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Insulin-like growth factor I (IGF-I) and its receptor (IGF-1R) in the rat anterior pituitary: IGF-I and its receptor (IGF-1R) in rat pituitary

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Few and controversial results exist on the cellular sites of insulin-like growth factor (IGF)-I synthesis and the type 1 IGF receptor (IGF-1R) in mammalian anterior pituitary. Thus, the present study analysed IGF-I and the IGF-1R in rat pituitary.
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  Insulin-like growth factor I (IGF-I) and its receptor(IGF-1R) in the rat anterior pituitary Elisabeth Eppler, Tanja Jevdjovic, Caroline Maake  and  Manfred Reinecke Division of Neuroendocrinology, Institute of Anatomy, University of Zu¨rich, Winterthurerstr. 190, CH-8057 Zu¨rich, Switzerland Keywords  : ACTH, adenohypophysis, gonadotrophs, growth hormone, IGF-I, type 1 IGF receptor Abstract Few and controversial results exist on the cellular sites of insulin-like growth factor (IGF)-I synthesis and the type 1 IGF receptor (IGF-1R) in mammalian anterior pituitary. Thus, the present study analysed IGF-I and the IGF-1R in rat pituitary. Reverse transcription-polymerase chain reaction revealed IGF-I and IGF-1R mRNA expression in pituitary. The sequences of both were identical to thecorresponding sequences in other rat organs.  In situ   hybridization localized IGF-I mRNA in endocrine cells. The majority of the growthhormone (GH) cells and numerous adrenocorticotropic hormone (ACTH) cells exhibited IGF-1R-immunoreactivity at the cellmembrane. At lower densities, IGF-1 receptors were also present at the other hormone-producing cell types, indicating aphysiological impact of IGF-I for all endocrine cells. IGF-I-immunoreactivity was located constantly in almost all ACTH-immunoreactive cells. At the ultrastructural level, IGF-I-immunoreactivity was confined to secretory granules in co-existence withACTH-immunoreactivity, indicating a concomitant release of both hormones. Occasionally, IGF-I-immunoreactivity was detected inan interindividually varying number of GH cells. In some individuals, weak IGF-I-immunoreactions were also detected also in follicle-stimulating hormone and luteinizing hormone cells. Thus, IGF-I seems to be produced as a constituent in ACTH cells, possiblyindicating its particular importance in stress response. Generally, IGF-I from the endocrine cells may regulate synthesis and   ⁄   orrelease of hormones in an autocrine   ⁄   paracrine manner as well as prevent apoptosis and stimulate proliferation. Production of IGF-I inGH cells may depend on the physiological status, most likely the serum IGF-I level. IGF-I released from GH cells may suppress GHsynthesis and   ⁄   or release by an autocrine feedback mechanism in addition to the endocrine route. Introduction Insulin-like growth factor (IGF)-I, a potent mitogenic hormone that induces growth and differentiation (Jones & Clemmons, 1995;Reinecke & Collet, 1998), is mainly produced in the liver, the principalsource of endocrine IGF-I. The main stimulus for synthesis and releaseof liver IGF-I is growth hormone (GH) released from the anterior  pituitary, and IGF-I specifically inhibits GH gene transcription andsecretion (Wallenius  et al  ., 2001) via a negative feedback mechanism.Using radioimmunoassay, reverse transcription-polymerase chainreaction (RT-PCR) or Northern blotting, the presence of IGF-I in the pituitary was demonstrated in rat (Yamaguchi  et al  ., 1990; Bach &Bondy, 1992; Olchovsky  et al  ., 1993; Gonza´lez-Parra  et al  ., 2001),mouse (Iida  et al  ., 2004) and human (Alberti  et al  ., 1991). In ovine pituitary, no IGF-I mRNA (Lu  et al  ., 1995; Adam  et al  ., 2000), but onlyIGF-I-immunoreactivity (Lu  et al  ., 1995) was detected. Few andcontroversial results exist on the cellular sites of IGF-I production. Inrat, IGF-I mRNA has been described as distributed throughout theanterior pituitary (Bach & Bondy, 1992; Michels  et al  ., 1993), and IGF-I-immunoreactive cells in human pituitary have been considered asnon-hormone-producing cells (Ren  et al  ., 1994).In contrast, in a mouse pituitary cell culture (Oomizu  et al  ., 1998) and in human pituitarytumours (Alberti  et al  ., 1991), IGF-I-immunoreactivity has beenconfined to endocrine cells. In cultured mouse pituitary cells, GH cellscontained IGF-I-immunoreactivity (Honda  et al  ., 1998). Besides theGH cells, the adrenocorticotropic hormone (ACTH) cells may also produce IGF-I, as IGF-I was synthesized and secreted from a mouseACTH-derived cell line (Schmidt & Moore, 1994), and recently IGF-ImRNA and peptide were detected in ACTH cells of a teleost (Melamed et al  ., 1999; Eppler   et al  ., 2006). Because in frog, IGF-I-immunore-activity was confined to prolactin (PRL)-immunoreactive cells (David et al  ., 2000), PRL cells are also candidates to produce IGF-I.To date, the presence of the IGF type 1 receptor (IGF-1R) in themammalian pituitary has been shown only by RT-PCR and at mouseGH and ACTH cells only (Honda  et al  ., 1998). Thus, there is noreliable information on the cellular sites of IGF-I and IGF-1R  production in mammalian adenohypophysis. The pituitary IGF-I production sites, however, imply important functional consequences.The presence of IGF-I in supporting cells would likely suggest itsfunction as a maintenance factor. This may be indicated by theobservation that in mice with disrupted IGF-I gene, structural andfunctional alterations occur in somatotrophs and lactotrophs (Stefane-anu  et al  ., 1999). The occurrence of IGF-I in endocrine cells, however,may imply paracrine   ⁄   autocrine-mediated regulations of endocrinecells by IGF-I (Melmed  et al  ., 1996; Oomizu  et al  ., 1998).The present study therefore analyses IGF-I and its receptor in the rat anterior pituitary. Northern blotting, RT-PCR, sequencing and  in situ hybridization were applied to study IGF-I at the mRNA level, andsingle- and double-labelling immunocytochemical techniques withantisera against mammalian IGF-I, GH, PRL, ACTH, follicle-stimula-ting hormone (FSH), luteinizing hormone (LH) and thyroid-stimulating Correspondence : Dr M. Reinecke, as above.E-mail: reinecke@anatom.unizh.ch  Received 26 July 2006, revised 4 October 2006, accepted 25 October 2006   European Journal of Neuroscience, Vol. 25, pp. 191–200, 2007   doi:10.1111/j.1460-9568.2006.05248.x ª  The Authors (2007). Journal Compilation  ª  Federation of European Neuroscience Societies and Blackwell Publishing Ltd  hormone (TSH) to localize the hormones at the light and ultrastructurallevel. The potential presence of the IGF-1R at the different endocrinecells was investigated using RT-PCR, sequencing, and single- anddouble-immunofluorescence. Materials and methods Tissue preparation  All animal experiments were approved by the Institutional AnimalWelfare Committee. Wistar rats (seven males, seven females, bodyweight 150–160 g, age 9 weeks) obtained from Charles