General and Comparative Endocrinology

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1 Generl nd Comprtive Endocrinology 172 (2011) Contents lists ville t ScienceDirect Generl nd Comprtive Endocrinology journl homepge: Insulin nd IGF-I effects on the prolifertion of n osteolst primry culture from se rem (Sprus urt) Encrnción Cpill, Águed Teles-Grcí, Lur Acerete 1, Isel Nvrro, Joquim Gutiérrez Deprtment of Physiology nd Immunology, Fculty of Biology, University of Brcelon, Av. Digonl 645, Brcelon 08028, Spin rticle info strct Article history: Aville online 3 April 2011 Keywords: Bone Fish Cell differentition Minerliztion Endocrine regultion Adipocytes Bone deformities in severl fish species, like gilthed se rem (Sprus urt), re currently mjor prolem in quculture. To gin knowledge of fish skeletl development, primry cell culture hs een estlished from se rem verter. The initil firolstic phenotype of the cells chnged to polygonl shpe during the culture, nd the ddition of n osteogenic medium promoted the deposition of minerls in the extrcellulr mtrix. Cell prolifertion ws nlyzed using the MTT ssy in control nd minerlizing conditions t different culture dys, up to dy 20. The cpcity of the cells to differentite into osteolsts ws evluted using Alizrin red stin. The cells showed slightly incresed prolifertion nd differentition in the presence of osteogenic medium. Furthermore, pluripotentility of these cells ws demonstrted y inducing them to differentite into dipocytes, nd the ccumultion of lipids into the cells ws detected with Oil Red O stining. Susequently, the effects of insulin (1, 10, 100 nd 1000 nm) nd IGF-I (0.1, 1 nd 10 nm) on cell prolifertion were evluted with the MTT ssy t dy 3. Both peptides significntly stimulted the prolifertion of the cells in dose-dependent mnner fter either 24 or 48 h of incution, with IGF-I pprently eing more potent thn insulin. In summry, primry culture of se rem osteolsts hs een chrcterized. This cellulr system cn e good model to study the process of osteolstogenesis in fish nd its endocrine regultion, which my help to improve the qulity of the product in quculture. Ó 2011 Elsevier Inc. All rights reserved. 1. Introduction Skeletl deformities in fish, cused y vriety of fctors including nutritionl, environmentl, physiologicl s well s genetic fctors, re currently mjor prolem in quculture. Gilthed se rem (Sprus urt) is one of the most importnt commercilized species in the Mediterrnen Se, nd operculr mlformtions or column devitions cn ffect up to 27% of the lrve produced [2], which cn result in importnt economic losses. During the lst decde, severl studies hve investigted some of the fctors responsile for inducing such deformities. For exmple, low level of phospholipids in reltion to the totl lipid content in the diet hs een ssocited with n increse in the percentge of mlformtions in se ss lrve [4]. Elevted rering temperture during the erly stges of development hs lso een relted to n increse in the level of deformities in slmon [34,38] nd se ss [1]. More interestingly, genetic study hs identified muttion in Corresponding uthor. Fx: E-mil ddress: ecpill@u.edu (E. Cpill). 1 Present ddress: Deprtment of Cell Biology, Physiology nd Immunology, Fculty of Medicine, Universitt Autonom de Brcelon, Bellterr 08913, Spin. the collgen 1A gene in model of dysplsi in zerfish tht resemles the humn disese, osteogenesis imperfect [13]. In ddition, few studies hve investigted the process of fish skeletl development, nlyzing minly one structure nd minerliztion [10 12]. More recently, gene expression studies in zerfish hve demonstrted tht the process of osteolstogenesis is similr to tht in mmmls nd hve identified mrkers of erly osteolst differentition [20]. However, reserch focused on osteolstogenesis in quculture-relted fish species is scrce. Only very recently, two models of osteolst-like cell cultures derived from fish