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A peak in gh-receptor expression is associated with growth activation in Atlantic salmon vertebrae, while upregulation of igf-I receptor expression is related to increased bone density

A peak in gh-receptor expression is associated with growth activation in Atlantic salmon vertebrae, while upregulation of igf-I receptor expression is related to increased bone density
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  General and Comparative Endocrinology 142 (2005) 163–$ - see front matter ©  2005 Elsevier Inc. All rights reserved.doi:10.1016/j.ygcen.2004.12.005 A peak in  gh-receptor  expression is associated with growth activation in Atlantic salmon vertebrae, while upregulation of igf-I receptor  expression is related to increased bone density Anna Wargelius a, ¤ , Per-Gunnar Fjelldal a , Susana Benedet b , Tom Hansen a , Björn Thrandur Björnsson a,b , Ulla Nordgarden a a Institute of Marine Research, Matre, N-5984 Matredal, Norway b Fish Endocrinology Laboratory, Department of Zoology/Zoophysiology, Göteborg University, Box 463, S-405 30 Göteborg, Sweden Received 23 September 2004; accepted 14 December 2004Available online 22 January 2005 Abstract Growth hormone (GH) and insulin-like growth factor-I (IGF-I) play major roles in the endocrine regulation of W sh growth, buttheir interdependency and mode of action has not been well elucidated. The GH–IGF-I system is essential for normal vertebralgrowth in mouse, but this has not been studied in W sh. To study the interplay between GH, IGF-I, and their receptors, postsmoltAtlantic salmon were studied during spring growth (January–June 2003). From January to June, W sh were sampled regularly forplasma and vertebral bone. The vertebra was collected from the same anterior–posterior position. The  growth hormone receptor  (  ghr )(There is no determined nomenclature of salmon genes but we stick to the nomenclature which is consequent for zebra W sh, where allgene names are named with small letters and in italic.) expression in the vertebrae peaked in the end of February coinciding with highlevels of plasma GH and IGF-I, and an increase of vertebral growth rate. From April to June, plasma IGF-I levels decreasedtogether with  ghr  expression in the vertebrae, while plasma GH did not decrease. In May and June, expression of the igf-I receptor ( igf  - Ir ) increased 4- to 5-fold, which coincided with an increase in bone density. The changes seen in gene expression of the IGF-I andGH receptors suggest that these hormones are involved in vertebral growth and bone density. ©  2005 Elsevier Inc. All rights reserved. Keywords: Atlantic salmon; Salmo salar ; IGF-I receptor; GH receptor; Osteoblast; Vertebrae; Growth 1. Introduction Growth hormone (GH) and insulin-like growth fac-tor-I (IGF-I) are hormones essential for skeletal growthin mammals (Daughaday, 2000; Ohlsson et al., 1998). In W sh, gill cartilage responds to GH and IGF-I in vivo byincreasing 35 S uptake (Cheng and Chen, 1995; Duan andInui, 1990; Gelsleichter and Musick, 1999; Marchantand Moroz, 1993; Moriyama et al., 1995; Tsai et al.,1994) and pharyngeal bone in rainbow trout increases itsturnover after GH administration (Takagi et al., 1992). GH and IGF-I regulate bone size and mass in mouse(Daughaday, 2000; Ohlsson et al., 1998). In W sh, thee V  ect of GH on bone may be seen in GH transgenic cohosalmon, which display a high degree of bony tissue mal-formations (Devlin et al., 1995). Furthermore, in W sh GHacts indirectly by stimulating IGF-I production andsecretion from the liver (Duan and Plisetskaya, 1993;Duguay et al., 1994). In mammals, GH also stimulatesIGF-I production in osteoblasts, and recently it has beenshown that IGF-I stimulation increases bone formationrate in mouse osteoblasts (Zhang et al., 2002). ¤ Corresponding author. Fax: +47 55236379. E-mail address: (A. Wargelius).  