Distribution of androgen receptor immunoreactivity in the spinal cord of wild-type,...

Distribution of Androgen Receptor-ImmunoreactiveCells in the Quail Forebrain and Their Relationshipwith Aromatase Immunoreactivity

J. Balthazart,1 A. Foidart,1 M. Houbart,1 G. S. Prins,2 G. F. Ball3

1 Laboratory of Biochemistry, University of Liege, 17 Place Delcour, Bat. L1, B-4020 Liege, Belgium

2 Department of Urology, University of Illinois, Chicago, Illinois 60616

3 Department of Psychology, Behavioral Neuroendocrinology Group, Johns Hopkins University,Baltimore, Maryland 21218

Received 7 July 1997; accepted 8 January 1998

the paraventricular (PVN), and the ventromedialABSTRACT: The distribution of androgen recep-(VMN) nuclei, but also in the tuberculum olfactorium,tor-like immunoreactive (AR-ir) cells in the quail brainthe nucleus accumbens/ventral striatum, the nucleuswas analyzed by immunocytochemistry with the use oftaeniae, the tuberal hypothalamus, the substantia griseathe affinity-purified antibody PG-21-19A raised againstcentralis (GCt), and the locus ceruleus. Cells exhibitinga synthetic peptide representing the first 21 N-terminala dense AR-ir label were also detected in the nucleusamino acids of the rat and human AR. This antibody isintercollicularis. Preincubation of the primary antibodyknown to bind to the receptor in the absence as well as inwith an excess of the synthetic peptide used for immuni-the presence of endogenous ligands, and it was thereforezation completely eliminated this nuclear staining. A sig-expected that a more complete and accurate character-nificant number of AR-ir cells in the POM, VMN, PVN,ization of AR-ir cells would be obtained in comparisonand tuberal hypothalamus also contained ARO-ir mate-with previous studies using an antibody that preferen-rial in their cytoplasm. These data confirm and extendtially recognizes the occupied receptor. Selected sectionsprevious studies localizing AR in the avian brain, andwere double labeled for aromatase (ARO) by a tech-raise questions about the possible regulation by andro-nique that uses alkaline phosphatase as the reportergens of the metabolizing enzyme aromatase. q 1998 Johnenzyme and Fast blue as the chromogen. AR-ir materialWiley & Sons, Inc. J Neurobiol 35: 323–340, 1998was detected in the nucleus of cells located in a variety ofKeywords: preoptic area; androgen receptor; aro-brain areas in the preoptic region and the hypothalamusmatase; immunocytochemistry; Japanese quailincluding the medial preoptic (POM), the supraoptic,

INTRODUCTION in part on the aromatization of testosterone (T) intoestrogenic metabolites at the neural target siteThe sexual differentiation and activation of male(MacLusky and Naftolin, 1981; Meisel and Sachs,sexual behavior in birds, as in mammals, depends1994; Balthazart, 1989). These locally formed es-trogens are thought to act primarily through specificCorrespondence to: J. Balthazart

Contract grant sponsor: NIMH; contract grant number: RO1 nuclear receptors (Kuiper et al., 1997). The distri-MH50388 bution of these receptors has been investigated in

Contract grant sponsor: Belgian FRFC; contract grant num-the avian and in particular the quail brain by autora-ber: 9.4565.96F

Contract grant sponsor: Government of the French Commu- diographic (Martinez-Vargas et al., 1976; Nordeennity of Belgium; contract grant number: 93/98-171 et al., 1987; Watson and Adkins-Regan, 1989) and

Contract grant sponsor: NATO; contract grant number:immunohistochemical (Gahr et al., 1987; Baltha-CRG910526

q 1998 John Wiley & Sons, Inc. CCC 0022-3034/98/030323-18 zart, 1989; Hutchison et al., 1990; Gahr, 1990)

323

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324 Balthazart et al.

methods. The T-metabolizing enzyme aromatase recognition of the occupied receptor by AR32 inimmunocytochemical studies may explain the lim-has been localized in the quail brain by radioenzy-

matic assays on micropunched nuclei (Schumacher ited distribution of the androgen receptors observedin the quail brain (see Balthazart et al., 1992b, forand Balthazart, 1987; Balthazart et al., 1990c) and

by immunohistochemistry (Balthazart et al., 1990a, discussion). The main goal of the present study wastherefore to reevaluate the distribution of androgen1990b; Foidart et al., 1995).

The Japanese quail Coturnix japonica has receptors with the use of another antibody (PG-21) that is specific for the N-terminal region ofemerged as an excellent model for the analysis of

steroid action in the brain, in relation with the sexual the androgen receptor and is known to bind to thereceptor in the absence as well as in the presencedifferentiation and activation of male copulatory be-

havior (for review, see Adkins, 1981; Balthazart, of endogenous ligands (Wood and Newman, 1993).Therefore, studies with this antibody should provide1989; Balthazart and Foidart, 1993, Balthazart and

Ball, 1995). There is evidence that some of the a more complete visualization of the distributionof androgen receptors to our previous work usingbehavioral effects of T in quail (e.g., the activation

of the crowing vocalization and strutting display) antibody AR32 that may have been markedly af-fected by the degree of receptor occupation.are due to the direct interaction of T or its andro-

genic metabolite, 5a-dihydrotestosterone (DHT), This study identified the presence of androgenreceptor-containing cells in many brain areas thatwith androgen receptors (Adkins, 1977; Adkins et

al., 1980; Adkins-Regan, 1981; Schumacher and are known to contain aromatase. A broad corpus offunctional studies carried out on a variety of mam-Balthazart, 1983). It also appears to be the case

that DHT and estradiol (E2) act synergistically to mals and birds, including the Japanese quail, hadestablished that preoptic aromatase activity is con-activate copulatory behavior in male quail (Adkins

et al., 1980; Schumacher and Balthazart, 1983; Bal- trolled by a synergistic action of androgens and es-trogens, with the most prominent role being playedthazart et al., 1985; Alexandre and Balthazart,

1986). The distribution of androgen receptors has by androgens in mammals, but by estrogens in birds(Roselli, 1991; Schumacher et al., 1987; Balthazartbeen described in a number of avian species by

autoradiographic methods using [3H]-T or [3H]- and Foidart, 1993). In quail in particular, we haddemonstrated that treatment of castrated males withDHT as ligands (for review, see Balthazart, 1983;