River Laboratories (Charles River, Iffa Credo, France) were kept at 25   Con a cycle of 12 h light : dark, and had free access to food anddrinking water. For tissue preparation, animals were anaesthetizedwith Pentothal (0.3 g/100 g body weight, Abbott Laboratories, S.A.,Baar, Switzerland) and bled by aostic puncture. For RNA preserva-tion, pituitaries and tissue specimens of liver were immediatelyexcised and frozen at   ) 80   C until later RNA isolation. For   in situ hybridization pituitaries were immersed in 4% neutral formalin, for immunofluorescence in acetic acid-free Bouin’s solution for 4 h.Thereafter, specimens were washed in 70% ethanol, dehydrated inascending series of ethanol and embedded in Paraplast plus (58   C).For semithin sections and electron microscopy, fixation was performedin a solution containing 2.5% paraformaldehyde (PFA), 0.1%glutaraldehyde (GA) and 0.01% picric acid for 4 h. The specimenswere washed in cacodylate buffer overnight, dehydrated in ascendingseries of ethanol and routinely embedded in LR White (Polysciences,Warrington, USA). RT-PCR for IGF-I and IGF-1R  Total RNA was extracted using a RNA extraction kit (NucleoSpinRNA II, Macherey-Nagel, Du¨ren; Germany). Two micrograms of RNA was reverse transcribed with M-MLV reverse transcriptase(Promega, Madison, WI, USA) in the presence of oligo (dT) primer (0.5  l m ) and 1  ·  reaction buffer [5  ·  (in m m ): Tris–HCl, 250, pH 8.3; KCl, 375; MgCl 2 , 15; dithiothreitol (DTT), 50]. The cDNAswere subjected to RT-PCR using the following primers: IGF-Isense: 5 ¢ -CAGTTCGTGTGTGGACCAAG-3 ¢ ; IGF-I antisense:5 ¢ -GTCTTGGGCATGTCAGTGTGG-3 ¢ ; IGF-1R sense: 5 ¢ -TGGCA-GAACTGCTGTCTGAG-3 ¢ ; and IGF-1R antisense: 5 ¢ -AACGCA-GGGTCTAGTTGAGC-3 ¢ . All amplifications were performed in aGeneAmp PCR System 9600 (Perkin Elmer, Norwalk, CT, USA)cycler in 10 m m  Tris–HCl, pH 8.3, 50 m m  KCl, 1.5 m m  MgCl 2  and0.001% gelatine, 0.2  l m  of each primer, 200  l m  of dNTPs and 1 U of Taq polymerase (Qbiogene, Basel, Switzerland). For IGF-I, one cycleof 1 min at 95   C, 30 s at 58   C, 30 s at 72   C; 31 cycles of 30 s at 95   C, 30 s at 58   C and 30 s at 72   C, followed by a final extensionstep of 5 min at 72   C; for IGF-1R, one cycle of 1 min at 95   C, 45 sat 63   C, 1 min at 72   C; 33 cycles of 45 s at 95   C, 40 s at 63   C and1 min at 72   C, followed by a final extension step of 5 min at 72   Cwere run. The PCR products were visualized by electrophoresis on1.5% ethidium bromide-stained agarose gels and purified using a PCR Purification Kit (Qiagen, Basel, Switzerland). PCR fragments weresequenced (Microsynth, Balgach, Switzerland) and compared with thedatabase. Generation of digoxigenin (DIG)-labelled IGF-I RNA probe  For synthesis of DIG-labelled RNA probes, IGF-I fragments weregenerated as described above by the use of the following IGF-I primers containing T3 or T7 RNA polymerase promoter sequences,respectively (sense: 5 ¢ -CTCTTCTACCTGGCGCTCTGAATTAAC-CCTCACTAAAGGGA-3 ¢ ; antisense: 5 ¢ -GGATCCTAATACGACT-CACTATAGGGCATGTCAGTGTGGC-3 ¢ ). The PCR product has asize of 302 bp plus the promoter’s sequences. Two-hundred nano-grams of the purified PCR product was transcribed  in vitro  using theDIG RNA labelling kit, and either T3 or T7 RNA polymerase (Roche,Rotkreuz, Switzerland) according to the protocol of the manufacturer.The integrity of the probe and efficiency of the labelling wereconfirmed by gel electrophoresis and dot blot. Northern blotting  Total RNA extracted