tissues hve een descried. In one study, two cell lines from se rem clcified tissues hve een chrcterized [28]. In the second study, Ytteorg nd coworkers [41] estlished n osteolst culture from slmon white muscle precursor cells nd studied the effects of polyunsturted ftty cids nd temperture on osteolst differentition. In mmmls, the mesenchyml stem cells (MSCs) from the one mrrow or other dult tissues like ft hve the ility to differentite into different cell types including osteolsts, chondrolsts, dipocytes nd myocytes [22,26]. The pluripotentility of these cells is lso reflected y the lrge quntity of regultory fctors controlling their fte tht hve een chrcterized. Among these re hormones, like insulin, s well s growth fctors, such s the /$ - see front mtter Ó 2011 Elsevier Inc. All rights reserved. doi: /j.ygcen

2 108 E. Cpill et l. / Generl nd Comprtive Endocrinology 172 (2011) pltelet-derived (PDGF), the firolstic (FGF) nd the insulin-like (IGF) growth fctors [6,24]. Both in fish s in mmmls, insulin nd IGF-I re structurlly nd functionlly relted peptides involved in mny regultory functions, such s growth, metolism nd reproduction, tht exert their ctions through specific tyrosine kinse receptors [35]. At the cellulr level, oth elicit vriety of iologicl responses including survivl, prolifertion nd differentition. Regrding the effects of insulin on osteolst cells in mmmls, using the humn osteosrcom cell line MG-63, insulin ws shown to stimulte osteolst prolifertion nd differentition in timend dose-dependent mnner through oth the phosphtidylinositol 3-kinse (PI3K) nd the mitogen-ctivted protein kinse (MAPK) signling pthwys [40]. Moreover, Yng nd coworkers [40] suggested tht the nolic effect of insulin on one could e direct, or indirectly medited through IGF-I, ecuse the expression of this growth fctor in cell culture ws incresed fter insulin stimultion. Furthermore, the stimultory effect of IGF-I on prolifertion nd differentition hs een demonstrted in primry culture of osteolstic cells from Holstein cttle [21]. In vivo, mice null for the igf-i gene exhiited severl developmentl normlities [37]; wheres osteolst-specific knock-out mice missing the igf-i receptor gene displyed reduced osteolst numer with decresed one formtion [42]. Otherwise, trnsgenic mice overexpressing IGF-I in osteolsts showed incresed one formtion despite unchnged osteolst prolifertion [43]. As in mmmls, the GH/IGF-I xis hs een demonstrted to e involved in the endocrine regultion of growth in fish [30]. Interestingly, insulin nd IGF-I hve lso een shown to directly stimulte crtilge growth in the eel [9]. In ddition, the mitogenic effects of IGF-I hve lso een demonstrted in primry cultures of trout myocytes in the presence of 2% nd 5% fetl ovine serum (FBS), wheres insulin ws not le to stimulte thymidine incorportion in these cells [7]. Similrly, prolifertion induced y IGF-I hs een reported in trout cultured dipocytes [3] s well s in the zerfish emryonic cell line ZF-4 [29]. Pozios nd coworkers [29] hve lso demonstrted using the ZF-4 cells tht these effects were medited through the PI3K nd MAPK signling pthwys. Nevertheless, studies on the prolifertive effects of insulin or IGF-I on fish osteolstic cells in culture hve not een performed yet. In the present study, we hve estlished primry culture of osteolsts from verter of se rem. The precursor cells otined hve een chrcterized nd their ility to differentite into osteolsts s well s into dipocyte-like cells hs een determined. Furthermore, the stimultory effects of insulin nd IGF-I on prolifertion hve een oserved. This cellulr system cn e good model to study one development in fish nd to identify the molecules involved in the process of osteolstogenesis. 