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Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania 18015
Address all correspondence and requests for reprints to: N. G. Simon, Ph.D., Department of Biological Sciences, 111 Research Drive, Lehigh University, Bethlehem, Pennsylvania 18015. E-mail: ngs0{at}lehigh.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Concerning sex differences, the intensity of AR immunostaining was greater in intact male vs. female rats in the posterior bed nucleus of the stria terminalis (BSTp) and periventricular nucleus, but not in the lateral septum (12), whereas in spinal motoneurons, qualitative immunostaining intensity did not differ between males and females (13). Using the number of positively labeled cells as an index, no significant sex difference was found between male and female cynomolgus monkeys in seven major limbic system areas, although possible differences in staining intensity were not reported (14). In mouse brain, a random sample of intact male and female mice (n = 3/group) was compared, and pronounced sex differences (males>>females) were found in the intensity of AR immunoreactivity in all receptor-positive regions using integrated particle density as a semiquantitative measure (15).
These mixed results have led to the suggestion of regional and/or species differences in neural AR regulation (5, 14, 16, 17). However, concerns about the properties of different AR antisera have limited efforts to resolve these issues. In particular, whether PG-21, the most widely used antiserum, recognizes unliganded AR remains a question (5, 6, 8, 18), which makes the interpretation of immunochemical findings regarding the effects of castration problematic. As a consequence, it is difficult to conceptualize how neural AR regulation may be, for example, linked to behavioral sensitivity to androgen.
In the present study, the effects of castration with or without T replacement on AR expression were assessed in male and female mouse brain in four regions that have been implicated in the regulation of male-typical behaviors: BSTp, medial preoptic area (MPO), and dorsal and ventral aspects of the lateral septum (LSD, and LSV, respectively) (see reviews in Refs. 7 and 19). Including females provided an immunocytochemical assessment of potential sexual dimorphisms in AR regulation that has not previously been reported and may be important for understanding a cellular mechanism linked to androgen sensitivity. The male-female comparisons are a useful model for assessing the latter, because there is a robust sex difference in behavioral sensitivity to androgen (reviewed in Ref.7). Western blot analyses were conducted with brain extracts prepared from comparably treated males and females to provide an additional test of quantitative differences in AR. The findings demonstrated that although there is a sexual dimorphism in AR density, regulatory responses to both gonadectomy and T treatment are essentially identical in both sexes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Reagents
Rabbit AR antiserum (PG-21) was a generous gift from Dr. Gail
Prins. The ABC staining kit was obtained from Vector Laboratories
(Burlingame, CA). Precast minigels, nonfat milk, and biotin-labeled mol
wt standards were purchased from Bio-Rad (Hercules, CA). Horseradish
peroxidase-conjugated antirabbit antiserum, nitrocellulose membranes,
enhanced chemiluminescence (ECL) detection reagents, and Hyperfilm were
obtained from Amersham (Arlington Heights, IL). TP was purchased from
Steraloids (Wilton, NH). Hemo De and Permount medium were obtained from
Fisher Scientific (Fairlawn, NJ). All other reagents were obtained from
Sigma Chemical Co. (St. Louis, MO).
ICC
Twenty-four hours after the injection of TP, mice were
anesthetized and perfused with PBS, followed by 4% formaldehyde in
PBS. The brains were postfixed for 24 h in the same fixative, and
40-µm frozen sections were cut on a rotary microtome. Sections were
treated with 0.15% Triton X-100/PBS for 40 min, then with 10% FBS/PBS
for 30 min. Free floating sections were incubated for 48 h at 4 C
in 1% BSA/PBS containing a peptide-based polyclonal AR antiserum
against N-terminal amino acids 1–21 (1 µg/ml). The sections were
washed and placed in biotinylated goat antirabbit IgG solution (1:400)
for 2 h, washed in PBS, and then incubated in
avidin-biotin-peroxidase complex solution (prepared according to the
manufacturer’s instructions) for 2 h. Nickel (0.16%)-intensified
diaminobenzadine (1 mg/ml) was used as the chromogen. After ICC
staining, sections were mounted on gelatin-treated glass slides,
air-dried, dehydrated through a graded ethanol series, cleaned in Hemo
De, and coverslipped with Permount medium. Although PG-21 is a fully
characterized AR antiserum (20), these were the initial studies with
mouse brain, and control procedures that included excess unlabeled
peptide and exclusion of primary antiserum were run. Staining was
absent under these conditions in all sections (not shown).
