General and Comparative Endocrinology 149 (2006) 166–172

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Insulin-like growth factors I and II in the sole Solea senegalensis: cDNA cloning and quantitation of gene expression in tissues

and during larval development

V. Funes, E. Asensio, M. Ponce, C. Infante, J.P. Cañavate, M. Manchado ¤

C.I.F.A.P. “El Toruño”, IFAPA, Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía, 11500 El Puerto de Santa María, Cádiz, Spain

Received 13 February 2006; revised 17 May 2006; accepted 24 May 2006Available online 11 July 2006

Abstract

Insulin-like growth factors I and II (IGF-I and IGF-II) play an important role as modulators of development, growth, and reproduc-tion. This study aimed to isolate the IGF-I and IGF-II cDNAs and determine their temporal expression pattern in diVerent organs andthroughout larval development in Senegal sole. The rapid ampliWcation of cDNA ends (RACE) was used to obtain both full-length IGFssequences. A high sequence similarity with other teleosts sequences was observed. Domains B and A revealed as the most evolutionary con-served. Steady-state copy numbers of IGF-I and IGF-II were also quantiWed in diVerent Senegal sole tissues by real-time PCR. IGF-I andIGF-II expressed ubiquitously with the highest mRNA levels in liver (88£ 106 molecules/�g total RNA) and gills (14.0£106 molecules/�gtotal RNA) respectively. IGF-II mRNA levels were higher than IGF-I in prehatching embryos and premetamorphic larvae with a signiW-cant drop before the commencement of eye migration in metamorphosis. The abundance of IGF-II transcripts correlated positively withthe growth rate during larval development. The putative role of IGF-II on metamorphosis and larval growth is discussed.© 2006 Elsevier Inc. All rights reserved.

Keywords: Solea senegalensis; Aquaculture; Insulin growth factors; IGF-I; IGF-II; Gene expression

1. Introduction

Insulin-like growth factors (IGF-I and IGF-II) are sin-gle-chain polypeptides with structural homology to proin-sulin. Mature IGF-I and IGF-II consist of B, C, A, and Ddomains (Jones and Clemmons, 1995). They are ubiqui-tously expressed and are essential for both fetal and postna-tal growth in mammals and Wsh (Duan, 1998; Jones andClemmons, 1995). Biological actions of IGFs are mediatedby the IGF receptors localized on the cell membrane. Twotypes of receptors have been reported. The type I IGFreceptor (IGF1R) has structural homology to the insulinreceptor with two � and � subunits and a tyrosine kinasedomain. The type II IGF receptor (IGF2R) is a single

* Corresponding author. Fax: +34 956 011324.E-mail address: manuel.manchado.ext@juntadeandalucia.es (M.

Manchado).

0016-6480/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.ygcen.2006.05.017

peptide chain identical to the mannose-6-phosphate recep-tor that binds lysosomal enzymes (Duan, 1998; Planaset al., 2001). In biological Xuids, IGFs usually bind to IGFbinding proteins (Jones and Clemmons, 1995; Reinecke andCollet, 1998).

IGF-I is produced mainly in the liver. Functional studiesin diVerent Wsh species indicate that its biological actionsare remarkably conserved throughout evolution. It is amediator of growth hormone (GH) action on a variety ofcells, playing an important role in promoting metabolismand growth, cell diVerentiation, reproduction, and osmo-regulation (Dubois and Callard, 1993; Maestro et al., 1997;McCormick, 1996; McCormick et al., 1992; Peterson et al.,2004, 2005). Moreover, an important role in apoptosis inhi-bition has been reported (Jones and Clemmons, 1995).IGF-II is an important fetal growth factor in mammals(Baker et al., 1993) but its function remains to be clariWed inWsh to date. Previous data in other teleosts indicate thatboth IGF-I and IGF-II express during early development

V. Funes et al. / General and Comparative Endocrinology 149 (2006) 166–172 167

promoting growth in a GH-dependent manner (Aysonet al., 2002; Gabillard et al., 2003a; Peterson et al., 2005).Both IGFs seem to exert their eVects in part in a paracrine/autocrine manner (Duan, 1998).

