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

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 ReineckeDivision of Neuroendocrinology, Institute of Anatomy, University of Zurich, Winterthurerstr. 190, CH-8057 Zurich, 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 thatinduces 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 anteriorpituitary, 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 thepituitary was demonstrated in rat (Yamaguchi et al., 1990; Bach &Bondy, 1992; Olchovsky et al., 1993; Gonzalez-Parra et al., 2001),mouse (Iida et al., 2004) and human (Alberti et al., 1991). In ovinepituitary, 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 mousepituitary 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 cells

contained IGF-I-immunoreactivity (Honda et al., 1998). Besides theGH cells, the adrenocorticotropic hormone (ACTH) cells may alsoproduce 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 (Melamedet al., 1999; Eppler et al., 2006). Because in frog, IGF-I-immunore-activity was confined to prolactin (PRL)-immunoreactive cells (Davidet al., 2000), PRL cells are also candidates to produce IGF-I.To date, the presence of the IGF type 1 receptor (IGF-1R) in the

mammalian 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-1Rproduction in mammalian adenohypophysis. The pituitary IGF-Iproduction 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 situhybridization 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: [email protected]

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 RiverLaboratories (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 situhybridization pituitaries were immersed in 4% neutral formalin, forimmunofluorescence 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, Duren; Germany). Two micrograms ofRNA was reverse transcribed with M-MLV reverse transcriptase(Promega, Madison, WI, USA) in the presence of oligo (dT) primer(0.5 lm) and 1 · reaction buffer [5 · (in mm): Tris–HCl, 250,pH 8.3; KCl, 375; MgCl2, 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 mm Tris–HCl, pH 8.3, 50 mm KCl, 1.5 mm MgCl2 and0.001% gelatine, 0.2 lm of each primer, 200 lm of dNTPs and 1 U ofTaq 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 at95 �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 PCRPurification 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 capillaryblotting, 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 mm Tris–HCl, pH 7.4, containing 150 mm

NaCl. The alkaline phosphatase-coupled antibody against DIG(Roche) was diluted 1 : 4000 in blocking solution and incubated for30 min at room temperature. After washing twice in 100 mm Tris–HCl, pH 7.4, 150 mm NaCl for 15 min and equilibration in detectionbuffer (100 mm Tris–HCl pH 9.5; 100 mm NaCl), the membrane wastreated with disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2¢-(5¢-chloro)tricyclo[3,3.1.13.7] 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 lm) were mounted on Super-Frost Plus slides(Menzel-Glaser, Germany) and dried overnight at 42 �C. Afterdewaxing 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 lg ⁄ mL proteinaseK (Qiagen) in 20 mm Tris–HCl, pH 7.4, 2 mm CaCl2 for 10 min at37 �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 mm EDTA, 275 lg ⁄ mL salmon sperm DNA, 250 lg ⁄ mL yeasttRNA] for 1 h at 54 �C. Hybridization was carried out in hybridizationmix (50% formamide, 1 · PBS, 2 · Denhardt’s, 25 mm EDTA,200 lg ⁄ mL salmon sperm DNA, 150 lg ⁄ mL tRNA, 10 mm 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 mm Tris–HCl, pH 7.4,150 mm NaCl) and treated with alkaline phosphatase-coupled anti-DIG antibody (1 : 4000 in P1) each for 1 h at room temperature. Afterwashing and equilibration in P3 (in mm: Tris–HCl, 100, pH 9.5; NaCl,100; MgCl2, 50; levamisole, 5), colour reactions were achieved bytreatment with 188 lg ⁄ mL 5¢-bromo-4¢-chloro-3¢-indolyl phosphate(BCIP) and 375 lg ⁄ 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 LtdEuropean 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 anteriorpituitary.

Immunohistochemistry of semithin sections

Serial semithin sections (1 lm) were cut using an Ultracut E(Reichert-Jung, Zurich, Switzerland), put onto glass slides andincubated with 10 mm gelatine in 10 mL PBS (pH 7.4) containing2% bovine serum albumin (BSA) for 30 min to reduce non-specificbinding. 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. Forthe detection of the primary antisera, the sections were incubated withbiotinylated goat anti-species IgG (1 : 100, BioScience Products,Emmenbrucke, 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 IntenSETM M silver enhancementkit (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 mm 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-rabbitIgG (1 : 50, BioScience Products) for 30 min, followed by incubation

with a streptavidin-gold-15 nm complex (1 : 50, Amersham) for 1 hat room temperature. Sections were rinsed in double-distilled waterand 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. Fordouble-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 ofthe IGF-1R, sections were incubated overnight at 4 �C with the rabbitIGF-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). Afterrepetitive rinses in PBS ⁄ 2% BSA, the sections were incubated for 2 hat room temperature with the antisera directed against the variouspituitary 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-1Rwere performed with a Zeiss Axioscope using the Axiovision 3.1software (Zeiss, Zurich, 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 References

