Brain Research 879 (2000) 174–182
www.elsevier.com / locate / bres
Research report
Androgen receptor-immunoreactivity in the forebrain of the Eastern
Fence lizard (Sceloporus undulatus)
a,
b
a
b
M.M. Moga *, B.M. Geib , D. Zhou , G.S. Prins
a
Terre Haute Center for Medical Education, Indiana University School of Medicine, Terre Haute, IN 47809, USA
b
Department of Urology, University of Illinois at Chicago, Chicago, IL 60612, USA
Accepted 25 July 2000
Abstract
Androgen receptor (AR) distribution in the lizard forebrain and optic tectum was examined using PG21 immunohistochemistry. In the
male Eastern Fence lizard, AR-immunoreactive (-ir) nuclei were observed in the medial preoptic area, ventromedial and arcuate
hypothalamic nuclei, periventricular hypothalamus, premammillary nucleus, bed nucleus of the stria terminalis, and ventral posterior
amygdala. Punctate immunostaining of neuronal processes (axons and / or dendrites) was concentrated in the cortex, hypothalamus, and
optic tectum. AR-ir nuclei in the female brain were confined to the ventral posterior amygdala and ventromedial hypothalamic nucleus.
The AR distribution in the lizard brain is similar to that reported for other vertebrate classes. Sex differences in AR-immunoreactivity may
contribute to sex-specific behaviors in the Eastern Fence lizard.  2000 Elsevier Science B.V. All rights reserved.
Theme: Other systems of the CNS
Topic: Comparative neuroanatomy
Keywords: Steroid receptor; Hypothalamus; Amygdala; Cortex; Reptile
1. Introduction
Male reproductive behavior is activated by circulating
sex steroids, particularly the androgens testosterone and
5a-dihydrotestosterone (DHT). Androgens elicit sex-specific behaviors by acting directly on androgen receptors in
the brain or through indirect activation of brain estrogen
receptors via androgen aromatization [32]. In whiptail and
anole lizards, androgens are more effective than estrogenic
metabolites in eliciting male sexual behavior [14,38,42].
The distribution of androgen receptor (AR) protein and
mRNA in the brain shows a similar pattern among the
different vertebrate classes, including bird [2,36], fish [19],
reptile [46], and mammal [21,35]. In each of these classes,
AR-positive cells are concentrated in the hypothalamus
and amygdala, specifically in brain nuclei that have been
implicated in male reproductive and aggressive behavior.
Comparative studies have proven useful in delineating
*Corresponding author. Tel.: 11-812-237-3420; fax: 11-812-2377646.
E-mail address: [email protected] (M.M. Moga).
species-specific AR-ir cell clusters, such as the song
control nuclei of songbirds [1,2], and have increased our
understanding of the neural basis of reproductive behaviors
[20].
To date, there has been no immunocytochemical study
of AR in the reptilian brain. An early study by Morrell et
al. [26] employed 3 H-testosterone autoradiography to
identify androgen-concentrating cells in the lizard brain.
Some of the androgen-concentrating cells which they
identified may actually have been estrogen-concentrating
cells, as circulating testosterone is aromatized to estrogen
in certain areas of the brain. Recently, Young et al. [46]
analyzed the distribution of AR mRNA in the brain of the
whiptail lizard using in situ hybridization. Although highly
sensitive, this method cannot distinguish between cytoplasmic and nuclear staining, or identify AR-positive
dendrites and axons. In the present study, we map the
distribution of AR in the forebrain of the Eastern Fence
lizard (Sceloporus undulatus) using immunocytochemistry.
The AR antibody employed in the present experiments
(PG21) is directed against amino acids 1–21 of the rat AR
[30]. The lizard AR shows a high degree of sequence
0006-8993 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved.
PII: S0006-8993( 00 )02771-2
M.M. Moga et al. / Brain Research 879 (2000) 174 – 182
homology with ARs in other species [47]. The DNAbinding and C-terminal ligand-binding domains of AR are
the most highly conserved [37]. However, positions 1–35
and 230–268 in the AR are far less variant than the rest of
the N-terminal domain [37]. A low degree of variance in
amino acids 1–21 of the AR may explain the ability of the
PG21 antibody to label ARs in diverse tissue types in a
variety of vertebrates, including mammal [11,13,18,21,
44,45,49], fish [15], bird [2,36], and amphibian [16].
