- Email: [email protected]
Androgen receptor immunoreactivity in specific neural regions in normal and hypogonadal male mice: effect of androgens
Brain Research 817 Ž1999. 19–24
Research report
Androgen receptor immunoreactivity in specific neural regions in normal and hypogonadal male mice: effect of androgens Stergios Apostolinas, Gopalan Rajendren, Areta Dobrjansky, Marie J. Gibson
)
Department of Medicine, Mount Sinai School of Medicine, Box 1055, 1 GustaÕe L. LeÕy Place, New York, NY 10029, USA Accepted 27 October 1998
Abstract This study examined the distribution and regulation of androgen receptor immunoreactivity ŽIR. in the brain of the hypogonadal Žhpg. male mouse, genetically deficient in GnRH. Five groups of animals were studied: intact, castrated, or castrated and testosterone propionate ŽTP.-treated normal adult male mice, and intact or TP-treated hpg adult male mice. All groups were studied 1 week after treatment. Five regions of the brain with high concentrations of androgen receptors in normal animals were examined, including the medial preoptic area, the lateral ventral septum, the ventromedial hypothalamus, the bed nucleus of the stria terminalis and the medial amygdala. The results showed that the congenital absence of GnRH results in minimal expression of androgen receptor-IR in mice in all regions examined. However, treatment with exogenous testosterone for 1 week was sufficient to induce the numbers of neurons containing androgen receptors, as detected by immunocytochemistry, into the range seen in normal male mice in all the areas studied except the VMH. Similar plasticity was also observed in normal males after 1 week of castration and TP replacement. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Androgen receptor; Brain; Hypogonadal; Mouse; Regulation; Castration; Testosterone
1. Introduction Androgens influence the development of the brain and male behavior either directly via interaction of the steroid with its receptor, or indirectly through aromatization to estrogens w1,23x. The androgen receptor is a 120 kDa protein that belongs to the steroid hormone receptor family. It contains an N-terminal that is important in transcriptional activation, a DNA-binding domain, a hinge region and a hormone binding C-terminal. It is encoded for by a single gene on chromosome Xq11–12 and it displays high affinity for testosterone and dihydrotestosterone w38x. The androgen receptor is present in various brain regions of many different species including humans w3,7,10,11,15,16, 19,22,29,31,33,37x. Hypogonadal mice Žhpg. have a spontaneously occurring deletion in the gene for gonadotropin-releasing hormone w24x, and as a result, the reproductive system in hpg
) Corresponding author. [email protected]
Fax:
q 1-212-534-2945;
E-mail:
mice remains infantile after birth w5x. The untreated adult males are infertile and fail to show normal male sexual behavior. The purpose of the present study was to evaluate the effects of the presence or absence of testicular androgens on the distribution of androgen receptors in both normal and hpg male mice.
2. Materials and methods
2.1. Animals and housing Both hpg and normal mice of the same strain ŽC3HrHeH= 101H. were bred and housed under a partially reversed lightrdark cycle Ž14:10 L:D; lights on 2300–1300 h. in a temperature controlled Ž24–268C. room. Food and water were available ad libitum. All procedures and animal care were in accord with the standards approved by the NIH Guide for the Care and Use of Laboratory Animals and were approved by the IACUC of the Mount Sinai School of Medicine.
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 1 1 8 0 - 9
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S. Apostolinas et al.r Brain Research 817 (1999) 19–24
2.2. Experimental groups Five groups of adult Ž3–4 months old. male mice were used: normal intact mice Ž n s 8., normal castrate mice Ž n s 4., normal testosterone propionate ŽTP.-treated castrate mice Ž n s 4., intact hpg mice Ž n s 9. and TP-treated intact hpg mice Ž n s 4.. TP-treated mice were given subcutaneous implants of 2-cm Silastic tubing Ži.d. 0.04 in.; o.d. 0.085 in.; Dow-Corning, Midland, MI. containing TP, and sealed at each end with silastic adhesive. This capsule size is reported to support plasma testosterone levels of approximately 110 ngrml in male mice of the same strain w6x. Surgeries and capsule implants were performed under chloral hydrate Ž380 mgrkg, i.p.. anesthesia 1 week before the experiment. Capsules were inserted in the intrascapular region via a small incision that was closed with a wound clip.
