Madhabananda Sar

The Human Glucocorticoid Receptor Isoform EXPRESSION, BIOCHEMICAL PROPERTIES, AND PUTATIVE FUNCTION

Alternative splicing of the human glucocorticoid receptor (hGR) primary transcript produces two receptor isoforms, hGRα and hGRβ, which differ at their carboxyl termini. The hGRα isoform conveys endocrine information to target tissues by altering patterns of gene expression in a hormone-dependent fashion. In contrast to hGRα, very little is known about the hGRβ splice variant. Using hGRα- and hGRβ-specific riboprobes on human multiple tissue Northern blots, we show that the hGRβ message has a widespread tissue distribution. We also prove by reverse transcriptase-polymerase chain reaction that the alternative splicing event underlying the formation of the hGRβ message occurs in these tissues. Because the hGRβ protein differs from hGRα at the extreme COOH terminus, we investigated several of the biochemical properties of hGRβ expressed in transfected cells. hGRβ does not bind the glucocorticoid agonist dexamethasone nor the glucocorticoid antagonist RU38486 in vivo. Moreover, in contrast to hGRα, hGRβ is located primarily in the nucleus of transfected cells independent of hormone administration. Finally, in the absence of hGRα, hGRβ is transcriptionally inactive on a glucocorticoid-responsive enhancer. However, when both isoforms are expressed in the same cell, hGRβ inhibits the hormone-induced, hGRα-mediated stimulation of gene expression. Thus, hGRβ potentially functions as a dominant negative inhibitor of hGRα activity.

Fetal testosterone insufficiency and abnormal proliferation of Leydig cells and gonocytes in rats exposed to di(n-butyl) phthalate

Adult male rats previously exposed on gestation days (GD) 12-21 to di(n-butyl) phthalate (DBP) have reproductive tract malformations, particularly agenesis of the epididymis, decreased sperm production, and Leydig cell hyperplasia and adenomas. Although similar effects are produced by the potent androgen receptor (AR) antagonist flutamide and are indicative of disruption of male sexual differentiation via an antiandrogenic mechanism, DBP is not an AR antagonist. The purpose of the study was to determine whether DBP causes pathologic changes and alterations in androgen status in the testis during the prenatal period of male reproductive tract differentiation. Pregnant CD rats were given corn oil, DBP (500 mg/kg/day), or flutamide (100 mg/kg/day) p.o. on GD 12-21. At GD 16-21, DBP caused hyperplasia of Leydig cells, many of which were 3beta-hydroxysteroid dehydrogenase- and/or AR-positive. Focal areas of hyperplasia had increased numbers of Leydig cells positive for proliferating cell nuclear antigen (PCNA). At GD 21, testis atrophy was apparent, seminiferous cords in DBP-exposed fetuses were enlarged and contained multinucleated gonocytes that, unlike controls, were PCNA-positive. DBP, but not flutamide, markedly decreased testicular testosterone levels at GD 18 and 21. Fewer epididymal ducts and reduced AR staining in some ducts were evident with DBP treatment, whereas decreased overall AR staining was seen with flutamide in the presence of mild Leydig cell hyperplasia. Leydig cell proliferation is likely a compensatory mechanism to increase testicular steroidogenesis triggered by testosterone insufficiency. The overall decrease in androgen concentration is not corrected and results in reproductive tract malformations. The multinuclearity and proliferation of gonocytes suggests an underlying Sertoli cell dysfunction.

