Sex-specific occurrence of androgen receptors in rat brain.

The metabolism and binding of [1, 2, 6, 7-3H] testosterone in male and female rat brain has been studied in an attempt to find an explanation for the relative androgen unresponsiveness characterizing the female hypothalamo-pituitary axis involved in regulation of hepatic steroid metabolism. The most significant sex differences in the pattern of [3H] testosterone metabolites recovered from several brain regions (including pituitary, pineal gland, and hypothalamus) after intraperitoneal administration of [3H] testosterone were the predominance of testosterone and androstenedione in male brain compared to the quantitative importance of 5alpha-androstane-3alpha, 17beta-diol, 5alpha-androstane-3beta, 17beta-diol, epitestosterone, and dihydroepitestosterone in female brain. One possible explanation for the androgen unresponsiveness of female rats is, therefore, the faster metabolism of testosterone to inactive compounds in female brain. Experiments both in vivo and in vitro showed the presence of high affinity, low capacity binding sites for [3H] testosterone in male pituitary, pineal gland, and hypothalamus (Kd values in the region of 1 X 10(-10) to 1 X 10(-9) M and number of binding sites 1.0 to 1.4 X 10(-14) mol per mg of protein). The steroid - macromolecular complexes generally had a pI of 5.1, were excluded from Sephadex G-200, were heat-labile, and were sensitive to protease. Competition experiments indicated the following order of ligand affinities: testosterone is greater than 5alpha-dihydrotestosterone and estradiol is greater than androstenedione is greater than corticosterone. No steroid-binding proteins of similar nature were found in pituitary, pineal gland, or hypothalamus from female rats. On the basis of these results it is suggested that the androgen unresponsiveness of female rats referred to above relates to the absence of receptor protein for androgens in female rat brain. In support of this hypothesis, 28-day-old female rats, which are known to be affected by androgens with regard to liver enzyme activities, were shown to contain receptor proteins for androgen in the brain. In conclusion, the relative androgen unresponsiveness of the female hypothalamo-pituitary axis is probably explained by the absence of receptor proteins for androgen in female hypothalamus and pituitary. The fast metabolism of testosterone in female rat brain also serves to decrease the availability of active androgen to potential receptor sites. It may be speculated that the presence of androgen receptors in male brain is the result of neonatal programming ("imprinting") by testicular androgen.     

Immunohistochemical localization of androgen receptors in female mole rat (Spalax leucodon) tissues

Androgens exert their effects through androgen receptors (AR) in tissues. We investigated the distribution of AR in female mole rat tissues. Tissues were excised, fixed with 10% formalin and embedded in paraffin. Sections were stained after microwave antigen retrieval for immunohistochemistry. Immunostaining of AR immunostaining was detected in the nucleus or cytoplasm of the cells in the cerebral cortex, cerebellum, anterior pituitary, lung, liver, uterus and skin. Granulosa and some thecal cells in the ovary, cardiac muscle cells and adipose cells exhibited a nuclear reaction for AR. In the kidney, labeling of AR was restricted to the cytoplasm of tubule cells. We found that AR could be detected using immunohistochemistry in the nucleus or cytoplasm or both in the presence of androgens.     

Characterization of the androgen receptors in the hypothalamus, preoptic area and brain cortex of the rat.

The specific androgen receptors for testosterone (T) (1) and 5alpha-dihydrotestosterone (DHT) in the cytosol fraction of the hypothalamus, preoptic area and brain cortex of the rat have been characterized using electrophoresis and isoelectric focusing in polyacrylamide gels. After labeling of the cytosol fractions in vivo and in vitro we were able to demonstrate androgen-receptor complexes moving with an electrophoretic mobility (R(f) of 0.5 in 3.25% acrylamide gels containing 0.5% agarose and 10% glycerol. Polyacrylamide gel electrophoresis was used as a quantitative assay for androgen receptors in the tissues. The hypothalamus, preoptic area and brain cortex were found to possess a single class of high affinity binding sites for androgens and the dissociation constants (K(D) were estimated to be 3.4, 4.3 and 2.6 X 10 (-10M) respectively. The binding capacities were 3.7 (hypothalamus), 3.5 (preoptic area) and 1.8 X 10 (-15) (brain cortex) moles of high affinity binding sites per mg protein. Like other androgen-receptor complexes, the testosterone-receptor complexes of the hypothalamus, preoptic area and brain cortex were temperature labile, sulfhydryl dependent and revealed a very slow rate of dissociation at o degrees C (t1/2 greater than 36 hr). The receptors in all the tissues had an isoelectric point of 5.8. The steroid specificity of the cytoplasmic androgen receptors was tested in vitro by the competing efficiency of different unlabeled steroids for (3H)-testosterone binding. In the three tissues in investigation the following order of affinity was found: DHT greater than T greater than Cyproterone acetate greater than progesterone greater than androstenedione greater than 17beta-estradiol. Cortisol did not effect androgen binding significantly. Thus, the physiochemical characteristics of the cytoplasmic androgen receptors of the hypothalamus, preoptic area and brain cortex are very similar, if not identical, to those of the androgen receptors described in the anterior pituitary, ventral prostate, epididymis and testis.     

