ORIGINAL PAPER

Jun Zheng Æ Kazuaki Nakamura Æ Yoko Maseki

Sofie M. E. Geelissen Æ Luc R. Berghman

Takafumi Sakai

Independent differentiation of mammotropes and somatotropesin the chicken embryonic pituitary gland

Analysis by cell distribution and attempt to detect somatomammotropes

Accepted: 14 September 2005 / Published online: 8 October 2005� Springer-Verlag 2005

Abstract It has been reported that mammotropes in arodent pituitary gland are derived from somatotropesvia somatomammotropes (SMTs), cells that produceboth growth hormone (GH) and prolactin (Prl). How-ever, no studies have been done on the transdifferenti-ation of somatotropes in the chicken pituitary gland. Inthis study, in order to determine the origin of mammo-tropes, we studied detail property of appearance ofchicken somatotropes, mammotropes and pit-1 cells andthen evaluated the existence of SMTs in the chickenembryonic pituitary gland. Immunohistochemical anal-ysis revealed that GH-immunopositive (GH-ip) cellsappeared on embryonic day (E) 14 and were mainlydistributed in the caudal lobe, while Prl-immunopositive(Prl-ip) cells appeared in the cephalic lobe of the pitui-tary gland on E16. In situ hybridization (ISH) and RT-PCR analysis showed that expression of GH and PrlmRNA starts at E12 in the caudal lobe and at E14 in thecephalic lobe respectively. Pit-1 mRNA was first de-tected on E5 by RT-PCR, and pit-1 mRNA-expressing

cells were found in the cephalic lobe on E8. Then withthe ontogeny of the chicken, these cells spread into bothlobes. Using a double staining method with ISH andimmunohistochemistry, we could not detect the exis-tence of SMTs in the chicken embryonic pituitary glandeven in the marginal region of either lobe. These resultssuggest that chicken somatotropes and mammotropesindependently appear in different lobes of pituitarygland and that transdifferentiation from somatotropesto mammotropes is not the central route for differenti-ation of mammotropes in the embryonic chicken pitui-tary gland.

Keywords Chicken Æ GH Æ Prl ÆSomatomammotrope Æ Pit-1

Introduction

The avian anterior pituitary gland consists of well-de-fined cephalic and caudal lobes that can be distinguishedby their morphological structure. Previous studies haveshown those growth hormone (GH)-producing cells,somatotropes, which are localized primarily in the cau-dal lobe, while prolactin (Prl)-producing cells, mam-motropes, exist mainly in the cephalic lobe (Jozsa et al.1979; Barabanov 1991; Berghman et al. 1988). Studieson somatotropes and mammotropes in the embryonicchicken pituitary gland have shown that somatotropescommence differentiation between embryonic day (E) 14and E16 in the caudal lobe (Thommes et al. 1987; Porteret al. 1995), while mammotropes first appear on E17 inthe cephalic lobe (Barabanov 1985; Woods et al. 1998).It has been reported that the GH mRNA level in thecaudal lobe increased on E16 and peaked on E20 (Bossiset al. 2000) and that the Prl mRNA level in the cephaliclobe increased on E18 and peaked on E20 (Kansakuet al. 1994). However, to our knowledge, there has been

J. Zheng Æ K. Nakamura Æ Y. Maseki Æ S. M. E. GeelissenL. R. Berghman Æ T. SakaiDepartment of Regulation-Biology, Faculty of Science, SaitamaUniversity, 255 Shimo-Ohkubo, Saitama, 338-8570 Japan

J. Zheng Æ K. Nakamura Æ Y. Maseki Æ T. Sakai (&)Laboratory of Cell Biology, Department of Regulation-Biology,Faculty of Science, Saitama University, 255 Shimo-Ohkubo,Saitama 338-8570, JapanE-mail: tsakai@post.saitama-u.ac.jpTel.: +81-48-8583422Fax: +81-48-8583422

S. M. E. GeelissenLaboratory of Comparative Endocrinology, Catholic University ofLeuven, Naamsestraat 61, 3000 Leuven, Belgium

L. R. BerghmanDepartment of Poultry science, Texas AM University,College Station, TX 778-2472, USA

Histochem Cell Biol (2006) 125: 429–439DOI 10.1007/s00418-005-0087-8

no detailed study on the relationship between somato-tropes and mammotropes in the embryonic chickenpituitary gland.

In rodents, many studies have shown that somato-tropes differentiate into mammotropes in the embryonicpituitary gland (Frawley et al. 1985; Borrelli et al. 1989).In addition, the existence of somatomammotropes(SMTs), cells that produce both GH and Prl, andestablishment of cell lines that produce both hormonesare strong evidence for this transdifferentiation (Go-luboff et al. 1969; Frawley et al. 1991; Inoue et al. 1991;Chaidarun et al. 1994). On the other hand, contraryresults have cast doubt on the existence of SMTs anddifferentiation of somatotropes into mammotropes(Begeot et al. 1984; Tierney et al. 2002).

It is well known that both somatotropes and mam-motropes express pituitary-specific transcription factor 1(pit-1) (Dolle et al. 1990; Cohen et al. 1996). In thespontaneous mutant Snell dwarf mouse (pit-1dw), pit-1deficiency leads to the ablation of thyrotropes, somato-tropes and mammotropes (Li et al. 1990; Sornson et al.1996) indicating that somatotropes and mammotropesare descendants of common pit-1-expressing precursorcells. Avian pit-1 has been cloned from turkey andchicken pituitary glands (Wong et al. 1992; Tanaka et al.1999). pit-1 mRNA was first detected on E5 in thechicken pituitary gland (Van et al. 2000) and was de-tected in both cephalic and caudal lobes by Northernblot analysis in the adult chicken (Tanaka et al. 1999).However, little is known about the detail property ofspatial appearance of pit-1-producing cells and pit-1mRNA expression in the embryonic chicken pituitarygland.