The Senegal sole (Solea senegalensis) is a XatWsh of highinterest in aquaculture. During larval development, itchanges from a symmetrical morphology to an asymmetric,benthic juvenile. This metamorphic process involves dra-matic morphological and physiological changes with thetranslocation of one eye to the opposite side of the headand an asymmetric skull and jaw development. Pro-grammed cell death or apoptosis induced by thyroxin hor-mone seems to play a key role in this tissue remodelling inXatWsh (Power et al., 2001). In S. senegalensis, eye migrationbegins towards 11–12 days after hatching (DAH) and endsaround 19–22 DAH. This time framework can oscillatedepending on the feeding regimes (Fernández-Díaz et al.,2001). During this period and under all circumstancesinvestigated, a weight gain reduction is detected (Fernán-dez-Díaz et al., 2001; Parra and Yúfera, 2001). Due to IGFsplaying a key role on nutritional condition, environmentaladaptation, embryonic development, and growth regula-tion of teleosts, it is important to elucidate the expressionpattern of both IGFs during Senegal sole development.

The goals of this study were (1) to establish the comple-mentary DNA (cDNA) sequence of IGF-I and IGF-II, (2)to study the expression of both IGFs in various organs and(3) to identify the gene expression pattern of both IGFsduring larval development.

2. Materials and methods

2.1. Animals

Senegal sole individuals (mean weight D 598§ 176 g, n D 3) wereobtained from CIFAP “El Toruño” facilities (El Puerto Santa María,Cádiz, Spain). They were sacriWed by immersion in tricaine methanesulfo-nate (MS-222). Liver, stomach, intestine, kidney, gill, and spleen were rap-idly dissected, frozen in liquid nitrogen and stored at ¡80 °C till use.

For larvae studies, eggs from a naturally spawning Senegal sole brood-stock (CIFPA “El Toruño”) were collected. Fertilized eggs were incubatedin a 150 L tank at 19–21 °C for 2 days. Newly hatched larvae were trans-ferred to a 400 L tank at an initial larval density from 45 to 50 larvae L¡1

with a 16L:8D photoperiod and a light intensity of 600–800 lux. Larvae werefed rotifers (Brachionus plicatilis) 3 DAH till 9 DAH. From 7 DAH enrichedartemia metanauplii were fed till the end of the experiment. Three pools ofprehatching embryos (»8 h after fertilization) and larvae from 2 to 22 DAHwere collected, washed with DEPC water, frozen in liquid nitrogen andstored at ¡80 C until analysis. Some larvae were simultaneously taken anddry weight was calculated for determination of growth rate. Body dry masswas determined by drying samples of approximately 15–30 larvae at 60 °Cduring 48 h. SpeciWc growth rate was calculated after logarithmic transfor-mation of larval body dry mass (W, �g) against time (D, days).

2.2. cDNA cloning and sequencing

Total RNA was isolated from 250 mg of S. senegalensis liver (IGF-I)and a pool of premetamorphic larvae (IGF-II) using the FastRNAProGreen Kit (Q-BioGene) for 40 s at speed setting six in the Fastprep FG120instrument (Bio101). All RNA isolation procedures were performed inaccordance with the manufacturer’s protocol.

The cDNA synthesis was achieved with the 3� and 5� RACE system forRapid AmpliWcation of cDNA Ends Kits (invitrogen, Rockville, MD, USA).First, the 3� end-cDNA was synthesized from 2.5�g of total RNA using theAdapter Primer (5�-GGCCACGCGTCGACTAG TAC[T]17-3�) and Super-Script II Reverse Transcriptase. AmpliWcation of the target cDNA was carriedout using as forward primers IGF1 (5�-TTGTGTGTG GAGAGAGAGGCTTTTATTTCA-3�) and IGF2 (5�-GACACGCTGC AGTTTGTGTGTGGAGA-3�), located in conserved sequences of the B domain. Primers weredesigned using Oligo v6.89 software (Medprobe, Oslo, Norway).

As reverse primer we used the Universal AmpliWcation Primer suppliedby the 3� RACE kit. PCR conditions involved denaturing at 94 °C for2 min, followed by 35 cycles of 30 s at 94 °C, 15 s at 58 °C or 45 °C (forIGF-I and IGF-II, respectively) and 1 min at 72 °C. PCR ampliWed prod-ucts were puriWed using the rapid PCR puriWcation kit (Marlingen Biosci-ence, Ijamsville, MD, USA) and subsequently cloned with the TOPO TACloning® kit for sequencing (Invitrogen). Positive clones were sequencedusing the BigDye Terminator v3.1 Cycle Sequencing kit in an ABI3130genetic analyzer (Applied Biosystems, Foster city, CA, USA).

To obtain the 5� end-cDNA of Senegal sole IGF-I and IGF-II, tworeverse speciWc primers (SseIGFI-2: 5�-GTGCCCTTGTCCGCCTTGTGC-3� and SseIGFII-2: 5�-CGCCGGTTGCTACCCCTGCTG-3�)were designed and used in reverse transcription. Each of these primers andthe Abridged Anchor Primer, provided in the 5� RACE kit, were used toamplify a speciWc product for each hormone. PCR conditions were 30 cyclesof 30 s at 94 °C, 15 s at 58 °C and 1 min at 72 °C. After PCR, the ampliWed 5�

cDNA was puriWed, cloned, and sequenced as described above.