Ball, 1990), and in one songbird species, the zebra testosterone increases the concentration of thearomatase mRNA, the number of aromatase-immu-finch, by binding assay methods (Harding et al.,

1984). In quail, comparative autoradiographic stud- noreactive (ARO-ir) cells and aromatase activity inthe preoptic area, and that all these effects can beies with [3H]-T and [3H]-DHT identified a wide-

spread distribution of labeled cells after injection of mimicked by treating birds with estrogens alone(for review, see Balthazart and Foidart, 1993; Bal-[3H]-T, but relatively few binding sites for [3H]-

DHT were detected (Watson and Adkins-Regan, thazart et al., 1996). However, these effects of es-trogens are clearly enhanced by a concurrent treat-1989). These results suggested that the large num-

ber of binding sites for [3H]-T were actually estro- ment with a nonaromatizable androgen such as 5a-dihydrotestosterone or methyltrienolone (R1881)gen receptors occupied by estrogens derived from

the aromatization of the tritiated T. More recently, that indicates a direct participation of androgens inthe control of aromatase (Schumacher et al., 1987;the distribution of androgen receptors was reana-

lyzed in quail by immunohistochemistry using the Balthazart et al., 1994; Harada et al., 1993). Thesefunctional data therefore suggested that aromatasepolyclonal antibody AR32 (Balthazart et al.,

1992b). However, this study identified relatively cells in the brain might contain both androgen andestrogen receptors. Previous studies indicated thatfew brain regions containing androgen receptor-im-

munoreactive (AR-ir) cells. Cells exhibiting immu- the cellular colocalization of aromatase and estrogenreceptors varies among different brain areas. Innoreactivity for the androgen receptor were limited

to the nucleus intercollicularis (ICo), and in much some areas such as the ventromedial nucleus of thehypothalamus, the cellular colocalization of thesesmaller numbers in preoptic-hypothalamic nuclei

such as the nucleus preopticus medialis (POM) two proteins is almost the rule; in other areas suchas the medial preoptic nucleus, it is quite rare (Bal-and the nucleus paraventricularis magnocellularis

(PVN). thazart et al., 1991). To our knowledge, no studyhas been completed to date (in quail or in any otherStudies in other species suggest that androgen

receptors are more widespread, but technical limita- species) that has analyzed the possible colocaliza-tion of androgen receptors and of aromatase. Thetions such as a high peripheral catabolism of [3H]-

DHT in autoradiographic studies or a preferential recent identification of a potential androgen re-


https://www.researchgate.net/publication/19468131_Estrogen_accumulation_in_zebra_finch_song_control_nuclei_Implications_for_sexual_differentiation_and_adult_activation_of_behavior?el=1_x_8&enrichId=rgreq-378a0425-7fb4-4003-8c12-2d9a4a6844a6&enrichSource=Y292ZXJQYWdlOzEzNjYyNTE2O0FTOjE1NDA3MDgzNjg0NjU5NEAxNDEzNzQ0NzU4OTM4

Androgen Receptors in Quail Brain 325

300 (1.65 mg/mL) in PBST. The sections were subse-sponse element at the 5* end of the brain specificquently processed according to the multiple-bridge tech-exon 1f of aromatase clearly suggests that such anique (Blaustein and Turcotte, 1989a, 1989b) to enhancecolocalization may be of functional significancethe detection of the binding sites for the first antibody.(Kato et al., 1997). Double-label immunocyto-They were incubated with the second antibody [goat anti-chemistry simultaneously visualizing androgen re-rabbit (GAR) diluted at 1/600 in PBST] for 90 min and

ceptors and aromatase was therefore used here to with the third antibody (PAP complex at 1/200 dilutedinvestigate whether androgen receptors are present in PBST) for 90 min. This procedure was repeated twice.in ARO-ir cells. The sections were rinsed extensively in PBST between

each step. The peroxidase was finally revealed by placingthe sections for 20 min in a solution of DAB (20 mg in50 mL PBST containing 20 mL H2O2 at 30%). SectionsMATERIALS AND METHODSwere mounted on microscope slides and coverslipped.

A total of five brains were stained under the specificconditions described above. Other subjects had been pre-The experiments reported here were carried out on a total

of 15 intact male Japanese quail (C. japonica) that were viously used to established these optimal conditions.These preliminary experiments included changes in thepurchased from a local breeder (C. Dujardin Farms,

Liernu, Belgium) when they were approximately 3 weeks concentration of the primary antibody (from 1/10 to 1/600), of the GAR (from 1/50 to 1/600), and of theold. They were maintained in our laboratory for another

3 weeks before euthanasia. Throughout their life in the PAP (from 1/100 to 1/500), modifications of the buffers(PBST or Tris buffer) , of the incubation time in DABbreeding colony and in the laboratory, the birds were

exposed to a photoperiod of 16:8 h light/dark cycle simu- (5–20 min), and, most important, of the number of suc-cessive incubations in the GAR and PAP (one to threelating stimulatory long summer days. Food and water

were available ad libitum. bridges) .Quail were injected intravenously with 100 mL of hep-

arin solution (Sigma H-7005; 20 mg/mL) and deeplyAntibody and Specificityanesthetized with Hypnodil (Janssen Pharmaceutica,

Beerse, Belgium; 50 mg/kg body weight) . They wereA rabbit polyclonal antibody (PG-21-19A) raised against

perfused through the heart with a saline solution (9 g/L;a synthetic peptide corresponding to the first 21 N-termi-

0.15 M NaCl) until the return blood was clear, and thennal amino acids of the rat androgen receptor was used in

with 400 mL of fixative (4% paraformaldehyde and 0.1%all experiments (see Prins et al., 1991, for details concern-

glutaraldehyde in 0.1 M phosphate buffer, pH 7.2) .ing the preparation and characterization of PG-21). The

Brains were dissected out of the skull and placed over-specificity of androgen receptor immunostaining in the

night in a 20% sucrose solution in 0.1 M phosphate buffer.quail brain was confirmed in control studies that included

The next day, they were frozen on powdered dry ice andthe omission of the primary antibody, GAR or PAP, and

stored in a freezer at 0757C until processed. At the timethe preincubation of the primary antibody with the 21

they were killed, sexual maturity was verified by measur-amino acid peptide used as antigen (AR 21) or with

ing the cloacal gland area, an androgen-dependent organanother 17 amino acid peptide (AR 462-478) correspond-