To clarify the possibility of transdifferentiation ofsomatotropes in the chicken pituitary gland, the presentstudy was designed to investigate the spatial and tem-poral appearance of somatotropes, mammotropes andpit-1 mRNA-expressing cells in the chicken pituitarygland during development and also to evaluate theexistence of SMT using double staining with immuno-histochemistry and in situ hybridization (ISH).

Materials and methods

Tissue sampling

All embryos were obtained from White Leghorn eggs,which were purchased from a commercial dealer (Sai-tama Poultry Laboratory, Saitama, Japan) and incu-bated at 38±1�C in a moisture incubator (P-800,SHOWA, Saitama, Japan). The age of the embryos atthe time of sacrificing (E) indicated the number of daysthat the eggs remained in the incubator. Embryos weresacrificed at various days up to E20 for immunohisto-chemistry, ISH and RT-PCR. Adult hens (White Leg-horn), about 1-year-old, were also obtained fromSaitama Pref. Agriculture and Forestry Research Center(Saitama, Japan). Tissues, including the embryonic

pituitary gland were immediately immersed in Bouin-Hollande fixative for immunohistochemistry and in 4%paraformaldehyde in 1/15 M phosphate buffer (pH 7.4)for ISH. For RT-PCR analysis, pituitary glands wereexcised immediately after sacrifice of embryos at variousembryonic days and immersed in ISOGEN (NipponGene, Toyama, Japan) and were then kept at �80�Cuntil analysis.

Production of monoclonal antiserum for chicken Prl

Production of a monoclonal antibody against re-combinant chicken Prl was performed essentially as de-scribed previously (Berghman et al. 1998). Briefly, micewere immunized s.c. at 3-week intervals with re-combinant chicken Prl (50 lg per animal per injection,total of four injections), and activated splenocytes werefused with myeloma cells by electrofusion (De Boer et al.1989). Hybridomas were screened for antibody produc-tion by an immunocytochemical protocol on sagittalparaffin sections of Bouin–Hollande sublimate-fixedchicken pituitary glands (Berghman et al. 1989). Fusedcells producing the antibodies that showed the sameimmunocytochemical staining property as that obtainedwith previously produced monoclonal antibodies againsta synthetic fragment of chicken Prl (Berghman et al.1992) were cloned by limiting dilution and used forascites production. The hybridoma line (5D7F11) thatproduced the desired results was selected and used in thisstudy.

Western blotting and absorption test

To evaluate the specificity of the monoclonal antibodyfor chicken Prl, we performed western blotting andabsorption test.

The protein extracts of the pituitary gland (30 lg)were separated by sodium dodecyl sulfate (SDS)-poly-acrylamide gel electrophoresis (PAGE) based on themethod of Laemmli as previous described (Maseki et al.2004). Briefly, the discontinuous slab gels consisted of4% stacking gel and 12.5% running gel. The proteinsample was dissolved by boiling for 1 min in a mixtureof 2% SDS, 5% 2-mercaptoethanol, 10% glycerol and0.025% bromophenol blue in 62.5 mM Tris–HCl, pH6.8. The proteins in the gel were electrotransferred ontoa polyvinyllidene difluoride (PVDF) membrane (PVDFProtein Sequencing Membrane, Bio-Rad laboratories)for 50 min at 120 mA in a semi-dry transfer apparatus(Model RB-310, Biocraft Co., Japan). The transferredmembrane was cut to two sheets, one of the membraneswas stained with 0.1% Amino Black 10B (Bio-RadLaboratories). For immunoblotting, the other mem-brane was blocked with 15% skim milk (Yukijirushi,inc., Tokyo, Japan) in PBS overnight and then cut in toeach lane. After washing with PBS, the membrane wasincubated overnight with anti Prl antibody [diluted

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1:16,000 with 10 mM PBS containing 1% normal horseserum and 0.4% Triton X-100 (TNBS)]. After incuba-tion, the membrane were washed extensively with PBSand then incubated with biotinylated goat anti-mouseIgG (Vector Laboratories, Inc., CA, USA) diluted 1:300in TNBS for 2 h. After being washed with PBS, themembrane was incubated with avidin-biotinylated HRP-complex (ABC, Vector Laboratories, Inc., CA, USA)for 30 min and then reacted with 0.02 % 3,3¢-diam-inobenzidine-tetrachloride and 0.006% hydrogen per-oxide in 50 mM Tris–HCl (pH 7.6) for approximately10 min, a permanent brown color reaction product wasproduced to identify Prl immunoreactivity.

All procedures were performed at room temperature.Specificity of the anti GH antibody used in this studywas also checked by Western blotting in the samemanner.

To examine the specificity of immunoreactivity, anabsorption test was performed as follows: The mono-clonal antiserum for chicken Prl was incubated over-night at room temperature with 1 lg/ml of purifiedchicken Prl (provided by National Hormone & PeptideProgram, NIDDK), and the mixture was centrifuged at12,000·g for 20 min at 4�C. The supernatant was usedas the primary antiserum.

