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M Sar

Publications and source records attributed to M Sar.

At least 55 records · Page 3Linked to original sources
PubMed1992

Androgen receptor phosphorylation, turnover, nuclear transport, and transcriptional activation. Specificity for steroids and antihormones.

Nuclear transport, phosphorylation, ligand binding, and degradation rate of the recombinant androgen receptor (AR) were analyzed in transfected COS cells in the presence of various steroids and antiandrogens. Transcriptional activation was assessed in CV1 cells by cotransfection with an androgen-responsive chloramphenicol acetyltransferase (CAT) reporter vector. Hormone binding specificity of recombinant AR was essentially identical to endogenous AR. AR localized in the nucleus in the presence of methyltrienolone (R1881, a synthetic androgen), dihydrotestosterone, testosterone, hydroxyflutamide, cyproterone acetate, estradiol, progesterone, and RU486. In the absence of hormone or with the antiandrogen, flutamide, AR remained largely in the cytoplasm with a perinuclear distribution. AR was degraded rapidly (t1/2 = 1 h) except in the presence of androgen (t1/2 = 6 h) which accounted for an apparent 2-4-fold androgen-induced increase in AR phosphorylation, indicating that AR phosphorylation was not enhanced by androgen. CAT activity was stimulated by R1881, dihydrotestosterone, testosterone, cyproterone acetate, estradiol, progesterone, and RU486 in a dose-dependent manner. The antiandrogens, flutamide and hydroxyflutamide, lacked agonist activity and inhibited R1881-induced activation of CAT and androgen stabilization of AR. Steroids and antiandrogens with moderate to low affinity for AR promoted both nuclear transport and transcriptional activation but only at high hormone concentrations. Hydroxyflutamide acted as a true antiandrogen since it lacked agonist activity and was an inhibitor of androgen-induced transcriptional activation.

Androgen Antagonists↗
PubMed1992

Methotrexate-induced overexpression of functional glucocorticoid receptors in Chinese hamster ovary cells.

We have used a modified cotransfection and selection strategy to create a series of mammalian cell lines that stably express high levels of intact glucocorticoid receptors. These cell lines were produced by subjecting Chinese hamster ovary (CHO) cells, which had been previously cotransfected with a glucocorticoid-responsive dihydrofolate reductase (DHFR) gene and the human glucocorticoid receptor gene, to growth in increasing concentrations of methotrexate (MTX). By linking the MTX selection process to glucocorticoid receptor function via the DHFR gene, stable cell lines resistant to a range of MTX concentrations (50 nM to 3 microM) were isolated that were strictly dependent upon glucocorticoids for growth. Quantitation of steroid binding capacity in MTX-resistant cells revealed a progressive increase in the number of glucocorticoid receptors as a function of increasing MTX concentration. This increase in receptor content was maximal at the highest level of MTX resistance examined (3 microM MTX) and represented a 25-fold elevation in glucocorticoid receptor number relative to CHO cells expressing only endogenous hamster receptor. The increases in steroid binding obtained after MTX selection were reflected by similar increases in the level of glucocorticoid receptor protein as determined by immunoblot analysis. Examination of glucocorticoid receptor structure by sucrose density gradient centrifugation revealed that oligomeric (9 S) steroid receptor complexes were formed at all levels of receptor expression. Subcellular localization of the glucocorticoid receptor protein by immunocytochemical staining revealed effective nuclear translocation of the overexpressed receptors in MTX-resistant cells. Functional transfection studies using a glucocorticoid-responsive reporter gene indicated that the additional glucocorticoid receptors in CHO cells were competent to activate transcription. To determine the molecular basis for the MTX-induced increases in functional glucocorticoid receptors, steady-state levels of glucocorticoid receptor mRNA were examined. MTX selection produced a 5- to 7-fold increase in transfected glucocorticoid receptor gene expression relative to untreated cells. MTX-resistant cells also expressed increased levels of a putative hamster glucocorticoid receptor mRNA species. Interestingly, the observed increases in receptor gene expression in these cells could not be accounted for by amplification of either the human or the hamster glucocorticoid receptor genes.