Differential Expression of Estrogen Receptor-β and Estrogen Receptor-α in the Rat Ovary

Immunohistochemical localization of two estrogen receptor (ER) subtypes, ERβ and ERα, was performed in neonatal, early postnatal, immature, and adult rats to determine whether ERα and ERβ are differentially expressed in the ovary. ERβ and ERα were visualized using a polyclonal anti-ERβ antibody and a monoclonal ERα (ID5) antibody, respectively. Postfixed frozen sections and antigen-retrieved paraffin sections of the ovary revealed nuclear ERβ immunoreactivity (IR) in granulosa cells, which was prevented when peptide-adsorbed antibody was used instead. In immature and adult rat ovaries, ERβ was expressed exclusively in nuclei of granulosa cells of primary, secondary, and mature follicles. Atretic follicle granulosa cells showed only weak or no staining. No specific nuclear ERβ IR was detected in thecal cells, luteal cells, interstitial cells, germinal epithelium, or oocytes. In neonatal rat ovary, no ERβ expression was found. In ovaries of 5- and 10-day-old rats, weak ERβ IR was observed in granulosa cells...

Brainstem catecholamine neurons are target sites for sex steroid hormones

Sex steroid hormones and catecholamines have physiological interactions in the brain. By the combined use of autoradiography and fluorescence histochemistry, steroid hormone target sites and catecholamine neurons were visualized simultaneously in the same tissue preparation. By this dual localization method, [3H]estradiol and [3H]dihydrotestosterone target sites were identified in nuclei of many catecholamine cell bodies in the brainstem, and catecholamine nerve terminals were observed near certain steroid hormone target neurons. These results suggest close anatomical interrelations between steroid hormone sites of action and catecholamine sites of production and action in the brain.

The Androgen Receptor: An Overview

Publisher Summary This chapter provides an overview of the androgen receptor (AR). AR is a ligand-activated transcriptional regulatory protein that mediates androgen-induced male sexual development and function. The chapter presents a schematic diagram that highlights the major functional domains of human AR. The centrally located DNA-binding domain contains nine highly conserved Cys residues common to the family of steroid receptors. This arrangement of Cys residues led to the confirmation of the requirement for zinc in the tertiary structure of this region of steroid receptors. It was recently noted that the AR protein is significantly stabilized by androgen binding. Pulse-chase experiments using 35S-labeled AR in transiently transfected monkey kidney COS cells showed rapid degradation of receptor in the absence of androgen (t1/2 = 1 hour at 37°C) or in the presence of nonandrogenic hormones. Androgen addition increased the AR protein half-life to 6 hours at 37°C. The sixfold androgen-induced stabilization of AR protein contrasts androgen-induced down-regulation of AR mRNA in tissues of the male reproductive tract.

Targeted Disruption of the Estrogen Receptor-α Gene in Female Mice: Characterization of Ovarian Responses and Phenotype in the Adult1

Targeted disruption of the mouse estrogen receptor-α gene (estrogen receptor-α knockout; ERKO) results in a highly novel ovarian phenotype in the adult. The ERKO mouse model was used to characterize ERα-dependent processes in the ovary. Visualization of the ovaries of 10-, 20-, and 50-day-old wild-type (WT) and ERKO mice showed that the ERKO phenotype developed between 20 and 50 days of age. Developmental progression through the primordial, primary, and antral follicle stages appeared normal, but functional maturation of preovulatory follicles was arrested resulting in atresia or in anovulatory follicles, which in many cases formed large, hemorrhagic cysts. Corpora lutea were absent, which also indicates that the normal biochemical and mechanical processes that accomplish ovulation were compromised. Northern and ribonuclease protection analyses indicated that ERKO ovary FSH receptor (FSHR) messenger RNA (mRNA) expression was approximately 4-fold greater than in WT controls. Ovarian LH receptor (LHR) mRNA ...

Gene Expression Profiling Following In Utero Exposure to Phthalate Esters Reveals New Gene Targets in the Etiology of Testicular Dysgenesis