Immunohistochemistry

After fixation, the tissue blocks were dehydrated with anascending ethanol series, immersed in methyl benzoateand benzene, and then embedded in PARAPLAST(Oxford, MO, USA). Sagittal sections of 7 lm inthickness were made and mounted on 3-Amin-opropyltriethoxysilane (ShinEtsu, Tokyo, Japan)-coatedslides for immunohistochemistry. The avidin-biotinperoxidase complex technique was used to determine thelocalization of the somatotropes and lactotropes as de-scribed elsewhere (Wada et al. 2003). Briefly, the sectionswere deparaffinized with xylene and rehydrated throughdescending concentrations of ethanol, and then treatedwith 0.5% sodium metaperiodate to block endogenousperoxidase for 10 min. Next, the sections were immersedin 1% normal horse serum and 0.4% Triton X-100 in10 mM phosphate-buffered saline (PBS) (TNBS) for 1 h,and incubated overnight with either GH antibody(1:1,000) or Prl antibody (1:16,000) in TNBS in a humidchamber. Mouse monoclonal antibodies raised againstpurified chicken GH and chicken recombinant Prl wereused in this study. Production and specificity of the GHantibody (IH7) were characterized and published pre-viously (Berghman et al. 1988), and that of antiPrlmonoclonal antibody (5D7F11) was showed in thisstudy.

For controls, sections were incubated with TNBS as asubstitute for GH antibody or Prl antibody. Afterincubation, the sections were washed extensively withPBS and then incubated with biotinylated goat anti-mouse IgG (Vector Laboratories, Inc., CA, USA) di-

luted 1:300 in TNBS for 2 h. After being washed withPBS, the sections were incubated with avidin-biotiny-lated HRP-complex (ABC, Vector Laboratories, Inc.,CA, USA) for 30 mins and then reacted with 0.02%3,3¢-diaminobenzidine-tetrachloride and 0.006% hydro-gen peroxide in 50 mM Tris–HCl (pH 7.6) for approx-imately 10 min, a permanent brown color reactionproduct was produced to identify GH or Prl immuno-reactivity. After processing, the sections were photo-graphed using a light microscope (BX60, Olympus,Tokyo, Japan) fitted with a digital camera (DP70,Olympus, Tokyo, Japan).

In situ hybridization

The tissue blocks were dehydrated with an ascendingethanol series and immersed in methyl benzoateand benzene, and then embedded in PARAP-LAST(OXFORD, MO, USA). Sagittal sections of7 lm in thickness were made and mounted on 3-Am-inopropyltriethoxysilane-coated slides. The sectionswere deparaffinized with xylene and rehydratedthrough descending concentrations of ethanol andwashed in PBS for 1 min, treated with 8–32 lg/mlproteinase K for 30 min at 37�C, and then fixed with4% paraformaldehyde in PB. After washing in PBSfor 1 min, the sections were incubated with 0.2 M HCland washed in PBS for 1 min. The sections were thentreated with 0.25% acetic anhydride in 100 mM tri-ethanolamine for 10 min washed in PBS for 1 minimmersed in a graded ethanol series (70, 80 and 90%)for 15 s each and then in 100% ethanol for 15 s twiceand dried for 20 min. Finally, DIG-labeled antisenseand sense cRNA probe: chicken GH (position 57–686,GenBank # M35609), chicken Prl (position 80–878,GenBank # J04614) and chicken Pit-1 (position 206–1130, GenBank # AF029892) were synthesized fromcDNA fragments, respectively, using a labeling kit(BOEHRINGER MANNHEM, GmbH, Germany).The probe was diluted 1 ng/ll with hybridizationbuffer (50% formamide, 3· SSC, 0.12 M PB, 1·Denhardt solution, 125 lg/ml tRNA, 100 lg/ml sal-mon sperm DNA, 10% dextran sulfate), and droppedon the tissue sections. The sections were covered withparafilm (American National Can Company, CT,USA) and incubated for 16 h at 55�C in a humiditychamber. The parafilm covers were removed by soak-ing the slides in 5· SSC and immersed in 2· SSC for30 min at 50�C. The sections were then treated withTNE [10 mM Tris–HCl (pH 7.6), 500 mM NaCl,1 mM EDTA (pH 8.0)] for 10 min and next withRNase A (50 lg/ml in TNE) for 30 min at 37�C. Thesections were immersed in TNE for 10 min at 37�Cand washed with 2· SSC for 20 min and then with0.2· SSC for 20 min twice at 50�C. The sections wereincubated for 5 min in buffer-1 (100 mM Tris–HCl(pH 7.5), 150 mM NaCl, 0.01% tween 20) at roomtemperature, immersed in 1.5% blocking reagent

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(BOEHRINGER MANNHEIM) in buffer-1 for 1 h at37�C, and then washed in buffer-1 for 5 min at roomtemperature. After washing, the sections were incu-bated with the chicken pituitary powder-absorbedalkaline phosphatase-conjugated anti-DIG antibody(Roche Diagnostics Corporation, Indianapolis, USA)diluted 1:1,000 in buffer-1 at 37�C. The sections werethen washed in buffer-1 for 15 min twice and in buffer-2 (100 mM Tris–HCl (pH 9.5), 100 mM NaCl, and50 mM MgCl2) for 3 min. A chromogen solution(337 lg/ml 4-Nitro blue tetrazolium chloride (NBT),175 lg/ml X-phosphate/5-Bromo-4-chloro-3-indolyl-phospate (BCIP) in buffer-2) was added, and the sec-tions were incubated until a visible signal was detectedat room temperature. The reaction was stopped byimmersing into a reaction stop solution [10 mM Tris–HCl (pH 7.6), 1 mM EDTA (pH 8.0)], and the sec-tions were mounted with 90 % glycerin in PBS, andviewed under a light microscope fitted with a digitalcamera.