Animals↗
PubMed1992

Domain functions of the androgen receptor.

Molecular cloning of the androgen receptor cDNA has facilitated analysis of structure/function relationships of this ligand activated transcription factor. Amplification of mutant androgen receptor DNA using the polymerase chain reaction has revealed single base and deletion mutations in the androgen receptor gene that cause the androgen insensitivity syndrome in rats and humans. Site directed mutagenesis of the NH2-terminal and hinge regions indicates specific sequences required for full transcriptional activation and nuclear targeting of the androgen receptor. Finally, transient transfection systems have shown that the antiandrogen cyproterone acetate is both an agonist and antagonist, while hydroxyflutamide acts only as an antagonist and thus is a pure antiandrogen.

Androgen Antagonists↗
PubMed1992

Progestin target cell distribution in forebrain and midbrain regions of the 8-day postnatal mouse brain.

The present study investigated the anatomical distribution of progestin target cells throughout the forebrain and midbrain regions of the 8-day postnatal female mouse. Female ICR mice were sc injected with 100 micrograms/100 g BW estradiol valerate on postnatal day 5 (birth = day 0). On postnatal day 8, treated mice were sc injected with 0.32 micrograms/100 g BW (Z)-17 beta-hydroxy-17 alpha-(2-[125I]iodovinyl)4-estren-3-one ([125I] progestin). For competition, additional estrogen-treated mice were each injected with 320 micrograms R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione; a potent synthetic progestin), 320 micrograms dihydrotestosterone, or 32, 160, or 320 micrograms corticosterone 1 h before [125I]progestin to show the specificity of [125I]progestin for the progestin receptor. Two hours after injection of [125I]progestin, the brains were removed, frozen, and processed for high resolution thaw-mount autoradiography. After 8-60 days of exposure, nuclear uptake and retention of [125I]progestin were detected in many brain regions, including the septum; bed nucleus of the stria terminalis; and preoptic area, periventricular nucleus, ventromedial nucleus, arcuate nucleus, and dorsomedial nucleus of the hypothalamus. In addition, labeling was seen in the cerebral cortex, caudate putamen, hippocampus, amygdala, and substantia nigra. Competition studies showed that excess R5020 prevented nuclear concentration of ligand, while dihydrotestosterone and corticosterone did not. The results indicate that the distribution of progestin target cells in extrahypothalamic regions of the developing brain is more extensive than that in the adult, while a similar topography was seen in the preoptic area and hypothalamus. The results further suggest that progestin action during brain development may influence the growth and development of target cells not only in the hypothalamus but also in regions of the brain previously not considered to be sites of hormone action.

Animals↗
PubMed1992

Single base mutations in the human androgen receptor gene causing complete androgen insensitivity: rapid detection by a modified denaturing gradient gel electrophoresis technique.

Mutations of the human androgen receptor (AR) gene impair normal sexual differentiation and development in karyotypic males, resulting in a spectrum of external genital phenotypes ranging from complete female to nearly complete male. Identification and characterization of these mutations can provide valuable information regarding the functional importance of specific amino acids of the AR. To screen for point mutations in the AR gene underlying the phenotypic abnormalities in the androgen insensitivity syndrome (AIS), the eight exons of the AR gene were amplified from genomic DNA using the polymerase chain reaction (PCR) and analyzed by denaturing gradient gel electrophoresis. A computer program, MELTMAP, was used to identify optimum sequences for denaturing gradient gel electrophoresis, and mutation detection sensitivity was enhanced by forming heteroduplexes between control and subject PCR products to create base mismatches. In seven families with complete AIS, single base mutations were found in the region of the AR gene encoding the steroid-binding domain of the receptor. The mutations that converted amino acid 774 from Arg to His and amino acid 864 from Asp to Gly were recreated using site-directed mutagenesis and the mutant ARs expressed in COS 7 and CV1 cells. In both cases, abnormalities of androgen binding and transcriptional activation were consistent with the observed sex phenotype. These results together with others reported previously demonstrate that single amino acid changes within the region encoded by exons D to H of the AR gene can alter androgen binding and are a common cause of complete androgen resistance. The strategy used herein, employing denaturing gradient gel analysis of heteroduplex PCR products, provides a valuable aid to rapid detection of single base mutations in AIS.