2. Mterils nd methods 2.1. Estlishment of primry cell culture from se rem verter Gilthed se rems ( S. urt) were otined from the fish frm Tinmenor S.L. (Cntri, Spin) nd mintined in the niml fcilities of the Fculty of Biology t the University of Brcelon. Fish were kept in 200-L fierglss tnks under 12 h light/ 12 h drk photoperiod nd fed d liitum twice dily. All niml hndling procedures were pproved y the Ethics nd Animl Cre Committee of the University of Brcelon, following the Europen Union, Spnish nd Ctln Government-estlished norms nd procedures. Primry cultures of se rem osteolsts were otined following the protocol tht Pominho nd coworkers [28] used for the estlishment of two one-derived cell lines with little modifictions. Six fish of n verge weight of 18 g were used for ech culture. Fish were killed y low on the hed, nd the verterl column ws removed nd clened of ll dherent tissues. After two wshes in phosphte uffered sline (PBS) with 1% ntiiotic/ntimycotic solution, smll frgments were otined mnully using sclpel. Next, two digestions of 30 nd 90 min, respectively, were performed with 0.125% Type II collgense in Hnk s lnced slt solution (HBSS) t 18 C with gittion. Susequently, the frgments otined were wshed with Dulecco s Modified Egle Medium (DMEM) supplemented with 1% ntiiotic/ntimycotic solution. Finlly, cells nd frgments were plted with complete growth medium () composed of DMEM with 10% FBS nd 1% ntiiotic/ntimycotic solution in 10-cm plte nd incuted t 23 C nd 2.5% CO 2. The medi ws chnged every 2 dys. After 1 week, the frgments were removed nd the cells collected with 0.25% trypsin EDTA nd plted into two new 10- cm pltes with fresh medi. From here, the cells were routinely su-cultured 1:2 to 1:4 every time the cells reched out 70 80% confluence in the pltes. The cells were counted nd plted ccordingly, for the different experiments, nd used for mximum of 10 pssges. All plsticwre for tissue culture ws otined from Nunc (Brcelon, Spin). The trypsin EDTA solution ws from Invitrogen (El Prt de Lloregt, Spin), nd ll the other regents were purchsed from Sigm Aldrich (Tres Cntos, Spin) unless otherwise stted Cell culture development chrcteriztion First, the ility of the precursor cells to proliferte nd differentite into osteolsts ws nlyzed. Once in suspension fter mild trypsiniztion, the cells were counted nd plted in 6-well pltes with t density of 10 5 cells per well. The next dy (considered dy 0), the medi ws chnged to grow the cells under control () or minerlizing conditions using n osteogenic medium (), which consisted of growth medium supplemented with 50 lg/ml of L-scoric cid, 10 mm -glycerophosphte nd 4 mm CCl 2 [26,28,41]. Ascoric cid stimultes the deposition of collgen mtrix, nd clcium nd glycerol phosphte re the minerl source for the formtion of hydroxyptite nodules within the collgen. The cells were mintined up to 20 dys with fresh or medi replcement every 3 4 dys. The development of the cells under oth growing conditions ws followed, nd imges were tken t different times using n Axiovert 40C inverted microscope (Zeiss, Germny) coupled to Cnon digitl cmer. Specific ssys to mesure the degree of prolifertion nd differentition of the cells were lso performed t different time points s explined elow Mesurement of cell prolifertion The methylthizolyldiphenyl-tetrzolium romide (MTT) ssy sed on the reduction of the tetrzolium slts y mitochondril reductses into formzn ws used to mesure cell prolifertion in the cell culture s ws previously done for se rem myocytes [23]. Briefly, on the selected dys (0, 5, 10, 15 nd 20), the cells were incuted for 3 h t 23 C in with finl concentrtion of 0.5 mg/ml of MTT. Then, the cells were wshed with PBS nd the lue formzn crystls formed resuspended in 250 ll of dimethyl sulfoxide (DMSO) per well. The prolifertion vlues were otined from the sornce mesured t 570 nm minus the mesurement t 650 nm, which corresponds to the ckground redings. Cells without MTT were used s nonspecific control, nd its vlue ws sutrcted from ll the dt. Dt re presented s fold chnge reltive to dy 0.