Dilution

(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, Zurich, 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., 2003

1 : 200 – –

Human FSH-b Mouse Ab11069, Abcam, Cambridge, UK Schwarz et al., 1986;Madersbacher et al., 1993

1 : 500 – –

Human LH-b Mouse Ab15226, Abcam, Cambridge, UK Berger, 1985;Schwarz et al., 1986

1 : 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., 2005

1 : 200 1 : 200 1 : 30

Mammalian 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


software (Bitplane AG, Zurich, 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 ratIGF-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 vastmajority 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 aband 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 anteriorpituitary (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 andpeptide level in endocrine cells of the mammalian, i.e. rat, anteriorpituitary. The Northern blot probe spanning exons 2 and 3 revealed agene expression pattern in the rat pituitary identical to that found in ratliver (Murphy et al., 1987; Shimatsu & Rotwein, 1987; Gosteli-Peteret al., 1994), and RT-PCR with IGF-I-specific primers resulted in aband at the expected size of 228 bp. Sequence analysis of the PCRproduct 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 humanpituitary 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 lm.



A major physiological role of IGF-I is to prevent the onset ofapoptosis 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 atall 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 shownpreviously (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 (Gonzalez-Parra et al., 2001). Incorrespondence, treatment of cultured rat pituitary cells with IGF-I hasbeen shown to prevent pituitary cell death induced by serumdeprivation (Fernandez 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 vivoapplication 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 lm) 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 thatcontain 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 lm.



secretion, did not modify the corticotroph responsiveness to CRH(Gianotti et al., 2001). In correspondence, in rat pituitary cultures IGF-I directly inhibited basal as well as theophylline-stimulated GHrelease, while the release of ACTH remained unaltered (Goodyeret al., 1984). Furthermore, IGF-I exerted no significant effect on thebasal or CRH-stimulated ACTH release from ovine pituitary (Lu et al.,1995). However, the presence of IGF-I in ACTH cells may suggestother physiological options. As discussed above, it is conceivable thatIGF-I in rat pituitary may serve as maintenance and proliferation factorwhen released from endocrine cells. On this basis one may speculatethat there may be a particular need for the ACTH cells thatconstitutively express IGF-I and exhibit the IGF-1R (Honda et al.,1998; this study). The involvement of the ACTH cells in coping withstressful situations may also stress the ACTH cells and, thus, requireIGF-I to protect against apoptosis. Experiments with stressfulsituations, such as social stress, may help to clarify this hypothesisand also the problem whether IGF-I in fact is constitutively expressedin the ACTH cells.While IGF-I does not seem to influence the production and release

of ACTH, it most likely influences PRL. No IGF-I was detected inPRL cells, but these exhibited the IGF-1R as shown in the presentstudy. In a subset of about 70% of human pituitary tumoursinvestigated, IGF-I not only inhibited GH release but also stimulatedPRL release (Atkin et al., 1994). In correlation, IGF-I increased PRLsecretion in a rat anterior pituitary cell culture (Lara et al., 1994) andthe PRL mRNA levels in the rat pituitary cell line GH4C1 (Castillo &Aranda, 1997). In primary cell cultures of female rat adenohypophy-sis, IGF-I incubation increased the proliferation of lactotrophs up tothreefold in a dose-dependent manner (Gutierrez et al., 2005).Pronounced changes were observed in the PRL cells of mice withdisrupted IGF-I gene (Stefaneanu et al., 1999). Here the number ofPRL cells was significantly reduced and the cells contained less PRLmRNA. While in normal mice, IGF-I stimulated PRL secretion, nosuch effect was found in mice with disrupted IGF-I gene (Stefaneanuet al., 1999). These effects under physiological conditions may well beraised by circulating IGF-I. However, based on the present results a

role of local IGF-I from neighbouring endocrine cells, e.g. the ACTHcells, is also conceivable.As demonstrated here, IGF-I peptide also occurred in a varying

number of GH cells in rat pituitary. This finding agrees with studies onrat GH3 cells that have been shown to express IGF-I mRNA and tosecrete IGF-I (Fagin et al., 1987, 1989), and with the presence of IGF-I-immunoreactive material in bovine GH cells (Noguchi et al., 1989).In further correspondence, cultured mouse pituitary cells containedboth GH- and IGF-I-immunoreactivities (Honda et al., 1998).IGF-I inhibited basal and GH–releasing hormone (RH)-stimulated

