The quantitative distribution of cytosolic androgen receptors in microdissected areas of the male rat brain: effects of estrogen treatment

Estrogen and androgen synergize in the regulation of various neuroendocrine functions. To determine a potential cellular basis of this synergism, we measured androgen receptor (AR) in the cytosol of 16 hypothalamic and limbic nuclei and subregions in castrated male rats and castrated rats treated with estradiol. Androgen receptor was measured by a previously validated in vitro binding assay using the synthetic androgen methyltrienolone [( 3H]R1881). Male Sprague-Dawley rats (250-350 g) were castrated 2 weeks before the implantation of a 2.5-cm Silastic capsule filled with crystalline 17 beta-estradiol. Control rats were sham implanted. Estrogen treatment lasted for 1 week, after which time the animals were killed, their brains were frozen and sectioned, and individual nuclei and subregions were removed by a tissue punch technique. Tissue from six rats were combined for each determination. The highest levels of AR were found in the ventromedial nucleus (16.5 +/- 1.4 fmol/mg protein), medial preoptic area (12.1 +/- 1.4 fmol/mg protein), bed nucleus of the stria terminalis (11.6 +/- 1.4 fmol/mg protein), lateral septum (11.4 +/- 1.4 fmol/mg protein), arcuate nucleus-median eminence (10.9 +/- 2.1 fmol/mg protein), and medial amygdala (10.3 +/- 0.9 fmol/mg protein). Estrogen treatment resulted in significant increases in AR in medial preoptic area (14.8 +/- 0.6 fmol/mg protein; P less than 0.05) and medial amygdala (14.6 +/- 1.2 fmol/mg protein; P less than 0.02). Subsequent studies using block-dissected hypothalamus-preoptic area, anterior pituitary, and prostate revealed significant estrogen-mediated elevations in AR in anterior pituitary cytosol [42.2 +/- 3.0 vs. 26.4 +/- 1.6 fmol/mg protein (control); P less than 0.01], but not in hypothalamus-preoptic area or prostate cytosols. Estrogen treatment had no effect on AR affinity. The binding of [3H]R1881 was specific for AR and was not affected by the addition of radioinert progesterone to the incubation tube. Estimates of AR concentration were similar regardless of whether [3H]R1881 or [3H]dihydrotestosterone was used as the ligand. In this study, we describe the distribution of AR throughout the hypothalamus and limbic areas using biochemical techniques. In addition, we have identified some cellular events that may mediate the synergistic actions of estrogen and androgen on the neuroendocrine system.     

Sex differences in androgen receptors and aromatase activity in microdissected regions of the rat brain

Males are generally more responsive than females to the behavioral and neuroendocrine actions of androgens. The present experiments were performed to determine whether these differences may result from sex differences in the number of androgen receptors (AR) in specific brain areas. For this reason, AR binding was compared in both cytosol (ARc) and cell nuclear KCl extracts (ARn) from microdissected brain regions of gonadectomized male and female rats treated with doses of testosterone (T) that produced equivalent physiological circulating androgen levels. In addition, microsomal aromatase activity was measured as a biochemical index of tissue responsiveness to T, since estrogen formation in certain brain areas is regulated by androgen. One week after exogenous T administration, males exhibited significantly higher levels of ARn than females in the bed nucleus of the stria terminalis, periventricular preoptic area, and ventromedial nucleus. Males also had significantly higher aromatase levels in these same areas plus the medial preoptic nucleus and anterior hypothalamus. There were no significant differences in ARn concentrations in eight other nuclei that were examined for significant sex differences in ARc levels observed under these experimental conditions. When ARc levels were compared in untreated gonadectomized male and female rats, males had greater levels of ARc in the bed nucleus of the stria terminalis only, indicating that new receptor synthesis may be responsible for the sex differences observed in T-treated rats. These results suggest that sex differences in neural responsiveness to androgens may be due in part to sex differences in ARn occupation in specific brain regions.     

Oxytocin content of microdissected areas of rat hypothalamus.