2. Methods
Male (n56) and female (n55) lizards (Sceloporus
undulatus) were purchased from Charles D. Sullivan Co.,
Inc. (Nashville, TN) during the spring / summer breeding
season (May through September). Nine of the lizards (5
males, 4 females) were classified as S. undulatus consobrinus (Southern Prairie subspecies); the other two individuals (experiments L8 and L18) were identified as S.
undulatus garmani (Northern Prairie subspecies). Animals
were maintained in captivity for 2–8 weeks in glass
aquaria (2 to 3 animals per aquaria) with peat moss as
substrate, and sticks and stones for climbing. Photoperiod
was maintained at 12 h light: 12 h dark. Room temperature
was 21–238C, with additional heat (up to 388C) provided
by a heat lamp situated at one end of the terrarium. Water
was available at all times. Animals were fed crickets 2 to 3
times per week, supplemented with reptile vitamins.
At the time of sacrifice, animals were given an overdose
of sodium pentobarbital followed by decapitation. The
dorsal skullcase was removed, and the brain was immersion-fixed in situ in 4% paraformaldehyde 0.1 M
phosphate buffer, pH 7.4, for 5–12 days. Following
fixation, the brains were removed from the skull and
embedded in 10% gelatin. The gelatin-brain blocks were
fixed in 4% paraformaldehyde for an additional 4–6 days.
Next, the brains were cut into 20 mm sections on a
Vibratome. One series of sections (one-of-three) was
processed for AR-immunocytochemistry, and a second
series was mounted on gel-coated slides and Nissl-stained
with thionin.
The immunocytochemical method was as follows. Sections were treated with 0.3% hydrogen peroxide in 0.01 M
phosphate-buffered saline (PBS), pH 7.4, for 10 min,
rinsed in PBS, treated with 0.5% sodium borohydride in
PBS for 5–10 min, and preincubated for 1 h in a PBS
solution containing 2% normal donkey serum (NDS) and
0.3% Triton X-100 (TX). Sections were then incubated
with the AR antibody (PG21), diluted 1:1,000–1:5,000
(0.2–1.0 mg / ml, final concentration) in PBS-NDS-TX, for
3–6 days at 48C. Next, the sections were rinsed in PBS,
incubated with a secondary antibody (biotin–SP-conjugated donkey anti-rabbit IgG; Jackson ImmunoResearch;
diluted 1:200 in PBS-NDS-TX) for 1–2 h at room
temperature, rinsed again, and reacted with ABC reagent
175
(Elite Standard ABC kit; Vector) for 1–2 h. After several
rinses in PBS, the sections were reacted in a solution of
0.05% 3,39-diaminobenzidine tetrahydrochloride (DAB;
Sigma)-0.025% nickel chloride-0.01% H 2 O 2 in 0.1 M Tris
buffer, pH 7.4, for 10 min. The sections were mounted on
gel-coated slides, dehydrated through alcohols and xylene,
and coverslipped with Permount (Fisher).
The specificity of the PG21 antibody was tested by, (1)
preabsorption of the primary antibody with its respective
peptide (AR21; amino acids 1–21 of the rat AR), (2)
preabsorption of the primary antibody with a peptide
derived from a different portion of the rat AR (AR462;
amino acids 462–478), and (3) omission of the primary
antibody. In the two preabsorption controls, sections were
incubated in 1 ml of diluted antibody (1:5000) preincubated with 370 ng of peptide (AR21 or AR462). No
immunocytochemical staining was observed in controls
where, (1) the primary antibody was omitted, or (2) the
primary antibody was preincubated with its respective
peptide (AR21). Staining was intact in control experiments
involving preabsorption with the distant, unrelated peptide
(AR462).
The distribution of AR-immunoreactivity in representative sections was plotted on a microscope (BH-2,
Olympus) with a digital stage readout head attached to a
computer with Neurolucida software (MicroBrightField,
Inc.). A camera lucida was used to add cytoarchitectural
details obtained from adjacent Nissl-stained sections.
3. Results
AR-immunoreactivity was observed in select brain areas
in both male and female S. undulatus. The distribution of
AR-immunoreactivity in the male Eastern Fence Lizard
brain was examined in six experiments. One male lizard
(experiment L2) was obtained and sacrificed in July, 1998;
two males (experiments L8 and L24) were obtained in
September, 1998 and sacrificed in November, 1998; and
three males (experiments L21, L22 and L25) were obtained
and sacrificed in June, 1999. The pattern of AR-ir labeling
was identical in these six experiments, but the number of
labeled cells and the intensity of the immunolabeling
varied by experiment. The following description specifically refers to representative experiment L8, which exhibited
the most robust and widespread labeling.
At rostral levels in experiment L8, AR-immunoreactivity was concentrated in the medial and dorsal cortices (Figs.
1A–D; 2A–B). AR-ir fibers in the medial cortex were
organized in a bilaminar pattern with both layers juxtaposed to the principal neuron layer (Fig. 2A). In the dorsal
cortex, AR-ir fibers displayed numerous varicosities (Fig.
