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General and Comparative Endocrinology 150 (2007) 355–363

Communication in Genomics and Proteomics

Isolation of Atlantic halibut pituitary hormones by continuous-elutionelectrophoresis followed by fingerprint identification, and assessment

of growth hormone content during larval development

Ingibjorg Eir Einarsdottir a,*, Liliana Anjos b, Jon Hildahl a,Bjorn Thrandur Bjornsson a, Deborah M. Power b

a Fish Endocrinology Laboratory, Department of Zoology/Zoophysiology, Goteborg University, Box 463, S-40530 Goteborg, Swedenb Centro de Ciencias do Mar, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal

Received 12 April 2006; revised 22 September 2006; accepted 27 September 2006Available online 13 November 2006

Abstract

Continuous-elution electrophoresis (CEE) has been applied to separate putative hormones from adult Atlantic halibut pitui-taries. Soluble proteins were separated by size and charge on Model 491 Prep Cell (Bio-Rad), where the homogenate runsthrough a cylindrical gel, and protein fractions are collected as they elute from the matrix. Protein fractions were assessedby SDS–PAGE and found to contain purified proteins of molecular size from 10 to 33 kDa. Fractions containing proteins withmolecular weights of approximately 21, 24, 28 and 32 kDa, were identified as putative growth hormone (GH), prolactin,somatolactin and gonadotropins, respectively. These were analyzed further by mass spectrometry and identified with peptidemass protein fingerprinting. The CEE technique was used successfully for purification of halibut GH with a 5% yield, andappears generally well suited to purify species-specific proteins often needed for research in comparative endocrinology, includingimmunoassay work. Thus, the GH obtained was subsequently used as standards and iodination label in a homologous radio-immunoassay, applied to analyze GH content through larval development in normally and abnormally metamorphosing larvae.As GH is mainly found in the pituitary, GH contents were analyzed in tissue extracts from the heads only. The pituitary GHcontent increases proportionally to increased larval weight from first feeding to metamorphic climax. No difference in relativeGH content was found between normal and abnormal larvae and it still remains to be established if GH has a direct rolein metamorphosis.� 2006 Elsevier Inc. All rights reserved.

Keywords: Flatfish; Hippoglossus hippoglossus; Growth hormone; Prolactin; Somatolactin; Fingerprinting; Larval development

1. Introduction

The Atlantic halibut (Hippoglossus hippoglossus) is thelargest flatfish species and is aquacultured around theNorth Atlantic. As other flatfish, the halibut undergoesa dramatic metamorphosis including extensive structural,functional and behavioral restructuring. Research intothe endocrine control of flatfish metamorphosis has

0016-6480/$ - see front matter � 2006 Elsevier Inc. All rights reserved.

doi:10.1016/j.ygcen.2006.09.010

* Corresponding author. Fax: +46 31 773 3807.E-mail address: ingibjorg.einarsdottir@zool.gu.se (I.E. Einarsdottir).

focused on the thyroid hormones which, appear to playan important role in this process (Miwa and Inui, 1991;Miwa et al., 1992; Yamano et al., 1994; Hiroi et al.,1997; Huang et al., 1998; Schreiber and Specker,1999a,b), while the role of pituitary hormones is unclear.In early stages of the Atlantic halibut, growth hormone(GH) producing somatotrophs, prolactin (PRL) produc-ing lactotrophs, somatolactin (SL) producing somatolac-totrophs and thyroid stimulating hormone (TSH)producing thyrotrophs are all evident soon after yolk sackabsorption and appear active (Einarsdottir et al., 2006).

356 I.E. Einarsdottir et al. / General and Comparative Endocrinology 150 (2007) 355–363

TSH stimulates the secretion of thyroxine (T4) andtriiodothyronine (T3), from thyroid follicles and these hor-mones, in turn, are the main regulatory hormones of theonset and development of metamorphosis in teleost fishand amphibia (Tata, 2000; Power et al., 2001), althoughtheir role in this process in cyclostomes is still poorlydefined and requires further studies (Youson and Sower,2001). The role of other pituitary hormones in fish meta-morphosis remains to be defined, and the general lack ofspecies-specific peptide hormones is a major barrier toprogress.

All pituitary hormones have been demonstrated in larval(Einarsdottir et al., 2006) and adult (Weltzien et al., 2003a)Atlantic halibut by immunohistochemistry. SL and GHhave been isolated by gel filtration chromatography andreverse phase high performance liquid chromatography(rpHPLC) and characterized on the basis of molecular size,N-terminal amino acid sequence (for SL) and immunoreac-tivity (Johnson et al., 1997; Einarsdottir et al., 2002). Plas-ma levels of GH (Einarsdottir et al., 2002) and SL(Johnson et al., 1997) have subsequently been quantifiedin adult fish with species-specific radioimmunoassays. ThecDNA sequences and deduced amino acid sequences forthe FSH-b subunit, LH-b protein and gonandotropin a-subunit are known (Weltzien et al., 2003b) and the nativeproteins have been isolated from the pituitaries and charac-terized (Weltzien et al., 2003c).