as described was used for isolation of poly ARNA (NucleoTrap, Macherey-Nagel). Ten micrograms of poly ARNA was electrophoresed on a 1% agarose gel containing 2  m formaldehyde, transferred to a nylon membrane (Roche) by capillary blotting, and fixed by UV cross-linking. A membrane was prehybrid-ized at 68   C in a commercial hybridization solution (DIG-Easy Hyb,Roche). Hybridization was performed in the same solution by addingDIG-labelled antisense RNA probe for IGF-I. After 16 h of incubationat 68   C, the membrane was washed twice for 10 min at roomtemperature in 1  ·  standard sodium citrate (SSC)   ⁄   0.1% sodiumdodecyl sulphate (SDS), and for 15 min at 68   C in0.1  ·  SSC   ⁄   0.1% SDS. Membrane was incubated in a 1% blockingreagent (Roche) in 100 m m  Tris–HCl, pH 7.4, containing 150 m m  NaCl. The alkaline phosphatase-coupled antibody against DIG(Roche) was diluted 1 : 4000 in blocking solution and incubated for 30 min at room temperature. After washing twice in 100 m m  Tris– HCl, pH 7.4, 150 m m  NaCl for 15 min and equilibration in detection buffer (100 m m  Tris–HCl pH 9.5; 100 m m  NaCl), the membrane wastreated with disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2 ¢ -(5 ¢ -chloro)tricyclo[3,3.1.1 3.7 ] decan}-4-yl)phenyl phosphate (CSPD;1 : 100 in detection buffer; Roche) for 15 min at 30   C and exposedto X-ray film (XAR; Kodak, Rochester, NY, USA) for 19 h. In situ  hybridization protocol  Paraffin sections (5  l m) were mounted on Super-Frost Plus slides(Menzel-Gla¨ser, Germany) and dried overnight at 42   C. After dewaxing and rehydration in a descending series of ethanol, sectionswere postfixed for 10 min with buffered 4% PFA   ⁄   0.1% GA.Pretreatment steps included a digestion with 20  l g   ⁄   mL proteinaseK (Qiagen) in 20 m m  Tris–HCl, pH 7.4, 2 m m  CaCl 2  for 10 min at 37   C, and an incubation with 1.5% triethanolamine   ⁄   0.25% aceticanhydride (Sigma, Buchs, Switzerland) for 10 min at room tempera-ture. The slides were prehybridized in prehybridization solution [50%formamide, 1  ·  phosphate-buffered saline (PBS), 2.5  ·  Denhardt’s,25 m m  EDTA, 275  l g   ⁄   mL salmon sperm DNA, 250  l g   ⁄   mL yeast tRNA] for 1 h at 54   C. Hybridization was carried out in hybridizationmix (50% formamide, 1  ·  PBS, 2  ·  Denhardt’s, 25 m m  EDTA,200  l g   ⁄   mL salmon sperm DNA, 150  l g   ⁄   mL tRNA, 10 m m  DTT,20% dextran sulphate and 200 ng DIG-labelled RNA probe) for 16 hat 54   C. After high-stringency washes, sections were blocked with1% blocking reagent (Roche) in P1 (100 m m  Tris–HCl, pH 7.4,150 m m  NaCl) and treated with alkaline phosphatase-coupled anti-DIG antibody (1 : 4000 in P1) each for 1 h at room temperature. After washing and equilibration in P3 (in m m : Tris–HCl, 100, pH 9.5; NaCl,100; MgCl 2 , 50; levamisole, 5), colour reactions were achieved bytreatment with 188  l g   ⁄   mL 5 ¢ -bromo-4 ¢ -chloro-3 ¢ -indolyl phosphate(BCIP) and 375  l g   ⁄   mL nitroblue tetrazolium chloride (NBT) (Roche)in P3 for 6 h at room temperature.192 E. Eppler   et al  . ª  The Authors (2007). Journal Compilation  ª  Federation of European Neuroscience Societies and Blackwell Publishing Ltd  European Journal of Neuroscience ,  25 , 191–200  Antisera used  Table 1 summarizes the antisera used for the localization of IGF-I andIGF-1R, and of the different hormone-producing cells in the anterior  pituitary. Immunohistochemistry of semithin sections  Serial semithin sections (1  l m) were cut using an Ultracut