2). At more caudal levels, AR-ir fibers were also found in
the lateral cortex (Fig. 1B, C). Cytoplasmic staining was
observed in a small number of neurons scattered in the
dorsal cortex. Ventral to the medial cortex, there was a
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M.M. Moga et al. / Brain Research 879 (2000) 174 – 182
Fig. 1. A series of camera lucida drawings illustrating the distribution of androgen receptor-immunoreactive nuclei (triangles) and fibers (small dots) in the
brain of a male Eastern Fence lizard (L8, rostral to caudal, A–E). AHA, anterior hypothalamic area; Arc, arcuate nucleus; AT, area triangularis; BST, bed
nucleus of the stria terminalis; DB, nucleus of the diagonal band; DCx, dorsal cortex; DL, dorsolateral thalamic nucleus; DLH, dorsolateral hypothalamic
nucleus; DM, dorsomedial thalamic nucleus; DVR, dorsal ventricular ridge; ExA, external nucleus of the amygdala; fb, forebrain bundles; fr, fasciculus
retroflexus; Hb, habenula; LA, lateral nucleus of the amygdala; LCx, lateral cortex; LG, lateral geniculate nucleus; LH, lateral hypothalamic area; MCx,
medial cortex; MPA, medial preoptic area; MPT, medial pretectal nucleus; MS, medial septal nucleus; NS, nucleus sphericus; oc, optic chiasm; opt, optic
tract; OTe, optic tectum; PG, pretectal geniculate nucleus; PrM, premammillary nucleus; Pv, periventricular hypothalamus; Rot, nucleus rotundus; S,
septum; StA, striatoamygdalar area; TV, tectal ventricle; VMH, ventromedial hypothalamic nucleus; VPA, ventral posterior amygdala.
M.M. Moga et al. / Brain Research 879 (2000) 174 – 182
177
Fig. 2. Photomicrographs of androgen receptor-ir fibers in the lizard brain (experiments L2 and L8). (A) AR-ir fibers in the medial cortex were
concentrated in two layers, one superficial and the other deep to the principal neuron layer (marked with an asterisk). (B) In the dorsal cortex, some AR-ir
fibers were oriented perpendicular to the brain surface (principal neuron layer indicated by asterisk) and many fibers were concentrated along the dorsal
brain surface. (C) AR-ir labeling in the preoptic area was diffuse and punctate (two AR-ir fibers are indicated by arrows). AHA, anterior hypothalamic
area; DCx, dorsal cortex; DVR, dorsal ventricular ridge; MCx, medial cortex; opt, optic tract; 3V, third ventricle. Scale bar5100 mm.
178
M.M. Moga et al. / Brain Research 879 (2000) 174 – 182
dense concentration of labeled fibers in the medial septal
nucleus (Fig. 1B).
Several cell groups in the amygdala contained AR-ir
nuclei. In many of the labeled cell nuclei, a darkly stained
AR-ir nucleolus was evident. One amygdalar cell group
with AR-ir nuclei formed a band of labeling ventral to the
striatum (Fig. 1B); this cell group corresponded at rostral
levels to the striatoamygdalar area, and at caudal levels, to
the medial / interstitial amygdala [7]. Labeled neurons in
this cell group (striatoamygdalar area, medial / interstitial
amygdala) were large in size and widely spaced. A few,
small, lightly stained AR-ir nuclei were observed in the
adjacent bed nucleus of the stria terminalis (Fig. 1B, C). A
dense cluster of darkly stained, AR-ir nuclei was found in
the ventral posterior nucleus of the amygdala (Figs. 1B, C,
3A) [7,25]. This cell group, composed of closely packed,
small neurons, formed a wedged-shaped protuberance at
rostral levels (Fig. 1B); it extended caudally as a thin band
of cells ventrolateral to the nucleus sphericus (Fig. 1C).
In the hypothalamus, some lightly stained AR-ir nuclei
were observed in the medial preoptic area, particularly in
the caudal dorsal portion (Fig. 1B). Scattered AR-ir fibers,
punctate in appearance, were present throughout the preoptic and anterior hypothalamic areas (Fig. 2C). Immediately
caudal to the optic chiasm, we observed a concentration of
AR-ir fibers in a dorsal section of the periventricular
hypothalamus (Fig. 1D). In Nissl-stained sections, this
hypothalamic area showed a distinct cytoarchitecture with
many small, dark-staining neurons as well as larger,
medium-staining neurons. Adjacent areas in the periventricular hypothalamus contained only large, medium-staining neurons. A cluster of lightly labeled AR-ir nuclei was
observed the ventromedial nucleus (Fig. 1D). Many darkly
stained, AR-ir nuclei with prominent, stained nucleoli were
present in the premammillary and arcuate nuclei (Figs. 1E;
3B, C). Scattered AR-ir fibers were found throughout the
hypothalamus.