Molecular properties of neurotensin receptors in rat brain. Identification of subunits by covalent labeling

Neurotensin binding sites in rat brain synaptic membranes were specifically and covalently labeled by two methods. In the first, a photoreactive and highly radioactive analogue of neurotensin, 125I-labeled azidobenzoyl[Trp11]neurotensin, was synthesized and used to photoaffinity label neurotensin receptors. In the second, the reversible association between neurotensin receptors and 125I-labeled[Trp11]neurotensin, a radioactive but nonphotoreactive analogue of neurotensin, was made irreversible by means of disuccinimidyl suberate, a bifunctional cross-linking reagent. Analysis of synaptic membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that using both methods the same two protein bands with apparent molecular weights of 49,000 and 51,000 were specifically labeled. Identical results were obtained with or without reduction of the photolabeled membranes by beta-mercaptoethanol before electrophoresis. Variation of the ligand concentration did not modify the relative labeling intensities of the two bands, indicating that the high- and low-affinity neurotensin binding sites previously detected in rat brain synaptic membranes have similar molecular structures. These results indicate that neurotensin receptors in rat brain may be composed of two different protein subunits with similar molecular weight of about 50,000, that are linked together by noncovalent bonds.     

Selective labeling of alpha-noradrenergic receptors in rat brain with [3H]dihydroergokryptine.

Using concentrations of [³H] dihydroergokryptine between 0.1 and 5 nM, saturable binding can be demonstrated in rat cerebral cortical membranes with a dissociation constant (K_D) of about 0.8 nM. α-Noradrenergic agonists and antagonists compete for the sites labeled by these low concentrations of [³H] dihydroergokryptine with relative potencies characteristics of classical α-noradrenergic receptors. The very low potency of serotonin in competing for these binding sites indicates that, in contrast to findings with higher concentrations of [³H] DHE, low concentrations do not label serotonin receptors. Moreover, the low potency of dopamine in competing for [³H] dihydroergokryptine binding in both striatal and cortical membranes indicates that no detectable portion of binding is associated with postsynaptic dopamine receptors.     

Sex differences in estrogen and androgen receptors in hamster brain.

The present study was conducted to measure the levels of estrogen and androgen receptors (ER and AR, receptively) simultaneously in the anterior pituitary (AP), and various brain regions from adult male and proestrous female hamsters. Medial preoptic area (MPOA), medial basal hypothalamus (MBH), lateral hypothalamus (LH), medial forebrain bundle (MFB), and amygdala (AMG) were identified and removed from 200-microns frozen brain sections by the Palkovits punch-out technique. ER and AR were determined by the in vitro binding assay using [3H]-estradiol and [3H]-methyltrienolone as the binding ligands. In males, high levels of AR were found in the MPOA, MBH, and AP. In females, the MPOA, MBH, LH, and AP contained high levels of ER. The males exhibited significantly higher levels of AR than females in the MPOA, MBH, and LH, whereas the ER levels in these areas were higher in females. In males, ER and AR contents in the AP were higher, but the contents in the AMG were lower as compared to those of females. The calculated ER/AR ratio in MPOA, MBH, and LH were lowest in males. On the contrary, the ratio in these areas were highest in females. These data suggest that sex differences in response to estrogen and androgen may in part be due to sex differences in ER and AR contents in specific brain regions.     