Oxytocin content has been measured by radioimmunoassay in microdissected hypothalamic nuclei. Equal concentrations of oxytocin were found in the supraoptic and the paraventricular nuclei, indicating that both are major sources of the hormone. The concentration of oxytocin in the median eminence was more than three times that in either the supraoptic or the paraventricular nuclei, and significant amounts of oxytocin were also found in the arcuate nucleus and in tow anterior hypothalamic nuclei.     

Regional distribution of nuclear T3 receptors in rat brain and evidence for preferential localization in neurons

We examined the distribution of nuclear T3 in mature rat brain with the aim of determining specific targets of thyroid hormones within this tissue. Saturation experiments, performed in 9 different structures of the brain and in 4 parts of the cortex, revealed the presence of a single class of binding sites with a mean Ka of 0.53 X 10(10) M-1. The highest concentrations of receptors were found in the amygdala (0.523 +/- 0.025 ng T3/mg DNA, Mean +/- SE) and the hippocampus (0.438 +/- 0.071 ng T3/mg DNA) while the lowest were in the brain stem (0.058 +/- 0.003 ng T3/mg DNA) and the cerebellum (0.079 +/- 0.026 ng T3/ml DNA). The receptor was not uniformally distributed within the cerebral cortex, its concentration being relatively high in the central sections and intermediate in the remaining portions. The cell type distribution of the T3 receptor was studied by separating glial and neuronal nuclei on a discontinuous sucrose gradient. There was no detectable specific T3 binding in the fraction of oligodendrocyte nuclei (approximately 95% pure). Conversely, the neuron-enriched fraction (approximately 60%) showed a significant increase in receptor concentration compared to total nuclei (35-40% neurons): 0.857 +/- 0.196 vs 0.511 +/- 0.095 ng T3/mg DNA (p less than 0.01) in the cortex and 0.425 +/- 0.018 vs 0.234 +/- 0.24 ng T3/mg DNA (p less than 0.01) in the forebrain. The absence of nuclear T3 receptors in oligodendrocytes may have important implications on the mechanism of action of thyroid hormone in myelination.     

Age-related changes in neurotensin content and receptors in various rat brain areas

Neurotensin immunoreactivity (NT-IR) is significantly reduced in striatum, nucleus accumbens and hippocampus but not in frontal cortex, hypothalamus and septum of 24-month-old male Sprague Dawley rats. Neurotensin binding in cortex, striatum, hypothalamus and hippocampus shows a rather uniform decline with age. The changes in NT-IR content in striatum and nucleus accumbens are of particular interest in view of the existence of a functional relationship between dopamine and neurotensin.     

Analysis of activated androgen receptors in rat brain and anterior pituitary and ventral prostate glands: nuclear binding and RNA polymerase activity

Cytosolic androgen receptors from neocortex, hypothalamus and anterior pituitary and ventral prostate glands were analysed by miniature isoelectric focusing and sucrose density-gradient centrifugation before and after precipitation of [3H]dihydrotestosterone (DHT)-bound complexes with ammonium sulphate. In the hypothalamus and neocortex (NH4)2SO4 precipitation appeared to cause heterodisperse peaks, and in the case of the ventral prostate there was a clear shift to a more basic isoelectric point. After sucrose density-gradient centrifugation all cytosols sedimented as large aggregates which appeared to dissociate into subunits in 0.4 M-KCl gradients. The functional significance of these altered forms was tested by nuclear uptake studies of cytosolic [3H]DHT-bound complexes, which could only be detected in brain and pituitary nuclei after prior precipitation with (NH4)2SO4, which also significantly increased extraction of ventral prostate [3H]DHT-bound complexes from the nucleus. The nuclei apparently responded to the (NH4)2SO4-precipitated and redissolved complexes by increased RNA polymerase activity. These results are consistent with the possibility that the neural androgen receptor is altered before interaction with the genome, and this alteration may be necessary for the action of the hormone to be expressed.     

