M .M. Moga et al. Brain Research 879 2000 174 –182
175
homology with ARs in other species [47]. The DNA- Elite Standard ABC kit; Vector for 1–2 h. After several
binding and C-terminal ligand-binding domains of AR are rinses in PBS, the sections were reacted in a solution of
the most highly conserved [37]. However, positions 1–35 0.05 3,39-diaminobenzidine tetrahydrochloride DAB;
and 230–268 in the AR are far less variant than the rest of Sigma-0.025 nickel chloride-0.01 H O in 0.1 M Tris
2 2
the N-terminal domain [37]. A low degree of variance in buffer, pH 7.4, for 10 min. The sections were mounted on
amino acids 1–21 of the AR may explain the ability of the gel-coated slides, dehydrated through alcohols and xylene,
PG21 antibody to label ARs in diverse tissue types in a and coverslipped with Permount Fisher.
variety of vertebrates, including mammal [11,13,18,21, The specificity of the PG21 antibody was tested by, 1
44,45,49], fish [15], bird [2,36], and amphibian [16]. 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
2. Methods derived from a different portion of the rat AR AR462;
amino acids 462–478, and 3 omission of the primary Male n56 and female n55 lizards Sceloporus
antibody. In the two preabsorption controls, sections were undulatus were purchased from Charles D. Sullivan Co.,
incubated in 1 ml of diluted antibody 1:5000 preincu- Inc. Nashville, TN during the spring summer breeding
bated with 370 ng of peptide AR21 or AR462. No season May through September. Nine of the lizards 5
immunocytochemical staining was observed in controls males, 4 females were classified as S
. undulatus consob- where, 1 the primary antibody was omitted, or 2 the
rinus Southern Prairie subspecies; the other two in- primary antibody was preincubated with its respective
dividuals experiments L8 and L18 were identified as S .
peptide AR21. Staining was intact in control experiments undulatus garmani Northern Prairie subspecies. Animals
involving preabsorption with the distant, unrelated peptide were maintained in captivity for 2–8 weeks in glass
AR462. aquaria 2 to 3 animals per aquaria with peat moss as
The distribution of AR-immunoreactivity in representa- substrate, and sticks and stones for climbing. Photoperiod
tive sections was plotted on a microscope BH-2, was maintained at 12 h light: 12 h dark. Room temperature
Olympus with a digital stage readout head attached to a was 21–238C, with additional heat up to 388C provided
computer with Neurolucida software MicroBrightField, by a heat lamp situated at one end of the terrarium. Water
Inc.. A camera lucida was used to add cytoarchitectural was available at all times. Animals were fed crickets 2 to 3
details obtained from adjacent Nissl-stained sections. times per week, supplemented with reptile vitamins.
At the time of sacrifice, animals were given an overdose of sodium pentobarbital followed by decapitation. The
3. Results
dorsal skullcase was removed, and the brain was im- mersion-fixed in situ in 4 paraformaldehyde 0.1 M
AR-immunoreactivity was observed in select brain areas phosphate buffer, pH 7.4, for 5–12 days. Following
in both male and female S . undulatus. The distribution of
fixation, the brains were removed from the skull and AR-immunoreactivity in the male Eastern Fence Lizard
embedded in 10 gelatin. The gelatin-brain blocks were brain was examined in six experiments. One male lizard
fixed in 4 paraformaldehyde for an additional 4–6 days. experiment L2 was obtained and sacrificed in July, 1998;
Next, the brains were cut into 20 mm sections on a two males experiments L8 and L24 were obtained in
Vibratome. One series of sections one-of-three was September, 1998 and sacrificed in November, 1998; and
processed for AR-immunocytochemistry, and a second three males experiments L21, L22 and L25 were obtained
series was mounted on gel-coated slides and Nissl-stained and sacrificed in June, 1999. The pattern of AR-ir labeling
with thionin. was identical in these six experiments, but the number of
The immunocytochemical method was as follows. Sec- labeled cells and the intensity of the immunolabeling
tions were treated with 0.3 hydrogen peroxide in 0.01 M varied by experiment. The following description specifical-
phosphate-buffered saline PBS, pH 7.4, for 10 min, ly refers to representative experiment L8, which exhibited
rinsed in PBS, treated with 0.5 sodium borohydride in the most robust and widespread labeling.
PBS for 5–10 min, and preincubated for 1 h in a PBS At rostral levels in experiment L8, AR-immunoreactivi-
solution containing 2 normal donkey serum NDS and ty was concentrated in the medial and dorsal cortices Figs.
