Quantitative distribution of nuclear androgen receptors in microdissected areas of the rat brain

The binding of androgens to specific high-affinity receptor sites in brain tissue is postulated as an initial event in the mechanism of central androgenic action. In an effort to assess the functional capacity of the androgen receptor system in the central nervous system, we measured the concentration of nuclear (ARn) as well as cytosolic androgen receptors (ARc) in 13 microdissected brain samples from intact male and female Sprague-Dawley rats. Tissues from 6 rats were combined for each determination and androgen receptor contents were measured with single-point in vitro assays that used saturating concentrations of high specific activity 3[H]dihydrotestosterone. We found that ARc levels tended to be higher in females than males although the general patterns of distribution were very similar. As expected, ARn concentrations were significantly higher in males than females. The highest concentrations of ARn (greater than 100 fmol/mg DNA) in males were measured in the ventromedial nucleus of the hypothalamus and medial amygdala; intermediate levels (50-100 fmol/mg DNA) were found in arcuate nucleus-median eminence, medial preoptic nucleus, periventricular preoptic area, bed nucleus of the stria terminalis, anterior hypothalamus, periventricular anterior hypothalamus, lateral septum, and parietal cortex, and low levels (less than 50 fmol/mg DNA) were measured in lateral preoptic nucleus and cortical amygdala. With the exception of the periventricular preoptic area (74 +/- 33 fmol/mg DNA), only very low concentrations of ARn were measured in females. These data provide the first quantitative profile of ARn in discrete brain nuclei and subregions of the rat.     

Sex differences in androgen-regulated cytochrome P450 aromatase mRNA in the rat brain

The conversion of testosterone to estradiol by cytochrome P450 aromatase (P450_AROM) in the medial preoptic area is required for full expression of male sexual behavior in rats. Preoptic P450_AROM activity is stimulated by androgens through an androgen-receptor mediated mechanism that regulates P450_AROM gene expression. The mechanism of enzyme induction appears to be sexually dimorphic in several species leading to greater testosterone-stimulated P450_AROM activity in males than in females. The present study was designed to determine whether the sex difference in androgen-regulated P450_AROM activity is manifested at the levels of mRNA expression. We compared the concentrations of P450_AROM mRNA and enzyme activity between five different treatment groups: intact males, castrated males (CX), ovariectomized females (OVX), CX males treated with dihydrotestosterone (CX+DHT), and OVX females treated with DHT (OVX+DHT). We found that unstimulated levels of P450_AROM mRNA and enzyme activity in both the preoptic area and medial basal hypothalamus were similar in the CX and OVX groups. However, when treated with equivalent doses of DHT, the levels of P450_AROM mRNA and enzyme activity in both brain regions were significantly higher in males than in females (i.e., CX+DHT group >OVX+DHT group). These results demonstrate that sex differences in the regulation of P450_AROM in brain are exerted pretranslationally by androgen and suggest that gender differences in androgen responsiveness play an important role in regulating gene expression in the adult rat brain.     

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.     

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.     

The regulation system of brain aromatase activity; distribution and changes with age of the aromatase activity of the rat brain

Sexual differentiation of brain structure and function is dependent on the hormonal environment during perinatal life. Recently, some studies have found the greatest aromatase activity in brain areas associated with sexual differentiation and sexual behavior, namely the hypothalamic and limbic structures. We have characterized the developmental and anatomical patterns of aromatase of aromatase activity in brains of fetal, neonatal, infantile and adult rats of both sexes. Aromatase activities in slices of brain were assayed by measuring the amount of 3H2O formed during the conversion of [1 beta-3H] androstenedione to estrogen. We have demonstrated major changes of the aromatase activity in the brain with age. Aromatase activities of both sexes reached peak values in the hypothalamus-preoptic area (HPOA) at least 3 days before birth. Thereafter, the activities declined to 3 weeks after birth. We have found the greatest amount of aromatase activity in HPOA and amygdala of both sexes. Aromatase activities in HPOA and amygdala of neonatal male rat were higher than adult male rat. The hippocampus, thalamus, pituitary, cerebral cortex and cerebellum all contained negligible aromatase activity. And, we studied HPOA in detail, aromatase activities in preoptic area and anterior part of hypothalamus were twice higher than that in posterior part of hypothalamus. Aromatase activity reached peak values at the critical period of the sexual differentiation of the brain in HPOA and amygdala, associated with sexual behavior and sexual differentiation. We have reported that aromatase activity was regulated by androgen. We suggested that aromatization didn't occur effectively in female rat, owing to scarcity of androgen, which was activator and substrate of aromatase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Divergence between ovarian aromatase activity, estrogen, and androgen levels in the cycling rat