(Sachs, 1967), and the mass of the testes.ing to a distant region of the androgen receptor. For these

Frozen brains were cut with a cryostat into 30-mm-preadsorbtion studies, the primary antibody was incu-

thick coronal sections that were collected in phosphate-bated for 90 min at room temperature with a 10- or 100-

buffered saline (PBS) (0.01 M ; NaCl: 0.125 M ; pH 7.2) .fold molar excess of the peptide. Adjacent sections were

For distribution studies, every third section in the preopticthen stained with either intact or preadsorbed antibody

area (between the tractus mesencephalicus and the ante-and the immunocytochemical signal observed in these

rior commissure) was saved; every sixth section waspairs of sections was compared.

saved from the remainder of the diencephalon, mesen-cephalon, and metencephalon. In the first brains that werecut, every section was saved to test a variety of staining Double Label with Aromataseconditions on adjacent sections. The plane of section wasadjusted to match as closely as possible the plane of the Free-floating sections of four subjects stained for AR

were washed in Tris-buffer saline (TBS) (Tris 0.05 M ,quail brain atlas (Bayle et al., 1974; Viglietti-Panzica etal., 1986). NaCl 0.15 M , pH 7.6) and incubated overnight in primary

antibody raised against recombinant quail aromatase (1/Sections were rinsed three times in PBS. They weretreated for 20 min at room temperature with a solution 200 at 47C in TBST) (see Foidart et al., 1995 for descrip-

tion concerning the preparation and specificity of thisof 0.6% H2O2 in PBS to block endogenous peroxidases.They were then rinsed three times in PBS containing antibody). On the next day, binding of the primary anti-

body was revealed by successive incubations in biotinyl-0.1% Triton X-100 (PBST) and preincubated in a 5%goat serum solution in PBST for 30 min. Sections were ated goat anti-rabbit immunoglobulins (Dako S/A, Den-

mark; 1/100 to 1/400 in TBST for 90 min) and in alka-incubated for 60 h at 47C with the primary antibody raisedagainst the androgen receptor (see below) diluted at 1/ line phosphatase-conjugated streptavidin (Dako S/A; 1/



Figure 1 Distribution of androgen receptor-immunoreactive (AR-ir) material in the rostralpart of the Japanese quail brain. Drawings collected at successive levels in the rostral to caudalaxis of the brain are presented from top to bottom and left to right. Drawings of actual quailbrain sections were prepared with a camera lucida, and distribution of AR-ir cells was thenplotted schematically on the drawings with the density of symbols adjusted to provide asemiquantitative representation of immunoreactive cells. Black circles represent cells containingAR-ir material in their nucleus; open stars refer to cells that contain immunoreactive materialin their cytoplasm; and small dots indicate the immunoreactive fibers and punctate structures.AA Å archistriatum anterior; Ac Å nucleus accumbens; AId Å archistriatum intermedium,



50 to 1/400 in TBST for 90 min). These sections were laris (ICo), androgen receptor-containing cellsfinally rinsed in TBS at pH 8.2 and the alkaline phospha- could be identified by the presence of immuno-tase activity was revealed by immersion for 10–15 min stained nuclei (see Figs. 1 and 2 for semischematicin Fast blue (BB salt; 10 mg/10 mL TBS 0.1 M , pH 8.2) maps illustrating this distribution). The AR-ir mate-containing 2 mg Naphthol AS-MS phosphate, 0.2 mL

rial filled these cell nuclei with the exception of aN,N-dimethylformamide, and 0.01 mL levamisole 1 M .

clear ovoid structure within the nucleus in somecases that presumably corresponds to the nucleolus,

Analysis of Sections but the cytoplasm of the cells was largely and oftencompletely devoid of immunoreactivity.Detailed observations of the distribution of the AR-ir

High densities of immunostained cells were firstmaterial were completed on sections obtained from thefive subjects stained under identical optimized conditions. identified throughout the rostral to caudal extentBrain sections were drawn with the assistance of a camera of the preoptic area and hypothalamus. They werelucida, and the position of AR-ir cells was recorded on primarily located in a medial periventricular loca-these drawings. A semischematic reconstruction of the tion and only rarely extended in the lateral parts ofdistribution of AR-ir cells was then prepared on drawings these areas. Labeled cells were present in a numberof the quail brain that was prepared independently based

of nuclei that were well defined in Nissl-stainedon a series of Nissl-stained sections.material, but the pattern of immunoreactivity didSections that were double-stained for aromatase werenot specifically define the boundaries. This was thesystematically examined for the presence of double-la-case in nuclei such as the supraoptic nucleus (SOv),beled cells in areas where the two neurochemical markers

coexisted, and the presence of such cells was confirmed [Fig. 1(B)] , medial preoptic nucleus (POM) [Figs.under the microscope at high magnification. 1(D,E) and 3(A,B,D)] , nucleus suprachiasmati-

cus, pars medialis (SCNm) [Fig. 1(E)] , nucleusanterior medialis hypothalami (AM) [Figs. 1(E,F)

RESULTS and 3(C)] paraventricular (PVN) [Fig. 1(F,G)] ,and ventromedial nuclei (VMN) [Fig. 1(G)] . In

Neuroanatomical Distribution of Nuclear the caudal hypothalamus, immunoreactive cellsAR-ir Material were present in the region of the nucleus inferior

hypothalami (IH) [Fig. 2(B)] and the nucleus tu-In several hypothalamic and telencephalic nuclei,as well as in the mesencephalic nucleus intercollicu- beris (Tu) [Fig. 2(C)] .