0.3 Triton X-100 TX. Sections were then incubated 1A–D; 2A–B. AR-ir fibers in the medial cortex were
with the AR antibody PG21, diluted 1:1,000–1:5,000 organized in a bilaminar pattern with both layers juxtap-
0.2–1.0 mg ml, final concentration in PBS-NDS-TX, for osed to the principal neuron layer Fig. 2A. In the dorsal
3–6 days at 48C. Next, the sections were rinsed in PBS, cortex, AR-ir fibers displayed numerous varicosities Fig.
incubated with a secondary antibody biotin–SP-conju- 2. At more caudal levels, AR-ir fibers were also found in
gated donkey anti-rabbit IgG; Jackson ImmunoResearch; the lateral cortex Fig. 1B, C. Cytoplasmic staining was
diluted 1:200 in PBS-NDS-TX for 1–2 h at room observed in a small number of neurons scattered in the
temperature, rinsed again, and reacted with ABC reagent dorsal cortex. Ventral to the medial cortex, there was a
176 M
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
dense concentration of labeled fibers in the medial septal experiment L8 November than in representative experi-
nucleus Fig. 1B. ments L2 July and L21 June. Conversely, more labeled
Several cell groups in the amygdala contained AR-ir cells were observed in the bed nucleus of the stria
nuclei. In many of the labeled cell nuclei, a darkly stained terminalis and striatoamygdalar area in experiment L2 than
AR-ir nucleolus was evident. One amygdalar cell group in L8 or L21. The intensity of the immunolabeling both
with AR-ir nuclei formed a band of labeling ventral to the AR-ir nuclei and fibers was greatest in L8, moderate in
striatum Fig. 1B; this cell group corresponded at rostral L2, and least in L21.
levels to the striatoamygdalar area, and at caudal levels, to AR-immunoreactivity in the female Eastern Fence lizard
the medial interstitial amygdala [7]. Labeled neurons in brain was examined in five experiments. Two female
this cell group striatoamygdalar area, medial interstitial lizards experiments L17 and L18 were obtained in
amygdala were large in size and widely spaced. A few, September, 1998 and sacrificed in November, 1998; and
small, lightly stained AR-ir nuclei were observed in the three females experiments L19, L20 and L23 were
adjacent bed nucleus of the stria terminalis Fig. 1B, C. A obtained and sacrificed in May, 1999. The distribution of
dense cluster of darkly stained, AR-ir nuclei was found in AR-immunoreactivity was identical for each case.
the ventral posterior nucleus of the amygdala Figs. 1B, C, In representative experiment L23, AR-ir nuclei were
3A [7,25]. This cell group, composed of closely packed, confined to the ventral posterior amygdala Fig. 4A and
small neurons, formed a wedged-shaped protuberance at the ventromedial hypothalamic nucleus. Labeled nuclei in
rostral levels Fig. 1B; it extended caudally as a thin band these areas were more lightly stained and fewer in number
of cells ventrolateral to the nucleus sphericus Fig. 1C. than in the male brain. No nuclear labeling was observed
In the hypothalamus, some lightly stained AR-ir nuclei in the medial preoptic area, the arcuate nucleus, the bed
were observed in the medial preoptic area, particularly in nucleus of the stria terminalis, or the striatoamygdalar area
the caudal dorsal portion Fig. 1B. Scattered AR-ir fibers, in any experiment involving female lizards Fig. 4B.
punctate in appearance, were present throughout the preop- The pattern of AR-ir fiber labeling in representative
tic and anterior hypothalamic areas Fig. 2C. Immediately experiment L23 female closely resembled that in L8
caudal to the optic chiasm, we observed a concentration of male. In both male and female, AR-ir fibers were
AR-ir fibers in a dorsal section of the periventricular concentrated in the medial cortex, dorsal cortex, medial
hypothalamus Fig. 1D. In Nissl-stained sections, this septum, area triangularis, dorsal portion of the periven-
hypothalamic area showed a distinct cytoarchitecture with tricular hypothalamus, habenular nuclei, optic tectum,
many small, dark-staining neurons as well as larger, lateral forebrain bundle, and the white matter surrounding
medium-staining neurons. Adjacent areas in the periven- the third ventricle.
tricular hypothalamus contained only large, medium-stain- ing neurons. A cluster of lightly labeled AR-ir nuclei was
observed the ventromedial nucleus Fig. 1D. Many darkly
4. Discussion