3 E. Cpill et l. / Generl nd Comprtive Endocrinology 172 (2011) Chrcteriztion of osteolst differentition Osteolst differentition ws evluted y nlyzing the deposition of minerls in the extrcellulr mtrix (ECM). At different dys during the culture (0, 5, 10, 15 nd 20), minerliztion of the ECM ws detected y Alizrin red stining in cells growing in control () or minerlizing conditions (). The cells were wshed with PBS, fixed for 15 min with 10% formlin nd stined with 2% Alizrin red t ph for 20 min with gentle shking. After wshing excessive dye with wter, the pltes were llowed to dry nd stored t 20 C. Pictures of the stined wells were otined with n Olympus digitl cmer. Quntifiction of the minerliztion ws done y cid extrction of the Alizrin red stin following the protocol of Gregory nd coworkers [15] with minor modifictions. After extrction of the dye with 10% cetic cid, the cells were heted t 85 C for 10 min, cooled on ice, nd centrifuged t 16,000g for 15 min. Then, the superntnt ws neutrlized with 10% mmonium hydroxide, nd liquots of the different smples were red in spectrophotometer t 405 nm. The dt from these redings re presented s fold chnge reltive to dy Chrcteriztion of dipocyte differentition To study the pluripotentility of the precursor cells, their ility to differentite into other cell types, like dipocytes, ws tested. For tht, the cells were counted nd plted into 6-well pltes t density of cells per well, nd the following dy (i.e., dy 0) the medi ws chnged to either or n dipogenic medium (AM). The AM medium [3,26] ws growth medium contining 10 lg/ml insulin, 0.25 lm dexmethsone, 0.5 mm 1-methyl-3- isoutylxnthine (IBMX) nd 5 or 10 ll/ml lipid mixture, designted s AM5 or AM10, respectively. The lipid mixture ws otined from Sigm Aldrich (Tres Cntos, Spin, L5146) nd contined minly cod liver oil nd cholesterol. Morphologicl chnges of the cells were oserved, nd imges were tken t different times using n inverted microscope coupled to cmer s descried ove. To evlute differentition, the ccumultion of neutrl lipids into the cells ws oserved y Oil Red O stining [18]. First, the cells were wshed with PBS, fixed for 1 h with 10% formlin nd stined with 0.3% Oil Red O prepred in 36% tri-ethyl phosphte for 2 h. Quntifiction of the differentition ws then mesured y extrction of the lipids with 2-propnol nd reding the smples in spectrophotometer t 490 nm. Susequent extrction of the proteins with 85% propylene glycol during 3 h t 60 C fter Comssie lue stining for 1 h ws used to correct for the cell content. The vlues were clculted s the sornce mesured t 490 nm divided y the mesurement corresponding to protein t 630 nm. Dt re presented s fold chnge reltive to dy 5 cells growing with Endocrine regultion of precursor cells prolifertion The effects of insulin nd IGF-I on cell prolifertion were nlyzed next. For these experiments, cells plted with in 6-well pltes t density of 10 5 cells per well were used. On dy 3, prolifertion of the cells ws stopped y chnging the to medium consisting on DMEM with 1% ntiiotic/ntimycotic solution nd only 0.02% FBS. After 24 h of strvtion, the medium ws chnged gin to DMEM contining ntiiotics, 2% FBS nd the pproprite mount of hormone. Porcine insulin ws otined from Sigm Aldrich (Tres Cntos, Spin), nd recominnt humn IGF-I ws purchsed from Bchem (Weil m Rhein, Germny). The finl concentrtions of peptide studied were: 1, 10, 100 nd 1000 nm for insulin, nd 0.1, 1 nd 10 nm for IGF-I. Prolifertion ws mesured y the MTT ssy dding the tetrzolium slts to the medi to end the experiments exctly fter 24 or 48 h of hormone stimultion. The dt were otined from the sornce redings s descried ove. The vlues re presented s fold chnge reltive to the corresponding control t ech time Sttisticl nlysis Results re presented s mens ± SEM. To perform the sttisticl nlyses, first it ws confirmed tht the dt (log-trnsformed) were normlly distriuted ccording to the Shpiro Wilk test nd tht presented homogeneity in the vrince ccording to Levene s test. Then, sttisticl differences were nlyzed y onewy nlysis of vrince (ANOVA) followed y Tukey s post hoc test. When the dt did not follow the ANOVA presumptions, the non-prmetric Kruskl Wllis followed y the Mnn Whitney tests were performed. In ddition, Student s t-test ws performed to compre t ech incution time, the effects on prolifertion etween equl concentrtions of insulin nd IGF-I (1 nd 10 nm). Differences were considered sttisticlly significnt t p < Results 3.1. Morphologicl chrcteriztion of the cell culture In the present study, we hve estlished primry culture of se rem osteolsts. The dy fter isoltion from fish verter, despite the lrge quntity of cell deris nd erythrocytes present in the medi, some osteolstic precursor cells were lredy ttched to the plte (dt not shown). One week fter isoltion, groups of cells within the sme plte were found growing t different densities depending on the time they detched from the one frgments (Fig. 1). The imges recpitulte wht cn e oserved throughout osteolst development once the cells re homogeneously replted fter removl of one frgments: isolted cells, to confluent cells strting to differentite. Morphologiclly, the cells were initilly mostly tringulr (Fig. 1, pnel A). Then, they (A) (B) (C) Fig. 1. Phse-contrst imges of precursor cells from verter of se rem 1 week fter isoltion. The three imges re from cells within the sme plte tht were growing t different densities; depending on the time since detchment from one frgments: (A) low, (B) medium nd (C) high. These imges were tken from representtive culture of four independent ones performed.