Double staining

At first, sections were stained by ISH with DIG-labeledantisense and sense cRNA probes for chicken GH or PrlmRNA. The sections were incubated until a visible sig-nal was detected at room temperature, and then thesections were treated with 0.5% sodium metaperiodateto block endogenous peroxidase. Next, the sections weresubjected to an immunostaining to detect Prl or GH-producing cells, respectively. The double stainingexperiments were conducted by combination of GH andPrl.

RT-PCR

Total RNA was extracted from tissues using ISOGENaccording to the manufacturer’s instruction manual.Traces of DNA contamination were removed by DNasedigestion. cDNA was synthesized from 4.5 lg totalRNA using a Ready-To-Go T-primed First-strand Kit(Amersham Pharmacia Biotech, Uppsala, Sweden). Thefollowing primers were designed to amplify chicken GHcDNA (fragment size: 630 bp): sense primer, 5¢-TCTCCTCTCCTCATCGCTGT-3¢; and antisense pri-mer, 5¢-TCAGATGGTGCAGTTGCTCT-3¢, based onthe chicken GH cDNA sequence (Zhvirblis et al. 1987).The following primer were designed to amplify chickenPrl cDNA (fragment size: 799 bp): sense primer, 5¢-CGGTTCTTCTGGTCTCCAAC-3¢; and antisense pri-mer 5¢-CAGCCAGAATTCACACAAGC-3¢, based onchicken Prl cDNA sequence (Watahiki et al. 1989). Thefollowing primer were designed to amplify the chickenPit-1 cDNA (fragment size: 311 bp): sense primer, 5¢-TCGCATCTTACAGACATGCAG-3¢: and antisenseprimer, 5¢-CCAGCTTAATTCTCCGCAGT-3¢, basedon the chicken Pit-1 cDNA sequence (Tanaka et al.

1999). Each PCR amplification (10 ll) of GH and Prlwas carried out with AmplyTaq Gold polymerse (RocheMolecular Systems Inc., NJ, USA). Reaction mixturescontained 0.1 lM of each primer, 200 mM of eachdNTP, 10 mM KCl, 20 mM Tris–HCl; pH 8.8, 10 mM(NH4)2SO4, 2 mM MgSO4, and 0.25 U of Amply TagGold polymerase and were overlaid with mineral oil.Reactions were performed using a Mini Cycler (MJResearch lnc., MA, USA). Initial template denaturationwas programmed for 10 min at 95�C. The cycle profilewas programmed as follows: 1 min at 94�C (denatur-ation) and 1 min at 55�C (annealing and extension).Forty-five cycles of the profile were run, and the finalextension step was increased to 10 min at 60�C. While,the PCR amplification (20 ll) of Pit-1 was performedusing Ex taq polymerase (Takara, Tokyo, JAPAN) bythe Takara EX taq protocol. The reaction mixturecontained 1 nM of each primer, 250 nM of each dNTP,1 U of Ex taq polymerase and reaction buffer, and theinitial template denaturation was programmed for 5 minat 94�C with the cycle profile programmed as follows:1 min at 94�C (denaturation), 1 min at 60�C (annealingand extension) and 30 s at 74�C (extension). Thirty-eightcycles of the profile were run. Reactions were carried outusing an iCycler (Bio-Rad Laboratories. CA, USA). Thefollowing primers were used to amplify the chicken b-actin cDNA as an internal control (fragment size:105 bp): sense primer, 5¢-TGGCACCACACTTTCTA-CAATGAG-3¢; and antisense primer, 5¢-TGTCATC-TTCTCTCTGTTGGCTTTG-3¢. We obtained a singleband in every stage.

Results

Analysis of specificity for antibodies

We checked the specificity of the anti-Prl antibody byWestern blotting and demonstrated that this antibodyreacts with a protein of about 25 kDa as expected(Fig. 1a). We also performed an absorption test withpurified chicken Prl provided by National Hormone &Peptide Program and found that the immunoreactivityof Prl in the cephalic lobe of the pars distalis of thepituitary gland completely disappeared when the ab-sorbed antibody was used (Fig. 1b, c), indicating thatthis monoclonal antibody specifically reacts with chickenPrl.

The anti GH antibody also reacted with a 22-kDaprotein in the extraction from adult chicken pituitarygland (data not shown).

Appearances and distributions of GHand Prl-immunopositive cells

No GH-immunopositive (GH-ip) cells were detected inthe pars distalis of the pituitary gland until E13 (datanot shown), and a few GH-ip cells first appeared in the

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ventral region of the caudal lobe of the pars distalis inthe pituitary gland at E14 (Fig. 2a). At E16 (Fig. 2b),the GH-ip cells had spread throughout most of thecaudal lobe of the pars distalis. The number of GH-ipcells in the caudal lobe had markedly increased com-pared to that on E14. On E18 (Fig. 2c) and E20(Fig. 2d), GH-ip cells were densely localized in thecaudal lobe of the pars distalis. Most of the GH-ip cellswere localized in the caudal region, though a few GH-ipcells were also found in the ventral region of the cephaliclobe of the pars distalis (Fig. 2c, d).