Image analysis
Sections were matched across the six groups according to the
method of Slotnick and Leonard (21). For each region, sections were
prepared from at least three animals. Images were taken with a CCD-72
video camera connected to a Macintosh computer equipped with
an AG-5 Scion frame grabber at a magnification of 20 x 3.3 using
an Olympus BH-2 microscope. Measurements of staining intensity were
taken in a matched 725 x 725-µm field in each area after the
images were thresholded by density slicing to the same value. The
mean background density for each image was determined after density
slicing the background area and was used as a correction factor. Data
generated by this approach include the total area of stained particles
and mean particle density, which were used for calculating integrated
particle density (IPD), defined by the following formula: IPD =
total area of stained particles x (mean particle density -
mean background density). The IPD measure provided a semiquantitative
index of average staining intensity within a given region and was used
as the unit of analysis. A similar approach for comparing AR
immunostaining across brain regions was employed by Menard and Harlan
(17).
Western blot and ECL detection
Twenty-four hours after gonadectomy and hormone administration,
the animals were killed by cervical dislocation, and blocks
containing the regions included in the ICC analysis plus hypothalamus
(which also exhibited strong AR staining in the presence of T; data not
shown) were isolated from the rest of the brain and minced on an iced
stage. Tissues were then placed in 10 mM Tris (pH 7.4),
0.12 M sucrose, 2.5 mM MgCl2, 5%
SDS, 1 mM phenylmethylsulfonylfluoride, and 0.02%
NaN3 and sonicated. Samples were then boiled for 7 min to
denature proteins and reduce proteinase activity and were centrifuged
at 7000 rpm for 2 min to pellet any undissolved debris. Supernatants
were saved, and protein concentrations were quantitated using the
Bio-Rad protein assay method. Each sample was adjusted to 60 µg total
protein before loading. Proteins were separated on Bio-Rad precast 10%
polyacrylamide mini gels. The buffers and electrophoresis procedure
have been described previously (22). Proteins were transferred to
nitrocellulose membranes by electroblotting. For immunoblot detection
the Amersham ECL detection system was used. In brief, blots were
blocked with 5% dry milk in TBS buffer (20 mM Tris, pH
7.6; 137 mM NaCl; and 0.1% Tween-20) for 1 h,
transferred into TBS buffer containing 1% dry milk and 0.1 µg/ml
PG-21 primary antiserum, and incubated for 1 h followed by washes
with TBS. Blots were then treated with horseradish
peroxidase-conjugated antirabbit IgG diluted in TBS buffer containing
1% dry milk for 1 h and again washed. Finally, blots were reacted
with ECL detection reagents, and exposed to Hyperfilm for 1 h.
Films were processed with Kodak GBX developer and fixative (Eastman
Kodak, Rochester, NY). The area and intensity of each band were
quantitated using NIH Image (version 1.59) installed in an Apple
Macintosh IIvx computer connected to a Fotodyne photographic system
(Fotodyne Inc., Hartland, WI). Three independent replicates were run in
each sex at each dose. Preliminary studies showed that the presence of
excess peptide completely blocked the immunoreactive bands (not
shown).
Data analysis
A two-way ANOVA was used to assess general trends in the ICC
results. A series of one-way ANOVAs was conducted to provide more
detailed information about effects within each group and each region.
The Western blot results were evaluated using ANOVA and trend analysis.
In all cases, post-hoc comparisons used Duncan’s new
multiple range test with 
= 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The results are shown graphically in Fig. 1
, and a representative series of
sections is presented in Fig. 2. This
pattern of group differences was consistent within each of the areas
analyzed. A two-way ANOVA showed that there were significant
differences among the six groups in the density of AR staining
independent of region [F(5,124) = 252; P < 0.001].
Duncan’s multiple range test demonstrated the INT-M, GDX-F+T, and
GDX-M+T groups did not differ from each other, but exhibited
significantly greater staining intensity than the INT-F, GDX-F, and
GDX-M animals (P < 0.05; the latter groups also did
not differ from each other). There also were significant overall
differences in AR immunostaining among the four regions [F(3,124) =
29.86; P < 0.001] independent of hormonal status.