Association of glucocorticoid receptors with prostate nuclear sites for androgen receptors and with androgen response elements

Ventral prostate glands of intact normal rats contained low levels (2500 molecules/cell) of high-affinity (dissociation constant (Kd) 0.57 nmol/l) glucocorticoid receptors (GR). Levels of GR increased 2.8-fold 1 day after castration, and 4.3-fold 3 days after castration. Nuclear GR increased from a normal value of 1150 molecules/nucleus to 5200 molecules/nucleus 3 days after castration. The greater increase in intranuclear GR was in that associated with oligomeric chromatin. Although nuclear GR never approached the normal population of nuclear androgen receptors (AR; approximately 16000 molecules/nucleus), the selective rise in chromatin-associated receptors ensured that almost 60% of chromatin sites remained occupied. GR associated with prostate nuclear structures in a similar manner to AR, and exogenous GR bound saturably and with high affinity (Kd 100 pmol/l) to a similar number of sites as did AR. Both steroid receptors apparently competed for the same sites. In DNA-cellulose competition analyses, synthetic oligonucleotides containing glucocorticoid response elements or putative androgen response elements competed similarly against immobilized non-specific DNA for both AR and GR. In view of these data and information from other sources, it is probable that the role of GR in the prostate should be assessed with a view to understanding its action under conditions of androgen deprivation.     

Corticosteroid receptors and glucocorticoid content in microdissected brain regions: correlative aspects

Stereoselective competition was used to determine (3H)-aldosterone binding to type I corticosteroid receptors, and (3H)-dexamethasone binding to type II receptors in punches obtained from 11 brain regions of short-term adrenalectomized (ADX) rats. It was observed that type I receptor binding was almost exclusive of the hippocampus (HIPPO), while type II receptor binding was more generally distributed among HIPPO, cerebral cortex, lateral septum, ventromedial and arcuate hypothalamic nuclei, with lower levels in 6 additional regions studies. We determined corticosterone (CORT) in brain punches from ADX rats, ADX rats receiving CORT for 5 days, intact rats and intact rats receiving ACTH for 5 days. We correlated (3H)-ligand binding with CORT content in punches obtained from identical brain regions and showed a significant positive correlation in the case of the ADX plus CORT group, for type II corticosteroid receptors. Similarly, a significant correlation emerged with type II sites, when binding capacity was correlated with percentage increases of CORT in brain areas of rats receiving ACTH. It is suggested that in situations where CORT levels are elevated, changes in CORT retention throughout the brain occur as a function of the type II glucocorticoid receptor, although at the level of the HIPPO, both receptors may provide appropriate control of the CNS-pituitary-adrenal axis, according to the physiological or stress levels of circulating hormone.     

Intranuclear distribution of androgen receptors in human prostate carcinoma

Androgen receptors in nuclei from human prostate carcinomas were characterized on the basis of their solubilization by, or resistance to, micrococcal nuclease. By this means, androgen receptors were assigned to three nuclear categories: those associated with nuclease-resistant structures, those associated with chromatin and those apparently uncommitted by association with either of these. Prostate carcinoma nuclei contained high concentrations (57-82% of total nuclear content) of nuclease-resistant androgen receptors. This was a different pattern from that observed previously for benign hypertrophic prostate epithelial nuclei which contained a variable high proportion of uncommitted androgen receptors. The differences could not be attributed to differential losses to cytosol, or to loss of functionality, as determined in vitro. The differences in distribution could reflect different responses of diseased cells to androgens, or the intervention of other factors more relevant to the disease process.     

Variation of cytoplasmic rat uterine androgen receptors

The number of testosterone binding sites present in rat uterine cytosol varied regularly during the estrous cycle, reaching a trough at metestrus and a peak at proestrus. Treating ovariectomized and adrenalectomized rats for 2 days with estradiol resulted in a 3-4-fold increase in the number of binding sites per uterus. Estradiol withdrawal induced a decrease in uterine androgen receptors. Testosterone or progesterone treatment also increased the number of these sites, but to a lesser degree. When administered together with estradiol, testosterone did not enhance the stimulatory effect of the latter, whereas progesterone even reduced it. Testosterone or progesterone did not prevent the number of receptors from declining after estradiol withdrawal. Thus the changes in the number of cytoplasmic androgen receptors in the uterus during the rat estrous cycle is mainly controlled by the rise and fall of the serum levels of estradiol.     