Prl-immunopositive (Prl-ip) cells were first detected inthe ventral region of the cephalic lobe of the pars distalisof the pituitary gland at E16 (Fig. 2e) and spread outinto the cephalic lobe at E18 (Fig. 2f). These cells weredensely localized in the caudal lobe of the pars distalis atE20 (Fig. 2g) and at the adult stage (Fig. 2h). Some Prl-ip cells were also detected in the anterior region of thecaudal lobe (Fig. 2g, h). No immunopositive cells weredetected in the control sections (data not shown).

GH, Prl, and pit-1 mRNA expression

GH mRNA expression was first detected by ISH in theventral region of the caudal lobe at E12 (Fig. 3a), and

GH mRNA-expressing (GH-ex) cells spread throughoutthe entire caudal lobe of the pars distalis at E16(Fig. 3b). Also, GH-ex cells were clearly observed in thecaudal lobe at E20 (Fig. 2c) and of the adult stage(Fig. 3d). These distributions of GH-ex cells coincidedwith the results obtained by immunohistochemistry.Specificity of ISH analysis was examined by using asense probe, and no signal was found in each stage (datanot shown).

On the other hand, Prl mRNA-expression (Prl-ex)cells were first detected in the dorsal region of the ce-phalic lobe at E16 (Fig. 3e), and the number of thesecells dramatically increased in the cephalic lobe of theanterior pituitary gland after E18 (Fig. 3f). Prl-ex cellswere observed in the cephalic lobe of E20 (Fig. 3g) andat the adult stage (Fig. 3h). These distributions of thePrl-ex cells also coincided with the results of immuno-histochemistry.

Cells expressing pit-1 mRNA were first detected inthe ventral region of the cephalic lobe of the pars distalisat E8 (Fig. 4a). After E10 (Fig. 4b), pit-1-mRNAexpressing cells showed homogeneous distributions inthe two lobes of the pituitary gland, and no differencewas found between cell densities in the cephalic andcaudal lobes of the pars distalis at E12, E14, E16 andE18 (Fig. 4c–f).

Fig. 1 Analysis of specificity ofthe monoclonal antibodyagainst chicken Prl Themonoclonal antibody used inthis study reacted with a 25-kDa protein corresponding tochicken Prl (a). Lane M(molecular weight marker).Many immunoreactive cells(arrowheads) were found in theE20 chicken anterior pituitarygland (b). Anti-chicken Prlantibody absorbed with purifiedchicken Prl (1 lg/ml) showedcomplete loss of theimmunoreactivity (c). Bar20 lm (b, c)

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Double staining for GH and Prl

Double staining using ISH and IHC with different colorssuccessfully showed discrete somatotropes or mammo-tropes in the section. Therefore the double stainingmethod used in this study made it possible to visualizethe colocalization of GH (Prl) mRNA and Prl (GH) in acell (Fig. 4). At E18, GH-ex cells were mainly distrib-uted in the caudal lobe of the chicken pituitary gland,while Prl-ip cells were mainly detected in the cephaliclobe (Fig. 5a). Despite careful examination for the

existence of SMTs, no double-stained cells were foundeven in the boundary region where both cells wereintermingled (Fig. 5c). An exchange experiment yieldedsame results (Fig. 5c, d).

RT-PCR

The results of RT-PCR analysis indicated that GH andPrl mRNA start to express at E12 and E14, respectively,and their expressions continue until adulthood. The

Fig. 2 Appearance of GH-ipcells (a–d) and Prl-ip cells (e–f)in the embryonic chickenpituitary gland. GH-ip cells(arrowheads) first appeared inthe ventral region of the caudallobe of the pars distalis in thepituitary gland at E14 (a). GH-ip cells had spread to mostregions of the caudal lobe of thepars distalis at E16 (b), E18 (c),and E20 (d). A few GH-ip cells(arrows) were also found at E20in the cephalic lobe of the parsdistalis (d) Prl-ip cells(arrowheads) first appeared inthe ventral region of thecephalic lobe of the pars distalisin the pituitary gland at E16 (e),and the number of Prl-ip cellshad markedly increased in theventral part of the cephalic lobeat E18 (f), E20 (g), and adultstage (h). A few Prl-ip cells(arrows) were also found in thecaudal lobe of the pars distalisat E20 (g) and adult stage (h).CA caudal lobe of the parsdistalis, CE cephalic lobe of thepars distalis, dashed lineboundary between the CA andCE. Bar 100 lm (a–g) and200 lm (h)

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results of RT-PCR analysis also revealed that pit-1mRNA expression starts at E5 (Fig. 6).

Discussion

In the present study, we investigated the ontogeny ofchicken somatotropes and mammotropes and theirmRNA expression during development. We found thatno SMTs existed in the chicken pituitary gland, andwe also demonstrated the detailed localization of

pit-1-expressing cells. To our knowledge, this is the firststudy in which a double-staining method was used to tryto detect SMTs in the chicken pituitary gland and also inwhich chicken-pit-1 expressing cells were investigated byISH.