Adolescent↗
PubMed1992

Ligand-dependent down-regulation of stably transfected human glucocorticoid receptors is associated with the loss of functional glucocorticoid responsiveness.

The effect of glucocorticoids on the regulation of stably transfected human glucocorticoid receptors has been examined. Exposure of a Chinese hamster ovary-derived cell line containing stably transfected human glucocorticoid receptor genes and glucocorticoid-responsive dihydrofolate reductase genes to 5 nM dexamethasone resulted in a rapid, time-dependent reduction in the level of glucocorticoid receptor protein to 50% of control levels within 5 h of steroid treatment. This decrease in receptor protein was persistent, with a maximal 70% reduction observed even after 4 weeks of dexamethasone treatment. Immunocytochemical analysis of the influence of dexamethasone on stably transfected glucocorticoid receptors revealed efficient translocation of receptors to the nucleus within 1 h of hormone treatment. However, upon longer exposure to dexamethasone (5 h), immunoreactive glucocorticoid receptors were localized primarily to the cytoplasm. By 24 h of treatment, glucocorticoid receptors were absent from the cytoplasm and the nucleus, suggesting that the ligand-induced loss of glucocorticoid receptors may be a cytoplasmic event. The decrease in transfected glucocorticoid receptor protein was largely reflected by similar changes in steady state levels of human glucocorticoid receptor mRNA; however, the effects of hormone on receptor protein levels were more profound than on receptor mRNA. There was an initial rapid reduction in transfected glucocorticoid receptor mRNA to 50% of control levels within 2 h of dexamethasone treatment. This reduction was followed by a transient rise in mRNA expression after 12 h of hormone treatment. With prolonged exposure to dexamethasone (> 12 h) a second, more gradual decline in human glucocorticoid receptor mRNA was observed. This biphasic pattern of glucocorticoid receptor gene expression was not reflected at the level of receptor protein, suggesting that both transcriptional and translational control mechanisms may be involved in ligand-dependent receptor regulation. When cells were removed from dexamethasone after up to 48 h of treatment, glucocorticoid receptor mRNA levels fully recovered within 12 h. Receptor protein recovered only partially during this same time period. Down-regulation of glucocorticoid receptor protein and mRNA levels by dexamethasone in stably transfected cells led to corresponding reductions in the hormone sensitivity to two glucocorticoid-regulated genes: a transiently transfected chloramphenicol acetyltransferase receptor gene and a stably integrated dihydrofolate reductase gene. These results demonstrate that stably transfected human glucocorticoid receptors are subject to ligand-induced down-regulation in a heterologous cell line. Moreover, glucocorticoid receptor autoregulation appears to be a highly conserved mechanism for attenuating cellular responsiveness to hormone.

Animals↗
PubMed1992

Complete androgen insensitivity due to deletion of exon C of the androgen receptor gene highlights the functional importance of the second zinc finger of the androgen receptor in vivo.

Androgen-dependent gene transcription is mediated by the androgen receptor (AR) through interaction of its central zinc finger region with specific DNA sequences on target genes. Failure of this receptor-mediated gene transcription results in end organ resistance to androgens-the androgen insensitivity syndromes. In a pair of siblings with complete androgen insensitivity who had supranormal levels of androgen binding in genital skin fibroblasts, polymerase chain reaction and Southern blot analysis of the androgen receptor gene confirmed by polymerase chain reaction and sequence analysis of AR cDNA, revealed an in-frame deletion of exon C encoding the second zinc finger of the receptor. The mutant receptor in cultured genital skin fibroblasts had normal androgen binding affinity and was localized in the nucleus but had markedly reduced DNA-binding affinity. When recreated in vitro and tested in a cotransfection assay system the mutant receptor failed to activate transcription of an androgen-responsive reporter gene. This naturally occurring mutation highlights the functional dependence of the AR upon its second zinc finger in vivo and explains the complete insensitivity to androgen manifest by the affected individuals despite increased androgen binding. The elevated AR levels in the subjects' genital skin fibroblasts further suggests a possible role for the second zinc finger in autoregulation of receptor levels in vivo.