Isolation of native hormones using conventional purifi-cation methods, such as gel filtration chromatographyand rpHPLC have been successfully used for many years.However, such methods require considerable expertiseand specialized equipment. They generally require largequantities of starting material involve numerous chroma-tography steps, and step-wise losses leading to relativelylow yield. Also, the acidified acetonitrile used in rpHPLCfrequently denatures or destabilizes the purified protein.

Proteomic methods for protein sequencing and identifi-cation is a valuable supplement to molecular approaches,as they confirm the amino acid sequence deduced fromcDNA and may also reveal important post-translationalmodifications. The tertiary structure of the pituitaryhormones, including disulphide bonds and subunit struc-tures, makes their production as recombinant proteinsoften difficult. The availability of a simple, single-step,high-yielding method for isolation of pituitary hormones(and other peptide hormones) from biological materialwould be an important innovation in the field of compara-tive endocrinology.

The aim of the study was to establish a stable source forhalibut GH to be used in a radioimmunoassay, by usingnovel methodology, a single-step continuous-elution elec-trophoresis (CEE). The GH was then used to improve anestablished halibut GH radioimmunoassay (Einarsdottiret al., 2002), and applied to series of normally and abnor-mally metamorphosing larvae, in order to elucidate the roleof GH in the endocrine control of the metamorphicprocess.

2. Materials and methods

2.1. Sampling

2.1.1. Pituitaries

The Atlantic halibut adults and larvae were raised by a commercialhalibut producer, Fiskey Ltd., Iceland. Pituitaries were sampled fromadult fish at Fiskey’s on-growth site in Thorlakshofn, southern Iceland.Fish in the range of 3–5 kg body weight were slaughtered and decapitatedfor commercial sale. The heads were kept on ice until the pituitary was dis-sected out, frozen on dry ice and stored for a year at �80 �C until analysis.

2.1.2. Larvae

Larvae were raised under standard commercial conditions, asdescribed by Einarsdottir et al. (2006). They were sampled with a dip-net at regular intervals from start of first feeding to end of metamorpho-sis. At sampling, myotome height of all larvae was measured to deter-mine their developmental stage (Saele et al., 2004). The study coversstages 5 (first feeding), 6 and 7 (premetamorphosis), 8 (start of metamor-phosis), 9 (metamorphic climax) and 10 (completed metamorphosis andsettlement). In the premetamorphic stages 5–7, only a single normal phe-notype can be detected. However, from stage 8 onwards, in addition tothe normal phenotype, an abnormal phenotype is found, primarily basedon incomplete eye migration, but also abnormal pigmentation. Thus, forstages 8, 9 and 10, normal and abnormal larvae were pooled separately.Larvae were pooled to make up five samples for each stage, with thenumber of larvae per pool decreasing at each stage as body massincreased. The number of larvae pooled per sample were as follows:25–28 for stage 5; 25 for stage 6; 13 and 14 for stage 7; 9 and 10 for stage8; 4 and 5 for stage 9; and 3 and 4 larvae for stage 10. For each stage, fivesamples were analyzed for GH. GH was measured in the heads andassumed to mainly represent the pituitary GH content. The pooled larvaewere counted and weighted, decapitated and head weight obtained imme-diately before extraction for GH determination carried out as describedbelow. Mean larval and head weights were calculated. Based on rate ofdevelopment and larval weight (W), specific growth rate for weight(SGRW) was calculated between stages, using the equationSRGW ¼ ½logðW 2 �W �1

1 Þ�ðd2 � d1Þ�1 where d indicates the number ofdays post start-feeding (DPSF; Einarsdottir et al., 2006) at the middleof a developmental stage. (d2 � d1) is thus the number of days betweendevelopmental stages and ðW 2 � W �1

1 Þ is the ratio between the larvalweights of two developmental stages.

2.2. Tissue processing

2.2.1. Pituitary extract preparation

About 0.4 g halibut pituitaries were homogenized in 2 ml 0.05 M Tris-base pH 8.6 with a manual glass–glass homogenizer and incubated on icefor 2 h. The homogenate was centrifuged at 16,000g for 20 min at 4 �C andthe supernatant collected and analyzed by SDS–PAGE (Laemmli, 1970)using a 12% polyacrylamide gel. The success of the protein extractionwas evaluated by Coomassie blue R staining of SDS–PAGE gels(Fig. 1A, lane 5), and Western blot analysis (data not shown), usinganti-halibut GH (1:30,000, tested in RIA; Einarsdottir et al., 2006). Thisconfirmed that pituitary hormones had been successfully extracted.