Differential distribution of androgen and estrogen receptors in rat pituitary cell populations separated by centrifugal elutriation

The distribution of 5 alpha-dihydrotestosterone (DHT)-binding sites within specific pituitary cell populations, as compared with the estradiol (E2)-binding sites distribution, could be estimated after gonadotrope cell enrichment. Centrifugal elutriation allowed the purification of large gonadotropes from 42-day-old male rat pituitaries; this population contained 5.3 +/- 1.0% of the cells, 54 +/- 3% of the LH, and less than 5% of PRL. A gonadotrope-depleted fraction was also defined that contained more than 92% of the total PRL and was thus called the lactotrope population though it still contained small gonadotropes and most somatotropes. DHT and E2 receptors were quantified by multidose saturation analysis. The association constants (Ka) ranged from 0.2-0.5 X 10(9) M-1 both for E2 and DHT independent of the cell population. The binding of DHT was mainly observed in the gonadotropes (50-100 fmol/10(6) cells), while it was very low in the other cells (0.9-3 fmol/10(6) cells). E2-binding sites measured within the same cell populations showed a broader distribution than DHT-binding sites. Whereas the ratio of binding DHT/E2 averaged 0.86 +/- 0.01 in the gonadotropes, it reached only 0.35 +/- 0.07 in the lactotropes. Thus, using enriched cell populations, our results demonstrate for the first time a quantitative selective distribution of androgen-binding sites as compared with the estrogen sites.     

Localization of androgen receptors and estrogen receptors in the same cells of the songbird brain.

Estrogens and androgens each have unique effects but act together for the neural differentiation and control of sexual behaviors in male vertebrates, such as the canary. The neuronal basis for these synergistic effects is elusive because the spatial relation between estrogen target cells and androgen target cells is unknown. This study localized estrogen receptor (ER)-containing cells by using immunocytochemistry and androgen receptor (AR)-containing cells by using autoradiography in the same sections of the male canary brain. Three cell types, those containing only ER, those containing only AR, and those containing both ER and AR, were found in tissue-specific frequencies. The midbrain nucleus intercollicularis exhibited the highest number of cells expressing both ER and AR, whereas ER and AR are expressed only in disjunctive cell populations in the forebrain nucleus hyperstriatalis ventrale, pars caudale. Synergistic effects of androgens and estrogens for the neural behavorial control could result from cells containing both ER and AR (intracellular) and from neural circuits containing ER and AR in different cells (intercellular).     

Relationship between sexual satiety and brain androgen receptors

Recently we showed that 24 h after copulation to satiety, there is a reduction in androgen receptor density (ARd) in the medial preoptic area (MPOA) and in the ventromedial hypothalamic nucleus (VMH), but not in the bed nucleus of the stria terminalis (BST). The present study was designed to analyze whether the ARd changes in these and other brain areas, such as the medial amygdala (MeA) and lateral septum, ventral part (LSV), were associated with changes in sexual behavior following sexual satiety. Males rats were sacrificed 48 h, 72 h or 7 days after sexual satiety (4 h ad libitum copulation) to determine ARd by immunocytochemistry; additionally, testosterone serum levels were measured in independent groups sacrificed at the same intervals. In another experiment, males were tested for recovery of sexual behavior 48 h, 72 h or 7 days after sexual satiety. The results showed that 48 h after sexual satiety 30% of the males displayed a single ejaculation and the remaining 70% showed a complete inhibition of sexual behavior. This reduction in sexual behavior was accompanied by an ARd decrease exclusively in the MPOA-medial part (MPOM). Seventy-two hours after sexual satiety there was a recovery of sexual activity accompanied by an increase in ARd to control levels in the MPOM and an overexpression of ARd in the LSV, BST, VMH and MeA. Serum testosterone levels were unmodified during the post-satiety period. The results are discussed on the basis of the similarities and discrepancies between ARd in specific brain areas and male sexual behavior.     