Androgen-Insensitivity Syndrome↗
PubMed1991

Progestin receptor cells in mouse cerebral cortex during early postnatal development: a comparison with preoptic area and central hypothalamus using autoradiography with [125I]progestin.

The distribution of progestin target cells in the cerebral cortex and the effect of estrogen treatment was assessed during the critical period of brain development and compared with the preoptic/central hypothalamic regions. [125I]progestin was injected into 0, 2, 8, and 12 day postnatal mice pretreated for 3 days with oil, 5 micrograms/100 g b, wt., or 100 micrograms/100 g b. wt. of estradiol dissolved in oil. Two hours after injection of radiolabeled ligand, brains were frozen and processed for thaw-mount autoradiography. At birth, labeled cells were detected in the deep (lamina VI) and intermediate (lamina V) layers of the lateral cortical regions, increased in laminae V-VI of the lateral cortex and laminae II-VI of the cingulate/paracingulate cortex at days 2 and 8, and decreased throughout the cortex by day 12. Pretreatment of animals with estradiol had no noticeable effect on the nuclear concentration of [125I]progestin in cortical cells, while estrogen weakly enhanced labeling in preoptic/central hypothalamic regions at day 2 and markedly augmented labeling in the 8 and 12 day brain. The results demonstrated that progestin receptor cells are present in the postnatal dorsal cortex, preoptic area, and hypothalamus and that the topography of cortical progestin target cells differs in part from that of estrogen target cells reported earlier.

Animals↗
PubMed1991

Transcriptional activation and nuclear targeting signals of the human androgen receptor.

The androgen receptor (AR) is a signal-transducing protein required for sexual differentiation, development, and expression of the male phenotype. A series of human AR deletion mutants were created either by site-directed mutagenesis using restriction enzyme digestion, the polymerase chain reaction, or, for a series of unidirectional NH2-terminal deletions, exonuclease III digestion. Receptor mutants were expressed in monkey kidney COS cells as truncated AR proteins between 20 and 107 kDa as revealed on immunoblots, where wild type AR was a doublet of 114 and 108 kDa. Subcellular localization by immunocytochemical staining demonstrated androgen-dependent nuclear uptake of AR from a perinuclear region of the cytoplasm. A nuclear targeting signal similar in sequence and position to the glucocorticoid receptor and homologous to the SV40 large T antigen was required for androgen-induced nuclear uptake of wild type AR. AR mutants lacking the NH2-terminal and/or steroid binding domains were constitutively nuclear with reduced transcriptional activity. Transcriptional activation by wild type AR was androgen-dependent in cotransfection studies of CV1 cells using the chloramphenicol acetyltransferase reporter gene linked to the mouse mammary tumor virus promoter. Deletion mutagenesis revealed within the NH2-terminal region a domain required for full transcriptional activity and within the steroid binding domain, an inhibitory function, deletion of which yielded a constitutively active receptor. Inhibition of wild type AR by coexpression with an inactive NH2-terminal fragment suggested competition for nuclear factors required for transcriptional regulation. These studies demonstrate a concerted interplay among the domains of the AR protein in regulating gene transcription.

Amino Acid Sequence↗
PubMed1991

Progestin receptor cells in the 8-day-old male and female mouse cerebral cortex: autoradiographic evidence for a sexual dimorphism in target cell number.