pars dorsalis; AIvÅ archistriatum intermedium, pars ventralis; AMÅ nucleus anterior medialis-hypothalami; APH Å area parahippocampalis; AVT Å area ventralis (TsaıF ) ; CA Å commissuraanterior; Cb Å cerebellum; CO Å chiasma opticum; CP Å commissura posterior; CPa Å com-missura pallii; DMA Å nucleus dorsomedialis anterior [rostralis] thalami; DMP Å nucleus dorso-medialis posterior [caudalis] thalami; DSD Å decussatio supraoptica dorsalis; DSM Å decus-satio supramamillaris; E Å ectostriatum; FLM Å fasciculus longitudinalis medialis; FPLÅ fasciculus prosencephali lateralis; GCt Å substantia grisea centralis; HA Å hyperstriatumaccessorium; HL Å nucleus habenularis lateralis; HM Å nucleus habenularis medialis; HpÅ hippocampus; HV Å hyperstriatum ventrale; IH Å nucleus inferioris hypothalami; ICoÅ nucleus intercollicularis; Imc Å nucleus isthmi, pars magnocellularis; Ipc Å nucleus isthmi,pars parvocellularis; IH Å nucleus inferioris hypothalami; LHy Å regio lateralis hypothalami;LoC Å locus ceruleus; ME Å eminentia mediana; ML Å nucleus mamillaris lateralis; MMÅ nucleus mamillaris medialis; MLd Å nucleus mesencephalicus lateralis, pars dorsalis; NÅ neostriatum; NIII Å nervus occulomotorius; nST Å nucleus striae terminalis; OM Å tractusoccipitomesencephalicus; OMd Å nucleus nervi occulomotorii, pars dorsalis; OMv Å nucleusnervi occulomotorii, pars ventralis; OV Å nucleus ovoıF dalis; PHN Å nucleus periventricularishypothalami; PL Å nucleus pontis lateralis; POM Å nucleus preopticus medialis; PVNÅ nucleus paraventricularis magnocellularis; PVO Å organum paraventriculare; PVT Å paleos-triatum ventrale; ROT Å nucleus rotundus; RPgc Å nucleus reticularis pontis caudalis, parsgigantocellularis; Ru Å nucleus ruber; SCNm Å nucleus suprachiasmaticus, pars medialis; SLÅ nucleus septalis lateralis; SM Å nucleus septalis medialis; SN Å substantia nigra; SOvÅ nucleus supraopticus, pars ventralis; SCv Å nucleus subceruleus ventralis; Tn Å nucleustaeniae; TIO Å tractus isthmo-opticus; TO Å tuberculum olfactorium; TPO Å area temporo-parieto-occipitalis; TSM Å tractus septomesencephalicus; Tu Å nucleus tuberis; VMN Å nu-cleus ventromedialis hypothalami.



Figure 2 Distribution of androgen receptor-immunoreactive (AR-ir) material in the caudalpart of the Japanese quail brain. See Figure 1 for details.

Numerous but weakly labeled immunoreactive observed in lateral parts of the septum and dorsalpart of the nucleus striae terminalis (nST) [Fig.cells, were also found in the tuberculum olfactor-

ium, nucleus accumbens, and paleostriatum ventrale 1(F)] . A large number of cells exhibiting high lev-els of immunoreactivity were also observed in the(PVT) [Fig. 1(A–E)]. A few stained cells were



ventral archistriatum (AA) [Figs. 1(B–D) and type of cytoplasmic AR-ir material were also ob-served in the GCt [Figs. 2(C–E) and 4(C,D)] .3(E)] and in the nucleus taeniae (Tn) [Figs. 1(B–

E) and 3(F)] . More caudally, the mesencephalicnucleus intercollicularis (ICo) [Figs. 2(B–D) and Specificity of Staining3(G–I)] contained the largest population of AR-ircells that was observed in the quail brain. These Two types of immunoreactive material were ob-

served in this study. In one case, nuclear stainingcells were also darkly labeled with immunoreactivematerial. They outlined the entire nucleus through- was found in several hypothalamic and telence-

phalic nuclei, as well as in the mesencephalic ICo.out its rostral to caudal extend. Some nuclear stain-ing was also observed in the substantia grisea cen- This immunoreactivity disappeared after omission

of either the primary antibody or the GAR or thetralis (GCt) [Fig. 2(B–F)] , the area ventralis ofTsai (AVT) [Fig. 2(D)] , the substantia nigra PAP in the staining sequence. It was also completely

suppressed after preadsorbtion of the primary anti-(SN), the locus ceruleus (LoC) [Fig. 2(G)] , andthe nucleus subceruleus ventralis (SCv) [Fig. 2(G)]. body with a 10-fold molar excess of the peptide

(AR21) used for immunization [Fig. 5(A,B)] . InThis nuclear staining was often associated witha much weaker staining of the cytoplasm sur- contrast, in control experiments using primary anti-

body that had been preincubated with peptide ARrounding the positive nuclei. This weak staining wasconfined to the cell perikarya and never extended in 462-478, a peptide that is part of the AR sequence

but distinct from the peptide used for immunization,cell processes far way from the labeled cell nuclei.no decrease in nuclear staining could be detected.This staining therefore appears to be specific for theAdditional Immunoreactive Material Isandrogen receptor.Also Observed in Cytoplasmic Location

In the second case, an exclusively cytoplasmicstaining was detected in cell processes and punctateThroughout the preoptic and hypothalamic region,

scattered fibers and punctate structures were also structures as well as in large stellate cells locatedin the AVT, SN, and lateral neostriatum (N). Thislabeled by antibody PG-21 [see arrows in Figs.

3(D) and 6(B)] . As indicated in Figures 1 and 2, cytoplasmic immunoreactivity was not fully elimi-nated by the preadsorbtion and a weak cytoplasmicthese immunoreactive fibers were primarily located

within all preoptic and hypothalamic cell groups staining was still observed in these conditions al-though with a decreased intensity [Fig. 5(A–D)].displaying nuclear staining, including the POM

[Fig. 1(D,E)] , SCNm [Fig. 1(E)] , AM [Fig. Preincubation of the primary antibody with 100-fold excess of peptide still failed to completely elim-1(E,F)] , PVN [Fig. 1(F,G)] , VMN [Fig. 1(G)] ,

IH [Fig. 2(B)] , and Tu [Fig. 2(C)] . These immu- inate this cytoplasmic staining. The nature of thiscytoplasmic material is therefore questionable (seenoreactive fibers were also observed in association

with the AR-ir cell nuclei located in the nucleus below).accumbens and paleostriatum ventrale [Fig. 1(A–E)], but they were not seen in the tuberculum ol- Colocalization of AR-ir Material withfactorium, lateral septum, ventral archistriatum in- Aromatasecluding the nucleus taeniae, or mesencephalic ICo[Fig. 2(B–D)]. Cells containing nuclear AR-ir material were pres-

ent in all areas containing ARO-ir cells in the quailIn addition, a high density of immunoreactivematerial filled the cytoplasm and cellular processes brain: namely, in the POM, nucleus striae terminalis