Age-related changes in the diurnal rhythm of serotonin turnover in microdissected brain areas of estradiol-treated ovariectomized rats

The purpose of this study was to determine whether 1) a diurnal rhythm in serotonin turnover is present in specific hypothalamic nuclei of middle-aged ovariectomized rats and 2) in middle-aged animals exposure to estrogen can induce the pattern of serotonin dynamics which appears necessary for the occurrence of an LH surge in young rats. Young (3-4 month old) and middle-aged (8-10 month old) rats which demonstrated estrous cyclicity were bilaterally ovariectomized. Seven days later half of the young and middle-aged animals received Silastic estradiol capsules. On day 9 post ovariectomy the groups were again divided; half of the rats in each group were killed at 0800, 1200, 1800, and 2400 h. Remaining animals were treated with pargyline (75 mg/kg BW, ip) at these times and were killed 10 min later, and the following brain areas were microdissected and analyzed for serotonin (5HT) and 5-hydroxyindole acetic acid: median eminence (ME), suprachiasmatic nucleus (SCN), medial preoptic nucleus (MPN), arcuate nucleus (AN), and globus pallidus. In young ovariectomized rats the SCN, MPN, and AN exhibited a diurnal rhythm in 5HT activity which was high during the light hours and low during the dark. The diurnal rhythm could not be detected in any hypothalamic nuclei of ovariectomized middle-aged animals. The loss in the circadian component of 5HT activity is not due to a loss in serotonergic function, since overall turnover rates were not reduced compared to young animals. Estrogen treatment modified the diurnal pattern of 5HT activity in the SCN, MPN, and AN in young rats but had no effect in the middle-aged rats. In young rats, estrogen induced a transitory rise in ME-5HT turnover at 1200 h, just before the expected onset of the LH surge. In middle-aged animals the increase in ME-5HT turnover did not occur until 1800 h and correlates with a delay in the initiation of the estradiol-induced LH surge. We conclude that: 1) there is a loss in the rhythm of 5HT activity in middle-aged rats and 2) the diurnal rhythmicity of 5HT turnover may be necessary for the maintenance of normal cyclic release of LH.     

Effects of estradiol on the diurnal rhythm of serotonin activity in microdissected brain areas of ovariectomized rats

The purpose of these studies was to determine whether diurnal rhythms in serotonin (5HT) activity are detectable in individual hypothalamic nuclei of ovariectomized rats and whether estradiol induces specific rhythms of 5HT which may be necessary to cyclic release of LH and/or PRL. Young (3- to 4-month old) rats were bilaterally ovariectomized and 7 days later half the animals received Silastic estradiol capsules. Two days later groups were again divided: half the animals in each group were killed at 0800, 1200, 1800, and 2400 h. The remaining animals received pargyline (75 mg/kg body weight, ip) at these times and were killed 10 min later. The median eminence (ME), suprachiasmatic nucleus (SCN), medial preoptic area (MPN), arcuate nucleus (AN), and globus pallidus (GP) were microdissected and assayed for 5HT by HPLC using electrochemical detection. A diurnal rhythm in 5HT turnover was found in the SCN, MPN, and AN of ovariectomized rats. 5HT turnover in these areas was significantly higher during the light hours (0800, 1200, and 1800 h) compared to the dark phase (2400 h). The ME and GP of ovariectomized rats did not exhibit a diurnal rhythm in 5HT activity. Exposure to estrogen altered the pattern of 5HT activity in all hypothalamic areas examined. In the ME, treatment with estradiol increased 5HT turnover at 1200 h, just before the predicted LH and PRL surge, and suppressed activity at all other times. In the SCN, estradiol reversed the 5HT rhythm: turnover was low during the light hours and high during the dark. In the AN and MPN, estradiol treatment increased 5HT activity and abolished the diurnal rhythm. 5HT activity in the GP was not altered by exposure to estrogen. We conclude from these data that specific brain nuclei exhibit diurnal rhythms in 5HT turnover and that the patterns of 5HT activity in specific hypothalamic nuclei exhibit individual and unique responses to the presence of estrogen. These data suggest that the estradiol-induced diurnal pattern of 5HT activity may be necessary for the induction of cyclic release of LH and/or PRL.     