pH 7.4.. Brains were removed, postfixed at 48C overnight in the same solution and then 50 mm coronal sections were obtained on a Vibratome. The sections were exposed to 0.1% H 2 O 2 to suppress endogenous peroxidase activity followed by incubation in 0.1 M PBS containing 1.5% normal donkey serum and 0.3% Triton-X. The sections were then incubated with a rabbit polyclonal anti-androgen receptor antibody Ž1:200, AR70, Novocastra Laboratories. for 72 h. The androgen receptor protein was visualized using a mixture of 3,3X-diaminobenzidine and 0.005% H 2 O 2 following incubation of the sections with 0.5% donkey anti-rabbit biotinylated antibody ŽJackson Immunoresearch. for 3 h and avidin biotin peroxidase solution ŽVector Laboratories. for 1 h. There was a complete lack of staining when the primary antibody was omitted from the incubations. The sections were mounted onto gelatincoated slides and cover-slipped using Permount.
2.3. Immunocytochemistry
2.4. Analysis
One week after castration andror TP treatment, the mice were deeply anesthetized and perfused with normal saline followed by 4% paraformaldehyde Žin 0.1 M PBS;
The regions where dense androgen receptor immunoreactivity ŽAR-IR. was observed in normal male brains were the following: the medial preoptic area ŽPOA., the
Fig. 1. Numbers Žmean " S.E.M.. of androgen receptor-positive ŽAR q . cells in specific brain regions in male mice, including the bed nucleus of the stria terminalis ŽBNST., medial preoptic area ŽPOA., ventromedial nucleus of the hypothalamus ŽVMH., lateral ventral septum and medial amygdala. )) p - 0.01 vs. normal intact ŽNorm., normal TP-treated castrate ŽCastrrTP., and TP-treated hpg ŽHpgrTP.mice. ) p - 0.01 vs. Norm mice. qq p - 0.01 vs. Norm, and CastrrTP. ap - 0.05 vs. HpgrTP mice.
S. Apostolinas et al.r Brain Research 817 (1999) 19–24 Fig. 2. Photomicrographs of androgen receptor immunoreactivity in the mPOA of representative male mice. ŽA. Normal intact. ŽB. Normal castrate. ŽC. Normal TP-treated castrate. ŽD. Hpg intact. ŽE. TP-treated intact hpg. Arrows indicate the third ventricle. Scale bar s100 mm in all figures.
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bed nucleus of the stria terminalis ŽBNST., the lateral ventral septum, the ventromedial nucleus of the hypothalamus ŽVMH. and the medial amygdala. These five brain regions were therefore chosen for analysis. For each region the two, preferably sequential, sections that had the highest number of positive cells on screening were used. One 0.0625 mm2 field on both the left and the right sides in each of the chosen sections was examined under 400 = magnification with an Olympus visible light microscope. The mean of the four areas was taken as the value for each animal per region. Slides were coded and the person counting the cells was unaware of the treatment group. Data were analyzed with a One-way ANOVA. If p - 0.05, post-hoc comparisons were made with Fisher’s LSD test.
3. Results Apart from the five regions chosen for analysis, there was only scattered AR-IR in other areas of the brains. AR-IR was confined to neuronal nuclei. In general, castrated normal and intact hpg male mice tended to have fewer labeled neurons than intact normal mice did in each of the regions studied except for the VMH ŽFig. 1.. After TP treatment both groups exhibited an increase in the number of AR positive cells in all the regions studied although this did not always reach significance. 3.1. BNST There were significant differences in this region with fewer AR positive cells in castrate normal and intact hpg mice than the other treatment groups Ž p - 0.01.. 3.2. mPOA Group differences in AR-IR cells were due to significantly fewer cells staining for AR in castrated normal and intact hpg mice Žp - 0.01. than normal mice ŽFig. 2.. With TP treatment both groups had more AR-IR cells Ž p - 0.01 and p - 0.05, respectively.. 3.3. Lateral Õentral septum Overall significant differences among groups reflected significantly fewer AR positive cells in both castrated normal and intact hpg mice as compared to intact normal mice Ž p - 0.01.. The increased numbers of AR positive cells when both groups were treated with TP did not reach significance. 3.4. VMH Despite similar trends of fewer AR positive cells in castrated normal and intact hpg animals than in other
groups, there were no significant differences among groups in the VMH ŽANOVA, p s 0.128.. 3.5. Medial amygdala The differences among groups in the medial amygdala were due to significantly fewer AR positive cells in the brains of hpg mice than in intact or TP-treated castrated normal animals Ž p - 0.01..