2.2.2. Larval extract preparation

The heads were homogenized on ice with a manual glass/glasshomogenizer in Tris–HCl, 0.05 M pH 8.6, 1 mM PMSF, with a larvae:buffer ratio of 1:5 (w/v). The homogenate was incubated for 1 h on icebefore it was spun at 14,000 g, at 4 �C for 1 h, after which the superna-tant was collected. In order to eliminate large, interfering molecules, thesupernatant was spun at 4 �C at 4000 g for 2–3 h in Ultrafree CL (Ami-con) with a molecular weight cut-off (MWCO) of 30 kDa. The filtratewas then concentrated by spinning the samples in Ultrafree CL (Amicon)with a MWCO of 5 kDa at 4000 g for 30 min at 4 �C. The volumes ofthe supernatants were noted and the GH content analyzed by RIA(see below).

16

24

35

kDa M 18 17 16 15 14 13

A

B

5

10

16

24

35

5473

M 1 2 3 4kDa

Fig. 1. (A) Coomassie blue staining of the PrepCell 491 fractionscontaining putative pituitary hormones separated by analytical SDS–PAGE (12%) under reducing condition. Lane 1, putative GH; lane 2,putative PRL; lane 3, putative GTH; lane 4 putative SL. The proteins wereapproximately 21, 24, 31 and 28 kDa. The isolated proteins were excisedfrom the gel and analyzed by MALDI-TOF MS spectrophotometry. Lane5, Coomassie blue staining of pituitary extract separated by analyticalSDS–PAGE (12%) under reducing condition. All lanes marked showmolecular weight markers with size indicated. (B) Western blot of PrepCell491 fractions using anti-GH. Lanes labeled 13–18 indicate fractionnumbers.

I.E. Einarsdottir et al. / General and Comparative Endocrinology 150 (2007) 355–363 357

2.3. Continuous-elution electrophoresis of pituitary extract

Purification and analytical separation of proteins from pituitaryextracts was carried out by preparative SDS–PAGE under denaturingcondition (Laemmli, 1970), with CEE using a Model 491 Prep Cell�

(Bio-Rad). In this method, the sample moves through a cylindrical gelwhere proteins separate into ring-shaped bands. Individual bands migrateaccording to size to the bottom of the gel into a collection chamber fromwhere they are pumped into an external fraction collector (Valkonen et al.,2001). The resolving gel was composed of 8% polyacrylamide in 0.375 MTris–HCl, pH 8.8. The stacking gel was 4% polyacrylamide in 0.125 MTris–HCl, pH 6.8. The dimensions (height · diameter, mm) of the resolv-ing gel and the stacking gel were approximately 65 · 28 mm and30 · 28 mm, respectively. Electrophoresis was carried out using 0.025 MTris–HCl; 0.19 M glycine; 0.1% SDS, pH 8.3 as the running buffer andsamples (1 ml of pituitary extract) were mixed with 1· sample buffer(100 mM Tris–HCl, pH 6.8; 200 mM DTT; 4% SDS; 0.2% bromophenolblue; 20% glycerol), heated at 100 �C for 5 min, centrifuged briefly, andrun at a constant power of 12 W over approximately 8 h maintaining aconstant temperature (18 �C). Proteins fractionated on the polyacrylamidegel were allowed to migrate off the end of the gel into the elution chamberand collected as 2.5-ml fractions at 8 �C. Fraction collection was initiatedwhen the ion front (bromophenol blue) had migrated out of the gel.

Every 10th fraction was analyzed by SDS–PAGE (12% polyacrylamidegels) and stained with silver nitrate in order to determine which fractionscontained the proteins of interest. Based on molecular size, putative pitu-itary hormones were estimated to appear within the first 32 fractions and

all these fractions were subsequently analyzed by analytical SDS–PAGEand silver stained. Fractions containing the same putative hormone, givinga single clear band, were pooled. In order to demonstrate that the halibutGH antiserum used, cross-reacts with the isolated GH, fractions contain-ing putative GH were analyzed with Western blot as described above(Fig. 1B) before they were pooled.

2.4. Mass spectrophotometry of purified proteins

The putative GH, PRL, SL and GTH fractions were concentrated withUltrafree-15 centrifugal filter device with a molecular cut-off of 5 kDa(Millipore, Bedford, UK) run on SDS–PAGE (12% polyacrylamide gels)and stained with Coomassie blue R (Fig. 1A, lanes 1–4). Bands containingapproximately 21, 24, 28 and 32 kDa proteins were excised from the geland placed in sterile ddH2O. Protein identification was conducted by theCentro de Genomica y Proteomica (Facultad de Farmacia, UCM, Spain).The proteins were subjected in-gel to trypsin digestion and the molecularweights of the peptides and amino acid sequences were analyzed byMALDI-TOF (Matrix Assisted Laser Desorption Ionization (MALDI)tandem Time-of-Flight (TOF) mass spectrophotometer), using the Post-Source Decay (PSD) technique. Unambiguously identified peptides werefurther examined by MS/MS fragmentation. The resulting amino acidsequences were identified both by peptide mass fingerprinting andMS/MS fragmentation. Database search using the peptide mass finger-print integrated with tandem mass spectrometry (MS/MS) was performedusing the MASCOT program (http://www.matrixscience.com; Perkinset al., 1999).