Binding studies and photoaffinity labeling identify two classes of phencyclidine receptors in rat brain

Binding and photoaffinity labeling experiments were employed in order to differentiate 1-(1-phenylcyclohexyl)piperidine (PCP) receptor sites in rat brain. Two classes of PCP receptors were characterized and localized: one class binds [3H]-N-[1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) with high affinity (Kd = 10-15 nM) and the other binds the ligand with a relatively low affinity (Kd = 80-100 nM). The two classes of sites have different patterns of distribution. Forebrain regions are characterized by high-affinity sites (hippocampus greater than frontal cortex greater than thalamus greater than olfactory bulb greater than hypothalamus), but some parts (e.g., hippocampus, hypothalamus) contain low-affinity sites as well. In the cerebellum only low-affinity sites were detected. Binding sites for [3H]PCP and for its photolabile analogue [3H]azido-PCP showed a regional distribution similar to that of the [3H]TCP sites. The neuroleptic drug haloperidol did not block binding to either the high- or the low-affinity [3H]TCP sites, whereas Ca2+ inhibited binding to both. Photoaffinity labeling of the PCP receptors with [3H]AZ-PCP indicated that five specifically labeled polypeptides of these receptors (Mr 90,000, 62,000, 49,000, 40,000, and 33,000) are unevenly distributed in the rat brain. Two of the stereoselectively labeled polypeptides (Mr 90,000 and 33,000) appear to be associated with the high- and low-affinity [3H]TCP-binding sites; the density of the Mr 90,000 polypeptide in various brain regions correlates well with the localization of the high-affinity sites, whereas the density of the Mr 33,000 polypeptide correlates best with the distribution of the low-affinity sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

beta-Adrenergic receptors in rat brain.

125I-Iodohydroxybenzylpindolol ([125I] IHYP), a potent beta-adrenergic receptor antagonist, has been used to study beta-adrenergic receptors in rat brain. Binding of [125I] IHYP (30 pM) to a membrane fraction min and dissociation took place with a half time of about 16 min. Phentolamine (10(-4) M) decreased non-receptor binding but it had no effect on the binding of [125I] IHYP to beta-adrenergic receptors in cortex, cerebellum or caudate. In the presence of phentolamine specific binding (defined as binding which was blocked by 0.3 muM dl-propranolol) represented 70-85% of total binding. The binding of [125I] IHYP was inhibited by beta-adrenergic agonists and antagonists. d-Stereoisomers were 2-3 orders of magnitude less potent than the corresponding 1-isomers. The denstiy of [125I] IHYP binding sites was studied in membrane fractions from cerebral cortex, cerebellum, and caudate nucleus by means of Scatchard analysis. The K(D) of [125I] IHYP was similar in the three regions studied, and the density of [125I] IHYP binding sites was approximately 50% greater in the cortex and caudate than in the cerebellum. The Hill coefficient for the binding of [125I] IHYP to membranes from cerebral cortex was 1.02. The properties of the binding of [125I] IHYP are similar to those which would be expected of binding to beta-adrenergic receptors in vitro.     

A new method for labeling and autoradiographic localization of androgen receptors

We have used a novel receptor labeling and autoradiographic technique to localize androgen receptors in the intact rat ventral prostate at the morphological level. Frozen slide-mounted prostate tissue sections (10 micron thick) were incubated with increasing concentrations of [3H]-R1881 in the absence and presence of excess unlabeled R1881. Tissue sections labeled in this way were subjected to concurrent biochemical and autoradiographic analysis. After incubation and washing to remove free [3H]-steroid, some of the sections were wiped from the slides for scintillation counting in order to characterize and quantitate [3H]-R1881 binding. Androgen receptors could indeed be labeled in slide-mounted tissue sections, and specific [3H]-R1881 binding to these receptors was high-affinity (Kd = 1 nM), saturable, and androgen-specific. All cellular androgen receptors appear to be retained, because receptor content in sections was comparable to the sum of receptors in subcellular fractions of homogenized tissue. Replicate labeled slide-mounted tissue sections were dried rapidly, apposed to dry emulsion-coated coverslips, and exposed in the dark for autoradiography. Silver grains were counted over nuclei or cytoplasm of epithelium or stroma to evaluate specific androgen receptor location. Autoradiographic analysis demonstrated androgen receptor localization almost exclusively in the epithelial nuclei, with little or none in the stroma. We discuss here the unique features and advantages of labeling androgen receptors in slide-mounted frozen tissue sections for autoradiographic localization.     

Immunohistochemical detection of androgen receptors in the canine uterus throughout the estrus cycle

Serum androgen levels in the bitch increase during proestrus and remain elevated until metestrus. To find out whether androgens can have a direct impact on the canine uterus, androgen receptors (AR) were identified immunohistochemically in uterine tissue. Androgen receptor distribution in the uterine horns, body and cervix was described during different cycle stages, during pregnancy and in the postpartum period. Nuclear staining for AR was observed in cells of the surface epithelium, glandular ducts, basal glands and stroma of the endometrium, and in myometrial smooth muscle cells. In addition, cytoplasmic staining was observed in epithelial cells from proestrus to early metestrus, when the cells were secretory active, and in stroma cells during pregnancy, suggesting a role for androgens in decidualization. During pregnancy and in the postpartum period nuclear staining for AR was nearly absent. During the estrus cycle stroma cells stained with higher intensities for AR than epithelial cells, supporting the idea that stroma cells mediate some effects of steroid hormones on epithelial cells in the genital tract. In contrast with earlier findings on estrogen receptor-alpha and progesterone receptors, no significant changes in androgen receptor expression were observed during the estrus cycle. Few correlations were found between the staining for AR and serum levels of the sex steroids. The present findings suggest that there is a basal expression of AR in the canine uterus throughout the estrus cycle that may not be influenced by sex steroid hormones.     