It is well known that pit-1 is a key factor for thedifferentiation of somatotropes, mammotropes andthyrotropes, and the precise temporal and spatialappearances of pit-1-ex cells in the rodent pituitarygland have been demonstrated (Ingraham et al. 1988;Nelson et al. 1988; He et al. 1989; Lin et al. 1994). On

Fig. 3 Microphotographs ofdistribution of GH mRNA-expressing cells (a–d) and PrlmRNA-expressing cells (e–h) inthe embryonic chicken pituitarygland. GH-ex cells (arrowheads)were first detected in the ventralregion of the caudal lobe at E12(a). The number of GH-ex cellsincreased markedly withdevelopment, and the cells hadspread from the ventral part todorsal part of the caudal lobe atE16, E20 and adult stage (b–d).A few GH-ex cells (arrows) werealso observed in the cephaliclobe at E20 and adult stage (c,d). Prl-ex cells (arrowheads)were first detected in the ventralregion of cephalic lobe at E16(e) and had spread to the caudallobe of the pars distalis at E18,E20 and adult stage (f–h). Prl-excells were mainly observed inthe cephalic lobe, although afew Prl-ex cells (arrows) werealso observed in the caudal lobeat E20 and adult stage (g, h).These observations almostcoincided with the results ofimmunohistochemical analysis.Bar 100 lm (a–c, e–g) and200 lm (d, h)

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the other hand, despite the fact that pit-1 has beencloned from turkey and chicken pituitary glands (Wonget al. 1992; Tanaka et al. 1999), not only the physio-logical role of pit-1 cells in the production of specificchicken pituitary hormones but also the ontogeny ofpit1 cells are not clear. We studied the differentiation ofpit-1 cells in the embryonic chicken pituitary gland andfound that pit1-ex cells appeared in the ventral region ofthe cephalic lobe at E8. In a previous study, we foundthat chicken TSHb mRNA-expressing cell appearedfrom E9 in the ventral region of the cephalic lobe, sug-gesting that pit-1 cell differentiation precedes thyrotropeappearance in the same region (Nakamura et al. 2004).This precedent expression of pit-1 was also found in thecase of somatotropes and mammotropes, indicating thatpit1-lineage cells differentiate at one to three days afterthe start of pit-1 expression in the chicken.

It has been shown that murine somatotropes andmammotropes appeared in the dorsal part of theembryonic anterior pituitary gland (Thommes et al.1987; Porter et al. 1995; Barabanov 1985; Woods et al.1998), and it is thought that, at least in rodents, somemammotropes are transdifferentiated from somato-tropes (Frawley et al. 1985). However, since these cells inthe murine pituitary gland are not strictly confined in a

region, it is difficult to evaluate the transdifferentiationof these cells only by their localization. On the otherhand, the advantage of using the chicken pituitary glandfor the study of pituitary cell differentiation is the strictdistribution of hormone-producing cells in a distinctlobe. In this study, we carefully examined the temporaland spatial distributions of these cells in the chickenembryonic pituitary gland using IHC and ISH, and wefound that somatotropes and mammotropes first ap-peared at different timings and in different lobes and thatthey remained throughout life in the lobe in which theyfirst appeared. These results strongly suggest that so-matotropes are not necessary for the differentiation ofmammotropes in the cephalic lobe and that these cellsindependently differentiate in each lobe.

In rodents, many immunohistochemical studieshave shown that SMTs account for 10–15% of cells inthe pituitary gland (Frawley et al. 1991) and are alsostrong evidence of the transdifferentiation of thesecells. On the other hand, many studies cast doubt onthe existence of SMTs in the rodent pituitary gland.For example, Taniguchi et al. reported that no SMTswere detected in the rat pituitary gland during latefetal and postnatal periods of rat pituitary gland(Taniguchi et al. 2001), and contrary opinions based

Fig. 4 Distribution of Pit-1mRNA-expressing cells in thechicken pituitary gland duringdevelopment, a E8, b E10, cE12, d E14, e E16, and f E18.Pit-1-ex cells (arrowheads) werefirst detected in the ventralregion of the pituitary gland atE8. With development, the cellsspread to both lobes, and nodifference between cell densitiesin the cephalic and caudal lobeswas found. Bar 100 lm

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on results of morphological experiments on the dif-ferentiation of mammotropes have also been reported(Begeot et al. 1984; Tierney et al. 2002). They claimedthat these contradictory results may be due to error intechnique of double staining method or the usedantibodies. Therefore, in the present study, to avoidtechnical errors, we tried to detect SMTs with ISHand IHC, and we found that somatotropes andmammotropes were localized in different regions andthat some of these cells were intermingled in theborder zone of the two lobes. However, we were un-able to verify the existence of SMTs even in thisborder zone. These results also suggest that transdif-ferentiation of mammotropes and somatotropes doesnot occur in the embryonic chicken pituitary gland.

Fu et al. recently reported results of a study focusedon the differentiation of mammotropes during chickenembryonic development (Fu et al. 2004). They examinedthe existence of SMTs in isolated embryonic chickenpituitary cells by a double immunostaining method

involving immunohistochemistry combined with animmunofluorescence method and found no SMTs be-tween E16 and E20. Moreover, treatment with cortico-sterone, which is known to stimulate differentiation ofmammotropes and somatotropes, increased the numberof SMTs by less than 1%, indicating that these cells wereprecious few even after stimulation. On the basis of theseexperimental findings, they concluded that mammo-tropes do not differentiate from somatotropes, at least inthe embryonic chicken pituitary gland. In the presentstudy, we used a double staining method involving ISHcombined with immunohistochemistry to visualize thecolocalization of GH (Prl) mRNA and Prl (GH) in theembryonic chicken pituitary gland on the same sectionsin vivo, and even in the boundary region where bothcells were intermingled, no double-stained cells werefound. This result strongly supports Fu’s conclusion.

Taken together, these results strongly suggest thatchicken somatotropes and mammotropes independentlydifferentiate in different lobes of the pituitary gland.