(nST), nucleus accumbens/PVT, VMN, and Tuof stellate cells that displayed an immunonegativenucleus. Cells labeled in this manner had a fairly (Fig. 6) . In all these areas including the POM, dou-

ble-labeled sections revealed that AR-ir nuclear ma-large size and were unevenly scattered in the lateralparts of the neostriatum [Figs. 1(A–G), 2(A), and terial was often present in the same cells as the

cytoplasmic ARO-ir signal. This colocalization was4(A)]. They were never observed in clusters ofsignificant density. At more caudal levels, similarly relatively frequent in all these brain areas except

in the nST, where the low number of AR-ir cellsstained cells were also detected in the two maindopaminergic areas, i.e., the area ventralis of Tsai precluded the presence of a high level of colocaliza-

tion with the very numerous ARO-ir perikarya. In(AVT), [Fig. 2(D)] and the avian homologue ofthe mammalian substantia nigra (SN) [Figs. 2(C– the POM, the double-labeled cells (AR-ir / ARO-

ir) were found in the entire nucleus, but they wereE) and 4(B)] that is centered on but definitely ex-tends beyond the limits of the nucleus tegmenti ped- present in higher density in the ventral part of the

nucleus. The density of the immunoreactive signalunculo-pontinus (TPc). A few cells containing this



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Figure 4 Major groups of neurons in the quail brain that contain androgen receptor-immunore-active material in their cytoplasm. (A) Scattered immunoreactive neurons in the lateral neostria-tum. (B) Ventral part of the substantia nigra. (C) Substantia grisea centralis. (D) Highermagnification of positive cells in the substantia grisea centralis. AQÅ aqueductus mesencephali.Magnification bar represents 500 mm in (C), 200 mm in (B), and 100 mm in (A,D).

for AR appeared to be somewhat variable from one portion (¢60%) of the ARO-ir cells, but in otherinstances [e.g., Fig. 6(A)] , the colocalization wassubject to the other, presumably as a consequence

of technical limitations of the immunocytochemical clear in a fraction of the ARO-ir cells ( large arrow-heads) but only suggested in others (small arrows).procedure used (sequential staining with two pri-

mary antibodies raised in the same species is techni- The present study therefore firmly establishes thepresence of nuclear androgen receptors in ARO-ircally challenging; see discussion below). In some

cases [e.g., Fig. 6(E,F)] , a colocalization with cells, but it is probably inadequate for quantifyingthis colocalization.dense nonambiguous AR was present in a large pro-

Figure 3 Major groups of neurons in the quail brain that contain androgen receptor-immunore-active material in their nucleus. (A) Medial preoptic area at the level of the of the organumvasculosum laminae terminalis (OVLT). (B) Rostral part of the medial preoptic area. (C)Rostral hypothalamus at the level of the chiasma opticum (CO). (D) Medial preoptic area atthe level of the medial preoptic nucleus; arrows indicate labeled fibers. (E) Archistriatumventrale. (F) Nucleus taeniae and surrounding archistriatum. (G) Rostral part of the nucleusintercollicularis. (H) Nucleus intercollicularis at the level of maximal extension of the nucleusmesencephalicus lateralis, pars dorsalis (MLd). (I) High magnification of immunoreactivecells in the nucleus intercollicularis. Asterisks indicate the third ventricle in (B,C); open starsindicate the ventriculus tecti mesencephali in (G,H). Magnification bar represents 500 mm in(G,H); 200 mm in (A–C,E,F,I) ; 100 mm in (D).



that contained nuclear AR-ir material, immunoreac-tive fibers and punctate structures were also ob-served. Three regions of the brain, SN, AVT, andlateral parts of the neostriatum were also found tocontain cells that exhibit a dense staining that wasprimarily cytoplasmic in our study. In nearly allbrain areas known to contain ARO-ir cells based onprevious studies (Balthazart et al., 1990a, 1990b;Foidart et al., 1995), we also observed AR-ir cells.In most of these areas, including the sexually dimor-phic POM, a large percentage of the ARO-ir cellswere found to contain AR-ir nuclei. We will firstdiscuss technical issues related to the specificity ofthe staining, and then we will discuss the functionalimplications of the AR-ir staining observed in thisstudy.

Specificity of Staining and CellularLocalization of the Receptors

The nuclear staining obtained throughout the brainwith the PG-21 antibody appears to be specific tothe androgen receptor, as demonstrated by controlexperiments, including the omission of the first anti-body and its preadsorbtion with the antigenic pep-tide. Preincubation with a peptide corresponding toFigure 5 Quail medial preoptic area (A,B) and areaanother part of the rat androgen receptor did notventralis of Tsai (C,D) illustrating the effects of pread-block immunostaining, because the antibody is di-sorbtion of antibody PG-21 with the peptide used forrected against only a fragment of the native recep-immunization on the immunocytochemical detection oftor; PG-21 is specific for the N-terminal region ofandrogen receptor-like immunoreactive structures. Sec-

tions in the left column (A,C) were immunostained by the androgen receptor of rat. As discussed below,procedures described in the text with antibody PG-21. the neuroanatomical distribution of this staining alsoSections in the right column (B,D) were stained with matches well with the available information (bothantibody that had been preadsorbed with a 10-fold excess autoradiographic and immunocytochemical studies)of peptide. The nuclear immunocytochemical signal ob- on the distribution of androgen receptors availableserved in the preoptic area (A) is completely abolished

for quail as well as other avian or mammalian spe-by the preadsorbtion (B); in contrast, the cytoplasmiccies. These converging lines of evidence clearly in-signal detected in the AVT (C) is clearly decreased butdicate that this immunoreactive signal actually rep-not completely abolished. Similarly, immunoreactive fi-resents a true functional androgen receptor.bers are still partially visible in the preoptic area after

In all diencephalic brain areas where we ob-preadsorbtion [arrows in (B)] . N III Å nervus occulomo-served androgen receptor nuclear staining, we alsotorius. Magnification bar Å 100 mm in (A,B) and 200

mm in (C,D). observed a weak staining in the associated cyto-plasm. The existence of cytoplasmic receptors forsteroids in the brain is still a matter of some contro-versy. Biochemical studies based on the extractionDISCUSSIONof nuclear and cytoplasmic fractions originally indi-cated that free receptors were present in the cellThe present study confirms and markedly expands

the data that were previously available on the distri- cytoplasm and were translocated into the nucleusupon steroid binding (Gorski et al., 1968). Morebution of the androgen receptors in the quail brain.