2.5. Radioimmunoassay

GH was measured in the larval extracts using the radioimmunoassay(RIA) described by Einarsdottir et al. (2002), with the modification thatGH purified by CEE was used for standards and radioactive labeling.The concentration of the GH stock solution was estimated by measuringtotal protein content absorbance at 240k using quartz micro-cuvettes andbovine serum albumin as a standard.

2.6. Statistical analysis

Results are presented as means ± SD. Statistical analysis was per-formed to reveal differences among stages and between phenotypes(normal and abnormal larvae). Comparison among normal larvae at dif-ferent stages (5–10) was carried out using one-way analysis of variance(ANOVA) after assessing the data for normality and homogeneity of var-iance, followed by a Tukey HSD post hoc test. For comparison betweenphenotypes (stages 8–10), a two-way ANOVA was applied. The analysiswas performed using SPSS 14.01 analytical software. Statistical signifi-cances were considered at p < 0.05.

3. Results

3.1. Hormone purification and identification

The hormones GH, PRL and SL were successfullypurified from halibut pituitary extracts (Fig. 1A, lanes 1,2 and 4) by CEE using SDS–PAGE under denaturing con-dition. A putative gonadotropin (GTH) was also isolated(Fig. 1A, lane 3) although in the absence of sequence dataits identity was not confirmed. Fractions 13 and 14 con-tained an approximately 21 kDa protein which was consid-ered to be putative GH. Fractions 15, 16 and 17 containeddecreasing amounts of GH as seen by Western blot(Fig. 1B), and were not included in the GH pool as theSDS–PAGE analysis and silver staining indicated that

358 I.E. Einarsdottir et al. / General and Comparative Endocrinology 150 (2007) 355–363

these fractions might also include other similar-sized pro-teins. The total GH content in the pool (fractions 13 and14) was 0.9 mg. Based on earlier RIA assessment of halibutpituitary GH content (45 mg GH per g pituitary tissue; IEEinarsdottir, unpublished results), the amount of pituitarytissue used (0.4 g) and the estimated total amount of GH inthat tissue (ca 18 mg), the yield of GH from pituitariesusing CEE purification appears to be about 5%.

Fraction 18 contained an approximately 24 kDa pro-tein, considered to be putative PRL. The proteins in frac-tions 23 and 24 were approximately 28 kDa, and thusconsidered to be putative SL, and the proteins in fractions29 and 30 were approximately 32 kDa, and considered tobe putative GTHs.

Table 1 lists the identity of the isolated proteins and thecorresponding molecular mass data of the tryptic peptidesidentified and their amino acid sequence. The respectiveMS fingerprint spectrums are presented in Fig. 2. Thesehave been deposited in database. For halibut GH, withan approximate molecular weight of 21 kDa (Fig. 1A,lane 1) three tryptic peptides were identified by full-scanMS. MASCOT blast search showed that the amino acidsequences had the highest match to the sequence predictedfrom the DNA of Atlantic halibut GH (BAC07253; Fig. 3).Sequence coverage of the protein was 12.8%. The nominalmass (Mr) for the protein was 21,366 Da.

Three tryptic peptides were identified by full-scan MS forthe halibut PRL (Fig. 1A, lane 2). The MASCOT Blastsearch indicated that the amino acid sequence of halibutPRL was most similar to the sequence predicted from thecDNA of PRL 2, in Nile tilapia (Oreochromis niloticus,E968574; Fig. 3). The sequence coverage of the isolated hal-ibut PRL was 21%. The Mr for the protein was 22,453 Da.

Eight tryptic peptides and sequences were obtained forhalibut SL (Fig. 1A, lane 4). The MASCOT blast searchfound that the amino acid sequences obtained for halibut

Table 1Principal characteristics of the halibut pituitary hormones purified by continu

Protein band Protein identified MASCOT Mr

(kDa)Start–End Mr obs

(Da)

Putative GH GH (fragment) 21,3 12–17 968.50Atlantic halibut 89–98 1253.60(Hippoglossus hippoglossus) 99–107 1009.54

Putative PRL TPRL2 22,4 6–22 1954.96Prolactin2 76–91 1758.95Tilapia nilotica 177–186 1189.63(Oreochromis niloticus)

Putative SL SL 27,1 39–50 1445.72Japanese flounder 51–60 1241.71(Paralichthys olivaceus) 83–92 1125.54

93–100 812.49199–208 1274.65209–218 1231.65213–218 780.44224–230 999.41

Molecular weight of proteins and the tryptic peptides generated and their aminoLaser Desorption Ionization (MALDI) tandem Time-of-Flight (TOF) mass sp

SL were most similar to the predicted amino acid sequenceof Japanese flounder (Paralichthys olivaceus) SL cDNA(A35793; Fig. 3). The sequence coverage of halibut SLwas 29%. The Mr for the protein was 27,113 Da.