Fig. 6 RT-PCR analysis of theexpression of Prl, GH and Pit-1mRNA in the chickenembryonic pituitary gland.Expression of GH, Prl, and Pit-1 mRNA was observed fromE12, E14 and E5, respectively.Chicken b-actin was used as aninternal control. M,100-bpDNA molecular weight marker.Numbers are embryonic days.A adult, N negative control

Fig. 5 Double stainingdetection of SMTs in the E18chicken pituitary gland. GH-excells (blue, arrowheads) weremainly distributed in the caudallobe of the chicken pituitarygland, while Prl-ip cells (brown,arrows) were detected in thecephalic lobe (a). In an enlargedpicture, no double-stained cellswere found even in theboundary region where bothGH-ex and Prl-ip cells wereintermingled (b). A replacementexperiment yielded the sameresults, Prl-ex cells (blue, arrows)and GH-ip cells (brown,arrowheads) were found in thecephalic and cephalic lobes,respectively (c). No doublestained cells were found in anenlarged picture (d). Bar 100 lm(a, c) and 20 lm (b, d). ISH (bluecolor), immunohistochemistry(brown color)

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These results also support the contrary opinionsregarding the existence of SMTs in the rodent pituitarygland. However, considering transdifferentiation of so-matotropes in the rodent pituitary gland were supportedby not only morphological but also gene targeting re-search, it may appear safe to conclude so far that Fu’sand our conclusion is confined to embryonic chickenpituitary gland.

Acknowledgements We are grateful to Dr. Veerle M. Darras(Catholic University of Leuven, Belgium) and Dr. Kiyoshi Shi-mada (Nagoya University, Japan), for their collaboration on thepreparation of chicken GH and Prl antibody.

Reference

Barabanov VM (1985) Immunohistochemical detection of prolac-tin in the hypophysis of the chick and chick embryo. Ontogenez16:118–126

Barabanov VM (1991) Determination of adenohypophysis cyto-differentiation during embryonic development. Ontogenez22:102–106

Begeot M, Hemming FJ, Dubois PM, Combarnous Y, Dubois MP,Aubert ML (1984) Induction of pituitary lactotrope differenti-ation by luteinizing hormone alpha subunit. Science 226:566–568

Berghman LR, van Beeumen J, Decuypere E, Kuhn ER, Vande-sande F (1988) One-step purification of chicken growth hor-mone from a crude pituitary extract by use of a monoclonalimmunoadsorbent. Comp Biochem Physiol B Biochem MolBiol 113:773–780

Berghman LR, Horsten G, Vandesande F (1989) Development of anovel screening device permitting immunocytochemicalscreening of numerous culture supernatants during hybridomaproduction. J Immunol Methods 125:225–232

Berghman LR, Grauwels L, Vanhamme L, Proudman JA, FoidartA, Balthazart J, Vandesande F (1992) Immunocytochemistryand immunoblotting of avian prolactins using polyclonal andmonoclonal antibodies toward a synthetic fragment of chickenprolactin. Gen Comp Endocrinol 85:346–357

Berghman LR, Devreese B, Verhaert P, Gerets H, Arckens L,Vanden Broeck J, Van Beeumen J, Vaudry H, Vandesande F(1998) The molecular characterisation of chicken pituitary N-terminal pro-opiomelanocortin (POMC). Mol Cell Endocrinol142:119–130

Bossis I, Porter TE (2000) Ontogeny of corticosterone-induciblegrowth hormone secreting cells during chick embryonic devel-opment. Endocrinology 141:2683–2690

Borrelli E, Heyman RA, Arias C, Sawchenko PE, Evans RM(1989) Transgenic mice with inducible dwarfism. Nature339:538–541

Chaidarun SS, Eggo MC, Stewart PM, Barber PC, Sheppard MC(1994) Role of growth factors and estrogen as modulators ofgrowth, differentiation, and expression of gonadotropin subunitgenes in primary cultured sheep pituitary cells. Endocrinology134:935–944

Cohen LE, Wondisford FE, Radovick S (1996) Role of Pit-1 in thegene expression of growth hormone, prolactin, and thyrotropin.Endocrinol Metab 25:523–540

De Boer M, Ossendorp FA, Van Duijn G, Ten Voorde GHJ, TagerJM (1989) Optimal conditions for the generation of monoclonalantibodies using primary immunisation of mouse splenocytes invitro under serum-free conditions. J Immunol Methods121:253–260

Dolle P, Castrillo JL, Theill LE, Deerinck T, Ellisman M, Karin M(1990) Expression of GHF-1 protein in mouse pituitaries cor-relates both temporally and spatially with the onset of growthhormone gene activity. Cell 60:809–820

Frawley LS, Boockfor FR, Hoeffler JP (1985) Identification byplaque assays of a pituitary cell type that secretes both growthhormone and prolactin. Endocrinology 116:734–737

Frawley LS, Boockfor FR (1991) Mammosomatotropes: presenceand functions in normal and neoplastic pituitary tissue. EndocrRev 12:337–355

Fu X, Nishimura S, Porter TE (2004) Evidence that lactotrophs donot differentiate directly from somatotrophs during chickembryonic development. J Endocrinol 183:417–425

Goluboff LG, Ezrin C (1969) Effect of pregnancy on the somato-troph and the prolactin cell of the human adenohypophysis. JClin Endocrinol Metab 29:1533–1538

He X, Treacy MN, Simmons DM, Ingraham HA, Swanson LW,Rosenfeld MG (1989) Expression of a large family of POU-domain regulatory genes in mammalian brain development.Nature 340:35–41