164 A. Wargelius et al. / General and Comparative Endocrinology 142 (2005) 163–168 The tissue-speci W c actions of plasma GH and IGF-Iare mediated through membrane-bound receptors; thegrowth hormone receptor (GHR) and the insulin-likegrowth factor type I receptor (IGF-IR) (Daughaday,2000). GHR and IGF-IR mRNA transcripts have beenidenti W ed in several W sh species. For GHR, these speciesinclude gold W sh (Lee et al., 2001), turbot (Calduch-Giner et al., 2001), black seabream (Tse et al., 2003), gilthead sea bream (Calduch-Giner et al., 2003), tilapia (Kajim- ura et al., 2004), coho salmon (NCBI Accession Nos.AF403539 and AF403540), masu salmon (AB071216),Japanese X ounder (AB058418), grass carp (AY283778),Southern cat W sh (AY336104), Japanese eel (AB180476and AB180477), common carp (AAT92555), and Atlan-tic salmon (Accession No. AY462105). Two isoforms of the IGF-IR have been identi W ed in coho salmon; SIR-5 and SIR-6   (Chan et al., 1997), and in rainbow trout; IGFRIa  and IGFRIb  (Greene and Chen, 1999), and oneisoform in Atlantic salmon; igfIr  (NCBI Acc. No.AY049954). The receptors are expressed at di V  erent lev-els in many tissues including gill cartilage (Chan et al.,1997; Greene and Chen, 1999; Nakao et al., 2002). So far,no studies on W sh have been carried out on the geneexpression of GHR and IGF-IR in the vertebrae duringgrowth in W sh.To further understand the endocrine regulation of vertebral growth, the  ghr 1  and igf  - Ir  expression was stud-ied in the vertebrae in relation to plasma levels of GHand IGF-I during spring growth in postsmolt Atlanticsalmon. 2. Materials and methods  2.1. Fish stock and rearing conditions In December 2002, 900 underyearling Atlanticsalmon ( Salmo salar  L.) smolts, with an average weightof 75g, were transferred from outdoor freshwater facili-ties for distribution among three seawater cages(5 £ 5 £ 8m) at the Institute of Marine Research, Matre(61°N, western Norway). One hundred W fty W sh in eachcage were individually PIT-tagged (Trovan tag ID100/01, LID 500 Hand Held Reader, Trovan, BTS Scandina-via AB). The ambient water temperature during thestudy changed from a minimum of 4.0°C in January to amaximum of 10.5°C in June. The salinity varied withdepth: from 17.2 § 3.2ppm at 1m to 31.1 § 0.9ppm at5m. The W sh were fed Bio-Optimal dry feed (BioMar,Trondheim, Norway) in excess, using a computer-oper-ated feeding system (ARE, Storebø, Norway). Three pel-let sizes (3, 4, and 6mm) were used as the W sh grewthroughout the experiment. To control sea lice infesta-tion, the W sh were given SLICE at a dose of 0.5% of bio-mass per day (Schering-Plough AS, Farum, Denmark)for 1 week in the middle of the experimental period.  2.2. Experimental setup, tissue sampling, and growth measurements The experimental period was from January to June2003. First sampling was carried out on January 13th,followed by samplings after 3, 6, 10, 15, and 22 weeks. Ateach sampling, all individually tagged W sh were anesthe-tized and measured for weight and length. Additionally,10 W sh from each cage were anesthetized with metomi-date hydrochloride (Wild-life Pharmaceuticals, CO,USA) according to Olsen et al. (1995), and killed with a blow to the head, before sampling of blood (10 W sh) andvertebral tissue (6 W sh). The fortieths vertebrae, found inthe vicinity of the adipose W n, were carefully dissectedout and immediately frozen on liquid nitrogen for lateranalysis of gene expression through real time PCR.Dorso-ventral diameter, lateral diameter, and length of the vertebrae were measured at each sampling ( n D 5).Blood was collected from the caudal vessels, using a hep-arinized syringe, plasma was obtained by centrifugationat 3000  g  , and stored at ¡ 80°C until analyses.  2.3. Bone density The vertebrae was defatted in hexane, dried overnightat 90°C, and weighed (Kacem et al., 2000). The volume of the vertebrae was estimated as (  £ ((dorso-ventraldiameter+lateral diameter)/4) 2 )) £ length.  