The putative halibut GTH was subject to tryptic diges-tion, but it was not possible to identify any peptides, prob-ably as a consequence of heavy glycosylation of theprotein.

3.2. Larval growth

Data showing larval weight, ratio between and headbody weight (H:B) as well as SGRW between developmen-tal stages are presented in Table 2 for both normal andabnormal phenotypes. For the normal larvae, weightincreased from 0.026 g at stage 5 to 0.307 g at stage 10(one-way ANOVA; p < 0.001). Overall, the abnormal lar-vae had a lower body weight than normal larvae (two-way ANOVA; p = 0.036), especially at stages 8 and 9.There was no interaction between stage and phenotype(p = 0.314). For the normal larvae, the fastest SGRW wasfound between developmental stages 7 and 8, with a lowerSGRW after the start of metamorphosis (between stages 8and 9).

For the normal larvae, H:B decreased from stage 5 tostage 8, and increased again at stage 10. Overall, the H:Bwas higher for abnormally metamorphosed larvae (two-way ANOVA, p = 0.006), and there was no interactionbetween stage and phenotype (p = 0.173).

3.3. GH content

The immunological similarity of the GH isolated byCEE and GH isolated previously by rpHPLC was com-pared and confirmed in the recently developed RIA(Einarsdottir et al., 2002).

ous electrophoresis and analyzed by MALDI-TOF

. Mr expt.(Da)

Mr calc.(Da)

Delta Miss Peptide sequence

HAITENQR1252.59 1252.61 �0.02 0 LIESWEFSSR1008.53 1008.54 �0.01 0 FLVAGFAER

1953.95 1953.97 �0.01 1 ISGSKLMMVLCVVAMCR1757.94 1757.92 0.03 0 EQALQVSESDLLSLAR1188.62 1188.61 0.00 0 DSHKIDSFLK

1444.72 1444.73 �0.02 1 CPSISQEKLLDR1240.70 1240.69 0.01 0 VIQHAELIYR1124.53 1124.53 0.00 0 NQAGYACITK811.48 811.48 0.00 0 ALPIPSSK

1273.64 1273.64 0.00 1 DYTLLSCFKK1230.64 1230.64 0.00 1 DAHKMEIFLK779.44 779.43 0.01 0 MEIFLK998.41 998.41 �0.01 1 QTDKYNCA

acid sequences. The cleavage products were analyzed on a Matrix Assistedectrophotometer.

766.0 1103.2 1440.4 1777.6 2114.8 2452.0

4.8E+4

0

10

20

30

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100

% In

ten

sity

968.

5032

1009

.576

0

1253

.644

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871.

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9935

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.667

8

805.

4455

990.

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828.

2993

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2132

.988

3

2198

.015

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2303

.141

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12HAITENQR17

89LIESWEFSSR98

99FLVAGFAER107

766.0 1072.6 1379.2 1685.8 1992.4 2299.0

4591.2

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886.

9922

877.

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6ISGSKLMMVLCVVAMCR22

76EQALQVSESDLLSLAR91

177DSHKIDSFLK186

766.0 1156.2 1546.4 1936.6 2326.8 2717.0

Mass (m/z)

9.6E+3

0

10

20

30

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% In

ten

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1241

.709

7

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39CPSISQEKLLDR50

83NQAGYACITK 92

93ALPIPSSK100

199DYTLLSCFKK208

209DAHKMEIFLK218

213MEIFLK218

224QTDKYNCA213

51VIQHAELIYR60

A

B

C

Fig. 2. MALDI-TOF spectrum of trypsin-cleaved proteins isolated from extracts of Atlantic halibut pituitaries indicating the respective peaks of peptidessequenced. (A) GH, (B) PRL, (C) SL.

I.E. Einarsdottir et al. / General and Comparative Endocrinology 150 (2007) 355–363 359

GH content is presented as total GH (ng) per head andas GH (ng) per head weight (g) in Table 2. For the normallarvae, GH content per head increased during development(one-way ANOVA; p < 0.001). However, GH content perhead weight was similar in all developmental stages, exceptfor a significant decrease at stage 10, compared with stage 9(Tukey HSD; p = 0.012). There were no statistical differ-ences in GH content per head, or in GH per head weight,

between normal and abnormal larvae (two-way ANOVA;p > 0.05).