2.3. RNA preparations and reverse transcription

Total RNA isolation was carried out as described above. RNA samplequality was checked electrophoretically and quantiWcation was performedspectrophotometrically. Each total RNA (1 �g) was reverse-transcribedusing the iScript™ cDNA Synthesis kit (Bio-Rad, Hercules, CA, USA).Reverse transcription reactions were performed in duplicate. Lack ofgenomic DNA contamination was conWrmed by PCR ampliWcation ofRNA samples without previous cDNA synthesis.

2.4. Real-time PCR

The absolute number of IGF-I and IGF-II mRNA molecules were mea-sured using real-time PCR ampliWcation. Two pairs of speciWc primers weredesigned using Oligo 6.89. Reactions were performed in a 25�l volume con-taining cDNA generated from 1ng of original RNA template (50-fold dilu-tion of RT reaction), 300 nM each of speciWc forward and reverse primer(IGF-I (for, 5�-CGGCGCCTGGAGATGTACTGTGC -3�; rev, 5�-GTGCCCTTGTCCGCCTTGTGC-3�) and IGF-II (for, 5�-GACGGAGACGCTGTGTGGGGGAGA-3�; rev, 5�- CGCCGGTTGCTACCCCTGCT-3�)),and 12.5�l of 2£ QuantiTect SYBR Green PCR Mix (BioRad). AmpliWedsignals were detected continuously with the Bio-Rad iCycler. The ampliWca-tion protocol used was as follows: initial 15 min denaturation and enzymeactivation at 95 °C, 40 cycles of 95 °C for 30s, 70 °C for 15 s and 72 °C for1 min. An absolute standard curve was constructed with speciWc IGF-I andIGF-II cRNA standards in the range of 102–109 RNA molecules. The num-ber of mRNA molecules was calculated from the linear regression of the stan-dard curve after retrotranscription and ampliWcation. PCRs were performedin triplicate. No primer-dimers were detected, and investigated transcriptsshowed optimal PCR eYciencies (0.91–96).

Kruskal–Wallis rank test and correlation analysis were carried outusing PRISM 4.0 (GraphPad Software, Inc.).

3. Results

3.1. Structure of Senegal sole IGF-I and IGF-II cDNAs

A cDNA of 668 nucleotides (nt) encoding Senegal soleIGF-I was isolated from liver by RACE-PCR (Accession

168 V. Funes et al. / General and Comparative Endocrinology 149 (2006) 166–172

No. AB248825). The open reading frame (ORF) of 560 ntwas followed by a 103-nt 3�-UTR (Fig. 1A). The B, C, A,and D domains constituting the 67-amino acids (aa) matureprotein were composed of 28, 10, 21, and 8 aa, respectively.The E-domain consisted of 74 aa. The six characteristic cys-teine residues involved in the formation of the disulWdebounds were conserved (CysB9, CysB18, CysA6, CysA7,CysA11, and CysA20). The B domain had a 1-aa deletion as inSparus aurata. C domain had a 2-aa deletion with respect toCyprinus carpio (Fig. 2).

For Senegal sole IGF-II, a cDNA sequence of 824 nt wasisolated from whole larvae (Accession No. AB248826). TheORF comprised 620 nt in length preceded by a 149-nt 5�-UTR and followed by a 57-nt 3�-UTR (Fig. 1B). The signalpeptide consisted of 47 aa. A total of 32, 11, 21, 6, and 91 aawere detected for B-, C-, A-, D-, and E- domains, respec-tively. The sequence exhibited the six characteristic cysteineresidues (CysB9, CysB21, CysA6, CysA7, CysA11, and CysA20).

Both Senegal sole IGFs revealed a high degree of similar-ity with those of other Wsh species (Fig. 2). At the amino acid

Fig. 1. Nucleotide and deduced amino acid sequences of Solea senegalensis (Sse) preproIGF-I (A) and preproIGF-II (B). Open reading frames are inuppercase. Signal peptides are shaded. IGF-I and IGF-II mature proteins consisting of B, C, A, and D domains are in boldfaced letters. The six conserved

cysteine residues are underlined.