Serum estrogen levels vary in a cyclic fashion during the rat estrous cycle, reaching peak values on proestrus and then rapidly declining. Daily measurements of ovarian aromatase activity appear to follow a similar pattern, suggesting a potential regulatory role for the enzyme. To examine this hypothesis, mature rats were killed at various times, and ovarian microsomes were assayed for aromatase activity, while androstenedione, testosterone, estradiol, and estrone levels were measured in ovarian cytosol by RIA. The ovarian aromatase activity reached a peak about 9 h after the onset of the light period on the day of proestrus, and then began to decrease gradually to 75% lower levels by the morning of estrus. Similarly, the estradiol levels also peaked at about 9 h, but then dropped sharply; at 18 h the estradiol levels were reduced 90-95%, while aromatase activity was still close to its peak value. In contrast, the androstenedione and testosterone levels remained high for 3-4 h after the estradiol levels began to decrease. Estrone levels varied considerably less than those of estradiol, reaching lower peak values, but then decreasing only 50-75%. The dissociation between product levels and enzyme activity cannot be explained by a change in the Km of aromatase, which was about 20 nM for androstenedione throughout this period. Therefore, it appears that the decrease in estradiol levels from the peak of the surge may in part be a consequence of a shift in the ratio of the estrogen products synthesized by the ovary, rather than a decrease in the ovarian capacity for estrogen production, although other factors appear to be involved as well.     

Sex differences in the concentrations of glucocorticoid receptors in rat liver and thymus.

The effects in rats of adrenalectomy, hypophysectomy, ovariectomy or combinations of these operations on the concentrations of glucocorticoid receptors in the cytosol of liver and thymus were measured. The concentrations of glucocorticoid receptors were lower in cytosols from liver and thymus of female than of male rats. After adrenalectomy, there was a significant increase in the concentrations of receptors measured in the cytoplasm from the liver and thymus of female rats and from the liver of male rats. After adrenalectomy or hypophysectomy, there was no sex difference in the concentration of glucocorticoid receptors in cytosols of liver or thymus. After ovariectomy, the concentration of receptors in cytosols from the thymus, but not from the liver, increased. Ovariectomized rats responded to adrenalectomy in the same way as intact male rats. The different responses shown by male and female rats to endocrine manipulation probably depend upon associated changes in plasma corticosterone concentrations which are influenced by the ovary. Differences in response between the liver and thymus probably reflect a preferential distribution of corticosterone to the liver rather than to the thymus.     

Behavioral action of estrogen in the male dove brain: area differences in codistribution of aromatase activity and estrogen receptors are steroid-dependent.

Brain aromatization of androgen to estrogen (E) and presence of estrogen-receptor (ER) containing cells (ERC) are required for the control of E-dependent neural events under-lying male sexual behavior. We examined whether (a) numbers of ERC and steroid-inducible aromatase activity are codistributed and directly correlated in the same brain areas of individual sexually active male doves and whether, (b) distribution of ERC is altered by a change in E formation in preoptic areas known to be involved in control of male behavior. To allow spatial correlation between ERC and aromatase activity, a new approach was used that combined in vitro measurement of aromatase activity (stereospecific formation of 3H2O from [1 beta-3H]-testosterone) in microdissected brain areas of one side of the brain and immunocytochemical localization of ERC (ER antibody H222Spy) in homologous contralateral areas of the same coronal brain section. The relationship between ERC and aromatase activity differs according to brain area in sexually active males: (a) large populations of ERC in preoptic areas, notably in nucleus preopticus medialis (6.5 +/- 0.8 ERC/5,000 microns2) and nucleus preopticus medialis, pars medianis (9.5 +/- 1 ERC/5,000 microns2) are codistributed with high steroid-dependent aromatase activity (greater than 100 fmol/mg tissue); (b) areas containing the nucleus interstitialis and ventromedial hypothalamic nuclei also have relatively high aromatase activity, but lower ERC density than POA; (c) the anterior hypothalamic nuclei have few ERC, but steroid-regulated aromatase activity; (d) the infundibular area contains elevated ERC and little steroid-inducible aromatase activity; (e) area basalis and neostriatum contain few or no ERC and no inducible aromatase activity. Castration of sexually active doves reduces aromatase activity in preoptic and posterior hypothalamic areas (by greater than 75%) to basal levels, but does not affect the distribution or number of ERC in brain areas containing steroid-regulated aromatase activity, notably in the preoptic area. The results show that steroid regulation of aromatase occurs in brain loci with high numbers of ERC. We suggest that steroid-inducible aromatase occurs in brain loci with high numbers of ERC. We suggest that steroid-inducible aromatase activity and ERC are codistributed in areas controlling male sexual behavior; thus the formation and action of E may occur in the same area. Regulation of the aromatase activity and supply of E, but not of number of cells containing ER, is one mechanism which accounts for changes in action of testosterone on estrogen target sites in the male brain.     

