Andrew N. Clancy

Immunohistochemical labeling of androgen receptors in the brain of rat and monkey

Androgen receptor antibodies have recently been developed using fusion proteins containing fragments of human prostatic androgen receptor. We have used a polyclonal antibody raised in rabbits to label androgen receptors in brain sections from male and female rats and monkeys. Free-floating frozen sections were incubated in primary antibody, and processed by the peroxidase-avidin-biotin complex method using biotinylated anti-rabbit IgG. Nickel intensified diaminobenzidine was used as the chromagen, and neurons were labeled in the amygdala, hippocampus, bed nucleus of stria terminalis, septum, preoptic area, in several hypothalamic nuclei including the supraoptic and paraventricular nuclei, in several brain stem motor nuclei and in cerebral cortex. Staining was most intense in cell nuclei but also occurred in cytoplasm and in some neuronal processes. Labeling was more restricted in monkey than in rat brain. Omitting the primary antibody or pre-incubating the primary antibody with rat prostatic cytosol for control purposes demonstrated the specificity of staining.

Androgen Receptors and Estrogen Receptors Are Colocalized in Male Rat Hypothalamic and Limbic Neurons that Express Fos Immunoreactivity Induced by Mating

Conversion of testosterone into estradiol is important for male rat sexual behavior, and both steroids probably contribute to mating. The distributions of neurons containing androgen receptors (AR) and estrogen receptors (ER) overlap, and many AR-immunoreactive (AR-ir) neurons express Fos immunoreactivity (Fos-ir) induced by mating. Because mating-induced Fos-ir in the male rat occurs mainly in AR-ir neurons, and because both steroids are important for mating, we hypothesized that (i) AR-ir and ER-ir are colocalized and that (ii) some of these neurons are activated during mating. We examined, in adjacent sections from the medial preoptic area (MPN) through the central tegmental field (CTF), the expression of ER-ir in: (i) AR-ir-containing neurons, and (ii) Fos-ir-expressive neurons. PG21 anti-AR, OA-11-824 anti-c-fos, H222 or 1D5 anti-ER primary antibodies were visualized, respectively, with cyanine-conjugated, fluorescein- or cyanine-conjugated, and fluorescein-conjugated secondary antibodies in male rats which were killed 1 h after ejaculating with a receptive female. In MPN, bed nucleus of the stria terminalis (BNST), and medial amygdala (MEA), 80–90% of ER-ir labeling occurred in AR-ir-positive neurons but only about 30% of AR-ir neurons were ER-ir-positive. No ER-ir was found in the CTF. This suggests the presence of three types of brain neurons sensitive to gonadal steroid hormones: neurons sensitive to androgens only, neurons sensitive to both androgens and estrogens, and neurons sensitive to estrogens only. About 50% of ER-ir labeling occurred in cells expressing mating-induced Fos-ir but only about 30% of Fos-ir neurons were ER-ir-positive. These findings suggest that, in the MPN, at least two different neuronal populations are activated during mating: the first contains AR-ir only and the second contains AR-ir and ER-ir. In the BNST and MEA, at least three hormonally sensitive populations are activated during mating: the two described above plus a third population which expresses ER-ir only.

Intracerebral Infusion of an Aromatase Inhibitor, Sexual Behavior and Brain Estrogen Receptor-Like Immunoreactivity in Intact Male Rats