AR-immunoreactivity was observed in a few other areas
of the diencephalon. AR-ir fibers were concentrated in a
cell-dense area located medial to the lateral geniculate
nucleus and dorsal to the forebrain bundles (Fig. 1C); this
region corresponded to the area triangularis [9]. Axonal
labeling was present throughout the lateral forebrain
bundle (Fig. 1B–E). Many AR-ir fibers coursed dorsally
through the white matter adjacent to the third ventricle, but
did not extend into the gray matter of the thalamus (Fig.
1E). A band of AR-ir labeling was observed in the
fasciculus retroflexus, ventral to the medial pretectal
nucleus [28] and dorsolateral to the caudal portion of the
nucleus rotundus (Fig. 1E). In the optic tectum, labeled
fibers were concentrated in the deep, ventricular layers 1–5
(Figs. 1E; 3D).
In the six experiments involving male lizards, the
number of AR-ir nuclei varied by experiment, although
this was not quantified. AR-ir nuclei in the arcuate nucleus
and periventricular hypothalamus were more numerous in
experiment L8 (November) than in representative experiments L2 (July) and L21 (June). Conversely, more labeled
cells were observed in the bed nucleus of the stria
terminalis and striatoamygdalar area in experiment L2 than
in L8 or L21. The intensity of the immunolabeling (both
AR-ir nuclei and fibers) was greatest in L8, moderate in
L2, and least in L21.
AR-immunoreactivity in the female Eastern Fence lizard
brain was examined in five experiments. Two female
lizards (experiments L17 and L18) were obtained in
September, 1998 and sacrificed in November, 1998; and
three females (experiments L19, L20 and L23) were
obtained and sacrificed in May, 1999. The distribution of
AR-immunoreactivity was identical for each case.
In representative experiment L23, AR-ir nuclei were
confined to the ventral posterior amygdala (Fig. 4A) and
the ventromedial hypothalamic nucleus. Labeled nuclei in
these areas were more lightly stained and fewer in number
than in the male brain. No nuclear labeling was observed
in the medial preoptic area, the arcuate nucleus, the bed
nucleus of the stria terminalis, or the striatoamygdalar area
in any experiment involving female lizards (Fig. 4B).
The pattern of AR-ir fiber labeling in representative
experiment L23 (female) closely resembled that in L8
(male). In both male and female, AR-ir fibers were
concentrated in the medial cortex, dorsal cortex, medial
septum, area triangularis, dorsal portion of the periventricular hypothalamus, habenular nuclei, optic tectum,
lateral forebrain bundle, and the white matter surrounding
the third ventricle.
4. Discussion
Our results show that AR-immunoreactivity is concentrated in limbic areas of the lizard forebrain, particularly
the medial cortex, amygdala, and hypothalamus, and that
this immunoreactivity is sexually dimorphic, with a wider
distribution of AR-ir nuclei present in the male brain as
compared to the female.
In the male fence lizard, we observed AR-ir cell nuclei
in the medial preoptic area, the ventromedial hypothalamic
nucleus, the arcuate nucleus, the caudal ventral part of the
periventricular hypothalamus, the premammillary nucleus,
the bed nucleus of the stria terminalis, the striatoamygdalar
area, and the ventral posterior amygdala. This distribution
is similar to the AR mRNA distribution reported for the
whiptail lizards, Cnemidophorus uniparens and C. inornatus [46]. In the whiptail lizard, AR is expressed in the
external amygdalar nucleus, the ventromedial hypothalamic nucleus, the medial preoptic area, the premammillary nucleus, and the periventricular hypothalamus. The
external amygdalar nucleus in the whiptail lizard most
likely corresponds to the ventral posterior amygdalar
nucleus in the fence lizard, based on its ventral location
and pattern of AR expression (cf., Fig. 1 with Figs. 3 and 4
M.M. Moga et al. / Brain Research 879 (2000) 174 – 182
179
Fig. 3. Photomicrographs of AR-ir nuclei and fibers in the male lizard brain (experiments L2 and L8). (A) A prominent cluster of AR-nuclei was observed
in the ventral posterior nucleus of the amygdala. (B) Darkly stained AR-ir nuclei and fibers were present in the premammillary nucleus. (C) AR-ir nuclei
were abundant in the arcuate nucleus and periventricular hypothalamus. (D) Punctate labeling was concentrated in the deep layers of the optic tectum
surrounding the tectal ventricle. Arc, arcuate nucleus; OTe, optic tectum; Pv, periventricular hypothalamus; PrM, premammillary nucleus; TV, tectal
ventricle, VPA, ventral posterior nucleus of the amygdala; 3V, third ventricle. Scale bar5100 mm.
in [46]). Young et al. [46] observed several additional
AR-positive cell clusters in the whiptail lizard that we did
not detect in the Eastern Fence lizard, such as the dorsal
lateral thalamic nucleus and the anterior portion of the
dorsal ventricular ridge. The in situ hybridization method
employed by Young et al. [46] is more sensitive than
immunocytochemistry, which could account for the addi-
tional AR cell populations detected in their study. Alternatively, there may be species differences in AR expression between whiptail and fence lizards.