4 110 E. Cpill et l. / Generl nd Comprtive Endocrinology 172 (2011) chnged to more spindle-like phenotype s they grew forming clusters of cells (Fig. 1, pnel B). Where the cell clusters ecme very dense, the cells ppered to strt differentiting nd chnged in shpe gin (Fig. 1, pnel C). After eliminting the one frgments nd strting the pssging, the cells were chrcterized morphologiclly t specific dys fter plting in control or minerlized conditions. For this, they were plted homogeneously in 6-well pltes t density of 10 5 cells per well with growth () or osteogenic () medi, respectively. Frequent oservtions were performed with n inverted microscope nd pictures tken t specific time points. Representtive imges from these cells re presented in Fig. 2. On dy 0, the cells showed the initil tringulr shpe previously descried (Fig. 2, pnel A) tht chnged into n elongted/firolstic shpe s the cells ecme confluent. This phenotype ws mostly mintined in the cells growing in during the first dys of the culture s it ws oserved y dy 5 (Fig. 2, pnel B). Then, it chnged y dy 10 to more polygonl shpe t which point they spontneously strted to differentite into osteolsts nd styed this wy until dy 20 (Fig. 2, pnels C E). The cells growing in, nd induced to differentite into osteolsts, chnged to polygonl/round shpe y dy 5 (Fig. 2, pnel F). As they continued to grow, the colestone-like ppernce of the culture turned into Dy 0 Dy 5 (A) (B) (F) more homogeneous pttern with diffuse cell limits (Fig. 2, pnels G I). In ddition, lck fiers nd spots ecme visile y dy 15, s deposits of minerls strted to ccumulte in the ECM. These minerl nodules, which were especilly oserved t dy 20 in the cells in, were igger in res of higher cell density (Fig. 2, pnel I) Prolifertion of cells in culture To investigte the prolifertion of the cells in culture, MTT ssys were performed in prllel to the morphologicl studies. As shown in Fig. 3, prolifertion of the cells significntly incresed 7 to 8-fold from dy 0 to dy 5 in cells growing in oth nd medi. From here nd until dy 20, the cells proliferted t constnt rte independent of the medi, despite cells growing in showed slightly higher sornce vlues t ll times (Fig. 3). This seems to indicte tht the cells were contct-inhiited once confluence ws reched, which occurred t out dy 3 ccording to visul oservtion Chrcteriztion of osteolst differentition To further chrcterize the differentition of cells into osteolsts, stining of the minerls deposited in the ECM ws performed with von Koss (dt not shown) nd Alizrin red. Higher deposition of minerls in the culture ws oserved s red stin in the wells of cells growing with in comprison to those growing in control conditions (Fig. 4). Susequent quntifiction of the Alizrin red stin fter its extrction supported this oservtion (Fig. 5). Sustined elevtion in minerl content from dy 0 to dy 20 ws oserved under oth growing conditions. In ddition, the increse versus the zero time ws lredy significntly higher y dy 5. More interestingly, the levels of stined minerls t dy 20 were found to e significntly different etween the cells cultured in medi nd those in medi, which ws in greement with the greter presence of minerl nodules oserved under the microscope in the ltter (Fig. 2) Chrcteriztion of dipocyte differentition Dy 10 (C) (G) To test the ility of these precursor cells to differentite into other cells types, confluent 6-well pltes were induced to differentite into dipocytes. An dipogenic medium contining two Dy 15 Dy 20 (D) (E) (H) (I) Prolifertion (Fold chnge) Time (dys) Fig. 2. Representtive phse-contrst imges of se rem osteolsts t different dys of the culture (0, 5, 10, 15 nd 20). The cells were growing in control (, pnels A E) or minerlizing conditions (, pnels F I) s descried in Section 2. The presence of minerl nodules is indicted y lck rrows. Fig. 3. Prolifertion profile of se rem osteolsts t different dys of culture (0, 5, 10, 15 nd 20). Prolifertion of cells growing in control () or minerlizing conditions () ws mesured using the MTT ssy, s descried in Section 2. Dt re mens ± SEM of 3 4 independent experiments with wells run in duplicte nd presented s fold chnge reltive to dy 0. Different letters indicte significnt differences t p < 0.05.