大鼠睾丸雄激素受体表达的增龄变化

应用免疫细胞化学及图像分析等方法,对出生到生后25月龄各发育阶段SD大鼠睾丸内雄激素受体(AR)的表达变化进行了系统的研究。发现(1)睾丸间质细胞：从出生到生后3周龄,AR阳性表达强度较弱;到生后1月龄,AR阳性表达强度显著增强,并达到峰值;在生后2月龄,AR阳性表达强度显著减弱,此后AR阳性表达又呈增强趋势。(2)肌样细胞：从出生到生后2周龄,AR阳性表达强度较弱;到生后3周龄,AR阳性表达强度显著增强,并一直维持到生后2月龄;从生后3月龄,AR阳性表达强度呈减弱趋势,到生后25月龄达到最低水平。(3)血管内皮细胞：从生后3周龄到生后2月龄AR阳性表达较强;生后3月龄,AR阳性表达强度明显减弱;生后25月龄,AR阳性表达强度与生后3月龄相比变化不明显。(4)支持细胞：在生后1月龄出现AR阳性表达,到性成熟后,支持细胞AR阳性表达随生精周期变化而变化,在生后25月龄未见AR阳性表达的支持细胞。(5)生精细胞：出生组,前精原细胞内有AR阳性表达;生后2周龄,精子细胞开始出现AR阳性表达;生后1月龄,精原细胞开始出现AR阳性表达;生后2月龄出现阳性表达的精子,生后25月龄未见阳性表达的生精细胞。     

Effects of gonadectomy and androgen treatment on aromatase activity in the fetal monkey brain

We studied the ability of neural tissues from fetal rhesus macaques to aromatize androgens to estrogens and assessed whether androgens are involved in the regulation of aromatase activity during development. Fetuses of both sexes, obtained on approximately Day 100 of gestation, were gonadectomized and immediately given intraabdominal Silastic capsules containing dihydrotestosterone. Sham-gonadectomized (intact) and gonadectomized, sham-implanted groups were also studied. Three weeks after the initial operation, the fetuses were delivered by cesarean section. To examine the developmental pattern of aromatase activity in the brain, fetuses were also delivered at two earlier times in gestation (at approximately 50 days and approximately 80 days). Whole-homogenates of preoptic area plus anterior hypothalamus (POA), hypothalamus (HT), amygdala (AMYG), and cerebral cortex (CTX) were incubated for 1 h in a phosphate buffer with saturating concentrations of [1 beta-3H] androstenedione. The amount of 3H2O formed was used as an index to estimate aromatase activity. The aromatase reaction exhibited Michaelis-Menten kinetics with an apparent Km of approximately 0.03 microM in all tissues from 120-day-old fetuses. Activity measured with the 3H2O assay closely corresponded with levels determined by product isolation. The highest levels of aromatase activity were found in the POA. Neither gonadectomy nor treatment with androgen affected aromatase in fetal tissue; activities in males were significantly greater than in females for the AMYG and CTX, but not for the POA or HT. The levels of aromatase activity detected in the HT and CTX of both sexes on Days 50 and 80 of gestation were at least ten times greater than the levels measured in adults.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ontogeny of androgen receptors in fetal guinea pig brain

Sexual differentiation of the guinea pig brain is androgen dependent. To understand the cellular mechanisms of androgen action, we studied the ontogeny of cytosolic (ARc) and nuclear (ARn) androgen receptors in the brains and anterior pituitaries of fetal, neonatal, and adult guinea pigs. Using cytosol from the hypothalamus-preoptic area-amygdala-septum of 60- to 65-day fetuses and nuclear preparations from 6-day-old neonates treated with testosterone propionate, validation studies revealed an AR with an apparent Kd of 1.9 +/- 1.1 (mean +/- SEM, n = 3) x 10(-10) M (ARc) and 3.4 +/- 3.2 (n = 3) x 10(-10) M (ARn). The cytosolic receptors were highly specific for androgens. After assay validation, AR content was determined from specific brain regions of fetuses obtained on Days 30, 40, 50, and 59 of gestation and on Days 6 and 120 postpartum. ARc differed significantly (p less than 0.05) between brain regions and times of gestation, but no sex differences were apparent. In contrast, ARn showed little difference between tissues or with gestational age, but there were significant differences between males and females, especially in late gestation and early postnatal life, with males having greater ARn binding (p less than 0.05). These data demonstrate the presence of ARc and ARn in the fetal brain and pituitary gland during the critical period of sexual differentiation (Days 30-37 of gestation), thus establishing the identity of cellular structures involved in androgen action.     