E(Reichert-Jung, Zu¨rich, Switzerland), put onto glass slides andincubated with 10 m m  gelatine in 10 mL PBS (pH 7.4) containing2% bovine serum albumin (BSA) for 30 min to reduce non-specific binding. After repetitive washing in PBS, five consecutive semithinsections were processed for immunohistochemistry using the follow-ing protocol: the first, third and fifth sections were incubated with anantiserum against IGF-I, and the second and fourth sections withantisera against classical hormones of the anterior pituitary (Table 1).Incubations were carried out at 4   C for 12 h in a humid chamber. For the detection of the primary antisera, the sections were incubated with biotinylated goat anti-species IgG (1 : 100, BioScience Products,Emmenbru¨cke, Switzerland, and Amersham Int., Little Chalfont, UK,respectively) for 30 min at room temperature, followed by incubationwith a streptavidin-gold-5-nm complex (1 : 100, Amersham) for 1 hat room temperature. After repetitive washes in double-distilled water,the sections were incubated with the IntenSE TM M silver enhancement kit (Amersham) for 10 min at room temperature, counterstained withmethylene blue   ⁄   azure and mounted with glycergel. Electron microscopy  Ultrathin sections were cut at 90 nm, transferred onto nickel grids(mesh size 100), treated with 50 m m  gelatine in 10 mL PBS   ⁄   2% BSAand rinsed in PBS. For single-labelling immunocytochemistry, thesections were incubated with a mouse ACTH antibody (Table 1)overnight followed by biotinylated goat anti-mouse IgG (1 : 50,Amersham) for 30 min, and incubation with streptavidin-gold-5 nmcomplex (1 : 50, Amersham) for 1 h at room temperature. For double-labelling immunocytochemistry, the sections were first incubated witha rabbit IGF-I antiserum (Table 1) overnight. After repeated washes inPBS, the sections were incubated with biotinylated goat anti-rabbit IgG (1 : 50, BioScience Products) for 30 min, followed by incubationwith a streptavidin-gold-15 nm complex (1 : 50, Amersham) for 1 hat room temperature. Sections were rinsed in double-distilled water and air-dried. Thereafter, the sections were incubated with the mouseACTH antibody overnight followed by biotinylated goat anti-mouseIgG (1 : 50) for 30 min. Thereafter, incubation with streptavidin-gold-5 nm complex (1 : 50) was performed for 1 h at room temperature.Sections were stained with uranyl acetate for 4 min. Single- and double-immunofluorescence  After dewaxing and rehydration as described above, microwaving(160 W, 15 min) in 0.01  m  citrate buffer (pH 6) was performed toallow unmasking of the antigen. Unspecific binding was reduced bytreatment with PBS   ⁄   2% BSA for 30 min at room temperature. For double-immunofluorescence of IGF-I and the gonadotropins, sectionswere incubated with a rabbit IGF-I antiserum (Table 1) at 4   Covernight. After repetitive rinses in PBS   ⁄   2% BSA, the sections wereincubated for 2 h at room temperature with the mouse FSH and LHantibodies (Table 1), respectively. For single-immunofluorescence of the IGF-1R, sections were incubated overnight at 4   C with the rabbit IGF-1R antiserum (Table 1). For double-immunofluorescence of theIGF-1R and the classical adenohypophyseal hormones, sections wereincubated overnight at 4   C with the IGF-1R antibody (Table 1). After repetitive rinses in PBS   ⁄   2% BSA, the sections were incubated for 2 hat room temperature with the antisera directed against the various pituitary hormones (Table 1). IGF-I and IGF-1R