Fig. 2. Amino acid alignment of proIGF-I (A) and proIGF-II (B) sequences. To maximize the alignment, gaps are introduced and indicated as dashes.Dots indicate identity. References for IGF-I and IGF-II sequences are as follows: Scopthalmus maximus, (Sma, Duval et al., 2002, only mature proteinsequence), Paralichthys olivaceus, (Pol, AJ010603) , Epinephelus coioides (Eco, AY513719), Dicentrarchus labrax (Dla, AY800248) , Oreochromis mossam-bicus (Omo, AF033796), Sparus aurata (Sau, AY996779), Oncorhynchus mykiss (Omy, M95183), and Cyprinus carpio (Cca, AF465830).

V. Funes et al. / General and Comparative Endocrinology 149 (2006) 166–172 169

level, mature IGF-I exhibited a percentage identity thatranged between 92.5% to S. aurata, and 74.6% to C. carpio.Higher similarity was detected for mature IGF-II with 92.9%for Paralichthys olivaceus, Epinephelus coioides, and S. aurataand 87.1% with C. carpio. Domains B and A in both IGFswere extremely conserved with respect to other teleosts. Acomparison in the amino acid sequence between IGF-I andIGF-II also revealed strong conserved regions between bothIGFs in the B, C, A, and D domains and in the seven cyste-ines. Amino acid sequence comparison between IGF-I andIGF-II in Senegal sole showed identities of 75, 30, 71.4 and50% in the B, C, A, and D domains, respectively.

3.2. Quantitation of Senegal sole IGF-I and IGF-II mRNA levels

3.2.1. Tissues expression patterns of IGF-I and IGF-IIWe used a quantitatively approach based on reverse tran-

scription followed by real-time PCR ampliWcation to investi-gate the steady-state levels of IGF-I and IGF-II transcriptsin liver, gills, stomach, intestine, kidney, and spleen. BothIGF-I and IGF-II were expressed in detectable amounts in

all these tissues (Fig. 3). The highest level of IGF-I transcriptswas detected in the liver (88£106§22£106/�g total RNA).The number of mRNA molecules ranged between 80–551-fold higher than in extrahepatic tissues.

IGF-II was mostly expressed in gills (14.0£106§ 4.6£106/�g total RNA) with the lowest values in liver(1.7£ 106§1.3£106/�g total RNA). IGF-II levels weremore abundant than those of IGF-I in all extrahepaticorgans investigated. Yet, the total number of IGF-I tran-scripts in liver was much higher than IGF-II mRNAs (from6.3 to 53.3-fold) in any other analyzed tissue.

3.3. IGF-I and IGF-II expression patterns during larval development

The absolute number of IGF-I and IGF-II mRNAs wasdetermined in samples extracted from pools of prehatchingembryos and whole larvae collected from 2 to 22 DAH(Fig. 4). Overall, IGF-II mRNAs were signiWcantly moreabundant throughout larval development than those ofIGF-I (3.8-fold as global mean; P < 0.05).

Fig. 3. IGF-I and IGF-II mRNA levels in diVerent S. senegalensis organs. Transcripts were quantitated using total RNA from organs of three juvenileSenegal soles. IGF-I and IGF-II (mean § SD) are indicated in grey and black bars, respectively. Letters indicate the diVerence between IGFS means ineach organ (P < 0.05).

Fig. 4. IGF-I and IGF-II mRNA levels during S. senegalensis larval development. Transcripts were quantitated in pools of prehatching embryos (E) and

whole larvae collected from 2 to 22 DAH (n D 3). IGF-I and IGF-II (mean § SD) are indicated in grey and black bars, respectively.

170 V. Funes et al. / General and Comparative Endocrinology 149 (2006) 166–172

IGF-I was expressed at a lower level in prehatchingembryos (0.030£ 106 molecules/�g total RNA). These levelsincreased 46.5-fold in 2 DAH larvae (1.3£ 106 molecules/�gtotal RNA) and maintained from 22 to 53-fold higher dur-ing larval development. No signiWcant diVerences (P > 0.05)in the transcript abundance throughout the premetamor-phic (mean 1.1£ 106 molecules/�g total RNA), metamor-phic (mean 0.9£ 106 molecules/�g total RNA), andpostmetamorphic stages (mean 0.9£ 106 molecules/�g totalRNA) were detected.

On the contrary, the IGF-II expressed at a higher levelduring premetamorphosis. A total of 7.4£106 molecules/�gtotal RNA was determined in 2 DAH larvae. These levelsmaintained high (2.2–3.6£106 molecules/�g total RNA)until 8 DAH, 3 days before the onset of eye migration. Thenumber of IGF-II molecules declined approximately 3.4-fold as global mean during metamorphosis (mean 1.2£106

molecules/�g total RNA).Growth rate during larval development was determined.