2.4. Radioimmunoassays Plasma levels of GH (Björnsson et al., 1994) and IGF-I (Moriyama et al., 1994) were measured using homologous salmonid radioimmunoassays validated forAtlantic salmon.  2.5. Real-time PCR Total RNA was extracted from vertebral tissue usingFastRNA Pro Green Kit (Qbiogene). RNA was DNAsetreated (Promega, 37°C for 30min) and then extractedonce more with phenol, pH 4.5. First strand cDNA wasreversely transcribed using a Reverse Transcription CoreKit (RT-RTCK-05, Eurogenetec) using 500ng RNA.For ampli W cation of igf  - Ir  (Acc. No. AY049954),  ghr (Accession No. AY462105), and elongation -  factor alfaela , Acc. No. AF321836), the following PCR primerswere used; 5  -TGAAGAGCCACCTGAGGTCACT-3  and 5  -TCAGAGGTGGGAGGTTGAGACT-3   for igf  - Ir ; 5  -TGGGAAGTTGAGTGCCAGACT-3   and5  -CACAAGACTACTGTCCTCCGTTGA-3   for  ghr ; 1 Side: 2 Thereis no determinednomenclature of salmon genes but we stick to the nomenclature which is consequent for zebra W sh, whereall gene names are named with small letters and in italic.  A. Wargelius et al. / General and Comparative Endocrinology 142 (2005) 163–168 165 and 5  -CCCCTCCAGGACGTTTACAAA-3   and 5  -CACACGGCCCACAGGTACA-3   for ela . Primerswere tested using conventional PCR and shown toamplify a single band of approximately 80bp. Real-timePCR was carried out on an ABI 7700 system. The fol-lowing Taqman probes were designed for igf  - Ir  (6-FAM-CGGGCTAAAGACCCGTCCCAGTCC),  ghr (6-FAM-TGGGAGAGCCAGCCAGCCTGC), and ela (6-FAM-ATCGGTGGTATTGGA). Thermal cyclingconditions were 50°C for 2min followed by 98°C for10min. Subsequently the reactions proceeded through40 cycles of 95°C for 15s followed by 60°C for 1min.Each reaction (25  l) contained 5  l cDNA diluted 1:1 indd H 2 O, 12.5  l Taqman Universal PCR master mix(ABI), and 0.9  M F/R primers. Each sample was run intriplicate and each stage contained six RNA replicas(samples from six di V  erent W sh). The e Y ciency of targets( igf  - Ir/grh ) in relation to reference ( ela ) was determinedusing a standard curve method together with a valida-tion experiment (ABI User Bulletin #2 for ABI 7700sequence detections system). In the validation experi-ment, 500, 250, and 125ng RNA were used for cDNAsynthesis and the slope of log input amount of RNA ver-sus  C  t  was 0.06 for  ghr/ela  and 0.06 for igf  - Ir/ela , whichis <0.1, which demonstrates that the e Y ciency betweentarget and reference is approximately equal. The relativeexpression level was calculated using the Comparative C  t  method (ABI User Bulletin #2 for ABI 7700 sequencedetection system). In all experiments no-template con-trols were run together with the samples.  2.6. Statistics Changes in length, dorso-ventral and lateral diameterof the fortieth vertebrae, plasma IGF-I levels, and bone igf  - Ir  expression were tested using one-way ANOVA,followed by post-hoc testing (Tukey’s HSD test forequal or unequal sample sizes). Bone density data fromFebruary to June were subjected to a simple regressionwith time as the predictor variable. As the plasma GHand bone  ghr  expression data were not normally distrib-uted, changes in plasma GH levels during the experimentwere analyzed by nonparametric statistics; Kruskal– Wallis ANOVA by ranks, followed by post-hoc testing(Kruskal–Wallis multiple comparisons of mean rankstest). Data analysis was performed using the Statistica6.1 (StatSoft, Tulsa, OK, USA) software package. 3. Results 3.1. Vertebral growth and bone density The size of the vertebrae increased throughout theexperiment, in which the lateral diameter signi W cantlyincreased in late February and length and dorso-ventraldiameter increased signi W cantly in late March (Fig. 1). Asimple regression analysis, excluding the January sam-ple, detected a signi W cant increase in bone density overtime (  p <0.04, r 2 D 0.17, Fig. 2). 