Ontogeny of cytosolic androgen receptors in the brain of the fetal rhesus monkey

In this study we compared the binding characteristics of methyltrienolone (R1881) in pooled cytosols from the hypothalamus-preoptic area-amygdala-septum (HPAS) of adult and fetal rhesus macaques. In addition, we studied the ontogeny of cytosolic androgen receptors (AR) in fetal neural tissue. Intact adult males and fetal rhesus monkeys of known gestational age were our experimental subjects. Fetuses were delivered by cesarean section at 50, 65, 80, and 150 days gestation. HPAS cytosols from 150-day fetuses and adult males were incubated with the synthetic ligand, [3H]R1881, for determining AR characteristics and to validate the assay. A single high affinity, low capacity receptor for R1881 was found in HPAS cytosols. The apparent dissociation constant was similar between adult and fetal HPAS (1.09 X 10(-10) vs. 1.59 X 10(-10) M, respectively). Binding specificity was determined by the addition of excess radioinert testosterone (T), 5 alpha-dihydrotestosterone, estradiol, or progesterone to the incubation tube. R1881 binding was displaced by the addition of excess amounts of T and dihydrotestosterone, but not of estradiol or progesterone. There were no differences between fetal and adult animals. Single point analyses of AR numbers in fetal animals showed significant age and regional differences (P less than 0.05). Since no sex differences were apparent, data from males and females were combined. In the hypothalamus-preoptic area there was a significant increase in AR throughout gestation [1.3 +/- 0.4 (+/- SE) fmol/mg protein; n = 7 (50 days gestation) vs. 6.2 +/- 0.3 fmol/mg protein; n = 4 (150 days of gestation); P less than 0.01]. These values differed significantly from adult male hypothalamic-preoptic area (14.1 +/- 0.3 fmol/mg protein; P less than 0.01; n = 3). AR levels in frontal and temporal cortex were high on day 50 of gestation, but showed a significant decline by day 150 (P less than 0.05). The administration of testosterone propionate (25 mg/kg.day) to pregnant animals from 40-50 days gestation, which resulted in elevated levels of serum T in female, but not male, fetuses had no effect on AR in any brain region studied. These studies confirm the presence of AR in fetal monkey brain. New information is provided on the changes in AR numbers in cortical and hypothalamic tissues during the critical period for sexual differentiation of the primate brain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Triiodothyronine receptors in adult rat brain: topographical distribution and effect of hypothyroidism

The binding properties (affinity constant Ka and number of binding sites) of the nuclear receptors for triiodothyronine (T3) were studied in selected brain areas of the adult rat: cerebral cortex, cerebellum, olfactory bulb, caudate nucleus, hippocampus, hypothalamus and pituitary. Normal rats were compared to hypothyroid animals. The Ka of the nuclear receptors in the different parts of the brain had a similar order of magnitude (10(9) M-1) except for the hypothalamus where the value was lower (6 X 10(7) M-1). In hypothyroid animals, two main changes were observed in the properties of nuclear receptors: the Ka in the cerebral cortex was reduced by half, whereas in the pituitary the value was three times higher than in controls. In contrast to the decreased affinity, hypothyroidism enhanced the maximal binding capacity, especially in the cerebral cortex, the olfactory bulb, the caudate nucleus and the hippocampus. Of particular interest were the effects of hypothyroidism on the pituitary receptors which showed a markedly increased affinity for T3 and a reduced number of binding sites; this observation could be related to the control feedback mechanism of thyroid-stimulating hormone secretion by thyroid hormones.     