The present study examined the number and distribution of progestin receptor cells in the 8-day-old male and female cortex and compared cortical labeling with that in the preoptic area and central hypothalamus. Eight-day postnatal mice (four males and four females), treated with estradiol, were each sc injected with 0.32 micrograms/100 g BW [125I]progestin (SA, 2200 Ci/mM). Brains were frozen 2 h after injection of [125I]progestin, sectioned, and processed for thaw-mount autoradiography. Cells with a nuclear concentration of radioactivity were localized in lamina VI of the lateral cortical regions of the male and female brain, while only a few cortical cells were seen in laminae II, III, and V of the suprarhinal, lateral, and cingulate/paracingulate regions. Comparison of the number of labeled cells revealed that the female cortex contained significantly more labeled cells than the male at three of the four levels investigated. Similarly, the number of target cells was higher in the female medial preoptic nucleus, but not in the arcuate nucleus and ventromedial hypothalamic nucleus, while the distributions of labeled cells in the male and female preoptic/hypothalamic regions were comparable. Injection of unlabeled progesterone or R5020 1 h before [125I]progestin reduced the nuclear concentration of radioactivity in all target regions and verified the specificity of [125I]progestin for the progestin receptor. The results of these studies indicate that mouse 8-day-old cortex and preoptic area in the female animal have more progestin receptor cells than those in the male and demonstrate that progestin receptor cells are localized in a region of the cortex known to contain few estrogen target cells. These results further suggest that a sexual dimorphism in progestin cell number may result in a differential effect of progestin on the cortex and preoptic area of the mouse, perhaps establishing a dimorphism in development and function.

Animals↗
PubMed1991

Immunocytochemical localization of the glucocorticoid receptor in rat brain, pituitary, liver, and thymus with two new polyclonal antipeptide antibodies.

The intracellular localization of the glucocorticoid receptor (GR) was studied in male rat brain, pituitary, liver, and thymus. Two new polyclonal anti-GR antibodies, GR 57 and GR 59, raised against two synthetic peptides (346-357 and 245-259) that correspond to unique regions of the amino-terminus of human GR were used. Vibratome sections (30-50 microns) of perfused brain and frozen sections (6-8 microns) of pituitary, liver, and thymus fixed in paraformaldehyde were incubated in preimmune serum, immunoserum, epitope-purified immunoserum, or peptide-absorbed immunoserum of either GR 57 or GR 59 and immunostained by the avidin-biotin peroxidase method. GR immunoreactivity (GR-ir) was primarily nuclear in brain, pituitary, liver, and thymus sections from intact rats. Adrenalectomy caused nuclear GR-ir to decrease and cytoplasmic GR-ir to increase. When adrenalectomized rats were treated with corticosterone (100 micrograms and 1 mg) or dexamethasone (1 microgram, 100 micrograms, and 1 mg), GR-ir was again predominantly nuclear. One microgram of corticosterone failed to cause nuclear GR-ir when administered to adrenalectomized rats. Immunoreactive neurons and glial cells were extensively distributed, with varied intensity, throughout the rat forebrain. The areas include cortex, septum, hippocampus, amygdala, thalamus, and hypothalamus. Cells with the strongest GR-ir were located in the caudate putamen, paraventricular, arcuate, and central amygdala nuclei, areas CA1-CA2 of the hippocampus, and laminae 4 and 5 of the cortex. In the pituitary, cells of the anterior and posterior lobes were GR immunoreactive, while those in the intermediate lobe were not. Hepatocytes of the liver and thymocytes and reticuloepithelial cells of the thymus were GR immunoreactive. The results show that GR can be localized immunocytochemically in numerous rat tissues using antipeptide polyclonal antibodies and correlated with the results of biochemical and ligand receptor studies.

Adrenalectomy↗
PubMed1990

A single base mutation in the androgen receptor gene causes androgen insensitivity in the testicular feminized rat.

The complete form of androgen insensitivity is an inherited X-linked syndrome in which genetic males fail to undergo masculinization in utero due to defective functioning of the androgen receptor (AR). The molecular basis of androgen insensitivity was investigated in the testicular feminized (Tfm) rat with this syndrome. AR mRNA size and amount, as well as nuclear AR protein revealed by immunocytochemistry, suggested normal expression of the AR gene in the Tfm rat. Sequence analysis of the AR coding region from Tfm and wild-type littermate male rats revealed a single transition mutation, guanine to adenine, within exon E, changing arginine 734 to glutamine within the steroid-binding domain of the AR. This arginine is highly conserved among the family of nuclear receptors and may be part of a phosphorylation recognition site. A recreated mutant AR (Arg734----Gln) expressed in COS cells had only 10-15% of the androgen-binding capacity of wild-type AR; the reduced androgen-binding capacity was similar to that of AR in tissue extracts of the Tfm rat. Stimulation of transcriptional activity by the recreated mutant AR was reduced relative to wild-type AR in cotransfection assays in CV1 cells using as reporter plasmid the mouse mammary tumor virus promoter linked to the chloramphenicol acetyltransferase gene. Thus, arginine 734 appears essential for normal AR function both in androgen binding and transcriptional activation. Absence of these functions results in androgen insensitivity and lack of male sexual development.