Ingraham HA, Chen RP, Mangalam HJ, Elsholtz HP, Flynn SE,Lin CR, Simmons DM, Swanson L, Rosenfeld MG (1988) Atissue-specific transcription factor containing a homeodomainspecifies a pituitary phenotype. Cell 55:519–529

Inoue K, Sakai T (1991) Conversion of growth hormone-secretingcells into prolactin-secreting cells and its promotion by insulinand insulin-like growth factor-1 in vitro. Exp Cell Res 195:53–58

Jozsa R, Scanes CG, Vigh S, Mess B (1979) Functional differen-tiation of the embryonic chicken pituitary gland studied byimmunohistological approach. Gen Comp Endocrinol 39:158–163

Kansaku N, Shimada K, Terada O, Saito N (1994) Prolactin,growth hormone, and luteinizing hormone-beta subunit geneexpression in the cephalic and caudal lobes of the anteriorpituitary gland during embryogenesis and different reproductivestages in the chicken. Gen Comp Endocrinol 96:197–205

Li S, Crenshaw EB, Rawson EJ, Simmons DM, Swanson LW,Rosenfeld MG (1990) Dwarf locus mutants lacking threepituitary cell types result from mutations in the POU domaingene pit-1. Nature 347:528–533

Lin SC, Li S, Drolet DW, Rosenfeld MG (1994) Pituitary ontogenyof the Snell dwarf mouse reveals Pit-1-independent and Pit-1-dependent origins of the thyrotropes. Development 120:515–522

Maseki Y, Nakamura K, Iwasawa A, Zheng J, Inoue K, Sakai T(2004) Development of gonadotropes in the chicken embryonicpituitary gland. Zoolog Sci 21:435–444

Nakamura K, Iwasawa A, Kidokoro H, Komoda M, Zheng J,Maseki Y, Inoue K, Sakai T (2004) Development of thyroid-stimulating hormone beta subunit-producing cells in thechicken embryonic pituitary gland. Cells Tissues Organs177:21–28

Nelson C, Albert VR, Elsholtz HP, Lu LI, Rosenfeld MG (1988)Activation of cell-specific expression of rat growth hormoneand prolactin genes by a common transcription factor. Science239:1400–1405

Porter TE, Couger GS, Dean CE, Hargis BM (1995) Ontogeny ofgrowth hormone (GH)-secreting cells during chicken embryonicdevelopment: initial somatotrophs are responsive to GH-releasing hormone. Endocrinology 136:1850–1856

Sornson MW, WuW, Dasen JS, Flynn SE, Norman DJ, O’ConnellSM, Gukovsky I, Carriere C, Ryan AK, Miller AP, Zuo L,Gleiberman AS, Andersen B, Beamer WG, Rosenfeld MG(1996) Pituitary lineage determination by the Prophet of Pit-1homeodomain factor defective in Ames dwarfism. Nature384:327–333

Tanaka M, Yamamoto I, Ohkubo T, Wakita M, Hoshino S,Nakashima K (1999) cDNA cloning and developmental alter-ations in gene expression of the two Pit-1/GHF-1 transcriptionfactors in the chicken pituitary. Gen Comp Endocrinol114:441–448

Taniguchi Y, Yasutaka S, Kominami R, Shinohara H (2001)Proliferation and differentiation of pituitary somatotrophs andmammotrophs during late fetal and postnatal periods. AnatEmbryol (Berl) 204:469–475

438

Thommes RC, Umporowicz DM, Leung FC, Woods JE (1987)Ontogenesis of immunocytochemically demonstrable somato-trophs in the adenohypophyseal pars distalis of the developingchick embryo. Gen Comp Endocrinol 67:390–398

Tierney T, Robinson IC (2002) Increased lactotrophs despite de-creased somatotrophs in the dwarf (dw/dw) rat: a defect in theregulation of lactotroph/somatotroph cell fate? J Endocrinol175:435–446

Van As P, Buys N, Onagbesan OM, Decuypere E (2000) Com-plementary DNA cloning and ontogenic expression of pitui-tary-specific transcription factor of chickens (Gallusdomesticus) from the pituitary gland. Gen Comp Endocrinol120:127–136

Wada R, Sakata I, Kaiya H, Nakamura K, Hayashi Y, KangawaK, Sakai T (2003) Existence of ghrelin-immunopositive and -expressing cells in the proventriculus of the hatching and adultchicken. Regul Pept 111:123–128

Watahiki M, Tanaka M, Masuda N, Sugisaki K, Yamamoto M,Yamakawa M, Nagai J, Nakashima K (1989) Primary structureof chicken pituitary prolactin deduced from the cDNA se-quence. Conserved and specific amino acid residues in the do-mains of the prolactins. J Biol Chem 264:5535–5539

Wong EA, Silsby JL, el Halawani ME (1992) ComplementaryDNA cloning and expression of Pit-1/GHF-1 from the domesticturkey. DNA Cell Biol 11:651–660

Woods KL, Porter TE (1998) Ontogeny of prolactin-secreting cellsduring chick embryonic development: effect of vasoactiveintestinal peptide. Gen Comp Endocrinol 112:240–246

Zhvirblis GS, Gorbulev VG, Rubtsov PM, Karapetian RV, Zhu-ravlev IV (1987) Genetic engineering of peptide hormones.Cloning and primary structure of cDNA of chicken growthhormone. Mol Biol (Mosk) 21:1620–1624

439