Premetamorphic larvae from 2 to 12 DAH (WD1.15+ 0.125D) grew faster than metamorphic (WD1.75+ 0.075D). When we compared speciWc growth rate betweentwo sampling days to the mean absolute amounts of IGF-IItranscripts during that period, a signiWcant and positivecorrelation (rD 0.97, P < 0.001) was found (Fig. 5).

4. Discussion

4.1. Sequence of IGF-I and IGF-II cDNAs

Senegal sole IGF-I and IGF-II cDNAs were cloned andsequenced using RACE-PCR. IGF-I and IGF-II matureproteins consisted of 67 and 70 aa, respectively. IGF-II hor-mones showed higher similarity levels with other teleoststhan IGF-I. The greatest conservation of the amino acidcomposition can be observed in the B and A domains,where the conserved cysteines and the residues that interact

Fig. 5. Correlation of IGF-II mRNA molecules and growth rate duringlarval development (r D 0.97, P < 0.001).

with IGF1R and IGFBPs in vertebrates are present (Duan,1998; Duval et al., 2002). This high degree of structural con-servation indicates the function of IGFs have been wellconserved during vertebrate evolution (Duan, 1998).

4.2. Expression in liver and other extrahepatic organs

In this study, we report the expression of IGF-I andIGF-II in diVerent tissues of Senegal sole. Real-time analy-sis revealed the liver as the major organ of IGF-I expres-sion. The total number of IGF-I mRNA copies wasestimated to be 88£106 molecules/�g total RNA. Thisvalue falls within the range of hepatic IGF-I transcriptsdescribed for other Wsh species such us rainbow trout(7.6£ 106) (Gabillard et al., 2003a) or tilapia (225£ 106)(Caelers et al., 2004). The diVerences in the number of liverIGF-I mRNAs may well due to species variation. However,other factors such us nutritional status, temperature orsalinity may also account for these diVerences (Gabillardet al., 2003a; McCormick, 1996; Meton et al., 2000).

The abundance of IGF-I transcripts in the liver was atleast two to three orders of magnitude higher than in extra-hepatic tissues. DiVerences in the steady-state copy numberof IGF-I mRNAs were similar to results reported for com-mon carp (Tse et al., 2002; Vong et al., 2003), channel cat-Wsh (LaTonya et al., 2005), or eurasian perch (Jentoft et al.,2004), although smaller diVerences were quantiWed in tila-pia (Caelers et al., 2004). The higher relative expression ofIGF in liver agrees with the observation of this organ as themain source of circulating IGF-I (Gabillard et al., 2003a).Moreover, the detection of IGF-I mRNAs in all extrahe-patic tissues analyzed supports the autocrine/paracrine roleof this hormone synthesized in parenchymal cells of diVer-ent organs (Perrot et al., 1999; Reinecke et al., 1997).

In contrast to IGF-I, IGF-II expressed at a lower level inliver (only 1.6£106 molecules/�g total RNA). The occur-rence of less IGF-II than IGF-I mRNAs could be consid-ered as a normal condition among Wsh species. Yet, thenumber of IGF-II transcripts was reduced in comparisonto those reported for rainbow trout and tilapia (7.0£ 106–87£106 molecules/�g total RNA) (Caelers et al., 2004;Gabillard et al., 2003a; Tse et al., 2002; Vong et al., 2003).IGF-I/IGF-II mRNAs ratio can Xuctuate signiWcantly inliver as a function of age being the IGF-II mRNA levelslower in common carp juveniles than in adults (Tse et al.,2002). This variation in the relative abundance of IGFsduring growth as well as species speciWc diVerences and thenutritional status could explain the IGF-II mRNA levelsfound in Senegal sole.

All extrahepatic tissues expressed IGF-II to a higherlevel than IGF-I. The total number of IGF-II mRNA cop-ies ranged between 14.0£ 106 and 3.5£ 106 molecules/�gtotal RNA in gill and kidney, respectively. This IGF-IIexpression pattern is fully consistent with data from com-mon carp juveniles (Tse et al., 2002) and partly with tilapia,with higher IGF-I mRNAs in brain, testes, and gill (Caelerset al., 2004). Interestingly, Degger et al. (2001) showed that,

V. Funes et al. / General and Comparative Endocrinology 149 (2006) 166–172 171

in juvenile Lates calcarifer individuals, kidneys and gillswere very eVective at accumulating radiolabelled IGF-II.The high level of IGF-II expression found in these organsin Senegal sole suggests a possible role of this hormone inosmoregulation.