4. Discussion

The present study demonstrates that CEE is an effectivealternative to conventional chromatography and rpHPLC,as a means of obtaining relatively high quantities of

Fig. 3. Identification of proteins by searching with tryptic peptide sequences against primary sequence databases, using the MASCOT search engine(http://www.matrixscience.com). Peptides resultant from trypsin cleavage of putative hormones purified from Atlantic halibut pituitaries, matchedsequences predicted for GH cDNA from Atlantic halibut (Hippoglossus hippoglossus; BAC07253), for the predicted amino acid sequence of PRL-2 cDNAfrom Nile tilapia (Oreochromis niloticus; E968574) and for the predicted amino acid sequence of SL cDNA from Japanese flounder (Paralichthys olivaceus;A35793) SL. The amino acid sequence of peptides resulting from trypsin cleavage of the isolated halibut proteins are shown in bold typeface.

Table 2Larval weight, head to larval weight ratio, specific growth rate for weight (SGRW) and GH content of normal (N) and abnormal (A) Atlantic halibutlarvae

Stage andphenotype

DPST Larval weight (g)� SGRW (% day�1)(stage interval)

Head weight:larval weight ratio�

GH (ng) head�1 GH (ng) headweight (g)�1

5 N 8.5 0.026 ± 0.001a 0.302 ± 0.014a 0.63 ± 0.07a 81.98 ± 9.08ab

6 N 23 0.040 ± 0.002a 1.29 (5N–6N) 0.266 ± 0.013b 0.74 ± 0.07a 66.88 ± 9.47ab

7 N 31 0.082 ± 0.004b 3.90 (6N–7N) 0.224 ± 0.012c 1.53 ± 0.36ab 80.72 ± 16.86ab

8 N 37 0.185 ± 0.012c 5.89 (7N–8N) 0.169 ± 0.014d 2.12 ± 0.18b 68.39 ± 10.29ab

8 A 37 0.161 ± 0.008 4.88 (7N–8A) 0.203 ± 0.021 1.85 ± 0.18 57.35 ± 7.779 N 45 0.253 ± 0.018d 1.70 (8N–9N) 0.181 ± 0.017d 4.24 ± 1.38c 91.54 ± 26.88a

9 A 45 0.229 ± 0.020 1.91 (8A–9A) 0.185 ± 0.018 3.53 ± 1.39 82.27 ± 26.4510 N 55 0.307 ± 0.031e 0.84 (9N–10N) 0.262 ± 0.018b 4.38 ± 0.68c 54.64 ± 7.57b

10 A 55 0.310 ± 0.018 1.31 (9A–10A) 0.281 ± 0.009 5.30 ± 0.83 61.19 ± 11.72

Stage 5: first feeding; stages 6 and 7: premetamorphosis; stage 8: start of metamorphosis; stage 9: metamorphic climax; stage 10: juvenile. Data given asmeans ± SD. Pooled larval samples (N = 5). DPST denotes the number of days post start-feeding at the middle of each developmental stage. Differentsuperscript letters denote statistical differences (p < 0.05) between stages.�There is a significant overall difference between normal and abnormal larvae.

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purified pituitary hormones (approx. 0.9 mg from 0.4 gpituitaries). Native proteins of GH, PRL and SL of Atlan-tic halibut were successfully isolated and the subsequentMALDI-TOF analysis of their amino acid sequences con-firmed their hormonal identity by similarity searchesagainst public databases. Importantly, the method doesnot cause loss of immunological activity as shown by thereactivity of GH in a homologous halibut GHradioimmunoassay.

One of the difficulties facing endocrinological researchon non-model organisms is the frequent lack of molecularresources. Hormonal analyses of tissues content and/or

plasma levels are important steps in elucidating the func-tion of endocrine systems, and homologous peptide hor-mones are most often needed to establish valid assaysystems.

For all but experts in protein purification, conventionalmethodologies which include several steps, including gel fil-tration and HPLC separation, represent a significant, oftentime-consuming hurdle for studies on endocrine function.Limited hormonal yield is also a major concern. TheCCE method contributes to the resolution of these prob-lems, as it is a single-step, rapid and cost-effective way ofpurifying pituitary hormones and other proteins to the

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level at which they can be characterized by MALDI-TOFand/or used to generate immunologically and biologicallyactive material (Anjos et al., 2005). In the present study,CEE was carried out under denaturing conditions whichis unlikely to yield biologically active GH as disulphidebridges important for maintenance of appropriate tertiarystructure would be eliminated. However, if the objectiveis to generate biologically active proteins, non-denaturingCEE conditions can be used.