Sex and species differences in plasma testosterone and in counts of androgen receptor-positive cells in key brain regions of Sceloporus lizard species that differ in aggression

We studied neuroendocrine correlates of aggression differences in adults of two Sceloporus lizard species. These species differ in the degree of sex difference in aggressive color signals (belly patches) and in aggression: Sceloporus undulatus (males blue, high aggression; females white, low aggression) and Sceloporus virgatus (both sexes white, lower aggression). We measured plasma testosterone and counted cells expressing androgen receptor-like immunoreactivity to the affinity-purified polyclonal AR antibody, PG-21, in three brain regions of breeding season adults. Male S. undulatus had the highest mean plasma testosterone and differed significantly from conspecific females. In contrast, there was no sex difference in plasma testosterone concentrations in S. virgatus. Male S. undulatus also had the highest mean number of AR-positive cells in the preoptic area: the sexes differed in S. undulatus but not in S. virgatus, and females of the two species did not differ. In the ventral medial hypothalamus, S. undulatus males had higher mean AR cell counts compared to females, but again there was no sex difference in S. virgatus. In the habenula, a control brain region, the sexes did not differ, and although the sex by species interaction significant was not significant, there was a trend (p=0.050) for S. virgatus to have higher mean AR cell counts than S. undulatus. Thus hypothalamic AR cell counts paralleled sex and species differences in aggression, as did mean plasma testosterone levels in these breeding-season animals.     

Genetic evidence for androgen-dependent and independent control of aromatase activity in the rat brain

To investigate the role of androgen receptors in the regulation of brain aromatase activity (AA) in adult rats, the levels of AA in discrete brain areas of androgen-insensitive testicular feminized (Tfm) rats were compared with those in their normal male littermates (NL). AA was measured in homogenates of brain tissue by using a radiometric assay that quantifies the production of 3H2O from [1 beta-3H]androstenedione as an index of estrogen formation. Initially, we assessed the capability of block-dissected tissues to aromatize androgens. We found that the AA in the amygdala and hypothalamus-preoptic area of Tfm rats was significantly lower (P less than 0.001) than the AA in NL despite the fact that circulating androgen concentrations in the Tfm were significantly higher. Kinetics studies demonstrated that the apparent Michaelis constant was equivalent for both groups (0.02-0.03 microM). Administration of testosterone propionate to castrated males produced 3 to 4-fold elevations of AA in NL, but did not affect brain AA in Tfm rats. To pinpoint specific sites where AA is affected in Tfm rats, we measured AA in 10 hypothalamic and limbic nuclei that were dissected from 300-micron frozen brain sections. Compared to NL, Tfm rats exhibited significantly lower levels of AA in all micro-dissected brain regions studied, except for the medial and cortical amygdala. These data provide genetic evidence for both androgen-dependent and independent regulation of AA in the rat brain.     

The regulation system of brain aromatase activity; effects of androgens on hypothalamic aromatase in the male rat

Recently, some studies have found the greatest aromatase activity in brain areas associated with sexual differention and sexual behavior, namely the hypothalamic and limbic structures. We studied the regulation of aromatase activity in the hypothalamic area of male rats, using a sensitive in vitro assay which measures the amount of 3H2O formed by tissue homogenates during the conversion of [1 beta-3H] androstenedione to estrogen. After castration, hypothalamic aromatase activity was significantly decreased (P less than 0.01), and seminal vesicle (SV) and prostate (PR) weights were also significantly decreased (P less than 0.01). Castrated male rats were given testosterone (T), 5 alpha-dihydrotestosterone (DHT), 5 alpha-androstane-3 alpha, 17 beta-diol (A3 alpha), 5 alpha-androstane-3 beta and 17 beta-diol(A3 beta) in various doses (200-1000 micrograms/day) for 10 days, and were given 600 micrograms/day T, DHT, A3 alpha and A3 beta for various durations (1-10 days). We found that T, DHT and A3 alpha but not A3 beta reversed the effects of castration on the hypothalamic aromatase activity. The order of this reversible effect of androgens was as follows: T greater than or equal to DHT greater than A3 alpha. T, DHT, A3 alpha and A3 beta increased SV and PR weights, and the order of this effect was as follows: DHT greater than T greater than A3 alpha much greater than A3 beta. We administered the antiandrogen (flutamide) to intact male rats (8 mg/day for 6 days). Flutamide decreased hypothalamic aromatase activity at the same level as that of castrated rats. Likewise, administration of both flutamide and T to castrated rats blocked the T-induced increase in hypothalamic aromatase activity and accessory sexual organ weight. From these results, we suggest that T, DHT and A3 alpha regulated hypothalamic aromatase activity, that T was the most effective of the androgens, and that was different from peripheral androgen target organs.     