Amino Acid Sequence↗
PubMed1990

Colocalization of atrial natriuretic factor (ANF) and estradiol in hypothalamic neurons by combined autoradiography-immunohistochemistry.

The distribution of estrogen target neurons which contain atrial natriuretic factor (ANF) in female rat hypothalamus was investigated by thaw-mount auto-radiography combined with immunocytochemistry using tritium-labeled estradiol and antibodies against ANF. Colocalization of the two hormones was found in the arcuate nucleus, periventricular nucleus, lateral ventromedial nucleus, ventral premammillar nucleus and lateral basal hypothalamus. The percentage of ANF containing cells which concentrate estradiol varies among the different hypothalamic nuclei with the highest number of ANF-positive cells showing nuclear concentration of 3H-estradiol (80-90%) in the nucleus premammillaris ventralis, but less (5-15%) in the other nuclei. These data, together with topographical correspondence in extrahypothalamic brain regions between sites of action of estradiol and production of ANF, suggest extensive interrelationships and modulatory effects of estradiol on ANF production and secretion in the brain, similar to the atrium of the heart.

Animals↗
PubMed1990

Evidence for direct action of estradiol on growth hormone-releasing factor (GRF) in rat hypothalamus: localization of [3H]estradiol in GRF neurons.

Sex steroids have been shown to influence the secretion of GH. There appears to be no good evidence of the effect of estradiol on the anterior pituitary, while the central site of estradiol action on the regulation of GH secretion is not known. The present investigation was carried out to determine whether some of the GH-releasing factor (GRF) neurons and somatostatin (SRIF) neurons in the hypothalamus and GH cells in the pituitary contain estradiol receptors. Colocalization of [3H]estradiol and antibodies to GRF or SRIF in brain and antibodies to GH in pituitary was studied to show interrelationships between estrogen target cells and peptidergic cells. Eight female Sprague-Dawley rats were ovariectomized, each rat was treated with colchicine, and 24-48 h later the animals were given an iv injection of [2,4,6,7,16,17-3H]estradiol (SA, 166 Ci/mM) at a dose of 0.5 micrograms/100 g BW. One hour after the injection, the rats were perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The hypothalami from the perfused rats and the pituitaries from unperfused rats were frozen in isopentane precooled in liquid nitrogen (-190 C) and processed for autoradiography. The brain autoradiograms were immunostained for GRF, SRIF, and tyrosine hydroxylase [TH; an enzyme for the synthesis of dopamine (DA)], and the pituitary autoradiograms were immunostained for GH by the avidin-biotin peroxidase method. The majority of GRF-containing neurons were found in the arcuate nucleus, with some scattered cells in the lateral region of the ventromedial nucleus and the basal lateral hypothalamus. In the central portion of the arcuate nucleus, 20-30% of GRF-containing neurons showed nuclear concentration of [3H]estradiol. In the anterior portion of the hypothalamus, 10-15% of immunoreactive GRF-containing neurons were labeled with [3H]estradiol. In the lateral basal hypothalamus and the lateral region to the ventromedial nucleus, a few GRF neurons showed nuclear concentration of radioactivity. In contrast, a few SRIF cells in hypothalamic periventricular nucleus showed nuclear labeling with [3H]estradiol. Dual immunostaining with GRF and TH antibodies revealed that the estradiol-labeled GRF neurons did not contain TH immunoreactivity. In addition, 80-90% of GH cells in the anterior pituitary showed nuclear concentration of [3H]estradiol. The present studies demonstrate for the first time that certain populations of GRF neurons are targets for estradiol and indicate that estradiol acts directly on certain hypothalamic GRF neurons. The results suggest that estradiol may have a role in the regulation of GH secretion by modulating GRF release and acting directly on the somatotrophs.