5 E. Cpill et l. / Generl nd Comprtive Endocrinology 172 (2011) Dy 0 Dy 10 Dy 20 Fig. 4. Representtive imges of se rem cells t different dys of the culture (0, 10 nd 20). Osteogenic differentition of the cells growing in control () or minerlizing conditions () ws detected y Alizrin red stin s descried in Section 2. higher levels of lipids were found in cells growing in AM10 thn in those cultured in AM5, these differences were only significntly different t dy Insulin nd IGF-I effects on precursor cells prolifertion Finlly, the endocrine regultion of prolifertion in the newly estlished se rem osteolst cell culture ws studied. The results otined with the MTT ssys performed fter 24 or 48 h of insulin or IGF-I stimultion t different doses re shown in Fig. 8. At 24 h post-stimultion, insulin showed cler dose response curve, with significnt increses in prolifertion t the concentrtions of 100 nd 1000 nm with respect to the control, nd etween ech concentrtion (Fig. 8, pnel A). After 48 h of incution with insulin, similr profile ws otined, ut the 10 nm dose lso showed significntly higher sornce vlues thn the control t this time (Fig. 8, pnel B). Furthermore, IGF-I cused significntly higher effects on prolifertion in comprison to the control t the doses of 1 nd 10 nm, with identicl results fter oth 24 nd 48 h of incution (Fig. 8, pnels A nd B). Despite the fct tht relile comprisons etween peptides cnnot e done, these dt suggest tht osteolst cells pper to e more sensitive to IGF-I thn insulin in terms of prolifertion ecuse equl concentrtions of oth peptides (1 nd 10 nm) gve significntly higher MTT vlues for IGF-I thn for insulin t either time. Minerliztion (Fold chnge) c Time (dys) different levels of lipids ws used, nmed AM5 nd AM10, with 5 or 10 ll/ml of lipid mixture, respectively. Fig. 6 shows representtive imges of the cells t dys 0, 5 nd 10 of culture. The control cells in mintined tringulr/elongted shpe t ll times (Fig. 6, pnels A C). However, the firolst-like phenotype of the cells growing in chnged completely in the dipose-induced cells y dy 5. These cells were round in shpe nd presented n enlrged cytoplsm with some lipid droplets, especilly those grown with 10 ll/ml of lipid (Fig. 6, pnels D nd F). At dy 10, higher lipid content nd droplets lrger in size thn t dy 5 were oserved in the cells growing in oth concentrtions of lipid (Fig. 6, pnels E nd G). Nevertheless, the mount of lipids tht ccumulted in the cells seemed to e relted to the quntity of lipids dded to the AM medi. To confirm this, the lipids in the cells were stined with Oil Red O, nd this dye ws susequently extrcted nd quntified. The results presented in Fig. 7 were prtilly in greement with visul oservtion. Although d cde Fig. 5. Differentition profile of se rem osteolsts t different dys of culture (0, 5, 10, 15 nd 20). Osteogenic differentition of the cells growing in control () or minerlizing conditions () ws mesured y extrction of Alizrin red s descried in Section 2. Dt re mens ± SEM of three independent experiments with wells run in duplicte nd presented s fold chnge reltive to dy 0. Different letters indicte significnt differences t p < de d e 4. Discussion Osteogenesis is the process of new one formtion. Bone growth nd minerliztion is mostly determined y the numer of osteolsts, which in turn is controlled y fctors tht regulte prolifertion nd differentition of precursor cells [6,24]. Among these molecules, insulin nd IGF-I re well known mitogenic fctors in mmmlin species, importnt for proper development of multiple tissues, including one [5,19,31]. Besides, nomlous one growth nd different types of deformities cn e relted to low one minerl content such s tht cused y low circulting IGF-I levels [37]. In fish lrve, skeletl mlformtions re undnt [2,8]; however, little is known out the process of osteolstogenesis nd its endocrine regultion in quculture-relted species. In the present study, we first estlished nd chrcterized n osteolst primry cell culture from se rem, n importnt commercilized fish species in the Mediterrnen Se. Next, we investigted the process of osteolst differentition nd determined the effects of insulin nd IGF-I on precursor cells prolifertion. The newly estlished cell culture derived from se rem verter gve rise to homogeneous popultion of cells with n initil firolst-like morphology similr to tht of mmmlin one