immunoreactivitieswere visualized by incubation with a fluorescein-isothiocyanate(FITC)-conjugated goat anti-rabbit IgG (1 : 100, Bioscience Prod-ucts), and the classical adenohypophyseal hormones by incubationwith Texas red-coupled antispecies IgGs (1 : 100, Bioscience Prod-ucts), for 30 min at room temperature in the dark. Sections weremounted with glycergel. Imaging  Conventional light and fluorescence microscopy of IGF-I and IGF-1R were performed with a Zeiss Axioscope using the Axiovision 3.1software (Zeiss, Zu¨rich, Switzerland). The IGF-1R localization indouble-immunofluorescence was analysed with a confocal laser-scanning microscope (Leica, Heidelberg, Germany) using the Imaris Table  1. Characterization of the antisera used for immunofluorescence (IF), immunohistochemistry (IHC) and immunocytochemistry (ICC)Antiserum against Host Code, source ReferencesDilution(IF) (IHC) (ICC)ACTH (1–39) Mouse M3501, Dako White  et al  ., 1985, 1987 1 : 100 1 : 50 1 : 30Human GH Guinea pig 4749–9509, Anawa Trading, Zu¨rich, Switzerland Portela-Gomes  et al  ., 2000 1 : 200 1 : 200 – Human PRL Goat Sc-7805, Santa Cruz, CA, USA Kline & Clevenger, 2001;Boon  et al  ., 2003;Antonson  et al  ., 20031 : 200 – – Human FSH- b  Mouse Ab11069, Abcam, Cambridge, UK Schwarz  et al  ., 1986;Madersbacher   et al  ., 19931 : 500 – – Human LH- b  Mouse Ab15226, Abcam, Cambridge, UK Berger, 1985;Schwarz  et al  ., 19861 : 200 – – Human TSH- b  Mouse M3503, Dako Rhodes & Chan, 2002 1 : 50 – – Human IGF-I Rabbit Sc 9013, Santa Cruz, CA, USA Bowen  et al  ., 2002;Bodo  et al  ., 20051 : 50 – – Human IGF-I Rabbit 116 (own antiserum) David  et al  ., 2000;Reinecke  et al  ., 2000;Zapf   et al  ., 2002;Jevdjovic  et al  ., 20051 : 200 1 : 200 1 : 30Mammalian IGF-1R Rabbit Sc-7952, Santa Cruz, CA, USA Camarero  et al  ., 2003 1 : 100 – –  IGF-I and its receptor (IGF-1R) in rat pituitary 193 ª  The Authors (2007). Journal Compilation  ª  Federation of European Neuroscience Societies and Blackwell Publishing Ltd  European Journal of Neuroscience ,  25 , 191–200  software (Bitplane AG, Zu¨rich, Switzerland). Transmission electronmicroscopy analysis of ultrathin sections was performed with a CM100 (Philips, Eindhoven, The Netherlands). Results IGF-I in male and female rats   No obvious differences in the distribution patterns of IGF-I mRNAand peptide or in the localization of the IGF-1 receptor (IGF-1R) at thedifferent subtypes of endocrine cells were observed between pituitariesof male and female individuals. IGF-I mRNA expression within the rat anterior pituitary  IGF-I mRNA was detected in rat pituitary by Northern blot analysiswith a rat IGF-I-specific probe (Fig. 1A) whereby the bands obtainedexhibited the expected sizes for rat IGF-I. IGF-I gene expression wasfurther revealed by RT-PCR with rat IGF-I-specific primers (Fig. 1B).Gene sequence comparison of the RT-PCR product (228 bp) with thedatabase revealed a 100% sequence identity with the corresponding rat IGF-I gene sequence for liver. By  in situ  hybridization with an IGF-I-specific antisense probe IGF-I mRNAwas localized in endocrine cellsscattered throughout the adenohypophysis (Fig. 1C), whereas thecorresponding sense probe as a negative control did not reveal anysignal (Fig. 1D). Immunohistochemical localization of IGF-I peptide  Both antisera used, i.e. 116 and Sc 9013, reacted with identical cellsalthough sometimes with slightly varying intensities. In all individualsinvestigated, consecutive semithin sections