Progestin receptors in rat brain: distribution and properties of cytoplasmic progestin-binding sites

Putative progestin receptors have been characterized in brain and pituitary tissue from untreated and estrogen-primed ovariectomized-adrenalectomized rats. The properties of these sites appear indistinguishable from those of cytoplasmic progestin receptors from the uterus: 1) sedimentation coefficient of 7S, which is reduced by half in the presence of 0.3 M KCl; 2) specificity of binding which strongly favors synthetic and natural progestins as opposed to glucocorticoids, androgens, and estrogens; 3) a dissociation constant for binding the synthetic progestin [3H]R5020 (17 alpha, 21-dimethyl-19-norpregna-4, 9-diene-3,20-dione) of 0.3 nM; and 4) similar rates of formation and dissociation of the [3H]R5020-receptor complexes. In these respects, the estrogen-inducible and noninducible receptors of the brain also appear to be indistinguishable from each other. Estrogen induction of progestin receptors is apparent in uterus (6-fold), pituitary (8-fold), mediobasal hypothalamus (4-fold), and preoptic area (4-fold), all estrogen receptor-containing areas. The corticomedial amygdala does not show an estrogen effect on progestin receptor levels even though it contains estrogen receptor sites. The midbrain of the rat doeogen-insensitive receptors in the brain is relatively low and of the same order of magnitude as in nonstimulated hypothalamus, preoptic area, and pituitary, yet variations are seen among the estrogen-insensitive structures, with lowest levels occurring in cerebellum (6-7 fmol/mg protein) and highest levels occuring in cerebral cortex (approximately 25 fmol/mg protein). These findings are discussed in relation to the actions of progesterone which do and do not require estrogen priming and in relation to intracranial progesterone implantation studies.     

Effect of spironolactone and potassium canrenoate on cytosolic and nuclear androgen and estrogen receptors of rat liver

Spironolactone and potassium canrenoate are diuretics that are used widely for management of cirrhotic ascites. The administration of spironolactone frequently leads to feminization, which has been noted less frequently with the use of potassium canrenoate, a salt of the active metabolite of spironolactone. The use of these two drugs has been associated with decreases in serum testosterone levels and spironolactone with a reduction in androgen receptor (AR) activity. This decrease in AR has been cited as the cause of the antiandrogen effect of these drugs. We therefore assessed the effect of both drugs on levels of androgen and estrogen receptors (ER) in the liver, a tissue that is responsive to sex steroids. Three groups of male rats (n = 12 rats each) were studied. Group 1 (control) received vehicle only; group 2 received spironolactone (5 mg/day); group 3 received potassium canrenoate (5 mg/day). After 21 days of treatment, the animals of all groups were killed and liver tissue was assayed for nuclear and cytosolic AR and ER, and for male specific estrogen binder (MEB), an androgen-responsive protein. Both drugs drastically decreased the nuclear AR content, as compared with the control group, but only spironolactone decreased cytosolic AR. When the total hepatic content of AR is considered, a highly significant decrease is observed only in rats treated with spironolactone. This reduction in hepatic AR content suggested loss of androgen responsiveness of liver. We confirmed this by assessing levels of MEB, and found that livers from group 2 animals had no detectable MEB activity, whereas livers from both group 1 and 3 had normal MEB activity. No changes were observed in nuclear ER and cytosolic ER of group 3 as compared with group 1. Nuclear estrogen receptor decreased and cytosolic ER increased in group 2, but with no change in total ER content. These results indicate that (a) only spironolactone appears to act as an antiandrogen in liver, resulting in a decrease in both AR and male specific estrogen binder content, and (b) neither drug results in elevated hepatic ER content, although spironolactone-treated animals show an altered subcellular localization.     

Reduced tissue androgen receptors in the congenitally obese male Zucker rat

Androgen receptor (AR) concentrations were measured in the prostates and livers of the infertile congenitally obese male Zucker rat and their fertile non-obese litter-mates. In obese rats AR concentrations were significantly lower in both the liver (p less than 0.001) and the prostates (p less than 0.01) of the obese rats compared with the non-obese rats, despite similar plasma testosterone concentrations in both phenotypes. These findings suggest that a potential cause for the infertility observed in the obese Zucker rat is a reduced sensitivity of target tissues to circulating androgens.