Nuclear AR-ir material was observed in a large recently, it was suggested that the majority of bothoccupied and unoccupied receptors are located innumber of cells located in several preoptic, hypotha-

lamic, and telencephalic nuclei. At more caudal lev- the cell nucleus (King and Greene, 1984; Welshonset al., 1984). This was demonstrated for estrogenels, many AR-ir cells were detected in the mesence-

phalic ICo. Scattered AR-ir–positive cells were also (Liposits et al., 1990), progesterone (Gasc et al.,1989), glucocorticoid (Brink et al., 1992), and an-present in the GCt and AVT. In many of these nuclei



Figure 6 Colocalization of androgen receptor-immunoreactive material (in brown) and aromatase-immunoreactive material (in blue). (A) Medial preoptic nucleus. Arrowheads indicate clearlydouble-labeled cells; arrows point to ARO-ir cells displaying a weak immunoreactivity for AR.(B) Insert showing two double-labeled neurons of (A) at higher magnification; arrows point to anandrogen receptor-immunoreactive fiber. (C) High magnification of one double-labeled neuron inthe medial preoptic nucleus. (D) Single- and double-labeled neurons in tuber. (E) Ventromedialnucleus of the hypothalamus. (F) Higher magnification of the same field showing double-labeledneurons. Magnification bar represents 50 mm in (A,D,E), and 25 mm in (B,C,F).



drogen receptors (Husman et al., 1990; Sar et al., within the pituitary which in intact subjects waspredominantly nuclear became predominantly cyto-1990; Tan et al., 1988). According to this model,

it was thought that unoccupied receptors were pres- plasmic in the castrates (Iqbal et al., 1995). Consis-tent with this finding, in Gambel’s white-crownedent in the nucleus but were more readily displaced

from the cell nucleus than occupied receptors that sparrows (Zonotrichia leucophyrs gambelii) , pre-treatment with testosterone just prior to perfusionwere tightly associated with chromatin (Gorski et

al., 1986). With the recent improvements in immu- greatly enhances the number of immunoreactivecells detected as compared to untreated intact birdsnocytochemical methodology, the presence of estro-

gen receptors in the cytoplasm was first confirmed or birds in a nonbreeding condition (Smith et al.,1996). Based on these published data and on thein the brain of guinea pigs (Blaustein and Turcotte,

1989b), and this finding was later extended to other control experiments performed here, there is there-fore every reason to conclude that the cytoplasmicmammalian species and other types of steroid recep-

tors (see Blaustein, 1992; Wood and Newman, staining associated with immunopositive cell nucleirepresents androgen receptors.1993; Blaustein et al., 1992, for a review). Estrogen

receptor immunoreactivity was even demonstrated In contrast, the staining observed in cell pro-cesses (fibers and punctate structures) and in theat the electron-microscopic level in axonal and den-

dritic processes of steroid-containing neurons large stellate cells of the AVT, SN, and lateral partsof the neostriatum is more problematic, and we do(Blaustein et al., 1992). Cytoplasmic immunoreac-

tive estrogen receptors have similarly been observed not think, at present, that this immunoreactive mate-rial represents the detection of androgen receptorsin the quail brain (Balthazart and Foidart, 1993).

The present data indicate that AR is also present in because (a) it is exclusively cytoplasmic; (b) pread-sorbtion with the synthetic peptide that the antibodythe neuronal perikarya in this species.

Current models of steroid hormone receptor ac- was grown against did not completely block theimmunoreactivity in these structures; (c) the texturetion suggest that unoccupied receptors are present,

at least in part, in the cytoplasm. When steroids of the staining in the large stellate cells is unusual(granular in nature) compared to that observed inbind to the receptors, the ligand-receptor complex

binds with high affinity to the chromatin, where it other parts of the brain in this study and in previousstudies in mammalian and avian species; and (d)acts as a transcription factor and initiates protein

synthesis. One could therefore expect regional dif- previous studies analyzing the distribution of ARin the quail brain either by autoradiography or byferences in the cellular localization of the receptor

if there are regional differences in the occupation immunocytochemistry did not identify receptors inthe SN, AVT, or lateral neostriatum (Watson andof the receptor. The PG-21 antibody appears to rec-

ognize both occupied and unoccupied forms of the Adkins-Regan, 1989; Balthazart et al., 1992b).Based on the current evidence, it therefore appearsreceptor (Prins and Birch, 1993; Wood and New-

man, 1993). However, the cellular localization of that this cytoplasmic immunocytochemical stainingdoes not represent functional androgen receptorsthe staining and the number of cells detected are

influenced by circulating levels of T in the plasma. and, rather, is related to either a variant of the recep-tor protein that is unable to bind androgens or aThese changes reflect both the autoregulation of an-

drogen receptors by T (total concentration of recep- completely different molecule whose sequenceshows some similarity with the peptide used to pro-tors increases when T is present) and the effects of

T on the subcellular localization of these receptors. duce antibody PG-21.In general, the presence of high T increases thenumber of cells detected by the antibody and in- Comparison with Previous Studies ofcreases the percentage of cells that exhibit immuno- Androgen Receptors in Quailreactivity in the nucleus as opposed to the cyto-plasm. For example, castration reduces the amount In general, the data obtained in the present study

are in agreement with previous work in mammalsof staining observed in cell nuclei in the hypothala-mus of hamsters and the rat prostate (Prins and or in avian species including quail. In an autoradio-

graphic study of the quail brain (Watson and Ad-Birch, 1993; Wood and Newman, 1993) and thiseffect is reversed by treatment with exogenous T. kins-Regan, 1989), [3H]-T–concentrating neurons

were found throughout the rostral diencephalon inIn male Brazilian opposums (Monodelphis domes-ticus) , castration 4 days prior to perfusion of the POM, PVN, and the medio-basal and tuberal hypo-

thalamus. In the telencephalon, radioactively la-animals in preparation for the immunocytochemicallocalization of AR-ir eliminated nearly all staining beled neurons were located ventrally and laterally

to the nucleus accumbens, in the lateral septum andin the brain and the intracellular pattern of staining



in the nucleus taeniae (which is considered to be 1986; Harada et al., 1993). The present distributionof AR provides an anatomical basis for these biolog-the homologous to parts of the mammalian amyg-

dala) . In the mesencephalon, the ICo was exten- ical effects of androgens.Many of the brain areas containing androgen re-sively labeled and a weaker signal was observed in

GCt. It should be noted, however, that all these ceptors are related to the control of reproductivebehavior. For example, the control of male-typicalareas were also found to concentrate [3H]estradiol.