High-affinity kainate and domoate receptors in rat brain.

Mammalian brain expresses receptors which bind the potent neurotoxins, kainate and domoate, with high affinity, and which form a subclass of ionotropic glutamate receptors. A new member of these receptors, expressed in both adult and embryonic CNS is compared in its ligand binding properties to its closely sequence-related homologs.     

Studies on the characterization of rat prostate androgen receptors

Summary Choline used as the sole carbon or carbon and nitrogen source induces in Pseudomonas aeruginosa an active transport system. The induction of the choline uptake is repressed by succinate independently of the presence of ammonium ion in the culture medium. The repression mediated by succinate was insensitive to cyclic AMP. Substitution for dibutyryl-cyclic AMP was without effect. Choline metabolites that also support the growth of Pseudomonas aeruginosa were poor inducer agents of the choline transport. Kinetic evidence and the employment of choline metabolites as effectors indicated that the choline uptake system of this bacterium is formed by at least two components: one of high affinity (Km=3 µM) and another of low affinity (Km=400 µM). Contrary to what occurs in the synaptosome system, the high affinity form for the choline uptake was not dependent on Na⁺ ions and is not inhibited by hemicholinium-3. Since Pseudomonas aeruginosa can utilize choline as the sole carbon and nitrogen source, the induction of the choline transport with two components in this bacterium may be related to its own strategy to survive and grow in an adverse environment.     

Co-localization of brain corticosteroid receptors in the rat hippocampus.

New developments in corticosteroid receptor research enabled us to perform a highly detailed study on the neuroanatomical topography of MR and GR in the rat hippocampus. Receptor immunocytochemistry was used to map the distribution of GR protein with the help of a monoclonal antibody raised against the purified rat liver GR-hormone complex. Furthermore, in situ hybridization with 35S-labeled RNA probes, which were transcribed from cDNAs complementary to either a fragment of the rat brain MR gene or to the rat liver GR gene, was applied to investigate the localization of MR and GR mRNA in the limbic brain. The pyramidal neurons of cell field Ca1 and CA2 and the granular neurons of the dentate gyrus showed marked GR immunoreactivity (GRir) as well as intense labeling of GR mRNA. The radiolabeled density of GR mRNA in cell fields CA3 and CA4 was considerable less, whereas low-to-almost-undetectable levels of GRir could be observed in these regions. MR mRNA appeared to be evenly distributed over all cell fields of the hippocampus and the dentate gyrus. The topography of GRir, GR mRNA and MR mRNA was found to agree with the cellular distribution of MR and GR binding sites in the hippocampus. Moreover, the microanatomy of MR and GR in the hippocampus appeared to overlap. Our data strongly suggest that MR and GR are co-expressed in the majority of pyramidal and granular neurons of the hippocampal formation. This assumption is based on coherence in the detection of different aspects of the receptor cycle of MR and GR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical demonstration of GABAB receptors in the rat gastrointestinal tract

Immunohistochemical localization of GABA_B-receptors was demonstrated in the rat gastrointestinal tract using a monoclonal antibody (GB-1) raised against the purified GABA_B-receptor. Immunoreactive staining for GABA_B-receptors was found in some populations of endocrine, muscular and neuronal components in the stomach and gut wall. Positive mucosal epithelial, probably endocrine, cells were distributed throughout the stomach and intestine. Double immunostaining indicated that such positive cells for GABA_B-receptors often co-possessed serotonin in the small intestine but not in the gastric body. In the muscular layer of the digestive canal, positive staining was seen as dotty granules punctuated on the surface of muscle fibers. In the enteric nervous system, positive neuronal somata were found in both submucosal and myenteric ganglia throught the entire canal extending from the stomach to the rectum. This is the first report to visualize the cellular localization of GABA_B-receptors in the gastrointestinal system of the rat, and should provide a fundamental basis for future studies on gastrointestinal functions regulated by GABA_B-receptors. Special issue dedicated to Dr. Kinya Kuriyama.