4. Discussion The present study demonstrates that intact adult male hpg mice have minimal expression of androgen receptor immunoreactivity in brain regions critical for male behavior. In comparison to normal males, the numbers of AR-IR neurons were significantly lower in the BNST, the mPOA, the lateral ventral septum, and the medial amygdala. Exogenously administered testosterone increased the number of AR-IR cells in these regions to the range seen in normal intact males. In a study of AR-IR sex differences in mice w22x that focused on the POA, BNST and lateral septum, the low levels of AR-IR present in normal female mice were similarly increased by testosterone treatment. These results suggest that the deficiency in AR-IR in regions critical for male behavior in hpg mice is a consequence of their undeveloped testes, and that normalization is readily achieved in the presence of circulating testosterone. The regions in which TP treatment induced expression of androgen receptors in hpg brain are critical for the expression of male sexual behavior. Lesions of the mPOA virtually abolish copulatory behavior in vertebrate males Žreview Ref. w27x.. Although less severe in impact, BNST lesions also impair copulatory behavior w20x. Fos expression, widely used as a marker of neuronal activation, is induced in specific neuronal regions by sexual behavior in male animals. Most consistently mentioned are the POA, BNST, and medial amygdala in rat w4,30x and hamster w12x. Further, Fos expression occurs within AR-IR neurons in POA, BNST and medial amygdala in both hamster w35x and rat w14x. In the latter case, almost all Fos-IR neurons within these three regions also contain AR-IR w13x. Male sexual behavior in rats is inhibited by direct application of an androgen receptor blocker in the mPOA or VMH w25x but not in the medial amygdala. Untreated male hpg mice display virtually no male sexual behavior. However, when adult hpg males are given TP capsules, some of them may eventually exhibit vigorous mounts and intromissions w21x. In contrast to normal males, however, the hpg male mice required longer exposure to testosterone. This dissociation between rapid normalization of AR-IR Ž1 week. vs. delayed or absent behavioral responses over weeks, may be analogous to findings in male hamsters. Testosterone is equally or more effective
S. Apostolinas et al.r Brain Research 817 (1999) 19–24
in inducing the expression of AR in prepuberal as in adult hamsters w26x, but even with similar circulating androgen levels, prepuberal males show fewer mounts, intromissions and ejaculations than adult males. Similarly, Lu et al. w22x point out that despite similar AR-IR levels in male and female mice, female mice are less sensitive to androgen stimulation of aggressive behaviors. They suggest that in male and female mice there is a common regulatory mechanism for induction of AR-IR, dependent on the presence or absence of androgen. Such a mechanism appears present also in the hpg mice, despite their profound lifelong deficit in gonadal hormones. Effects of androgens on behavior, in contrast, appear to depend on factors additional to induction of AR-IR. For example, activation of AR in the neonatal period associated with ‘organization’ of the substrate for sexual behavior may induce other gene productŽs. whose presence is necessary during a subsequent period of hormonal ‘activation’. Based on the extensive overlap in the distribution of neurons containing androgen and estrogen mRNA, Simerly et al. w32x suggested possible co-expression of androgen and estrogen receptors in individual neurons. This has been borne out for the hamster w36x, and in the rat about 30% of AR-IR neurons are also positive for ER in the mPOA, BNST and the medial amygdala w13x. Depriving neonatal male rats of estrogen by administration of an aromatase inhibitor affects the distribution of estrogen receptor cells in the adult animals w2x but does not affect forebrain AR levels, which are similar in treated and control groups. The general finding of decreased numbers of AR-IR neurons in specific neural regions in castrate normal males, and increases with additional androgen treatment is consistent with previous reports of mouse w22x, rat w14,28x, ferret w18x, opossum w17x, and hamster w26x. There are interspecies differences, however. For example, in the ferret w18x, considerably fewer AR-IR cells are present