once with 2 µg/g ody weight porcine GH () or 8 µg/g (). Plsm levels of porcine GH were mesured y specific rdioimmunossy. Vlues re men ± SEM (n = 6). For sttisticl nlysis, vlues were nturl-log trnsformed. Symols shring the sme letters re not significntly different from ech other for given time point (Fisher s PLSD, P < 0.05).

26 1.5 () * FW 1/2 SW 1.0 GHR/ARP () FW 1/2 SW IGF-I (ng/ml) (c) * FW 1/2 SW K BP (ng/ml) Dys fter trnsfer Shimizu et l., Fig. 1

27 1.5 () c c 1.0 GHR/ARP 0.5 GH dose Medium FW SW FW SW FW SW Dys fter trnsfer (injection) (1) (2) (3) 1.5 () d GHR/ARP c GH dose Medium FW SW FW SW FW SW Dys fter trnsfer (injection) (1) (2) (3) Shimizu et l., Fig. 2

28 120 () IGF-I (ng/ml) c 20 GH dose Medium FW SW FW SW FW SW Dys fter trnsfer (injection) (1) (2) (3) () c IGF-I (ng/ml) c c 20 GH dose Medium FW SW FW SW FW SW Dys fter trnsfer (injection) (1) (2) (3) Shimizu et l., Fig. 3

29 400 () K BP (ng/ml) GH dose Medium FW SW FW SW FW SW Dys fter trnsfer (injection) (1) (2) (3) 400 () c K BP (ng/ml) GH dose Medium FW SW FW SW FW SW Dys fter trnsfer (injection) (1) (2) (3) Shimizu et l., Fig. 4

30 800 d 600 pgh (ng/ml) 400 c 200 d c c 0 GH dose Medium FW SW FW SW FW SW Dys fter trnsfer (injection) (1) (2) (3) Shimizu et l., Fig. 5