Post-hoc comparisons showed that BSTp and
LSV, although not different from each other, exhibited more
intense staining than MPO and LSD (P <
0.05). The latter two regions did not differ significantly. The
group x region interaction also was statistically significant
[F(15,124) = 3.51; P < 0.01]. This appeared to be
primarily due to a very strong induction of AR in GDX-F+T in
LSV. The one-way ANOVA on results from this region provides
more detailed information regarding this point.
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). LSD. The ANOVA showed that the groups differed significantly in the intensity of AR staining [F(5,36) = 37.98; P < 0.01]. Post-hoc comparisons revealed that GDX-M+T, INT-M, and GDX-F+T did not differ from each other and had significantly denser staining than INT-F, GDX-M, or GDX-F. Differences between INT-F and GDX-M were not significant, nor did GDX-M and GDX-F differ from each other. However, AR immunoreactivity in INT-F was significantly greater than that in GDX-F.
BSTp. ANOVA revealed significant differences among the groups in AR immunoreactivity [F(5, 22) = 105.2; P < 0.01]. Duncan’s test showed that IPD was greater in GDX-M+T than in all other groups. Males and GDX-F+T, although not differing significantly from each other, had more intense staining than INT-F, GDX-M, and GDX-F. Within these three conditions, AR immunoreactivity was significantly greater in INT-F than in GDX-M or GDX-F.
MPO. ANOVA demonstrated significant differences in the IPD of AR immunoreactivity [F(5, 30) = 55.88; P < 0.01]. Post-hoc comparisons showed that the GDX-M+T group exhibited significantly higher immunoreactivity than all other groups. GDX-F+T and GDX-M+T did not differ from each other, but were significantly higher than INT-F, GDX-M, and GDX-F. Significantly greater immunoreactivity was seen in INT-F than in GDX-M and GDX-F, and these latter two groups did not differ from each other.
Intragroup effects
INT-M. ANOVA of the results summarized in Fig. 1 showed that
there were significant differences in AR immunoreactivity among the
four regions [F(3, 20) = 4.09; P < 0.05]. The
intensity of AR-positive staining was highest in BSTP and
was significantly greater than that seen in LSD and MPO,
but not in LSV. The AR-positive staining intensity was
significantly higher in the latter region than in LSD.
Values in the MPO and LSD did not differ from each
other.
INT-F. There were no significant differences in the density of AR staining across the four regions [F(3, 20) = 3.05; P = NS].
GDX-M. The ANOVA showed significant overall differences among the four regions [F(3, 20) = 7.97; P < 0.01]. Post-hoc comparisons revealed that IPD was significantly greater in LSv than in the other regions. Values in BSTp, LSD, and MPO did not differ significantly from each other.
GDX-F. ANOVA of the results summarized in Fig. 1 showed that
there were no significant differences in the density of AR-positive
staining among the four regions [F(3, 20) = 1.33; P =
NS].
GDX-M+T. There were significant regional differences in AR
immunoreactivity [see Fig. 1; F(3, 20) = 18.7; P <
0.01]. Post-hoc comparisons revealed that values in all
regions differed significantly from each other, with the rank order
being BSTp > LSv > MPO >
LSD.
GDX-F+T. ANOVA revealed significant differences in AR density among the regions [F(3, 20) = 7.35; P < 0.01]. Post-hoc comparisons showed that AR immunoreactivity in LSV and BSTp did not differ, whereas staining intensity was significantly greater than in LSD. The LSV also was significantly higher in IPD than MPO.