The amino acid sequences of three peptides obtainedfrom the trypsin-cleaved native GH match that deducedfrom the cDNA sequence of mature Atlantic halibut GH(Einarsdottir et al., 2002). For two of the peptides, 89LIES-WEFSSR98 and 99FLVAGFAER107, the match is com-plete, while for the peptide 12HAITENQR17, the first twoamino acids, histidine (H) and alanine (A), differ, beingglutamine (Q) and proline (P), in the deduced GH aminoacid sequence. The deduced amino acid sequence of halibutGH (Einarsdottir et al., 2002) has a 99.5% similarity withBarfin flounder (Verasper moseri; Peyush et al., 2000),81.7% similarity with Japanese flounder (P. olivaceus;Momota et al., 1988) and 74.2% similarity with plaice(Pleuronectes platessa; Nagler et al., 1991) GH. All fourGHs have glutamine in their first position and, except forplaice, have proline in their second position in thesequence. Thus, glutamine and proline appear to be thedominating amino acids in position one and two, respec-tively, for flatfish GH. The codons for glutamine and pro-line are CAG and CCA, respectively, CAT or CAC forhistidine and GCA for alanine. This indicates a possiblesingle nucleotide polymorphism (SNP) in both codons,possibly reflecting allelic variation in the halibut GH gene.

The halibut PRL peptide fragments generated sharegreatest similarity with Nile tilapia PRL2. In Nile tilapia(O. niloticus) and Mozambique tilapia (O. mossambicus),two molecular forms of mature PRL exists, tiPRL1 andtiPRL2, which are 188 (tiPRL188) and 177 (tiPRL177) ami-no acids long, respectively (Specker et al., 1985; Yamagu-chi et al., 1988; Swennen et al., 1991); Nile tilapia: NCBIAccession Nos. CAA00720 and CAA00722, Mozambiquetilapia: NCBI Accession Nos. B28106 and A28106. The dif-ferent isoforms in Nile tilapia, and probably also inMozambique tilapia, are encoded by different genes andshare only 69% amino acid identity (Yamaguchi et al.,1988; Rentier-Delrue et al., 1989) and have been demon-strated to have different osmoregulatory functions in Niletilapia (Auperin et al., 1994), but not in Mozambique tila-pia (Specker et al., 1985). The molecular mass for the20 kDa PRL and 24 kDa PRL were later calculated to be19,584 Da and 20,836 Da, respectively, after amino acidsequence determination (Yamaguchi et al., 1988). In com-parison, the molecular mass for halibut PRL was calculat-ed to be 22,453 Da. Interestingly, in the trypsin-cleavedpeptide 6ISGSKLMMVLCVVAMCR22 corresponding topart of the signal peptide of halibut PRL, all 17 amino acidresidues match those of the Nile tilapia PRL177 signal pep-tide, and 16 out of 17 amino acid residues in the Mozam-

bique tilapia PRL177 signal peptide (94% identity;Accession No. P09318). Curiously, no significant matcheswith other teleost PRL signal peptides were found. The sig-nificance of the high level of conservation of the halibutand tilapia signal peptides is unclear.

The eight peptides obtained from the trypsin-cleavedhalibut SL matched four main domains in the aminoacid sequence of Japanese flounder (P. olivaceus) SLpredicted from cDNA (Ono et al., 1990). The domainscomprised amino acids 39–60, 83–100, 199–218 and124–130. Four of the seven cystein residues in the flounderSL are found within these four domains (Ono et al., 1990).The halibut SL molecular mass (27.1 kDa) is comparablewith that of the flounder SL, which is 28 kDa in size andis composed of 231 amino acids, including a 24 amino acidsignal peptide (Ono et al., 1990). Two SL molecules havebeen identified in halibut pituitary extracts, one 24.2 kDaand one 26.3 kDa proteins (Johnson et al., 1997), of whichthe 24.2 kDa protein had the N-terminal sequence similarto the 230 amino acid sequence deduced from two isolatedhalibut SL genes by Iraqi et al. (1993). The 24.2and 26.3 kDa proteins were supposed to be a nongycosy-lated and a glycosylated form of the SL, respectively(Johnson et al., 1997).

GTHs genes in Atlantic halibut have been sequencedand the native proteins purified and characterized(Weltzien et al., 2003b,c). Two different GTHs have beenidentified, follicle stimulating hormone (FSH) and luteiniz-ing hormone (LH), each consisting of heterodimers of acommon a-subunit and separate FSHb and LHb subunits.Molecular mass analyzed by SDS–PAGE were 33 and32 kDa for FSH and LH, respectively (Weltzien et al.,2003c). For comparison, SDS–PAGE molecular mass inour study was approximately 32 kDa. In halibut, thea-subunit is probably glycosylated (Weltzien et al.,2003b). In the present study, the approach taken for theMALDI-TOF analyses did not consider possible glycosyl-ation and, therefore, no amino acid sequences could beobtained for these hormones.

Size and charge heterogeneity can result from differenttypes of post-translational modifications, glycosylationand phosphorylation of PRL and GH are major modifica-tions in mammals (Ray et al., 1989; Sinha, 1995; Garcia-Barros et al., 2000), reptiles (Noso et al., 1992), and birds(Corcoran and Proudman, 1991; Aramburo et al., 1992).In Atlantic cod, GH charge heterogeneity due to phosphor-ylation of the native pituitary hormone has been reported(Skibeli et al., 1990). In the present study, a single formof each hormone was observed in the halibut pituitaryand no modified forms were identified. However, it is notpossible to rule out their existence and alternativeapproaches will be required to address this question.