3.2. Vertebral tissue expression of ghr and igf-Ir in relation to plasma GH and IGF-I levels Plasma levels of GH and  ghr  expression in the verte-bral body are shown in Fig. 3. A signi W cant (  p <0.05)GH plasma peak at the end of February coincided witha signi W cant (  p <0.05) increase in  ghr  expression in bone.The continuous high levels of GH (March–June) coin-cided with a signi W cantly (  p <0.05) lowered expression of the  ghr  (May and June) in bone.Plasma levels of IGF-I and expression of igf  - Ir  in ver-tebral tissue are shown in Fig. 4. IGF-I plasma levels reached a signi W cant peak (  p <0.05) in the beginning of February and decreased to the initial level in April. InMay–June, IGF-I plasma levels remained stable, while Fig. 1. Length, dorso-ventral diameter, and lateral diameter of verte-brae number 40 measured at each sampling (mm, mean § SE, n D 5).Fig. 2. Bone density of vertebrae number 40 measured at each sam-pling (mg (mm 3 ) ¡ 1 , mean § SE, n D 5).  166 A. Wargelius et al. / General and Comparative Endocrinology 142 (2005) 163–168 transcripts for its cognate receptor in the vertebral tissueincreased signi W cantly (  p <0.05). 4. Discussion Growth of Atlantic salmon is greatly in X uenced byenvironmental changes in temperature, and photoperiod(Eriksson and Lundqvist, 1982). Also dynamic changesin the GH/IGF-I axis has been observed in response totemperature and photperiod manipulation, in several W sh species (Gabillard et al., 2003; Larsen et al., 2001;Mingarro et al., 2002). In line with this, we see in thisstudy dynamic changes in gene expression of GH–IGF-Ireceptors, in relation to size and bone density of the ver-tebrae.One of the most important W ndings in the presentstudy is that plasma GH probably has a role in growthactivation in bony tissues, evaluated from the increase in  ghr  in response to a plasma peak of GH. However,  ghr expression signi W cantly decreases after an initialresponse to GH, even though plasma concentrations of GH are maintained at a high level throughout the exper-imental period. This may be the reason why GH in vitroshows little (Cheng and Chen, 1995) or no e V  ect on pro-teoglycan incorporation in gill cartilage in W sh (Duan andInui, 1990; Gray and Kelley, 1991), since GH might havean initial role in inducing growth, but might need otherendocrine factors, such as IGF-I to be able to initiatematrix production. This is further in line with bonegrowth regulation in mice, where IGF-I ¡ / ¡  mice exhibitgreater impairment in bone accretion than mice de W cientin GH (Mohan et al., 2003). The hypothesis that IGF-I isimportant in bone matrix production is strengthened bythe present study, where a signi W cant increase in IGF-IRgene expression was followed by a signi W cant increase inbone density the following month. Interestingly, theincrease in gene expression of IGF-I receptor and bonedensity occurred together with a signi W cantly lowered  ghr expression. These results are in line with studies in mouse,where overexpression of IGF-I is found to result inincreased trabecular bone formation without increasedosteoblast proliferation (Zhao et al., 2000) and disruptionof IGF-I signaling in osteoblasts signi W cantly reducesbone formation rate (Zhang et al., 2002). The decrease in  ghr  expression could be explained by the fact that IGF-Ican mediate increased bone density independently of GH.These results suggest that growth regulation in the verte-bral bone of W sh is similar to that in mammals. If this isthe case, then initial growth phase (March–April) wouldbe characterized by cell recruitment shown in this studyby low bone density. The next phase, acellular bonerecruitment phase (May and June) would be character-ized by low proliferation and increased matrix produc-tion, which is evident in this study as bone density and igf  - Ir  expression increase.Interestingly, the igf  - Ir  upregulation seen in the verte-bral body in the present study was not seen in muscle tis-sue (Ulla Nordgarden, pers. communication), which caneither be due to the tetraploidy of salmon, or alternatively,growth is regulated through di V  erent mechanisms in mus-cle tissue. Di V  