The distribution of AR-immunoreactivity in the lizard
brain is similar to that observed in the brains of other
vertebrates. In the mammalian brain, AR-ir nuclei are
present in the bed nucleus of the stria terminalis; the
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M.M. Moga et al. / Brain Research 879 (2000) 174 – 182
Fig. 4. Photomicrographs of AR-ir nuclei and fibers in the female lizard brain (experiment L23). (A) In the ventral posterior amygdala, most of the AR-ir
nuclei were lightly stained. (B) A few AR-ir fibers, but no AR-ir nuclei, were observed in the arcuate nucleus and periventricular hypothalamus. Arc,
arcuate nucleus; NS, nucleus sphericus; Pv, periventricular hypothalamus; VPA, ventral posterior nucleus of the amygdala; 3V, third ventricle. Scale
bar5100 mm.
medial preoptic area; the arcuate, ventromedial and
paraventricular hypothalamic nuclei; the ventral premammillary nucleus; the medial and cortical amygdalar nuclei;
the CA1 hippocampus; and the cerebral cortex
[10,12,21,34,43,45,49]. A similar pattern is observed in the
bird brain, with AR-ir nuclei concentrated in the medial
preoptic, paraventricular and ventromedial hypothalamic
nuclei; the infundibular hypothalamus (corresponding to
the mammalian arcuate nucleus); the nucleus taeniae (avian
homologue of the mammalian amygdala); the nucleus
intercollicularis; and several telencephalic nuclei involved
in birdsong [1,2,36]. In the fish brain, AR-ir nuclei are
located predominantly in the dorsal and lateral parts of the
ventral telencephalon (possibly corresponding to the mammalian lateral septum and amygdala, respectively [8]), the
preoptic, periventricular and tuberal hypothalamus, the
dorsal telencephalon, and the optic tectum [19]. Across the
different vertebrate classes, AR-ir neurons are found
consistently in certain brain areas, such as the amygdala,
preoptic area and arcuate hypothalamus; these areas are
necessary for reproductive behavior and / or hormone release [24,27,32,41]. AR-immunoreactivity in taxon-specific
areas, such as the magnocellular nucleus of the anterior
neostriatum [1], the supraoptic nucleus [13], the optic
tectum [19] and the nucleus of the lateral olfactory tract
[21], may be involved in more specialized aspects of
reproductive behavior (e.g., song production, intruder-chasing) or in other adaptive behaviors (e.g., water homeostasis).
We observed dark-staining of the nucleolus in many
AR-ir nuclei located in the amygdala and hypothalamus,
suggesting that androgens may activate nucleolar processes
(e.g., transcription of ribosomal genes) in the lizard brain.
Several other studies have noted steroid receptor immunostaining of nucleoli [6,17,23]. For example, Kessels et al.
[23] observed nucleolar staining in breast carcinoma cells
(MCF7) immunostained for the estradiol receptor. In
osteoblasts and osteocytes, an antibody to the steroid
1,25-dihydroxyvitamin D3 receptor labels primarily the
nuclear chromatin but also the nucleolus [6]. AR-ir nucleoli have not previously been described, possibly due to
masking of nucleolar immunoreactivity by intense nuclear
AR-immunoreactivity. In support of this possibility, we
could not distinguish AR-ir nucleoli in darkly stained
nuclei. Most of the AR-ir cell nuclei in our experiments
were lightly stained which we attributed to the relatively
M.M. Moga et al. / Brain Research 879 (2000) 174 – 182
long fixation times (9–18 days) involved in post mortem
immersion fixation and gelatin blocking.