Animals↗
PubMed1990

Localization of neuropeptide-Y immunoreactivity in estradiol-concentrating cells in the hypothalamus.

Considerable evidence shows that gonadal steroids exert a facilitatory influence on levels and release of neuropeptide-Y (NPY) from the hypothalamus. However, it is not known whether gonadal steroids act directly on NPY-producing cells in the arcuate nucleus (ARC) of the hypothalamus to produce these facilitatory effects on NPY or whether they act on other cells that have a modulatory influence via synapses on ARC NPY cells. We applied the combined method of steroid autoradiography and immunocytochemistry to assess the localization of [3H]estradiol in relation to NPY-producing cells in the hypothalamus. Rats (n = 6) were bilaterally ovariectomized and injected intracerebroventricularly with colchicine. Twenty-four hours later each rat received an iv injection of 17 beta-[2,4,6,7,16,17(-3)H]estradiol (SA, 166 Ci/mmol) at a dose of 5.0 micrograms/kg BW. One hour after the injection of [3H]estradiol, the rats were perfused with 4% paraformaldehyde; brains were removed, frozen in isopentane precooled in liquid nitrogen (-190 C), sectioned, and processed for autoradiography. The autoradiograms were then incubated with specific antibodies for NPY immunostaining by the avidin-biotin-peroxidase method. The results revealed NPY-immunopositive cells in the ARC, striatum, hippocampus, amygdala, and cerebral cortex and a few cells in the median eminence. NPY-immunoreactive fibers were also detected in the internal layer of the median eminence. The largest number of neurons showing NPY immunoreactivity in the cytoplasm was detected in the ARC, and only in this nucleus did we observed colocalization of [3H]estradiol and NPY immunoreactivity in neurons. A population of NPY-immunopositive cells in the ARC (10-20%) exhibited nuclear [3H]estradiol; the majority of these cells were located in the lateral and ventral portions of the ARC. Since gonadal steroids stimulate hypothalamic NPY levels and release, this anatomical evidence of colocalization is suggestive of a direct genomic modulation of NPY neurosecretion by steroids in a subpopulation of hypothalamic NPY-immunopositive neurons.

Animals↗
PubMed1990

Immunohistochemical localization of the androgen receptor in rat and human tissues.

Immunohistochemical localization of the androgen receptor (AR) was performed in reproductive tissues, submaxillary gland, pituitary, and brain of the rat and in human prostate. AR was visualized using either of two polyclonal antibodies raised against peptides with sequences derived from rat and human AR. Tissue sections of 6-8 microns, frozen in isopentane and fixed in paraformaldehyde, were stained using immunoglobulin G fractions of immune, preimmune, and peptide-adsorbed immune sera in the avidin-biotin peroxidase procedure. AR was prominent in nuclei of acinar epithelial cells of epididymis, ventral prostate, seminal vesicle, and ductus deferens from the intact rat. Androgen withdrawal, 3 days after castration, resulted in the loss of receptor immunostaining, which was restored within 15 min of androgen administration. Stromal cell staining was absent or weak in the ventral prostate of intact rats, but was more evident in the epididymis. AR was confined to nuclei of cells within and bordering the interstitial compartment of the testis, including Sertoli cells, peritubular myoid cells, and interstitial cells, and was undetectable in germ cells. Submaxillary gland epithelial cells and a population of rat anterior pituitary cells showed strong nuclear staining of AR. In rat brain, AR was present in the medial preoptic, arcurate, and ventromedial nuclei of the hypothalamus, the medial nucleus of the amygdala, the CA-1 hippocampus, and the cortex. AR was prominent in acinar epithelial cells in human benign prostatic hyperplasia and was also present in stroma of fibromuscular benign hyperplasia. Heterogeneous staining was observed in stromal and epithelial cells of prostatic adenocarcinoma. The results of these studies indicate that AR can be detected immunohistochemically in a variety of tissues and cell types using antipeptide polyclonal antibodies. The presence of AR in tissues correlated with their known androgen responsiveness.

Animals↗