Copulatory behavior was studied in five groups of sexually experienced, gonadally intact male rats in which: (i) the nonsteroidal aromatase inhibitor Fadrozole (CIBA-Geigy CGS 16949A) was delivered bilaterally (0.756 microgram in 6.0 microliters saline in 24 h to each side) into the medial preoptic area (POM) together with normal saline given s.c. via osmostic minipumps (n = 10); (ii) normal saline was delivered bilaterally into POM together with the same dose of Fadrozole s.c. (n = 9); (iii) Fadrozole was delivered bilaterally into the lateral preoptic area together with normal saline given s.c. (n = 6); (iv) Fadrozole was delivered bilaterally into the cerebral cortex together with normal saline given s.c. (n = 6), and (v) unoperated controls (n = 14). Mounting and ejaculation were significantly decreased in rats receiving Fadrozole in POM compared with the behavior of rats in the other 4 groups. Few differences occurred between rats in the latter 4 groups, all of which continued to mate. The H222 and ER-715 anti-estrogen receptor (ER) antibodies were used to examine the distribution of ER immunoreactive (ERir) neurons in hypothalamic and limbic sites in gonadectomized controls and in some of the rats in groups i, ii and v. Since labeling of ERir neurons in rat brain with the H222 anti-ER antibody is reported to be inhibited by estrogen, it was used here to identify regions (with staining) where the aromatization of testosterone (T) into estradiol (E2) had been suppressed. Intense H222 ERir nuclear neuronal labeling was confined to the POM of males receiving Fadrozole in POM, and significantly more labeled neurons were found in the POM of these rats than in the POM of rats treated with saline in POM. In contrast, the ER-715 antibody, which is reported to stain neurons independently of hormonal status, labeled neuronal nuclei in hypothalamic and limbic regions of all groups, demonstrating the presence of ER. These findings show that conversion of T into E2 in the POM of the male rat is important for male rat copulatory behavior and that H222 ERir nuclear neuronal labeling can be used to identify the neurons in POM that were affected by Fadrozole.

Fos Induced by Mating or Noncontact Sociosexual Interaction Is Colocalized with Androgen Receptors in Neurons within the Forebrain, Midbrain, and Lumbosacral Spinal Cord of Male Rats☆

This study was designed to determine the extent to which Fos immunoreactivity (induced either by mating or noncontact sociosexual interaction) and androgen receptor (AR) immunoreactivity are colocalized in brain and spinal cord of male rats. Some males (Mated) were allowed to mate to ejaculation; others (Social Controls) were placed with females but physical contact was prevented by a wire mesh screen; remaining males (Isolated) were placed alone in the test jar for the duration of the test period. After testing, brains and spinal cords were examined for AR and Fos immunoreactivity (ir). PG21 anti-AR and anti-c-fos primary antibodies were visualized by fluorescence microscopy using cyanine-conjugated and fluorescein-conjugated secondary antibodies. In both brain and spinal cord, the number of Fos-ir neurons varied according to group: Mated males > Social Controls > Isolated males. Fos was highly localized in subsets of AR-ir neurons within the medial preoptic nucleus, bed nucleus of the stria terminalis, dorsomedial nucleus of the amygdala, and central tegmental field. Fos was also localized in subsets of AR-ir neurons within the L5, L6, and S1 segments of the spinal cord. Spinal cord concentrations of AR-ir and Fos-ir neurons were greatest in Lamina X, and the vast majority of Fos-ir neurons in the dorsal part of Lamina X were also AR-ir. Thus, in both brain and spinal cord, androgen-sensitive neurons are active during mating, and transmission of sexually relevant information from cord to brain is probably accomplished via hormone-sensitive spinal neurons.

Androgen receptor immunoreactivity and mating-induced Fos expression in forebrain and midbrain structures in the male rat

The distribution of androgen receptor immunoreactive-neurons, mapped with the PG21 anti-androgen receptor antibody, was compared in male rat brains with the distribution of Fos-immunoreactive neurons induced by mating. In gonadally intact, but not in castrated male rats, substantial numbers of androgen receptor-containing neurons were present in a variety of forebrain and midbrain regions. The PG21 antibody apparently had a higher affinity for occupied than for non-occupied androgen receptors. Androgen receptor-immunoreactive regions included the medial preoptic area and other forebrain areas previously identified as containing androgen receptors, the dorsal and ventral periaqueductal gray, and a midbrain region that included the lateral part of the central tegmental field, part of the caudal zona incerta, the subparafascicular nucleus of the thalamus and the peripeduncular nucleus. Fos-expressive neurons were essentially absent in non-mated males but were present in the brains of rats which mated to ejaculation. All brain regions in which androgen receptor-immunoreactive neurons were counted also expressed Fos immunoreactivity after mating, and there was considerable overlap between the distributions of androgen receptor- and Fos-immunoreactive neurons. In a second experiment, we used immunofluorescent techniques to document the intraneuronal co-localization of Fos with androgen receptor immunoreactivity in the medial preoptic area, medial amygdala, and central tegmental field. In these regions mating-induced Fos immunofluorescence was exclusively localized in androgen receptor-immunofluorescent neurons. However, not all androgen receptor neurons were Fos expressive, suggesting that only some androgen-sensitive neurons were activated during mating. These results are consonant with the view that hormone actions on forebrain and midbrain structures influence the neuronal activity correlated with mating.