revealed that the vast majority of the ACTH cells (Fig. 2A) contained IGF-I-immunoreac-tivity (Fig. 2B). While in numerous individuals GH cells (Fig. 2C)did not contain IGF-I-immunoreactivity (Fig. 2B), in some individualsa minority of the GH cells (Fig. 2D) exhibited IGF-I-immunoreactivity(Fig. 2E). In the semithin sections, infrequent IGF-I-containing cells(Fig. 2B, double arrows) were detected that were neither ACTH(Fig. 2A, double arrows) nor GH (Fig. 2C, double arrows) cells. Bythe use of double-immunofluorescence, in a number of individualssome FSH (Fig. 3A–C) and LH (Fig. 3D–F) cells were found tocontain IGF-I-immunoreactivity (Fig. 3B and E). Ultrastructural localization of IGF-I  At the ultrastructural level, ACTH cells exhibited the typical shape(Fig. 4A) and exhibited ACTH-immunoreactivity within their granules(Fig. 4B). By the use of the double-immunogold technique on thinsections IGF-I-immunoreactivity was co-localized with ACTH-immu-noreactivity in secretory granules (Fig. 4C). Expression of IGF-1R mRNA RT-PCR in rat pituitary with rat IGF-1R-specific primers revealed a band at the expected size (Fig. 5A). Gene sequence comparison of thePCR product with the database revealed a 100% identity with thecorresponding IGF-1R gene sequence for other rat organs. Immunohistochemical localization of the IGF-1R  IGF-1R-immunoreactivity was distributed throughout the rat anterior  pituitary (Fig. 5B) where it was localized at all types of endocrinecells investigated (Fig. 6). The highest density of IGF-1R-immuno-reactions was observed at ACTH (Fig. 6A) and GH (Fig. 6B) cells,and at the gonadotrophs, i.e. FSH (Fig. 6D) and LH (Fig. 6E) cells.At PRL cells (Fig. 6C) IGF-1R-immunoreactivity was localizedoccasionally, and at thyroid-stimulating hormone (TSH) cells veryrarely (Fig. 6F). Discussion The present study is the first to localize IGF-I at the mRNA and peptide level in endocrine cells of the mammalian, i.e. rat, anterior  pituitary. The Northern blot probe spanning exons 2 and 3 revealed agene expression pattern in the rat pituitary identical to that found in rat liver (Murphy  et al  ., 1987; Shimatsu & Rotwein, 1987; Gosteli-Peter  et al  ., 1994), and RT-PCR with IGF-I-specific primers resulted in a band at the expected size of 228 bp. Sequence analysis of the PCR  product approved that the gene sequence of IGF-I in the pituitary isidentical to that published for other rat organs (Shimatsu & Rotwein,1987).  In situ  hybridization localized IGF-I mRNA scatteredthroughout the adenohypophysis with varying densities in a distribu-tion pattern equivalent to that of the peptide. IGF-I-immunoreactivityoccurred in different subpopulations of the endocrine cells, but wasnot detected in supporting cells. In rat, IGF-I mRNA was evenlydistributed throughout the anterior pituitary (Bach & Bondy, 1992;Michels  et al  ., 1993) and thought to be expressed either by allendocrine cells or by folliculo-stellate cells (Michels  et al  ., 1993). Incontrast, IGF-I-immunoreactive cells in human pituitary were des-cribed as non-hormone-producing cells (Ren  et al  ., 1994), and in amouse pituitary cell culture (Oomizu  et al  ., 1998) and in human pituitary tumours (Alberti  et al  ., 1991) IGF-I-immunoreactivity wasconfined to endocrine cells. The present study indicates that thesynthesis of IGF-I in rat pituitary occurs in the endocrine cells. Fig . 1. Expression of insulin-like growth factor (IGF-I) mRNA. (A) Nor-thern blot. Bands are shown at the expected transcript sizes. (B) RT-PCR withrat IGF-I-specific primers [100 bp molecular weight marker (MWM)].