mrrow stem cells [26]. This shpe chnged to more compct/ polygonl shpe chrcteristic of osteolsts, s tht previously descried for differentited humn MSCs derived from one mrrow or treculr one [26,32], or in immortlized rt cell lines of the osteolstic linege [16]. In ddition, these phenotypic chnges throughout development followed the profile lredy reported for two different osteolst-like cell cultures derived from fish tissues, one from se rem clcified tissues nd nother one from slmon muscle stellite precursor cells [28,41]. To etter understnd one growth in fish, we were then interested in studying the endocrine regultion of osteolsts prolifertion in our cell culture. Prolifertion of the precursor cells incresed from dy 0 to dy 5, nd then it reched plteu until dy 20. This suggested tht growth ws inhiited upon contct t confluence. This ehvior is similr to tht of different cultured cells descried y severl uthors, which include one cells

6 112 E. Cpill et l. / Generl nd Comprtive Endocrinology 172 (2011) (A) Dy 0 (B) (D) (F) Dy 5 AM5 AM10 (C) (E) (G) Dy 10 AM5 AM10 Fig. 6. Representtive phse-contrst imges of se rem dipocyte-like cells t different dys of culture (0, 5 nd 10). The cells were growing in control () or dipogenic conditions with 5 or 10 ll/ml lipid mixture (AM5 nd AM10, respectively), s descried in Section 2. Lipid content (Fold chnge) AM5 AM10 c 5 10 Time (dys) Fig. 7. Differentition of se rem dipocyte cells t different dys of culture (5 nd 10). Adipogenic differentition of the cells growing under control () or dipogenic conditions with 5 or 10 ll/ml lipid mixture (AM5 nd AM10, respectively) ws mesured y extrction of the Oil Red O stin s descried in Section 2. Dt re mens ± SEM of 2 3 independent experiments with wells run in duplicte nd presented s fold chnge reltive to dy 5 cells growing with. Different letters indicte significnt differences t p < 0.05 with dys 5 nd 10 nlyzed seprtely. [3,28]. According to this, dy 3 cells were used for the susequent hormonl studies. Altogether, our dt demonstrted tht oth insulin nd IGF-I were le to significntly stimulte in dosedependent mnner the prolifertion of the osteolst precursor cells in culture. The mitogenic effects we oserved for IGF-I re in greement with those reported y mny uthors working with fish cells of different origin. For exmple, Cstillo nd co-workers [7] showed significnt increse in thymidine uptke in trout muscle cells fter 24 h of incution with IGF-I t doses similr to those used in our study. Similr results were lso oserved in emryonic ZF-4 cells where IGF-I, nd lso IGF-II, stimulted DNA synthesis, nd the numer of BrdU positive cells fter 22 nd 42 h of tretment [29]. Nevertheless, there is no informtion to dte regrding the prolifertive effects of IGF-I on fish one-derived cells. In comprison to mmmlin studies, our results re lso in ccordnce to the IGF-I growth-promoting effects on cultured osteolsts either from humn tii or rt verter reported recently [17,21,39]. On the other hnd, one study in humn mrrow stroml cells did not find differences in prolifertion with IGF-I, perhps due to the very long exposure to this growth fctor (28 dys) done to these cells [36]. Whether insulin stimultes cell prolifertion in mmmlin s well s in fish tissues is controversil. Wheres some uthors hve detected mitogenic ctivity in response to insulin, for exmple, in the emryonic zerfish ZF-4 [29] or in the humn MG-63 osteosrcom [40] cell lines, other uthors hve een unle to oserve ny effects even t high doses of insulin or fter long incution times [7]. In our studies, we oserved dose response increse in prolifertion fter 24 or 48 h of incution with insulin t concentrtions s low s 10 nm, in greement with oservtions y Yng nd coworkers [40], who lso reported time response increse. Overll, these dt support role for insulin in regulting growth tht could e tissue specific. Furthermore, the low doses t which IGF-I ws used, in comprison to insulin, were lredy effective in stimulting osteolst prolifertion. These results re in concordnce with previous studies in fish where IGF-I ws more potent thn insulin in ctivting growth s well s most metolic pthwys [7,29]. This oservtion is in greement lso, with the higher undnce of IGF-I receptors over those of insulin reported in severl fish species nd tissues [27], indicting more importnt functionl role for this growth