In contrast, the same study revealed a very limited copulatory behavior involves the actions of steroidsin the preoptic region; there are dramatic effectsdistribution of [3H]-DHT-binding sites that were

restricted to the ICo, SL, PVN, Tn, LHy, and GCt. of estrogen in this region and smaller effects ofandrogens (Balthazart and Surlemont, 1990), sug-Because aromatase activity is widespread in the

quail brain and is present in most of the nuclei gesting that the synergism between androgens andestrogens necessary for the activation of male copu-exhibiting [3H]-T uptake, these data suggested that

most of the binding sites observed by [3H]-T auto- latory behavior occurs, in part, in this brain region.The androgen receptors detected in more caudalradiography were actually binding sites for estro-

gens, and that the distribution of androgen receptors areas such as the AM, PVN, and VMN have alsobeen implicated in the control of reproductive and/is rather limited in the quail brain. This idea was

subsequently reinforced by an immunocytochemical or aggressive behavior, but definitive evidence inavian species is lacking (see Barfield et al., 1978;study that used a rabbit polyclonal antibody (AR

32) and only identified limited populations of AR- Barfield, 1979; Balthazart et al., 1992b, for discus-sion). The negative feedback effects of testosteroneir cells in POM, AM, PVN, VMN, Tu, and ICo

(Balthazart et al., 1992b). on gonadotropin secretion have been localized tothe tuberal hypothalamus (Stetson, 1972; Follett,The present study indicates that these previous

reports underestimated the number of androgen re- 1973; Wilson, 1970), but these studies employedtestosterone, so the identity of the testosterone me-ceptors in the quail brain. In the areas that were

previously identified as containing androgen recep- tabolite that is mediating these effects is unknown.The possible actions of androgens in these braintors, more immunoreactive cells were detected in

the present study than in previous studies and some areas in the preoptic region and the diencephalonhave been discussed in detail previously (Arnold etadditional areas in the quail brain that contain an-

drogen receptors have now been identified. This dis- al., 1976; Barfield et al., 1978; Balthazart et al.,1992b).tribution agrees well with data previously reported

for a variety of mammalian and avian species (e.g., In addition to the brain areas just discussed,where androgen receptors had been previously de-Iqbal et al., 1995; Choate and Resko, 1992; Clancy

et al., 1992, 1994; Arnold et al., 1976; Barfield et tected, we also observed AR-ir in the nucleus ac-cumbens and the contiguous paleostriatum ventrale.al., 1978; Nastiuk and Clayton, 1995; Smith et al.,

1996). The binding of T had been identified in this regionin the brain of male domestic fowl (Barfield et al.,1978) but it was not known if these sites representedFunctional Implications of the Pattern ofandrogen or estrogen receptors. AutoradiographicDistributionstudies in quail found estrogen receptors in this re-gion but little evidence for androgen receptorsAndrogen receptors in the quail brain are present in

brain areas that have been implicated in a variety of (Watson and Adkins-Regan, 1989). The data pre-sented here indicate that this brain region does in-biological processes, often related to reproduction.

Many of the effects of testosterone are known to deed contain cells expressing the androgen receptorprotein. This brain area has been implicated in theinvolve the conversion of testosterone to an estrogen

via the action of the metabolizing enzyme aromatase control male sexual behavior, especially appetitivecomponents of this behavior, in a number of verte-(Balthazart, 1989). However, in other cases, a di-

rect action on the androgen receptor of testosterone brate species (Everitt et al., 1989; Everitt, 1990,1995). In quail, lesion studies suggest a role initself or its 5a-reduced metabolite, DHT, appears

to be mediating the biological effectiveness of T. consummatory aspects of male sexual behavior(Balthazart and Ball, 1997), but not in appetitiveEven in cases where aromatization has been impli-

cated, there is almost always evidence for a syner- responses. However, further investigations are on-going concerning the role played by this nucleus ingism between the action of estrogens and androgens

in the activation of the biological response (Adkins quail male sexual behavior.A significant population of AR-ir cells were alsoet al., 1980; Schumacher and Balthazart, 1983; Bal-

thazart et al., 1994, 1985; Alexandre and Balthazart, identified in the olfactory tubercle. This brain area



contains a variety of neuropeptides (gonadotropin- 1983). The use of different reporter enzymes (per-oxidase and alkaline phosphatase) also precludedreleasing hormone, b-endorphin); (Van Gils et al.,

1993, 1994) and to receive a dense catecholaminer- any cross-reaction at this level, and this techniqueallowed us to successfully demonstrate the presencegic input (Ball et al., 1995); however, androgen

effects in this area have not been described. of androgen receptors in aromatase cells located inmost if not all aromatase cell clusters. As stated inA limited number of AR-ir cells were identified

in catecholaminergic areas in the brain stem, such the results, technical limitations presumably associ-ated with the use of primary antibodies raised inas SN and LoC. There are well-described steroid

hormone effects on catecholaminergic neurotrans- the same species made it impossible to quantify thedegree of colocalization between ARO and AR. Itmission in birds and mammals (see, namely, Hull,

1995; Hull et al., 1995; Barclay and Harding, 1988, can only be said that in sections that appeared tobe optimally stained, the majority (¢60%) of the1990; Balthazart et al., 1992a, and references in-

cluded in these articles) . Estrogen and androgen ARO-ir cells contained unambiguous AR, while inother cases, a weak AR immunoreactivity only wasreceptors have also been identified in these and

other catecholaminergic areas, and in some cases present in a large number of ARO-ir cells. Whetherthis reflects true biological variation or, more proba-were found to be present specifically within cate-

cholaminergic neurons (Heritage et al., 1980; Grant bly, technical limitations will be ascertained onlywhen better techniques for the detection of theseand Stumpf, 1975; Herbison et al., 1996; Skinner

and Herbison, 1997). The AR-ir cells identified two antigens become available.Despite these limitations, the present study estab-here suggest that androgens could be acting directly

on catecholaminergic neurons. This notion should lishes for the first time the presence of AR in (whatappears to be a large fraction of) brain aromatasebe further explored in double-label immunocyto-

chemical studies attempting to colocalize AR with cells. This contrasts with our previous work indicat-ing that there is anatomical specificity in the degreetyrosine hydroxylase immunoreactivity.to which aromatase cells do or do not contain estro-gen receptors (Balthazart et al., 1991; Foidart et al.,Colocalization of Androgen Receptors1996).and Aromatase