erential expression of homologous genes isreported in rainbow trout, where the homologous gene igf  - Irb  shows a steady state expression in response to feed-ing after a time of starvation, whereas the other isoform, igf  - Ira  showed a decrease in expression (Chauvigne et al.,2003). On the other hand, previous studies show tissue-speci W c IGF-I a Y nity at the receptor level. Red and car-diac muscle tissue decrease their a Y nity for IGF-I whenplasma IGF-I levels increase (Banos et al., 1997; Moon etal., 1996), whereas trout adipose and skeletal muscle tissueincrease their a Y nity for IGF-I when plasma levelsincrease (Parrizas et al., 1995; Planas et al., 2000).In the present study, peak levels of IGF-I in plasmawere observed before peak GH levels. This could be dueto initial GH binding to hepatic receptors, thus loweringthe plasma levels of available hormone (Björnsson,2002), followed by receptor saturation and consequently Fig. 3. Plasma levels of GH (mean § SE, n D 30) and tissue-speci W cgene expression of  ghr  (mean § SE, n D 6) in vertebral bone (relativeexpression). The relative expression of  ghr  was normalized to ela .Fig. 4. Plasma levels of IGF-I (mean § SE, n D 30) and tissue-speci W cgene expression of igf  - Ir  (mean § SE, n D 6) in vertebral bone (relativeexpression). The relative expression of  ghr  was normalized to ela .  A. Wargelius et al. / General and Comparative Endocrinology 142 (2005) 163–168 167 increased plasma GH levels. The following decline inplasma IGF-I may in part be due to receptor binding, asthe IGF-IR gene expression was upregulated in the ver-tebrae, or it could be due to a phenomenon called GHresistance where high levels of GH decrease IGF-Iexpression in salmon hepatocyte culture (Pierce et al.,2004) and IGF-I levels in plasma (Duan et al., 1995). Earlier studies show that liver-derived IGF-I a V  ectsmouse bone growth while it does not a V  ect vertebral(trabecular bone) bone growth (Sjögren et al., 2002).Furthermore, the IGF ¡ / ¡  (IGF knock out) mouse dis-plays a signi W cantly reduced axial skeleton (Daughaday,2000). These data suggest that osteoblasts of the axialskeleton rely mostly on locally produced IGF-I. Unfor-tunately, in the present study, local transcription of IGF-I was not measured within the vertebrae. However, theupregulation of igf  - Ir  expression suggests that the IGF-Iexpression might have been locally induced, as plasmalevels of IGF-I did not change. Also, studies in mousehave shown that locally produced IGF-I is crucial forvertebral growth (Lupu et al., 2001; Reinecke et al., 2000;Sjögren et al., 1999; Yakar et al., 1999).In conclusion, the growth activation in the vertebrae of Atlantic salmon is characterized by a signi W cant increasein  ghr  gene expression followed by increased growth rate.Two months after growth activation, an increased igf  - Ir expression and a signi W cantly reduced  ghr  gene expressionoccurs together with increased bone density. While a peakin plasma GH levels coincides with increased  ghr  geneexpression, the increase in igf  - Ir  expression in the verte-brae is not re X ected in plasma level of IGF, suggestingthat plasma GH has an e V  ect on its receptor within thevertebrae, while locally synthesized IGF-I could be medi-ating most of the IGF-I signaling in the vertebrae. Acknowledgments The authors thank Barbro Egnér for radioimmunoas-say analysis of plasma GH and IGF-I levels. This workhas been carried out with W nancial support from Norwe-gian research council (NFR) and the Swedish ResearchCouncil for Environment, Agricultural Sciences, andSpatial Planning (FORMAS). References Banos, N., Moon, T.W., Castejon, C., Gutierrez, J., Navarro, I., 1997.Insulin and insulin-like growth factor-I (IGF-I) binding in W sh redmuscle: regulation by high insulin levels. Regul. Pept. 68, 181–187.Björnsson, B.Th., Taranger, G.L., Hansen, T., Stefansson, S.O., Haux,C., 1994. 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