Age-related and sex-related alterations in beta-adrenergic receptors in different regions of rat brain

The binding of (-)[3H]dihydroalprenolol, an antagonist of norepinephrine, to beta-adrenergic receptors in different regions of the brain of male and female rats of various ages was measured. The binding to the synaptosomal fraction of corpus striatum, hypothalamus, cerebral cortex, cerebellum and the brainstems shows a significant decrease in the binding in old rats of both sexes. Only in the female corpus striatal region, the binding in the adult and the old is the same. In the case of females, the highest binding is seen in the young. In the male, an increase in binding occurs up to adulthood, after which it declines, suggesting a definite sex-related difference in the beta-adrenergic receptor.     

Differential regulation of brain aromatase by androgen in adult and fetal ferrets

The contribution of androgens to the regulation of aromatase activity (AA), measured by quantifying the in vitro formation of [3H]estrone from 19-[3H]hydroxyandrostenedione precursor, was studied in equivalent microdissected brain regions of adult and fetal ferrets. In adulthood, AA was similar in the bed nucleus of the stria terminalis, medial (M) and lateral (L) preoptic area (POA), medial (MA) and lateral amygdala (LA), ventromedial hypothalamus (VMH), and parietal cortex of gonadectomized males and females given no concurrent steroid treatment. Daily sc injections of the androgen dihydrotestosterone propionate significantly stimulated AA in MPOA, MA, and VMH of adult males and in MA of females; a similar trend was seen in MPOA and VMH of females. By contrast, no evidence of androgenic regulation of AA was obtained in these three brain regions microdissected from fetuses killed on embryonic day 35 (E35; 41-day gestation). Transplacental administration of the antiandrogen flutamide beginning on day E24 failed to affect AA in MPOA, LPOA, MA, LA, or parietal cortex, although this treatment significantly reduced AA in bed nucleus of the stria terminalis of fetal males. The results suggest that the responsiveness of aromatizing enzymes to androgenic induction is similar in several subcortical brain regions of adult ferrets of both sexes. In breeding males such an action of androgen may augment the neural production of estrogen, which has previously been implicated in the control of sexual behavior and the feedback regulation of LH secretion. By contrast, androgen apparently contributes minimally to the regulation of AA in brain regions of fetal ferrets, particularly in the MPOA, in which a sexually dimorphic nucleus differentiates in males around E37 in response to estrogen produced locally.     

Sex differences in peripheral vascular adrenergic receptors

Although the incidence of many vascular diseases differs in men and women, sex differences in vascular physiology have not been extensively examined in human in vivo studies. The present study compared finger blood flow responses of normal men and women with brachial artery infusions of adrenergic agonists and with other neurally and nonneurally mediated procedures. In response to phenylephrine and clonidine, men showed significant dose-related vasoconstriction while women did not. In response to isoproterenol, men showed significant dose-related vasodilation while women did not. There were no sex differences in response to intra-arterial nitroglycerin or digoxin or to reactive hyperemia, procedures that do not act through adrenergic receptors. These data show that the sensitivity and/or density of peripheral vascular adrenergic receptors is lower in women than in men. There were no sex differences in response to reflex vasoconstriction or to intra-arterial tyramine, suggesting that neurally released norepinephrine acts at alpha-adrenergic receptors that are spatially removed from those that respond to circulating catecholamines.     