in the BNST as compared to rodent. In that animal, the same androgen doses that increase the density of AR-IR in POA, lateral septum, medial amygdala and arcuate region to greater than that in normal, are insufficient to affect the density in the BNST. The guinea pig appears to respond somewhat differently. An early study in guinea pig found no effect of castration on the distribution or intensity of AR-IR in the brain, although staining was abolished in the prostate within 4 days of gonadectomy w8x. When this group subsequently evaluated cytosol and nuclear extracts of AR w9x, nuclear AR declined significantly in all regions studied after castration. In the hamster the decline in nuclear AR-IR cells observed in castrated males is accompanied by an increase in cytoplasmic AR-IR w34x, but similar to other findings in the mouse w22x we saw virtually no cytoplasmic AR-IR in any of the treatment groups. In summary this study describes for the first time the distribution and regulation of AR-IR in the brain of hpg male mice. Induction of AR-IR by exogenous androgens was comparable to that seen in normal castrate male
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mouse, and similar to findings in female mice w22x. The studies here are consistent with the hypothesis that androgen receptor immunoreactivity in the adult mouse brain is regulated by circulating androgens, and does not depend on prior exposure in the postnatal period.
Acknowledgements Supported by NIH grants NS 20335 and T32 DK 07645.
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S. Apostolinas et al.r Brain Research 817 (1999) 19–24 forebrain and midbrain structures in the male rat, Neuroscience 75 Ž1996. 161–171. A.E. Herbison, Neurochemical identity of neurones expressing oestrogen and androgen receptors in sheep hypothalamus, J. Reprod. Fertil. 103 Ž1995. 271–283, Suppl. 49. A.E. Herbison, D.C. Skinner, J.E. Robinson, I.S. King, Androgen receptor-immunoreactive cells in ram hypothalamus: distribution and co-localization patterns with gonadotropin-releasing hormone, somatostatin and tyrosine hydroxylase, Neuroendocrinology 63 Ž1996. 120–131. 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 Ž1995. 1–18. M.L. Kashon, J.A. Arbogast, C.L. Sisk, Distribution and hormonal regulation of androgen receptor immunoreactivity in the forebrain of the male European ferret, J. Comp. Neurol. 376 Ž1996. 567–586. M.L. Kashon, C.L. Sisk, Pubertal maturation is associated with an increase in the number of androgen receptor-immunoreactive cells in the brains of male ferrets, Dev. Brain Res. 78 Ž1994. 237–242. Y.C. Liu, J.D. Salamone, B.D. Sachs, Lesions in medial preoptic area and bed nucleus of stria terminalis: differential effects on copulatory behavior and noncontact erection in male rats, J. Neurosci. 17 Ž1997. 5245–5253. I. Livne, A.-J. Silverman, M.J. Gibson, Reversal of reproductive deficiency in the hpg male mouse by neonatal androgenization, Biol. Reprod. 47 Ž1992. 561–567. S.-F. Lu, S.E. McKenna, A. Cologer-Clifford, E.A. Nau, N.G. Simon, Androgen receptor in mouse brain: sex differences and similarities in autoregulation, Endocrinology 139 Ž1998. 1594–1601, Abstract. N.J. MacLusky, F. Naftolin, Sexual differentiation of the central nervous system, Science 211 Ž1981. 1294–1303. A.J. Mason, J.S. Hayflick, R.T. Zoeller, W.S. Young III, H.S. Phillips, K. Nikolics, P.H. Seeburg, A deletion truncating the gonadotropin-releasing hormone gene is responsible for hypogonadism in the hpg mouse, Science 234 Ž1986. 1366–1371. M.Y. McGinnis, G.W. Williams, A.R. Lumia, Inhibition of male sex behavior by androgen receptor blockade in preoptic area or hypothalamus, but not amygdala or septum, Physiol. Behav. 60 Ž1996. 783–789. L.R. Meek, R.D. Romeo, C.M. Novak, C.L. Sisk, Actions of testosterone in prepubertal and postpubertal male hamsters: dissocia-