(C)  In situ  hybridization with IGF-I antisense-specific probe. Numerousendocrine cells in the anterior pituitary express IGF-I mRNA. (D) No signalscan be obtained by  in situ  hybridization with IGF-I sense control. Scale bar:100  l m. 194 E. Eppler   et al  . ª  The Authors (2007). Journal Compilation  ª  Federation of European Neuroscience Societies and Blackwell Publishing Ltd  European Journal of Neuroscience ,  25 , 191–200  A major physiological role of IGF-I is to prevent the onset of apoptosis and promote cell proliferation (LeRoith & Roberts, 2003).Thus, IGF-I released from the different endocrine cells may havewidespread local functions exerted in either autocrine or paracrinemanner. This hypothesis gets support by the presence of the IGF-1R at all endocrine subpopulations as shown in the present study. Inagreement, a stimulatory effect of IGF-I on the proliferation of alltypes of cultured mouse endocrine pituitary cells has been shown previously (Oomizu  et al  ., 1998). Apoptosis has been demonstrated tooccur in rat pituitary (Drewett   et al  ., 1993), and there is some evidencethat it is prevented by IGF-I (Gonza´lez-Parra  et al  ., 2001). Incorrespondence, treatment of cultured rat pituitary cells with IGF-I has been shown to prevent pituitary cell death induced by serumdeprivation (Ferna`ndez  et al  ., 2004). Based on the present results, it is assumed that the protective and proliferative effects of IGF-I in therat pituitary may be exerted not only by circulating but also by localIGF-I released from pituitary endocrine cells.In the adenohypophysis of all individuals investigated, IGF-I-immunoreactivity was found in the vast majority of the ACTH cells, possibly indicating that IGF-I is constitutively synthesized in ACTHcells. To date, there is little evidence for a co-existence of ACTH andIGF-I in the mammalian pituitary. Only the mouse pituitary cortico-troph tumour cell line AtT-20 has been shown to synthesize andsecrete IGF-I (Schmidt & Moore, 1994). The present study also showsthat ACTH- and IGF-I-immunoreactivities occur in the same secretorygranules, indicating a concomitant release of both hormones. Thereseems to be no effect of IGF-I on ACTH secretion as  in vivo application of IGF-I in human, although leading to a decreased GH Fig . 2. Localization of insulin-like growth factor (IGF-I) peptide in adrenocorticotropic hormone (ACTH) and growth hormone (GH) cells. Consecutive semithin(1  l m) sections were stained with a mouse ACTH antibody (A), a rabbit antiserum directed against IGF-I (B and E) and a guinea pig GH antiserum (C and D)(Table 1) followed by biotinylated goat anti-species antisera visualized with 5 nm gold-coupled streptavidin and silver enhancement. (A–C) All ACTH cells (A)contain IGF-I immunoreactivity (B). Examples marked by single arrows. No GH cells (C) exhibit IGF-I immunoreactivity (B). Double arrows point to cells that contain IGF-immunoreactivity (B), but neither ACTH- (A) nor GH- (C) immunoreactivities. (D and E) Infrequent GH cells (D) in a different individual displayIGF-I-immunoreactivity (E). Scale bar: 25  l m. IGF-I and its receptor (IGF-1R) in rat pituitary 195 ª  The Authors (2007). Journal Compilation  ª  Federation of European Neuroscience Societies and Blackwell Publishing Ltd  European Journal of Neuroscience ,  25 , 191–200
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