4.3. Expression during larval development

The expression of IGF-I and IGF-II seems to be devel-opmentally regulated. Several studies have detected highlevels of expression of IGF-II in early embryogenesis fol-lowed by a decrease in the levels of expression of IGF-IIand an increase in the levels of expression of IGF-I(Duguay et al., 1996; Gabillard et al., 2003b; Greene andChen, 1999; Perrot et al., 1999). Although IGF-I concentra-tion is generally lower than IGF-II, it seems that it plays akey role in fetal growth and development (Liu et al., 1993).In the present study, we quantiWed the amounts of IGF-Iand IGF-II transcripts in prehatching embryos and Senegalsole larvae. IGF-I and IGF-II mRNAs were detected in allsamples analyzed. IGF-I expressed at a lower level inembryos (0.03£ 106 molecules/�g total RNA) andincreased from 22 to 53-fold after hatching. In rabbitWsh,IGF-I transcripts remained undetectable in unfertilizedeggs and during embryogenesis. However, it expressed to asteady level in larvae (Ayson et al., 2002). In contrast, IGF-I transcripts were detected throughout all these stages ingilthead seabream rainbow trout and channel catWsh in atissue-speciWc and age-dependent manner (Greene andChen, 1999; Perrot et al., 1999; Peterson et al., 2005). Thisearly detection of IGF-I mRNAs in Wsh eggs has been asso-ciated to transcripts stored maternally that would enablethe development in a growth-hormone independent mannerin the marine environment (Greene and Chen, 1997; Perrotet al., 1999).

On the other hand, IGF-II expression was higher thanIGF-I in embryos and during premetamorphosis. A sig-niWcant drop in IGF-II mRNAs was detected just beforemetamorphosis. This reduction in the IGF-II levels couldassociate to the apoptotic processes that control meta-morphosis. IGFs not only stimulate cell proliferation butalso have a broad and potent ability to prevent cell deathduring normal development (Vincent and Feldman, 2002).The direct or indirect participation of IGF-II in metamor-phosis is supported by two facts: (i) reduction of IGF-IIlevels coincides with the beginning of the proteolytic pro-cesses that favour tissue remodelling as the Wrst steps formetamorphosis. This drastic cellular changes coincidewith maximal protease activity and a reduction in the lar-val growth rate (Fernández-Díaz et al., 2001) and (ii)diVerent feeding regimes modiWed the timing of metamor-phosis in Senegal sole. Larvae with a faster growth begantheir transformation earlier in spite of the time required tocomplete the transformation was similar (Fernández-Díazet al., 2001). If we consider that expression and produc-tion of IGFs are inXuenced by nutrition and physicalactivity, IGF-II constitutes a target that could mediate the

mechanisms that underlie the metamorphosis in Senegalsole larvae.

DiVerent growth rates are observed during early devel-opment of Senegal sole. In premetamorphosis, larvae growat almost twice the rate of metamorphosis and accumulateenergetic reserves in tissues to be used during metamorpho-sis (Fernández-Díaz et al., 2001; Parra and Yúfera, 2001).Although IGF-I has been proven to promote growth in tel-eosts (Chen et al., 2000; McCormick et al., 1992), no varia-tion in mRNA levels during Senegal sole larvaldevelopment was detected. On the other hand, a signiWcantpositive correlation between growth rate and the number ofIGF-II transcripts was determined. The importance ofIGF-II on Wsh larvae growth was also demonstrated inrainbow trout, where it mediates the eVect of temperatureon growth during embryonic development (Gabillard et al.,2003c). In bovine, faster-developing embryos producedin vivo had more IGF-II transcription than their in vitrocounterparts and more than the slower in vivo producedembryos (Gutiérrez-Adán et al., 2004). A diVerent IGF-IIexpression pattern (three IGF-II peaks from 1 to 16 DAH)was described in S. aurata larvae (Perrot et al., 1999). How-ever, in this seabream species a constant growth ratethroughout this larval period was reported (Parra and Yúf-era, 2001). The possible role of IGF-II on larval growth andmetamorphosis as well as its regulation in Senegal soledeserves further investigation.

Acknowledgments

We thank the anonymous reviewers for their commentsand suggestions to improve the manuscript. We are alsograteful to technicians Aniela Crespo and Eugenia Zuastifor their laboratory help. This work was supported by pro-ject PIA 03-069 (IFAPA, CICE, Junta de Andalucía,Spain).

References

Ayson, F.G., de Jesus, E.G.T., Moriyama, S., Hyodo, B., Funkenstein, B.,Gerter, A., Kawauchi, H., 2002. DiVerential expression of insulin-likegrowth factor I and II mRNAs during embryogenesis and early larvaldevelopment in rabbitWsh, Siganus guttatus. Gen. Comp. Endocrinol.126, 165–174.