GH expression and secretion in rat dispersed anterior pituitary cells(Sheppard & Bala, 1986), rat and mouse pituitary cell cultures(Yamashita & Melmed, 1986; Simes et al., 1991; Lara et al., 1994;Honda et al., 2003; Pazos et al., 2004), human somatotrophinomas(Atkin et al., 1994), MtT ⁄ S somatotrophic cells (Niiori-Onishi et al.,1999), the rat pituitary cell line GH3 (Castillo & Aranda, 1997) and ina teleost in vitro model (Fruchtman et al., 2000). In correspondence,in vivo experiments in rat showed an inverse relationship between thecirculating IGF-I and the pituitary GH levels (Abe et al., 1983;Tannenbaum et al., 1983). The suppressive action of IGF-I on GHsynthesis and secretion is exerted at the pituitary and not thehypothalamic level (Fletcher et al., 1995), and probably mediated viatype 1 IGF receptors. These have been identified in rat (Rosenfeldet al., 1984; Werther et al., 1989; Olchovsky et al., 1993), ovine(Adam et al., 2000) and mouse (Oomizu et al., 1998) pituitary,particularly on GH cells (Yamamoto et al., 1993; Honda et al., 1998).Thus, there is evidence for a negative feedback mechanism, which isgenerally thought to be exerted on the pituitary GH cells by liver-derived IGF-I (Jones & Clemmons, 1995; Melmed et al., 1996;Reinecke & Collet, 1998; Reinecke, 2004).The present study shows that IGF-I-immunoreactivity is present in

GH cells, with the number of GH cells containing IGF-I exhibitingmarked interindividual differences and that the GH cells showparticularly pronounced equipment with the IGF-1R. The first findingis consistent with data recently obtained in a bony fish species, thetilapia (Eppler et al., 2006). As postulated for tilapia, the pronounced

Fig. 3. Localization of insulin-like growth factor (IGF-I) peptide in gonadotrophic cells. Double-immunofluorescence with antisera against follicle-stimulatinghormone (FSH; A, red), luteinizing hormone (LH; D, red) and IGF-I (B and E, green) (Table 1) visualized with Texas red-conjugated goat anti-mouse IgG for theantibodies against the gonadotrophs and FITC-coupled anti-rabbit IgG for the IGF-I antiserum. (C and F) Merge. Yellow cells contain the gonadotrophic hormonesand IGF-I in coexistence. Erythrocytes show unspecific yellow-orange fluorescence. Scale bar: 15 lm.



Fig. 4. (A) Electron micrograph of an ACTH cell at low magnification. The frame marks the area shown in (B) at higher magnification. Scale bar: 1 lm.(B) Immunocytochemical identification of the ACTH cell. ACTH-immunoreactivity (5 nm gold particles) occurs in the secretory granules. (C) Ultrastructur-al localization of IGF-I-immunoreactivity. ACTH and IGF-I localized with specific antisera (Table 1). ACTH- (5 nm gold particles) and IGF-I- (15 nm goldparticles) immunoreactivities occur in the same secretory granules. (B and C) Scale bar: 75 nm.



interindividual differences in the IGF-I content within the GH cellsmay indicate that synthesis and release of IGF-I by GH cells dependon the physiological status, most likely the serum IGF-I level. Becausethe nutritional status influences the amount of serum IGF-I (Under-wood, 1996), starvation experiments may help to test this hypothesis.Thus, IGF-I released from rat GH cells may suppress GH synthesis

and ⁄ or release by an autocrine feedback mechanism in addition to thewell-established endocrine route. This idea agrees with a previousstudy on mouse pituitary cells (Honda et al., 2003) that has shown thatGH stimulates the expression of IGF-I, and has raised the hypothesisthat GH may first stimulate IGF-I expression in somatotrophs in anautocrine manner and that, in turn, the elevated IGF-I levels within thepituitary may reduce GH synthesis. Indeed, there is evidence for anautocrine or paracrine role of IGF-I in GH regulation of rat becauseelevated pituitary IGF-I levels in streptozotocin-diabetic rats mostlikely caused a suppression of GH synthesis and secretion (Olchovskyet al., 1991).Several in vivo studies have shown that oestrogen may regulate GH