Western blot
The results are shown in Fig. 3. A
dominant 97-kDa band, which was regulated by androgen, was detected in
all groups. The values reflect the integrated density of this band,
which was calculated by multiplying intensity x area. A two-way
ANOVA showed that there was a significant sex difference in the optical
density of the AR band [F(1, 32) = 8.9; P < 0.01]. As
shown in the histogram, this effect was due primarily to the difference
in normal and residual AR band densities in males and females when
intact and after castration, respectively. The induction of AR in both
sexes 24 h after exogenous TP administration was essentially
identical. There also was a significant difference among the dosages
independent of sex [F(6, 32) = 22.9; P < 0.01],
whereas the sex x treatment interaction was not significant
[F(6, 32) = 1.18; P = NS]. Trend analyses were
employed to assess whether there was a linear response to the
increasing doses of TP (in this analysis, data from the intact animals
were excluded because of the sex difference in endogenous circulating
androgen). In both males and females, a significant linear trend was
evident through the 1000-µg dose [males: F(1, 12) = 33.62;
P < 0.001; females: F(1, 12) = 49.81; P
< 0.001], demonstrating that AR can be induced beyond levels seen in
intact males in neural tissue. A second band at approximately 55 kDa
also was detected (not shown). The identity of this band is unclear,
because integrated density did not differ significantly among the
groups, which would have been expected if the band represented a
proteolytic fragment of the receptor.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous efforts to understand neural AR regulation in males had led to the suggestion that PG-21 did not recognize unliganded AR (5, 6, 8, 18). This position was derived from observations of AR after androgen withdrawal or treatment in, for example, BST and several androgen-responsive spinal nuclei and conflicting reports on whether castration did or did not cause a loss of AR immunoreactivity in neural target cells (c.f. Ref. 10 vs. Ref.11). The present study, by combining both ICC and Western blot analyses, provides some insight into this question. The former consistently showed a loss of AR immunoreactivity 24 h after castration in both males and females in the four regions reported and has been observed throughout the mouse brain (Lu, S., and N. G. Simon, unpublished observations). Because the Western analyses yielded ratios of AR band densities that mirrored the ICC data, it seems reasonable to conclude that the loss of immunoreactivity in gonadectomized animals in the ICC study reflected a steep decline in AR protein levels rather than an inability of PG-21 antiserum to detect unoccupied AR. More specifically, if unoccupied receptor conformation in situ had made the targeted epitope unavailable for antibody recognition and thus caused the decreased staining in the ICC experiment, then the Western blot analyses, where proteins were denatured, would have shown more intense AR bands in the gonadectomized male and female conditions. Combined, these observations suggest that there is a rapid loss of AR in neural target tissue as circulating androgens decrease, which also has been seen in peripheral tissues such as ventral prostate (23, 24), and that androgen serves to stabilize the receptor. Support for the latter is seen in biochemical studies that showed an effect of ligand on AR half-life (1, 25, 26). One implication of this perspective about AR loss in the absence of androgen is that earlier equilibrium binding studies that used an interval of 24 h or more after castration before conducting the assays may have underestimated the normal endogenous AR population (27, 28, 29, 30). Exchange assays thus appear to be necessary for accurate quantitation of AR (31, 32).
Males and females exhibited comparable responses to gonadectomy and TP replacement, which bears on the question of whether AR regulation by androgen is a factor in the sexual dimorphism in neural and behavioral sensitivity to androgen. The effects were similar across the four regions, particularly in response to TP treatment. Dose-response effects did not differ between males and females in the Western analyses; there was a linear response to the increasing level of androgen. These observations demonstrate that there is a common regulatory mechanism for AR in male and female neural tissue in mice and that the major factor is the presence or absence of androgen, findings that are consistent with autoregulation of AR by androgen. The progressive increase in AR band density that accompanied higher TP doses demonstrates that augmentation of AR levels is possible and is in keeping with recent immunochemical results. In one of these studies, AR expression also was increased beyond that seen in normal males 24 h after treatment (5), whereas in the other, enhanced AR levels were found 14 days after exposure to an anabolic androgen cocktail (17). Because androgen binding slows receptor degradation and extends the AR complex half-life (1, 26), it is likely that stabilization of AR is a contributing factor in the up-regulation of receptor levels. At the same time, de novo AR synthesis is required for a constant supply of receptor protein (23). Given that AR functions as a transcription factor, it is likely that alterations in its cellular level have direct effects on target gene expression.