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tion of effects on reproductive behavior and brain androgen receptor immunoreactivity, Horm. Behav. 31 Ž1997. 75–88. R.L. Meisel, B.D. Sachs, The physiology of male sexual behavior, in: E. Knobil, J.D. Neill ŽEds.., The Physiology of Reproduction, Raven Press, New York, 1994, pp. 3–105. C.S. Menard, R.E. Harlan, Up-regulation of androgen receptor immunoreactivity in the rat brain by androgenic–anabolic steroids, Brain Res. 622 Ž1993. 226–236. L. Puy, N.J. MacLusky, L. Becker, N. Karsan, J. Trachtenberg, T.J. Brown, Immunocytochemical detection of androgen receptor in human temporal cortex: characterization and application of polyclonal androgen receptor antibodies in frozen and paraffin-embedded tissues, J. Steroid Biochem. Mol. Biol. 55 Ž1995. 197–209. G.S. Robertson, J.G. Pfaus, L.J. Atkinson, H. Matsumura, A.G. Phillips, H.C. Fibiger, Sexual behavior increases c-fos expression in the forebrain of the male rat, Brain Res. 564 Ž1991. 352–357. M. Sar, D.B. Lubahn, F.S. French, E.M. Wilson, Immunohistochemical localization of the androgen receptor in rat and human tissues, Endocrinology 127 Ž1990. 3180–3186. 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 Ž1990. 76–95. 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 Ž1996. 1075–1080. 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 Ž1993. 925–938. R.I. Wood, S.W. Newman, Mating activates androgen receptor-containing neurons in chemosensory pathways of the male Syrian hamster brain, Brain Res. 614 Ž1993. 65–77. R.I. Wood, S.W. Newman, Androgen and estrogen receptors coexist within individual neurons in the brain of the syrian hamster, Neuroendocrinology 62 Ž1995. 487–497. S.S. Wu, P.W. Nathanielsz, T.J. McDonald, Immunocytochemical distribution of androgen receptors in the hypothalamus and pituitary of the fetal baboon in late gestation, Dev. Brain Res. 84 Ž1995. 278–281. Z.-X. Zhou, C.-I. Wong, M. Sar, E.M. Wilson, The androgen receptor: an overview, Rec. Prog. Horm. Res. 49 Ž1994. 249–274.
Research report
Androgen receptor immunoreactivity in specific neural regions in normal and hypogonadal male mice: effect of androgens Stergios Apostolinas, Gopalan Rajendren, Areta Dobrjansky, Marie J. Gibson
)
Department of Medicine, Mount Sinai School of Medicine, Box 1055, 1 GustaÕe L. LeÕy Place, New York, NY 10029, USA Accepted 27 October 1998
Abstract This study examined the distribution and regulation of androgen receptor immunoreactivity ŽIR. in the brain of the hypogonadal Žhpg. male mouse, genetically deficient in GnRH. Five groups of animals were studied: intact, castrated, or castrated and testosterone propionate ŽTP.-treated normal adult male mice, and intact or TP-treated hpg adult male mice. All groups were studied 1 week after treatment. Five regions of the brain with high concentrations of androgen receptors in normal animals were examined, including the medial preoptic area, the lateral ventral septum, the ventromedial hypothalamus, the bed nucleus of the stria terminalis and the medial amygdala. The results showed that the congenital absence of GnRH results in minimal expression of androgen receptor-IR in mice in all regions examined. However, treatment with exogenous testosterone for 1 week was sufficient to induce the numbers of neurons containing androgen receptors, as detected by immunocytochemistry, into the range seen in normal male mice in all the areas studied except the VMH. Similar plasticity was also observed in normal males after 1 week of castration and TP replacement. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Androgen receptor; Brain; Hypogonadal; Mouse; Regulation; Castration; Testosterone
1. Introduction Androgens influence the development of the brain and male behavior either directly via interaction of the steroid with its receptor, or indirectly through aromatization to estrogens w1,23x. The androgen receptor is a 120 kDa protein that belongs to the steroid hormone receptor family. It contains an N-terminal that is important in transcriptional activation, a DNA-binding domain, a hinge region and a hormone binding C-terminal. It is encoded for by a single gene on chromosome Xq11–12 and it displays high affinity for testosterone and dihydrotestosterone w38x. The androgen receptor is present in various brain regions of many different species including humans w3,7,10,11,15,16, 19,22,29,31,33,37x. Hypogonadal mice Žhpg. have a spontaneously occurring deletion in the gene for gonadotropin-releasing hormone w24x, and as a result, the reproductive system in hpg
) Corresponding author. [email protected]
Fax:
q 1-212-534-2945;
E-mail:
mice remains infantile after birth w5x. The untreated adult males are infertile and fail to show normal male sexual behavior. The purpose of the present study was to evaluate the effects of the presence or absence of testicular androgens on the distribution of androgen receptors in both normal and hpg male mice.