In two avian species that have been studied insome detail, the Japanese quail and the ring dove,It was also interesting to observe that AR-ir cells are

present in most brain areas that had been previously physiological experiments indicate that estrogenicmetabolites of T are mostly responsible for the in-shown to contain ARO-ir neurons (Balthazart et al.,

1990a, 1990b; Foidart et al., 1995). Some sections crease in aromatase activity that is observed whencastrated males are treated with T. However, therewere therefore double labeled for both aromatase

and AR, and their analysis revealed the existence is a synergism between androgens and estrogens inthe induction of this response (Balthazart and Foid-of a frequent colocalization between the enzyme

and the receptor. Although the two antibodies used art, 1993; Balthazart et al., 1996; Hutchison andSteimer, 1986; Hutchison et al., 1989). For exam-in this study were raised in rabbit, the cellular local-

ization of the immunocytochemical signals detected ple, it has been demonstrated in quail that eitherDHT or the pure nonaromatizable androgen methyl-in double-labeled section provides independent evi-

dence for the specificity of the reaction. The brown trienolone (R1881) enhances the effects of estro-gens on aromatase activity, aromatase immunoreac-chromogen indicative of AR was limited to the cell

nuclei in the areas of interest, while the blue staining tivity, and aromatase mRNA concentration in thepreoptic area (Schumacher et al., 1987; Harada etindicative of aromatase was exclusively cyto-

plasmic. Double-label immunocytochemical studies al., 1993; Balthazart et al., 1994). These non-aromatizable androgens have, however, little or nohave been previously carried out with primary anti-

bodies raised in the same species (VanNoorden and effect on aromatase by themselves. This is very dif-ferent from the situation observed in mammalianPolak, 1985; Herbison et al., 1996; Zhou et al.,

1994), and it is clearly established that as long as studies, where androgens appear to directly increasearomatase activity and estrogenic metabolites arediaminobenzidine is used in high concentration dur-

ing the visualization of the first sequence of reac- ineffective in isolation, although they act synergisti-cally with nonaromatizable androgens to increasetion, this compound will effectively block all re-

maining antigenic sites so that the reagents used in the activity of the enzyme (Roselli et al., 1987;Roselli and Resko, 1984; Connolly et al., 1990; Ro-the second immunocytochemical sequence will not

cross-react with unoccupied sites on the primary selli, 1991).It was therefore somewhat unexpected to observeantibody from the first sequence (Vandesande,



roanatomical Aspects, Neurotransmitters and Neuro-that ER are not often colocalized in the same cellspeptides. Balthazart, J., Ed. Karger, Basel, pp. 148–where ARO-ir is observed, but that AR are present167.in these cells. What does this mean from the hor-

BALL, G. F., CASTO, J. M., and BALTHAZART, J. (1995).monal regulation of aromatase activity by gonadalAutoradiographic localization of D1-like dopamine re-sex steroids? These data concerning the anatomicalceptors in the forebrain of male and female Japanese

localization of AR and ER in the quail brain suggest quail and their relationship with immunoreactive tyro-that the effects of estrogen may be transsynaptic in sine hydroxylase. J. Chem. Neuroanat. 9:121–133.nature (see Balthazart et al., 1996, for a detailed BALTHAZART, J. (1983). Hormonal correlates of behav-discussion), while the synergistic effects of andro- ior. In: Avian Biology. Farner, D. S., King, J. R., andgen might be direct. This interpretation is indepen- Parkes, K. C., Eds. Academic Press, New York, pp.

221–365.dently supported by recent molecular studies dem-BALTHAZART, J. (1989). Steroid metabolism and the acti-onstrating that a potential androgen response ele-

vation of social behavior. In: Advances in Comparativement has been detected at the 5* end of the brainand Environmental Physiology. Vol. 3. Balthazart, J.,specific exon 1f of aromatase, while no estrogenEd. Springer Verlag, Berlin, pp. 105–159.response element has been detected at this level so

BALTHAZART, J., and BALL, G. F. (1995). Sexual differ-far (Kato et al., 1997). This notion should now beentiation of brain and behavior in birds. Trends Endo-

tested by direct stimulation with androgenic and/or crinol. Metab. 6:21–29.estrogenic steroids of isolated aromatase cells in BALTHAZART, J., and BALL, G. F. (1997). Neuroendo-culture. crine regulation of appetitive and consummatory as-

pects of male sexual behavior in Japanese quail. In:Perspectives in Avian Endocrinology. Etches, R., andThis work was supported by a grant of the NIMHHarvey, S., Eds. Society for Endocrinology, Bristol,(R01 MH50388) to GFB and JB, and by grants from theUK, pp. 241–255.Belgian FRFC (No. 9.4565.96F) and the Government of

BALTHAZART, J., and FOIDART, A. (1993). Brainthe French Community of Belgium (Action Concerteearomatase and the control of male sexual behavior. J.no. 93/98-171) to JB. The authors thank Dr. P. LeprinceSteroid Biochem. Mol. Biol. 44:521–540.(Department of Physiology, University of Liege) for his

BALTHAZART, J., FOIDART, A., ABSIL, P., and HARADA,help in searching for possible homologies of PG21 withN. (1996). Effects of testosterone and its metabolitesother peptide sequences. The collaboration between JBon aromatase-immunoreactive cells in the quail brain:and GFB was supported by NATO Collaborative Re-Relationship with the activation of male reproductivesearch Grant CRG910526. AF is Research Associate ofbehavior. J. Steroid Biochem. Mol. Biol. 56:185–200.the Belgian FNRS.

BALTHAZART, J., FOIDART, A., and HARADA, N. (1990a).Immunocytochemical localization of aromatase in thebrain. Brain Res. 514:327–333.

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