The augmentation of AR levels and the significant up-regulation of this protein within 24 h of androgen administration have implications for efforts to understand behavioral changes associated with anabolic steroid abuse and mechanisms underlying neural target tissue sensitivity to androgen. Extended exposure to anabolic steroids has been linked to a set of personality changes, including "roid rage" (33, 34, 35). The elevated levels of AR seen in response to higher doses of androgen may represent part of the cellular events that underlie these changes. In this context, chronic, high level anabolic androgen treatment significantly increased the aggressiveness of pubertal male hamsters (36) and the optical density of AR in rat brain (17). At the same time, the comparable increases in AR immunoreactivity in females and males suggest that changes in the level of this protein are not sufficient to produce parallel changes in behavioral sensitivity to androgen. A prominent example of this dissociation is the induction of male-typical aggression in adult female mice. The activation of this response in females is a direct, androgen-dependent effect (37, 38, 39). Sixteen to 21 days of androgen exposure were required before aggression toward a stimulus male was seen, yet the present results showed that AR levels were dramatically increased in females within 24 h. This suggests that changes in AR content alone are not sufficient for the induction of male-typical behaviors. Rather, increased cellular AR content probably triggers progressively enhanced (or suppressed) transcription of other androgen-regulated genes (40), which, in combination, lead to the expression of behaviors such as aggression. A comparable view regarding the lack of a simple relationship between AR immunoreactivity and responsiveness to the masculine sexual behavior-promoting effect of T recently was expressed based on work in male hamsters (41), although it was noted that aromatization of T to estradiol was a cautionary factor in their conclusions.
Three additional issues require attention. One is the observed molecular weight of AR (97 kDa), the second is the absence of any apparent regional differences in AR regulation, and the third is the apparent lack of cytoplasmic immunostaining. Regarding the mol wt, 97 kDa is consistent with that expected based on the derived amino acid sequence for AR (4, 42). In addition, Puy et al. (43) isolated human neural AR from temporal lobe, and Western analysis demonstrated that it was a 98-kDa protein. However, other investigators have reported AR as an approximately 110-kDa molecule (44, 45, 46). Although explanations for this modest discrepancy probably include differences in extract preparation and electrophoresis conditions, the most important points are that the 97-kDa protein observed in the Western-ECL blots was immunoreactive with PG-21, a fully characterized anti-AR antiserum, and that this band was selectively regulated by androgen. Regional differences in AR regulation were not observed, at least qualitatively. This should be viewed in the context of the 24-h sampling point used in the study. Caution is necessary before generalizing this finding, however, because other groups have found regional variation in up-regulation at shorter or longer intervals using different measurement systems and when comparing animals before and after puberty (5, 17, 47, 48). Future work that examines multiple time points and uses directly comparable analytical methods can help clarify this issue. Third, cytoplasmic staining was not detected in intact or T-treated males and females and was negligible in the castrated groups. This observation indicated that the decreased intensity of nuclear immunoreactivity seen 24 h after castration was due to a loss of AR, a conclusion strongly supported by the Western blot results. In this context, increased cytoplasmic immunostaining has been reported 2 weeks or more postcastration (10, 11), and others have noted some staining in this compartment (5, 49). Although the former is not inconsistent with the present results because of the intervals employed, the latter may be due to species, tissue, or methodological considerations.
In closing, a pronounced, androgen-dependent sexual dimorphism in endogenous AR populations was described in four regions of the mouse brain. This sexual dimorphism was not due to an inability of PG-21 to recognize unliganded AR, because Western blot analyses showed effects on AR expression that were fully consistent with the ICC findings. The central role of androgen as a determinant of AR expression was demonstrated in both male and female neural tissue. Finally, the virtually identical effects of androgen administration on the level of immunoreactivity in both sexes indicate that there is a common mechanism of AR regulation in mouse brain.
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Acknowledgments |
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Footnotes |
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2 Current address: National Institute of Environmental and Health
Sciences, 111 TW Alexander Drive, P.O. Box 12233, MD E4–01, Research
Triangle Park, North Carolina 27709.
3 Current address: Laboratoire de Ethologie et Neurobiologie,
Université Louis Pasteur, URA CNRS 1295, 7 rue de la
Université, 67000 Strasbourg, France.
Received September 15, 1997.
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-dihydrotestosterone-receptor complexes formed by
human skin fibroblasts. J Steroid Biochem 19:561–565[CrossRef][Medline]
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, Females; 
, males. The presence of excess peptide
completely blocked the immunoreactive bands in other runs (not shown).
See text for details of the statistical analyses.