2. Materials and methods
2.1. Animals and housing Both hpg and normal mice of the same strain ŽC3HrHeH= 101H. were bred and housed under a partially reversed lightrdark cycle Ž14:10 L:D; lights on 2300–1300 h. in a temperature controlled Ž24–268C. room. Food and water were available ad libitum. All procedures and animal care were in accord with the standards approved by the NIH Guide for the Care and Use of Laboratory Animals and were approved by the IACUC of the Mount Sinai School of Medicine.
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 1 1 8 0 - 9
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S. Apostolinas et al.r Brain Research 817 (1999) 19–24
2.2. Experimental groups Five groups of adult Ž3–4 months old. male mice were used: normal intact mice Ž n s 8., normal castrate mice Ž n s 4., normal testosterone propionate ŽTP.-treated castrate mice Ž n s 4., intact hpg mice Ž n s 9. and TP-treated intact hpg mice Ž n s 4.. TP-treated mice were given subcutaneous implants of 2-cm Silastic tubing Ži.d. 0.04 in.; o.d. 0.085 in.; Dow-Corning, Midland, MI. containing TP, and sealed at each end with silastic adhesive. This capsule size is reported to support plasma testosterone levels of approximately 110 ngrml in male mice of the same strain w6x. Surgeries and capsule implants were performed under chloral hydrate Ž380 mgrkg, i.p.. anesthesia 1 week before the experiment. Capsules were inserted in the intrascapular region via a small incision that was closed with a wound clip.
pH 7.4.. Brains were removed, postfixed at 48C overnight in the same solution and then 50 mm coronal sections were obtained on a Vibratome. The sections were exposed to 0.1% H 2 O 2 to suppress endogenous peroxidase activity followed by incubation in 0.1 M PBS containing 1.5% normal donkey serum and 0.3% Triton-X. The sections were then incubated with a rabbit polyclonal anti-androgen receptor antibody Ž1:200, AR70, Novocastra Laboratories. for 72 h. The androgen receptor protein was visualized using a mixture of 3,3X-diaminobenzidine and 0.005% H 2 O 2 following incubation of the sections with 0.5% donkey anti-rabbit biotinylated antibody ŽJackson Immunoresearch. for 3 h and avidin biotin peroxidase solution ŽVector Laboratories. for 1 h. There was a complete lack of staining when the primary antibody was omitted from the incubations. The sections were mounted onto gelatincoated slides and cover-slipped using Permount.
2.3. Immunocytochemistry
2.4. Analysis
One week after castration andror TP treatment, the mice were deeply anesthetized and perfused with normal saline followed by 4% paraformaldehyde Žin 0.1 M PBS;
The regions where dense androgen receptor immunoreactivity ŽAR-IR. was observed in normal male brains were the following: the medial preoptic area ŽPOA., the
Fig. 1. Numbers Žmean " S.E.M.. of androgen receptor-positive ŽAR q . cells in specific brain regions in male mice, including the bed nucleus of the stria terminalis ŽBNST., medial preoptic area ŽPOA., ventromedial nucleus of the hypothalamus ŽVMH., lateral ventral septum and medial amygdala. )) p - 0.01 vs. normal intact ŽNorm., normal TP-treated castrate ŽCastrrTP., and TP-treated hpg ŽHpgrTP.mice. ) p - 0.01 vs. Norm mice. qq p - 0.01 vs. Norm, and CastrrTP. ap - 0.05 vs. HpgrTP mice.