Baker, J., Liu, J.P., Robertson, E.J., Efstratiadis, A., 1993. Role of insulin-like growth factors in embryonic and postnatal growth. Cell 75, 73–82.

Caelers, A., Berishvili, G., Meli, M.L., Eppler, E., Reinecke, M., 2004.Establishment of a real-time RT-PCR for the determination of abso-lute amounts of IGF-I and IGF-II gene expression in liver and extra-hepatic sites of the tilapia. Gen. Comp. Endocrinol. 137, 196–204.

Chen, J.Y., Chen, J.C., Chang, C.Y., Shen, S.C., Chen, M.S., Wu, J.L., 2000.Expression of recombinant tilapia insulin-like growth factor I andstimulation of juvenile tilapia growth by injection of recombinantIGFs polypeptides. Aquaculture 181, 347–360.

Degger, B., Richardson, N., Collet, C., Upton, Z., 2001. Production, in vitrocharacterisation, in vivo clearance, and tissue localisation of recombi-nant barramundi (Lates calcarifer) insulin-like growth factor II. Gen.Comp. Endocrinol. 123, 38–50.

Duan, C., 1998. Nutritional and developmental regulation of insulin-likegrowth factors in Wsh. J. Nutr. 128, S306–S314.

172 V. Funes et al. / General and Comparative Endocrinology 149 (2006) 166–172

Dubois, W., Callard, G.V., 1993. Culture of intact sertoli/germ cell unitsand isolated Sertoli cells from Squalus testis. II. Stimulatory eVects ofinsulin and IGF-I on DNA synthesis in premeiotic stages. J. Exp. Zool.267, 233–244.

Duguay, S.J., Lai-Zhang, J., Steiner, D.F., Funkenstein, B., Chan, S.J., 1996.Developmental and tissue-regulated expression of IGF-I and IGF-IImRNAs in Sparus aurata. J. Mol. Endocrinol. 16, 123–132.

Duval, H., Rousseau, K., Elies, G., Le Bail, P.Y., Dufour, S., Boeuf, G.,Boujard, D., 2002. Cloning, characterization, and comparative activityof turbot IGF-I and IGF-II. Gen. Comp. Endocrinol. 126, 269–278.

Fernández-Díaz, C., Yúfera, M., Cañavate, J.P., Moyano, F.J., Alarcón, F.J.,Díaz, M., 2001. Growth and physiological changes during metamorpho-sis of Senegal sole reared in the laboratory. J. Fish Biol. 58, 1–13.

Gabillard, J.C., Weil, C., Rescan, P.-Y., Navarro, I., Gutiérrez, J., 2003a.EVects of environmental temperature on IGF-I, IGF-II and IGF type Ireceptor expression in rainbow trout (Oncorhychus mykiss). Gen.Comp. Endocrinol. 133, 233–242.

Gabillard, J.C., Duval, H., Cauty, C., Rescan, P.-Y., Weil, C., Le Bail, P.-Y.,2003b. DiVerential expression of the two GH genes during embryonicdevelopment of rainbow trout Oncorhynchus mykiss in relation withthe IGFs system. Mol. Rep. Dev. 64, 32–40.

Gabillard, J.C., Rescan, P.-Y., Fauconneau, B., Weil, C., Le Bail, P.-Y.,2003c. EVect of temperature on gene expression of the Gh/Igf systemduring embryonic development in rainbow trout (Oncorhynchusmykiss). J. Exp. Zool. 298A, 134–142.

Greene, M.W., Chen, T.T., 1997. Temporal expression pattern of insulin-like growth factor mRNA during embryonic development in a teleost,rainbow trout (Onchorynchus mykiss). Mol. Mar. Biol. Biotechnol.1997, 144–151.

Greene, M.W., Chen, T.T., 1999. Quantitation of IGF-I, IGF-II, and multi-ple insulin receptor family member messenger RNAs during embry-onic development in rainbow trout. Mol. Rep. Dev. 54, 348–361.

Gutiérrez-Adán, A., Rizos, D., Fair, T., Moreira, P.N., Pintado, B., De laFuente, J., Boland, M.P., Lonergan, P., 2004. EVect of speed of develop-ment on mRNA expression pattern in early bovine embryos culturedin vivo or in vitro. Mol. Rep. Dev. 68, 441–448.

Jentoft, S., Aastveit, A.H., Andersen, Ø., 2004. Molecular cloning andexpression of insulin-like growth factor-I (IGF-I) in Eurasian perch(Perca Xuviatilis): lack of responsiveness to growth hormone treatment.Fish Physiol. Biochem. 30, 67–76.

Jones, J.I., Clemmons, D.R., 1995. Insulin-like growth factors and theirbinding proteins: biological actions. Endocr. Rev. 16, 3–34.