Effects of the nonsteroidal aromatase inhibitor, Fadrozole, on sexual behavior in male rats

The new nonsteroidal aromatase inhibitor, Fadrozole (CGS 16949A, CIBA-Geigy Corp.), was tested for its ability (i) to inhibit the conversion of testosterone (T) to estradiol (E2) in brain and (ii) to suppress male sexual activity. Sprague-Dawley rats were castrated and immediately given sc Silastic T-implants and osmotic minipumps delivering 2.5 mg/kg/day Fadrozole (N = 4), 0.25 mg/kg/day Fadrozole (N = 4), or water (N = 4 controls). T-implants were removed after 6 days and, 3 days later, 3H-T (1 microCi/g) was given as an iv bolus. No 3H-E2 was detected in hypothalamic or amygdaloid nuclear pellets from Fadrozole-treated males but this metabolite predominated in controls. However, nuclear concentrations of 3H-T and [3H]dihydrotestosterone were similar in all groups. In another group of males (N = 18), brain aromatase activity was reduced by more than 96% at the 0.25 mg/kg dose level. Additional castrated, T-implanted males received minipumps delivering 0.25 mg/kg/day Fadrozole (six males) or water (six behaviorally matched controls) and were tested weekly with receptive females. After 2 weeks, ejaculations were reduced by 77% compared with controls (P less than 0.01) and, after 4 weeks, intromissions were also significantly reduced (P less than 0.05) but less so (48%). Radioenzymatic estimates of plasma aromatase inhibitor levels remained elevated throughout Fadrozole treatment. These males were then given Silastic E2 implants: intromissions increased significantly in 1 week (P less than 0.01), but ejaculations remained below control values. Results supported the view that aromatization is important for sexual behavior in male rats and suggested that Fadrozole has utility for studying the mechanisms by which testosterone affects behavior.

Androgen receptor and mating-induced Fos immunoreactivity are co-localized in limbic and midbrain neurons that project to the male rat medial preoptic area

Two studies were designed to document neuronal colocalization of androgen receptor immunoreactivity and mating-induced Fos immunoreactivity (AR-ir, Fos-ir) in brain of male rats and to examine the extent to which limbic and midbrain neurons that project to the preoptic area are androgen sensitive and activated by mating. Brains from male rats, killed 1 h after ejaculating with receptive females, were examined for Fos-ir and AR-ir and compared with those from control rats not given access to females. PG21 anti-AR and anti-c-fos primary antibodies were visualized by fluorescence microscopy using cyanine-conjugated and fluorescein-conjugated secondary antibodies. In mated males (Expt. 1), Fos-ir and AR-ir were colocalized in neurons of the medial preoptic nucleus (MPN), the dorsal medial amygdala (dMEA), the central tegmental field (CTF), the bed nucleus of the stria terminalis, the anterior hypothalamus, the lateral hypothalamus, and the ventral premamillary nucleus. In Expt. 2, male rats received a unilateral injection of the retrograde tracer FluoroGold (FG) in the preoptic area and four days later were killed after ejaculating with receptive females. Brains were subsequently examined for FG transport, Fos-ir and AR-ir. Fluorogold-containing neurons were present in dMEA and CTF as well as in other hypothalamic and limbic regions known to project to the MPN. In dMEA and CTF, nuclear colocalization of AR-ir and mating-induced Fos-ir was present in a proportion of FG-containing neurons. Sexually relevant information may be carried through the brain by an interconnected network of hormone-sensitive neurons.