AR cytoplasmic labeling was observed with the PG21
antibody in the Eastern Fence lizard brain. The punctate
staining pattern of the AR-ir fibers was consistent with a
synaptic localization on either axon terminals and / or
dendrites. Glial processes may also possess steroid receptors [22], but the length and diameter of the AR-ir
fibers in the lizard brain makes a glial origin unlikely. We
believe this cytoplasmic immunostaining to be specific for
AR for the following reasons. (1) In control experiments,
AR-immunoreactivity in both cell nuclei and neuronal
processes was completely abolished when the PG21 antibody was preabsorbed with its immunogenic peptide
(AR21) or when the primary antibody was omitted. (2)
Cytoplasmic staining of steroid receptors is well documented and has been reported for the estrogen [4,5,29],
androgen [18,44], progestin [3,31], and glucocorticoid [33]
receptors. In the guinea-pig hypothalamus of ovarectomized females, estrogen receptor-immunoreactivity is present in dendrites and axon terminals, as well as in the cell
soma [5]. A punctate staining pattern, similar to that
observed in the present study, has been demonstrated for
membrane glucocorticoid and estrogen receptors
[29,33,40]. Future studies will examine the ultrastructure
and possible neuronal origin of the AR-ir fibers in the
fence lizard brain.
A sex difference in AR-immunoreactivity was detected
in the hypothalamus and amygdala. AR-ir nuclei were
abundant in the hypothalamus of the male fence lizard but
were restricted to the ventromedial nucleus in the female.
AR-immunostaining in the amygdala of the female lizard
was limited to the most ventral portion of the ventral
posterior nucleus; staining in the male was more extensive
and spanned the ventral posterior nucleus, the
striatoamygdalar area and the bed nucleus of the stria
terminalis. Few studies have examined the brains of both
sexes for possible sex differences in AR protein or mRNA
expression. Six-day-old male rats show robust AR immunostaining of the ventral premammillary nucleus; this
nucleus is unstained in female rats of the same age [45]. In
the fetal mouse (E16), an intense AR mRNA signal is
present in the male embryo brain with only weak AR
signal detected in the female brain [48]. These and the
present results suggest that a sex comparison of ARimmunoreactivity in the brain of an adult bird or mammal
would be worthwhile.
Sex differences in the brain are generally correlated with
sexually dimorphic behaviors [20]. Male and female S.
undulatus show sex differences in size, skin coloring, and
aggressive behavior [39]. Males have blue belly patches
which they display during frequent aggressive encounters.
Females are physically larger than males, lack blue coloration on their belly, and display little or no aggression. The
marked sexual dimorphism in brain AR-immunoreactivity
in this species may underlie sex differences in its behavior.
181
Acknowledgements
This work was supported by the Terre Haute Center for
Medical Education, Indiana University School of Medicine. We are grateful to Dr Diana Hews for introducing us
to Sceloporus lizards.
References
[1] J. Balthazart, A. Foidart, E.M. Wilson, G.F. Ball, Immunocytochemical localization of androgen receptors in the male songbird
and quail brain, J. Comp. Neurol. 317 (1992) 407–420.
[2] J. Balthazart, A. Foidart, M. Houbart, G.S. Prins, G.F. Ball,
Distribution of androgen receptor-immunoreactive cells in the quail
forebrain and their relationship with aromatase immunoreactivity, J.
Neurobiol. 35 (1998) 323–340.
[3] J.D. Blaustein, J.C. King, D.O. Toft, J. Turcotte, Immunocytochemical localization of progestin receptor-immunoreactivity in guinea pig
hypothalamus and preoptic area, Brain Res. 474 (1988) 1–15.
[4] J.D. Blaustein, J.C. Turcotte, Estrogen receptor-immunostaining of
neuronal cytoplasmic processes as well as cell nuclei in guinea pig
brain, Brain Res. 495 (1989) 75–82.
[5] J.D. Blaustein, M.N. Lehman, J.C. Turcotte, G. Greene, Estrogen
receptors in dendrites and axon terminals in the guinea pig hypothalamus, Endocrinology 131 (1992) 281–290.
[6] G. Boivin, P. Mesguich, J.W. Pike, R. Bouillon, P.J. Meunier, M.R.
Haussler, P.M. Dubois, G. Morel, Ultrastructural immunocytochemical localization of endogenous 1,25-dihydroxyvitamin D3 and its
receptors in osteoblasts and osteocytes from neonatal mouse and rat
calvaria, Bone and Mineral 3 (1987) 125–136.
[7] L.L. Bruce, T.J. Neary, The limbic system of tetrapods: a comparative analysis of cortical and amygdalar populations, Brain Behav.
Evol. 46 (1995) 224–234.
[8] A.B. Butler, W. Hodos, Comparative Vertebrate Neuroanatomy:
Evolution and Adaptation, Wiley–Liss, New York, 1996.
[9] A.B. Butler, R.G. Northcutt, Architectonic studies of the diencephalon of Iguana iguana, J. Comp. Neurol. 149 (1973) 439–462.
[10] J.V.A. Choate, O.D. Slayden, J.A. Resko, Immunocytochemical
localization of androgen receptors in brains of developing and adult
male rhesus monkeys, Endocrine 8 (1998) 51–60.