fctor in lower vertertes. Nevertheless, further studies will e required to investigte the effects nd mechnisms of these peptides, oth in prolifertion nd on osteolst differentition. In terms of differentition, n importnt chrcteristic of one tissue, nd therefore of n osteolst cell culture, is the presence of n ECM uilt up of collgen nd the non-collgenous proteins responsile for mtrix mturtion nd minerliztion. As expected, the presence of minerl nodules ws oserved in our cell culture y phse-contrst microscopy fter dys growing in osteogenic medium. To further chrcterize the level of minerliztion long the culture, detection of mtrix minerliztion ws performed

7 E. Cpill et l. / Generl nd Comprtive Endocrinology 172 (2011) A Prolifertion (Fold chnge) B Prolifertion (Fold chnge) Control Control using Alizrin red stin. The level of minerliztion of the ECM incresed over time, nd ws significntly higher thn tht oserved in control conditions t stges of finl differentition. This finding is similr to tht reported in previously estlished osteolst cell cultures y other uthors [26,28,33]. Overll, these results confirm tht the primry culture estlished from se rem verter in the present work elongs to cells tht cn e considered osteolsts. Therefore, this cell system cn e good model with which to study osteolstogenesis in fish nd chrcterize the expression of key moleculr mrkers involved in the process. The multilinege potentil of mmmlin MSCs derived from one mrrow or other dult tissues like ft hs een well descried [26]. This is very importnt ecuse, for exmple, ft is recently eing used for the therpeutic regenertion of different tissues, including one [22]. To determine the pluripotentility of our se rem precursor cells, their ility to differentite into dipocyte-like cells ws tested using n dipogenic medium, which effectiveness hs een previously demonstrted in trout predipocyte cells [3]. The ddition of this dipogenic medium to confluent cell pltes cused rpid morphologicl chnge to the cells, which c d c Insulin IGF-I Concentrtion (nm) Concentrtion (nm) * * * * Insulin IGF-I Fig. 8. Effects of insulin (1, 10, 100 nd 1000 nm) nd IGF-I (0.1, 1 nd 10 nm) on the prolifertion of se rem osteolst precursor cells. On dy 3, fter 24 h of strvtion (0.02% FBS in medi), stimultion with the different doses of peptides ws performed in medi with 2% FBS for 24 (A) or 48 h (B). Prolifertion of the cells ws mesured using the MTT ssy s descried in Section 2. Dt re mens ± SEM of 3 4 independent experiments with wells run in duplicte nd presented s fold chnge reltive to the corresponding control t ech time. Different letters indicte significnt differences t p < 0.05 with insulin nd IGF-I nlyzed seprtely ut with the control in ech cse. Asterisks indicte significnt differences t p < 0.05 etween insulin nd IGF-I t the sme concentrtion (1 nd 10 nm). ecme round-shped nd ccumulted lipids within their enlrged cytoplsm. This morphology, chrcteristic of the mture dipocyte, is in greement with tht previously descried in dipose-induced stem cells or other mmmlin dipocyte cell lines like the 3T3-L1 cells [14,26,32], s well s in recently estlished primry cultures of differentited pre-dipocytes from severl fish species [3,25]. These dt suggest tht the one-derived cells isolted in our study could e considered mesenchyml stem cells, similr to the stellite cells isolted from slmon muscle, which re lso pluripotent, s they were le to differentite into osteolsts [41]. Interestingly, ecuse it is well known in mmmls tht the dipogenic nd osteolstogenic progrms re competitively lnced to control cell fte, this could e good model to study these regultory mechnisms nd the molecules involved, which in fish re still poorly known. In conclusion, in the present study we hve estlished nd chrcterized cell culture with the cpcity to differentite into mture osteolsts, nd lso dipocyte-like cells. We hve identified IGF-I s well s insulin s regultors of precursor cell prolifertion, nd hve estlished the tools to further investigte the process of osteogenesis in fish. This reserch will e very importnt to etter understnd one growth nd the development of skeletl deformities in fish, nd to help improve the qulity of quculture products in future. Acknowledgments The uthors would like to thnk Crlos Mzorr from Tinmenor S.L. 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