S. Apostolinas et al.r Brain Research 817 (1999) 19–24 Fig. 2. Photomicrographs of androgen receptor immunoreactivity in the mPOA of representative male mice. ŽA. Normal intact. ŽB. Normal castrate. ŽC. Normal TP-treated castrate. ŽD. Hpg intact. ŽE. TP-treated intact hpg. Arrows indicate the third ventricle. Scale bar s100 mm in all figures.
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bed nucleus of the stria terminalis ŽBNST., the lateral ventral septum, the ventromedial nucleus of the hypothalamus ŽVMH. and the medial amygdala. These five brain regions were therefore chosen for analysis. For each region the two, preferably sequential, sections that had the highest number of positive cells on screening were used. One 0.0625 mm2 field on both the left and the right sides in each of the chosen sections was examined under 400 = magnification with an Olympus visible light microscope. The mean of the four areas was taken as the value for each animal per region. Slides were coded and the person counting the cells was unaware of the treatment group. Data were analyzed with a One-way ANOVA. If p - 0.05, post-hoc comparisons were made with Fisher’s LSD test.
3. Results Apart from the five regions chosen for analysis, there was only scattered AR-IR in other areas of the brains. AR-IR was confined to neuronal nuclei. In general, castrated normal and intact hpg male mice tended to have fewer labeled neurons than intact normal mice did in each of the regions studied except for the VMH ŽFig. 1.. After TP treatment both groups exhibited an increase in the number of AR positive cells in all the regions studied although this did not always reach significance. 3.1. BNST There were significant differences in this region with fewer AR positive cells in castrate normal and intact hpg mice than the other treatment groups Ž p - 0.01.. 3.2. mPOA Group differences in AR-IR cells were due to significantly fewer cells staining for AR in castrated normal and intact hpg mice Žp - 0.01. than normal mice ŽFig. 2.. With TP treatment both groups had more AR-IR cells Ž p - 0.01 and p - 0.05, respectively.. 3.3. Lateral Õentral septum Overall significant differences among groups reflected significantly fewer AR positive cells in both castrated normal and intact hpg mice as compared to intact normal mice Ž p - 0.01.. The increased numbers of AR positive cells when both groups were treated with TP did not reach significance. 3.4. VMH Despite similar trends of fewer AR positive cells in castrated normal and intact hpg animals than in other
groups, there were no significant differences among groups in the VMH ŽANOVA, p s 0.128.. 3.5. Medial amygdala The differences among groups in the medial amygdala were due to significantly fewer AR positive cells in the brains of hpg mice than in intact or TP-treated castrated normal animals Ž p - 0.01..