[11] P. Ciofi, J.E. Krause, G.S. Prins, M. Mazzuca, Presence of nuclear
androgen receptor-like immunoreactivity in neurokinin B-containing
neurons of the hypothalamic arcuate nucleus of the adult male rat,
Neurosci. Lett. 182 (1994) 193–196.
[12] A.N. Clancy, R.W. Bonsall, R.P. Michael, Immunohistochemical
labeling of androgen receptors in the brain of rat and monkey, Life
Sci. 50 (1992) 409–417.
[13] A.N. Clancy, C. Whitman, R.P. Michael, H.E. Albers, Distribution
of androgen receptor-like immunoreactivity in the brains of intact
and castrated male hamsters, Brain Res. Bull. 33 (1994) 325–332.
[14] D. Crews, V. Traina, F.T. Wetzel, C. Muller, Hormonal control of
male reproductive behavior in the lizard, Anolis carolinensis: role of
testosterone, dihydrotestosterone, and estradiol, Endocrinology 103
(1978) 1814–1821.
[15] K.D. Dunlap, H.H. Zakon, Behavioral actions of androgens and
androgen receptor expression in the electrocommunication system of
an electric fish, Eigenmannia virescens, Horm. Behav. 34 (1998)
30–38.
[16] S.B. Emerson, A. Greig, L. Carroll, G.S. Prins, Androgen receptors
in two androgen-mediated, sexually dimorphic characters of frogs,
Gen. Comp. Endocrinol. 114 (1999) 173–180.
´ A. Gonzalez
´
[17] O.M. Echeverrıa,
Maciel, A.M. Traish, H.H. Wotiz, E.
´
Ubaldo, G.H. Vazquez-Nin,
Immuno-electron microscopic localiza-
182
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
M.M. Moga et al. / Brain Research 879 (2000) 174 – 182
tion of estradiol receptor in cells of male and female reproductive
and non-reproductive organs, Biol. Cell 81 (1994) 257–265.
L.M. Freeman, B.A. Padgett, G.S. Prins, S.M. Breedlove, Distribution of androgen receptor immunoreactivity in the spinal cord
of wild-type, androgen-insensitive and gonadectomized male rats, J.
Neurobiol. 27 (1995) 51–59.
D. Gelinas, G.V. Callard, Immunolocalization of aromatase- and
androgen receptor-positive neurons in the goldfish brain, Gen.
Comp. Endocrinol. 106 (1997) 155–168.
J. Godwin, D. Crews, Sex differences in the nervous system of
reptiles, Cell. Molec. Neurobiol. 17 (1997) 649–669.
J. Iqbal, J.J. Swanson, G.S. Prins, C.D. Jacobson, Androgen
receptor-like immunoreactivity in the Brazilian opossum brain and
pituitary: distribution and effects of castration and testosterone
replacement in the adult male, Brain Res. 703 (1995) 1–18.
C.L. Jordan, Glia as mediators of steroid hormone action on the
nervous system: an overview, J. Neurobiol. 40 (1999) 434–445.
M.M. Kessels, B. Qualmann, H.H. Thole, W.D. Sierralta, Subcellular
localization of estradiol receptor in MCF7 cells studied with
nanogold-labelled antibody fragments, Eur. J. Histochem. 42 (1998)
259–270.
P.A. Kingston, D. Crews, Effects of hypothalamic lesions on
courtship and copulatory behavior in sexual and unisexual whiptail
lizards, Brain Res. 643 (1994) 349–351.
´
´
´
E. Lanuza, C. Font, A. Martınez-Marcos,
F. Martınez-Garcıa,
Amygdalo-hypothalamic projections in the lizard Podarcis hispanica: a combined anterograde and retrograde tracing study, J.
Comp. Neurol. 384 (1997) 537–555.
J.I. Morrell, D. Crews, A. Ballin, A. Morgenthaler, D.W. Pfaff,
3
H-Estradiol, 3 H-testosterone and 3 H-dihydrotestosterone localization in the brain of the lizard Anolis Carolinensis: an autoradiographic study, J. Comp. Neurol. 188 (1979) 201–224.
C.B. Nemeroff, C.A. Lamartiniere, G.A. Mason, R.E. Squibb, J.S.
Hong, S.C. Bondy, Marked reduction in gonadal steroid hormone
levels in rats treated neonatally with monosodium L-glutamate:
further evidence for disruption of hypothalamic–pituitary–gonadal
axis regulation, Neuroendocrinology 33 (1981) 265–267.
R.G. Northcutt, Forebrain and midbrain organization in lizards and
its phylogenetic significance, in: N. Greenberg, P.D. MacLean
(Eds.), Behavior and Neurology of Lizards, NIMH, Rockville,
Maryland, 1978, pp. 11–64.