Relationships between aromatase and estrogen receptors in the brain of teleost fish

Teleost fish are known for exhibiting a high aromatase activity mainly due to the expression of the cyp19b gene, encoding aromatase B (AroB). Recent studies based on both in situ hybridization and immunohistochemistry have demonstrated in three different species that this activity is restricted to radial glial cells. In agreement with measurements of aromatase activity, such aromatase-expressing cells are more abundant in the telencephalon, preoptic area, and mediobasal hypothalamus, although positive cells are also found in the midbrain and hindbrain. Comparative distribution of AroB and estrogen receptor (ERalpha, ERbeta1, and ERbeta2) expression indicates that the preoptic region and hypothalamus are major target for locally produced estradiol (E2) which is likely involved in controlling expression of genes implicated in neuroendocrine regulations. However, AroB and ER have never been reported to be co-expressed in the same cells which is intriguing given that, at least in some species, AroB is strongly up-regulated by E2 itself in agreement with the presence of an estrogen-responsive element (ERE) in the proximal promoter of the cyp19b gene. In vivo data in zebrafish have shown that E2 up-regulates AroB only in radial glial cells. This is in agreement with in vitro transfection experiments indicating that this ERE is functional, but not sufficient, as the E2 regulation of AroB only occurs in glial cell contexts, suggesting a cooperation between ER and so far unidentified glial-specific factors. These data also suggest that radial glial cells may express low amounts of ER that escaped detection until now. The expression of AroB in radial cells, well known for their roles in neurogenesis and now considered as progenitor cells, suggests that local E2 production within these cells could influence the well-documented capacity of the brain of teleosts to grow during adulthood.     

The ontogeny of sex differences in estrogen-induced progesterone receptors in rat brain

The sex and age-related changes in the concentration of cytosol progesterone receptors (CPR) induced by estrogen (E) treatment in rat brain and pituitary were investigated by a modification of the Palkovits punch procedure using fresh tissue. Young male and female gonadectomized rats, 15, 21, 30, and 42 days of age, were treated for 44 h by a single sc injection of synthetic E. [Moxestrol (Ru2858)]. Adult gonadectomized animals were treated for 72 h by three injections of estradiol benzoate. Cytosol from pituitary and from punches of ventromedial nuclei (VMN), medial preoptic area (mPOA), arcuate nucleus (ARC), and cerebral cortex was labeled with 0.4 nM [3H]promegestrone (Ru5020) to maximize signal to noise and specificity of labeling of CPR. The developmental patterns of CPR differed across brain regions and between males and females. In VMN, females showed higher CPR levels after estrogen priming at 15, 21, and 42 days of age and in the adult; in ARC, females showed higher CPR levels after E priming at 15, 21, and 30 days of age, but not at 42 days or in the adult. In mPOA and pituitary, no consistent sex differences in CPR induction were found. Cortex showed no induction of CPR by E priming. Radioautography of [3H]Ru5020 uptake in VMN of E-primed 15-day-old male and female rats revealed significantly higher labeling in females, thus showing that the CPR levels in vitro reflect a difference in hormone retention in vivo. Female VMN contained more neurons with a higher labeling index than male VMN. Before puberty (approximately = 30 days), there was a decline in CPR levels induced by E priming in both sexes in pituitary, ARC, mPOA, and VMN. At 30 days, the female greater than male sex difference disappeared and tended to reverse in mPOA and VMN, only to be restored again by 42 days of age. Thus, the CPR induction by E priming may reflect underlying changes in E-sensitive brain regions associated with the preparation for puberty, as well as underlying sex differences in response to estrogen programmed by perinatal exposure to testosterone.     

Sex differences in myocardial contractility in the rat

Summary We examined the intrinsic contractile performance of papillary muscles removed from the left ventricle of male and female Wistar rats. Muscles were studied isometrically and isotonically, stimulated at 0.1 Hz, perfused with Tyrode's solution having an external calcium concentration=2.4 and maintained at 30°C. In addition, we examined muscle response to changes in external calcium, added norepinephrine or verapamil and alterations in contraction frequency. No significant change in peak isometric development tension was observed between male and female preparations. However, muscles from male rats showed a significantly greater isometric time-to-peak tension and time to 1/2 relaxation with a depression of both the maximum rate of tension rise and maximum rate of tension decay. Isotonically, although peak shortening showed no difference between male and female preparations, the maximum velocities of shortening and relaxation were significantly depressed in muscles from male rats. Muscles from male animals also displayed significant prolongation of the time-to-peak shortening and time-to-peak velocity of shortening. These differences in papillary muscle performance were found over a wide range of muscle lengths, stimulus frequencies and bath concentrations of calcium, norepinephrine and verapamil. Thus differences in intrinsic contractile performance between papillary muscle from male and female rats have been characterized.Supported in part by NIH grants #HL 21933-02, #HL 07071-05 and a Herman Raucher Investigatorship Award of the New York Heart Association to Dr. J. M. Capasso.     

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.