The GH previously used in the halibut GH radioimmu-noassay (Einarsdottir et al., 2002), has been purified frompituitaries by size exclusion gel chromatography andrpHPLC. The GH yield has been low and the acetonitrileused in the rpHPLC has rendered the GH unstable, even

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at �80 �C, limiting the usage of the GH-RIA to help eluci-date the role of GH in this species. The CEE methodologyappears to have solved both problems. Utilizing CEE-puri-fied GH in the RIA, the present study demonstrates thathalibut larvae contain measurable amount of GH in theirhead region from an early stage, confirming previousimmunohistochemical and in situ hybridization observa-tions (Einarsdottir et al., 2006).

The gradual increase of larval head GH content untilmetamorphic climax appears to be well correlated to bodygrowth and thus a presumed increase in pituitary gland size.This is in line with data on silver sea bream (Sparus sarba) lar-vae, where larval GH mRNA abundance increases progres-sively until 35–46 days post hatch (Deane et al., 2003). Also,in Japanese flounder, GH content, assessed indirectly bymeasuring cell volume, increases continuously during larvaland juvenile development (Tanaka et al., 1995).

Little is known about the role of GH in regulatory pro-cesses in fish development. GH has been suggested to beantimetamorphic on some processes in amphibian develop-ment, but to a much less extent than PRL (Takada andKasai, 2003). In the present study, the larval pituitaryGH content does not exhibit any significant, metamorpho-sis-related changes, but rather a steady increase correlatedto growth. Nonetheless, a role for GH in metamorphosiscannot be excluded. As somatotrophs normally store largeamounts of GH (mg g�1 range), while plasma levels are low(ng ml�1 range), significant changes in secretion rate andplasma levels may well occur without significant changesin pituitary GH content. This has been observed duringAtlantic salmon parr-smolt transformation (Agustssonet al., 2001). Furthermore, changes in the tissue distribu-tion and/or density of GH receptors may be important reg-ulatory mechanism for GH action, even when secretionrate is steady. Such changes in GH receptor expressionhave recently been quantified during halibut metamorpho-sis. Reduced GH receptor mRNA expression was observedin abnormally metamorphosed halibut larvae compared tonormal larvae, indicating a role for GH in metamorphosis(J. Hildahl, unpublished data). In the present study, GHcontent per head weight did not differ between normaland abnormal larvae.

The larval growth rate follows a sigmoid pattern withthe most rapid growth between stages 7 and 8, after whichweight growth rate slows down. The metamorphic processin halibut larvae starts at stage 8 and climaxes at stage 9(Saele et al., 2004; Einarsdottir et al., 2006). The decreasein SGR during the later stages thus coincides with the onsetof metamorphosis and may reflect a trans-allocation ofenergy from growth towards the metamorphic processes,but may also be due to decreased feed intake during meta-morphosis (H. Smaradottir, pers. comm.).

The decrease in head to body weight ratio until the startof metamorphosis (stage 8) reflects a change in body shape,with a faster growth of the body than the head during theperiod of high SGR and a slower body growth after meta-morphic climax (stage 9). In comparison, Saele et al. (2004)

noted an increase in body depth from first feeding untiljuvenile settlement, when the larvae adapted a more elon-gated form. In the present study, the lower body weightof the abnormal larvae, together with a higher head tobody ratio, especially at the start of metamorphosis, indi-cates a slimmer body shape of this phenotype. This maybe due to endogenous changes in regulation of growthand development, even if this is not reflected in GH con-tent, and/or a transitory difficulty of these fish to obtainfood.

In conclusion, continuous-elution electrophoresis allowsa simple one-step purification of pituitary hormones, over-coming problems associated with traditional column chro-matography methods. It has thus enabled the use ofhomologous GH-RIA to examine the role of GH duringdevelopment and metamorphosis of Atlantic halibut. GHcontent is found to increase proportionally with larvalgrowth, and does not differ between normally and abnor-mally metamorphosing larvae. While this does not indicatea direct role for GH in the metamorphic process, it doesnot exclude it.

Acknowledgments

We thank Heiðdıs Smaradottir and Arnar Jonsson atFiskey Ltd., Iceland, for excellent logistic and scientific col-laboration. This work has been carried out within the pro-ject ‘‘Arrested development: The Molecular and EndocrineBasis of Flatfish Metamorphosis’’ (Q5RS-2002-01192) withfinancial support from the Commission of the EuropeanCommunities. However, it does not necessarily reflect theCommission’s views and in no way anticipates its futurepolicy in this area. This project was further supported bythe Swedish Council for Agricultural and ForestryResearch (FORMAS) and Pluriannual funding to CCMARby the Portuguese Science and Technology Council.

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