LaTonya, A.C., Wang, S.Y., Wolters, W.R., Peterson, B.C., Waldbieser,G.C., 2005. Molecular characterization of the insuling-like growth fac-tor-I (IGF-I) gene in channel catWsh (Ictalurus punctatus). Biochim Bio-phys Acta 1731, 139–148.

Liu, J.P., Baker, J., Perkins, A.S., Robertson, E.J., Efstratiadis, A., 1993.Mice carrying null mutations of the genes encoding insulin-like growthfactor I (Igf-I) and type 1 IGF receptor (Igf1r). Cell 75, 59–72.

Maestro, M.A., Mendez, E., Parrizas, M., Gutierrez, J., 1997. Characteriza-tion of insulin and insulin-like growth factor-I ovarian receptors dur-

ing the reproductive cycle of carp (Cyprinus carpio). Biol. Reprod. 56,1126–1132.

McCormick, S.D., 1996. EVects of growth hormone and insulin-likegrowth factor I on salinity tolerance and gill Na+, K+-ATPase inAtlantic salmon (Salmo salar): interaction with cortisol. Gen. Comp.Endocrinol. 101, 3–11.

McCormick, S.D., Kelley, K.M., Young, G., Nishioka, R.S., Bern, H.A.,1992. Stimulation of coho salmon growth by insulin-like growth fac-tor-I. Gen. Comp. Endocrinol. 86, 398–406.

Meton, I., Caseras, A., Canto, E., Fernandez, F., Baanante, I.V., 2000. Liverinsulin-like growth factor-I mRNA is not aVected by diet compositionor ration size but shows diurnal variations in regularly-fed gilthead seabream (Sparus aurata). J. Nutr. 130, 757–760.

Parra, G., Yúfera, M., 2001. Comparative energetics during early develop-ment of two marine Wsh species, Solea senegalensis (KAUP) and Spa-rus aurata (L). J. Exp. Biol. 204, 2175–2183.

Perrot, V., Moiseeva, E.B., Gozes, Y., Chan, S.J., Ingleton, P., Funken-stein, B., 1999. Ontogeny of the insulin-like growth factor system(IGF-I, IGF-II, and IGF-1R) in gilthead seabream (Sparus aurata):expression and cellular localization. Gen. Comp. Endocrinol. 116,445–460.

Peterson, B.C., Waldbieser, G.C., Bilodeau, A.L., 2004. IGF-I and IGF-IImRNA expression in slow and fast growing families of USDA103channel catWsh (Ictalurus punctatus). Comp. Biochem. Physiol. Part A139, 317–323.

Peterson, B.C., Bosworth, B.G., Bilodeau, A.L., 2005. DiVerential geneexpression of IGF-I, IGF-II and toll-like receptors 3 and 5 duringembryogenesis in hybrid (channel x blue) and channel catWsh. Comp.Biochem. Physiol. Part A 141, 42–47.

Planas, J.V., Castillo, J., Navarro, I., Gutiérrez, J., 2001. IdentiWcation of atype II insulin-like growth factor receptor in Wsh embryos. Endocrinol-ogy 142, 1090–1097.

Power, D.M., Llewellyn, L., Faustino, M., Nowell, M.A., Bjornsson, B.T.,Einarsdottir, I.E., Canario, A.V., Sweeney, G.E., 2001. Thyroid hor-mones in growth and development of Wsh. Comp. Biochem. Physiol. CToxicol. Pharmacol. 130, 447–459.

Reinecke, M., Collet, C., 1998. The phylogeny of the insulin-like growthfactors. Int. Rev. Cytol. 183, 1–94.

Reinecke, M., Schmid, A., Ermatinger, R., LoYng-Cueni, D., 1997. Insulin-like growth factor I in the teleost Oreochromis mossambicus, the tilapia:gene sequence, tissue expression, and cellular localization. Endocrinol-ogy 138, 3613–3619.

Tse, M.C., Vong, Q.P., Cheng, C.H., Chan, K.M., 2002. PCR-cloning andgene expression studies in common carp (Cyprinus carpio) insulin-likegrowth factor-II. Biochim. Biophys. Acta 1575, 63–74.

Vincent, A.M., Feldman, E.L., 2002. Control of cell survival by IGF signal-ing pathways. Growth Horm. IGF Res. 12, 193–197.

Vong, Q.P., Chan, K.M., Cheng, C.H., 2003. QuantiWcation of commoncarp (Cyprinus carpio) IGF-I and IGF-II mRNA by real-time PCR:diVerential regulation of expression by GH. J. Endocrinol. 178,513–521.