4. Discussion The present study demonstrates that intact adult male hpg mice have minimal expression of androgen receptor immunoreactivity in brain regions critical for male behavior. In comparison to normal males, the numbers of AR-IR neurons were significantly lower in the BNST, the mPOA, the lateral ventral septum, and the medial amygdala. Exogenously administered testosterone increased the number of AR-IR cells in these regions to the range seen in normal intact males. In a study of AR-IR sex differences in mice w22x that focused on the POA, BNST and lateral septum, the low levels of AR-IR present in normal female mice were similarly increased by testosterone treatment. These results suggest that the deficiency in AR-IR in regions critical for male behavior in hpg mice is a consequence of their undeveloped testes, and that normalization is readily achieved in the presence of circulating testosterone. The regions in which TP treatment induced expression of androgen receptors in hpg brain are critical for the expression of male sexual behavior. Lesions of the mPOA virtually abolish copulatory behavior in vertebrate males Žreview Ref. w27x.. Although less severe in impact, BNST lesions also impair copulatory behavior w20x. Fos expression, widely used as a marker of neuronal activation, is induced in specific neuronal regions by sexual behavior in male animals. Most consistently mentioned are the POA, BNST, and medial amygdala in rat w4,30x and hamster w12x. Further, Fos expression occurs within AR-IR neurons in POA, BNST and medial amygdala in both hamster w35x and rat w14x. In the latter case, almost all Fos-IR neurons within these three regions also contain AR-IR w13x. Male sexual behavior in rats is inhibited by direct application of an androgen receptor blocker in the mPOA or VMH w25x but not in the medial amygdala. Untreated male hpg mice display virtually no male sexual behavior. However, when adult hpg males are given TP capsules, some of them may eventually exhibit vigorous mounts and intromissions w21x. In contrast to normal males, however, the hpg male mice required longer exposure to testosterone. This dissociation between rapid normalization of AR-IR Ž1 week. vs. delayed or absent behavioral responses over weeks, may be analogous to findings in male hamsters. Testosterone is equally or more effective
S. Apostolinas et al.r Brain Research 817 (1999) 19–24
in inducing the expression of AR in prepuberal as in adult hamsters w26x, but even with similar circulating androgen levels, prepuberal males show fewer mounts, intromissions and ejaculations than adult males. Similarly, Lu et al. w22x point out that despite similar AR-IR levels in male and female mice, female mice are less sensitive to androgen stimulation of aggressive behaviors. They suggest that in male and female mice there is a common regulatory mechanism for induction of AR-IR, dependent on the presence or absence of androgen. Such a mechanism appears present also in the hpg mice, despite their profound lifelong deficit in gonadal hormones. Effects of androgens on behavior, in contrast, appear to depend on factors additional to induction of AR-IR. For example, activation of AR in the neonatal period associated with ‘organization’ of the substrate for sexual behavior may induce other gene productŽs. whose presence is necessary during a subsequent period of hormonal ‘activation’. Based on the extensive overlap in the distribution of neurons containing androgen and estrogen mRNA, Simerly et al. w32x suggested possible co-expression of androgen and estrogen receptors in individual neurons. This has been borne out for the hamster w36x, and in the rat about 30% of AR-IR neurons are also positive for ER in the mPOA, BNST and the medial amygdala w13x. Depriving neonatal male rats of estrogen by administration of an aromatase inhibitor affects the distribution of estrogen receptor cells in the adult animals w2x but does not affect forebrain AR levels, which are similar in treated and control groups. The general finding of decreased numbers of AR-IR neurons in specific neural regions in castrate normal males, and increases with additional androgen treatment is consistent with previous reports of mouse w22x, rat w14,28x, ferret w18x, opossum w17x, and hamster w26x. There are interspecies differences, however. For example, in the ferret w18x, considerably fewer AR-IR cells are present in the BNST as compared to rodent. In that animal, the same androgen doses that increase the density of AR-IR in POA, lateral septum, medial amygdala and arcuate region to greater than that in normal, are insufficient to affect the density in the BNST. The guinea pig appears to respond somewhat differently. An early study in guinea pig found no effect of castration on the distribution or intensity of AR-IR in the brain, although staining was abolished in the prostate within 4 days of gonadectomy w8x. When this group subsequently evaluated cytosol and nuclear extracts of AR w9x, nuclear AR declined significantly in all regions studied after castration. In the hamster the decline in nuclear AR-IR cells observed in castrated males is accompanied by an increase in cytoplasmic AR-IR w34x, but similar to other findings in the mouse w22x we saw virtually no cytoplasmic AR-IR in any of the treatment groups. In summary this study describes for the first time the distribution and regulation of AR-IR in the brain of hpg male mice. Induction of AR-IR by exogenous androgens was comparable to that seen in normal castrate male
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mouse, and similar to findings in female mice w22x. The studies here are consistent with the hypothesis that androgen receptor immunoreactivity in the adult mouse brain is regulated by circulating androgens, and does not depend on prior exposure in the postnatal period.
Acknowledgements Supported by NIH grants NS 20335 and T32 DK 07645.
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