T.C. Pappas, B. Gametchu, C.S. Watson, Membrane estrogen
receptors identified by multiple antibody and impeded ligand
labeling, FASEB J. 9 (1995) 404–410.
G.S. Prins, L. Birch, G.L. Greene, Androgen receptor localization in
different cell types of the adult rat prostate, Endocrinology 129
(1991) 3187–3199.
W.G. Rossmanith, S. Wolfahrt, A. Ecker, E. Eberhardt, The demonstration of progesterone, but not of estrogen, receptors in the
developing human placenta, Horm. Metab. Res. 29 (1997) 604–610.
B.D. Sachs, R.L. Meisel, The physiology of male sexual behavior,
in: E. Knobil, J. Neil (Eds.), The Physiology of Reproduction,
Raven Press, New York, 1988, pp. 1393–1485.
F.N.A. Sackey, C.S. Watson, B. Gametchu, Cell cycle regulation of
membrane glucocorticoid receptor in CCRF-CEM human ALL cells:
correlation to apoptosis, Am. J. Physiol. 36 (1997) E571–583.
[34] M. Sar, D.B. Lubahn, F.S. French, E.M. Wilson, Immunohistochemical localization of the androgen receptor in rat and human
tissues, Endocrinology 127 (1990) 3180–3186.
[35] R.B. Simerly, C. Chang, M. Muramatsu, L.W. Swanson, Distribution
of androgen and estrogen receptor mRNA-containing cells in the rat
brain: an in situ hybridization study, J. Comp. Neurol. 294 (1990)
76–95.
[36] G.T. Smith, E.A. Brenowitz, G.S. Prins, Use of PG-21 immunocytochemistry to detect androgen receptors in the songbird brain, J.
Histochem. Cytochem. 44 (1996) 1075–1080.
[37] J.W. Thornton, D.B. Kelley, Evolution of the androgen receptor:
structure–function implications, BioEssays 20 (1998) 860–869.
[38] R.R. Tokarz, Hormonal regulation of male reproductive behavior in
the lizard Anolis sagrei: a test of the aromatization hypothesis,
Horm. Behav. 20 (1986) 364–377.
[39] M.B. Vinegar, Comparative aggression in Sceloporus virgatus, S.
undulatus consobrinus, and S. u. tristichus (Sauria: Iguanidae),
Anim. Behav. 23 (1975) 279–286.
[40] C.S. Watson, B. Gametchu, Membrane-initiated steroid actions and
the proteins that mediate them, Proc. Soc. Exp. Biol. Med. 220
(1999) 9–19.
[41] J.M. Wheeler, D. Crews, The role of the anterior hypothalamus–
preoptic area in the regulation of male reproductive behavior in the
lizard, Anolis carolinensis: lesion studies, Horm. Behav. 11 (1978)
42–60.
[42] S.M. Winkler, J. Wade, Aromatase activity and regulation of sexual
behaviors in the green anole lizard, Physiol. Behav. 64 (1998)
723–731.
[43] R.I. Wood, R.K. Brabec, J.M. Swann, S.W. Newman, Androgen and
estrogen receptor-containing neurons in chemosensory pathways of
the male Syrian hamster brain, Brain Res. 596 (1992) 89–98.
[44] R.I. Wood, S.W. Newman, Intracellular partitioning of androgen
receptor immunoreactivity in the brain of the male Syrian hamster:
effects of castration and steroid replacement, J. Neurobiol. 24
(1993) 925–938.
[45] M. Yokosuka, G.S. Prins, S. Hayashi, Co-localization of androgen
receptor and nitric oxide synthase in the ventral premammillary
nucleus of the newborn rat: an immunohistochemical study, Dev.
Brain Res. 99 (1997) 226–233.
[46] L.J. Young, G.F. Lopreato, K. Horan, D. Crews, Cloning and in situ
hybridization analysis of estrogen receptor, progesterone receptor,
and androgen receptor expression in the brain of whiptail lizards
(Cnemidophorus uniparens and C. inornatus), J. Comp. Neurol. 347
(1994) 288–300.
[47] L.J. Young, J. Godwin, M. Grammar, M. Gahr, D. Crews, Reptilian
sex steroid receptors: amplification, sequence and expression analysis, J. Steroid Biochem. Molec. Biol. 55 (1995) 261–269.
[48] W.-J. Young, C. Chang, Ontogeny and autoregulation of androgen
receptor mRNA expression in the nervous system, Endocrine 9
(1998) 79–88.
[49] L. Zhou, J.D. Blaustein, G. De Vries, Distribution of androgen
receptor immunoreactivity in vasopressin- and oxytocin-immunoreactive neurons in the male rat brain, Endocrinology 134 (1994)
2622–2627.