secretion (Chowen et al., 2004), the amount of circulating IGF-I(Jorgensen et al., 2004), and also IGF-I mRNA and IGF-I binding in ratpituitary (Michels et al., 1993) although the evidence is conflicting(Honda et al., 2003). In the present study, no obvious difference wasobserved between male and female individuals, and this was also truefor the portion of GH cells containing IGF-I-immunoreactivity. Thus,the interindividual differences in the IGF-I content within the GH cellsare unlikely caused by differences in the oestrogen status. Because allrats were of the same age (9 weeks old), age also may not be the reasonfor the different degrees of GH ⁄ IGF-I coexistence observed.However, there is also evidence for effects of IGF-I on further

subpopulations of the pituitary endocrine cells, particularly thegonadotrophs. The exposure of rat pituitary cells to IGF-I markedlystimulated basal LH and FSH release (Pazos et al., 2004). Further-more, IGF-I augmented the basal and Gn–RH-stimulated release ofLH from rat anterior pituitary explants or dispersed cells in vitro(Kanematsu et al., 1991; Soldani et al., 1994; Xia et al., 2001). Inagreement with these investigations, the present study shows thepresence of the IGF-1R at LH and FSH cells. Because IGF-I-immunoreactivity was localized only in infrequent LH or FSH cells insome individuals, under physiological conditions these effects may beexerted either by circulating or by IGF-I released from neighbouring

A

B

Fig. 5. Molecular biological characterization and immunofluorescence local-ization of the type 1 insulin-like growth factor receptor (IGF-1R). (A) RT-PCR with rat IGF-1R-specific primers [100 bp molecular weight marker(MWM)]. (B) IGF-1R-mmunoreactivity visualized with a specific antiserum(Table 1) followed by FITC-coupled anti-rabbit IgG. Scale bar: 25 lm.

Fig. 6. Localization of IGF-1R-immunoreactivity at adenohypophyseal cells. Double-immunofluorescence of IGF-1R (yellow-green dots) and the diverseendocrine cells (A–F, red). IGF-1R and pituitary hormone immunoreactivities were identified with specific antisera (Table 1) visualized with FITC-coupled anti-rabbit IgG for the IGF-1R and Texas red-conjugated goat anti-species IgGs for the hormones. Erythrocytes show unspecific yellow-orange fluorescence.(A) ACTH. Scale bar: 15 lm. (B) GH. Scale bar: 12 lm. (C) PRL. Scale bar: 12 lm. (D) FSH. Scale bar: 15 lm. (E) LH. Scale bar: 8 lm. (F) TSH. Scalebar: 15 lm.



GH or ACTH cells. It is also conceivable that IGF-I is expressed ingonadotrophs only during certain reproductive phases, as is indicatedby a study in pig where IGF-I increased the basal LH release with theefficacy changing during the oestrous cycle (Whitley et al., 1995). Theinvestigation of the IGF-I expression in FSH and LH cells duringpuberty and reproductive phase will help to clarify this topic.

In summary, the present study has shown that all subpopulations ofthe endocrine cells exhibit the type 1 IGF receptor, particularly the GHand ACTH cells, and the gonadotrophs. Furthermore, subpopulationsof the endocrine cells, the GH and ACTH cells, and also lesspronounced the gonadotrophs, contain IGF-I. Our results suggestparticular physiological functions of local IGF-I in the rat pituitary.Physiological experiments, such as using stress conditions and foodrestriction, and the investigation of IGF-I in pituitary during puberty orreproductive phases as well as in vitro experiments with well-characterized pituitary cell lines are now needed to clarify the impactof local IGF-I in the pituitary.

Acknowledgements

This work was supported by the Hartmann Muller-Foundation for MedicalResearch (Grant no. 972). We gratefully appreciate the skilful technicalassistance of Mrs Elisabeth Katz.

Abbreviations

ACTH, adrenocorticotropic hormone; BSA, bovine serum albumin; DIG,digoxigenin; DTT, dithiothreitol; FSH, follicle-stimulating hormone; GA,glutaraldehyde; GH, growth hormone; IGF-I, insulin-like growth factor I; IGF-1R, type 1 insulin-like growth factor receptor; LH, luteinizing hormone; PBS,phosphate-buffered saline; PFA, paraformaldehyde; PRL, prolactin; RH,releasing hormone; RT-PCR, reverse transcription-polymerase chain reaction;SDS, sodium dodecyl sulphate; SSC, standard sodium citrate; TSH, thyroid-stimulating hormone.

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