Estrogen in the medial preoptic area of male rats facilitates copulatory behavior.

Mating was studied in sexually experienced, gonadally intact male rats assigned to two surgical groups matched on the basis of mean mounting frequency during behavioral screening trials conducted prior to the study. Estradiol (E(2)) was delivered bilaterally into the medial preoptic area (MPO) of experimental males by means of hormone-coated implants, and fadrozole was given sc (0.25 mg/kg/day) via osmotic minipumps to block E(2) formation from testicular testosterone throughout the brain. Control males received blank bilateral implants in the MPO and sc fadrozole. After the completion of behavioral testing, immunocytochemical comparisons of the brains from experimental and control rats were made using the H222 antiestrogen receptor (ER) antibody, whose labeling is inhibited by the presence of E(2). The histology demonstrated that E(2) was confined exclusively to the MPO of experimental males but was absent throughout the brains of controls. In controls, mounting decreased significantly by the 7th day after surgery compared with presurgical levels and did not recover. In contrast, on all postsurgical days, the mounting frequency of the experimental group was significantly higher than that of controls. Although experimental males also showed an initial, significant postsurgical decline in mounting frequency, it recovered completely by the 28th postoperative day. Ejaculations declined significantly after surgery in both groups but, unlike in controls whose performance remained low, ejaculations in experimental males partially recovered and were significantly higher than in controls during the postoperative period. Results showed that ER-containing neurons in the MPO influence male rat copulatory behavior.

Estradiol in the male rat amygdala facilitates mounting but not ejaculation

Mating activates estrogen sensitive neurons in several regions of male rat brain, including the medial amygdala (MEA). Infusion of the aromatase inhibitor, Fadrozole, into the MEA reduced mating, presumably by inhibiting conversion of testosterone (T) to estradiol (E(2)). We investigated whether administering E(2) locally into the amygdala (AMG) would maintain sexual behavior in male rats given systemic Fadrozole to eliminate E(2) elsewhere in the brain. Gonadally intact male rats were divided into two matched groups, based on ejaculatory performance in weekly tests with receptive females. All males received 0.29 mg/kg/day sc Fadrozole and bilateral implants to AMG. E(2)-in-AMG males (N=6 experimentals) received implants tipped with a cured mixture of E(2) in Silastic Medical Adhesive, whereas Vehicle-in-AMG males (N=6 controls) received implants tipped with cured adhesive alone (without E(2)). In E(2)-in-AMG males, postoperative mount and intromission frequency did not differ significantly from pretreatment baseline levels, but ejaculation frequency declined significantly (P<.01). Conversely, in Vehicle-in-AMG males, postoperative mounts and intromissions (P<.01) and ejaculations (P<.01) declined significantly. Postoperative mount and intromission frequency of Vehicle-in-AMG males was significantly lower than that of E(2)-in-AMG males (P<.01), but ejaculation frequency did not differ significantly between groups. This suggests that E(2)-sensitive AMG neurons are important for sexual arousal but not ejaculatory performance.

Distribution of androgen receptor-like immunoreactivity in the brains of intact and castrated male hamsters

The distribution of androgen receptor-like (AR) immunoreactivity was mapped in brains of (a) intact, sham-castrated and (b) castrated male hamsters. The pattern of AR-immunoreactive (AR-ir) staining was, in general, similar to that reported for gonadal steroid autoradiography of the male hamster brain. Moreover, with one exception, AR-like staining was similar in intact and castrated males, and occurred in the medial preoptic area, bed nucleus of stria terminalis, amygdala, hippocampus, thalamus, and several hypothalamic nuclei including the periventricular, supraoptic, and ventromedial nuclei, and median eminence. However, while AR-ir labeling was virtually absent in the lateral septum of intact males, it was clearly present in the lateral septum of castrated males. The view that androgen receptors in brain generally decline after castration received no support from this study.