Abstract Male reproductive tract abnormalities associated with testicular dysgenesis in humans also occur in male rats exposed gestationally to some phthalate esters. We examined global gene expression in the fetal testis of the rat following in utero exposure to a panel of phthalate esters. Pregnant Sprague-Dawley rats were treated by gavage daily from Gestational Days 12 through 19 with corn oil vehicle (1 ml/kg) or diethyl phthalate (DEP), dimethyl phthalate (DMP), dioctyl tere-phthalate (DOTP), dibutyl phthalate (DBP), diethylhexyl phthalate (DEHP), dipentyl phthalate (DPP), or benzyl butyl phthalate (BBP) at 500 mg/kg per day. Testes were isolated on Gestational Day 19, and global changes in gene expression were determined. Of the approximately 30 000 genes queried, expression of 391 genes was significantly altered following exposure to the developmentally toxic phthalates (DBP, BBP, DPP, and DEHP) relative to the control. The developmentally toxic phthalates were indistinguishable in their effects on global gene expression. No significant changes in gene expression were detected in the nondevelopmentally toxic phthalate group (DMP, DEP, and DOTP). Gene pathways disrupted include those previously identified as targets for DBP, including cholesterol transport and steroidogenesis, as well as newly identified pathways involved in intracellular lipid and cholesterol homeostasis, insulin signaling, transcriptional regulation, and oxidative stress. Additional gene targets include alpha inhibin, which is essential for normal Sertoli cell development, and genes involved with communication between Sertoli cells and gonocytes. The common targeting of these genes by a select group of phthalates indicates a role for their associated molecular pathways in testicular development and offers new insight into the molecular mechanisms of testicular dysgenesis.

[13] Autoradiographic techniques for localizing steroid hormones

Publisher Summary Autoradiography is one of the most sensitive techniques for the localization of hormones or other substances in tissue structures or compartments. This is attributable to the use of radioisotopic labels and the photographic exposure-related amplification. The technique is so sensitive that the number of tagged molecules occupying an individual cell or even a subcellular organelle can be determined, without disrupting the topographic relationships of the tissue components. In general, slices or surfaces of intact specimens containing the label are used and exposed to radiation sensitive material, and subsequently, the localized radiation effect on the photographic film and the histological structure can be viewed simultaneously. Thus, information can be gained through autoradiography, which may not be obtainable otherwise, provided the technical approach used did not betray the authenticity of the information gleaned from the object. In autoradiography two major objectives exist—to obtain useful localization and interpret the meaning of the localization. In the case of steroids and similar small molecular weight substances, which are not covalently bound to macromolecules, loss and redistribution easily occur during tissue preparation and photographic emulsion application. Special prerequisites have been established for the autoradiography of diffusible substances (a) to exclude all solvents and fluids for the histological treatment of the tissue or (b) use a fixative, which when penetrating into the tissue does not translocate the labeled molecules and tissue constituents but immobilizes the hormones at their original sites by specific linkage among tissue components, fixatives, and hormones or by trapping of the hormone to such a degree so as to prevent loss and translocation during tissue treatment subsequent to fixation.

Steroid hormone target sites in the brain: The differential distribution of estrogen, progestin, androgen and glucocorticosteroid

Abstract Nuclear concentration of steroid hormones or their metabolites exists in neurons in selective areas of the mammalian brain. The distribution of estrogen target cells in different mammalian species appears to follow a similar pattern that involves selective nuclear groups in the forebrain, midbrain and lower brainstem. The distribution of androgen target cells in the rat brain largely corresponds to the distribution of estrogen target cells, with differences existing, however, in certain structures, such as lateral septum, ventromedial hypothalamus, hippocampus, cortex, epithalamus, lower brainstem and spinal cord. The distribution of progestin in the guinea-pig hypothalamus seems restricted to the preoptic and infundibular region, where it overlaps with estrogen localization. Corticosterone target cells are accumulated in extrahypothalamic regions, and not in the hypothalamus, in contrast to the sex steroid. The synthetic “glucocorticosteroid” dexamethasone, however, is found in neurons and glial cells throughout the rat brain, differing from the natural glucocorticosteroid corticosterone. Localization of different hormones in neurons of identical regions suggests that the same neuron may be addressed by different hormones. On the other hand, the differential distribution of the various types of hormones is likely to reflect differences in their action. Both estrogens and androgens appear to act on neural structures that are identical to or closely associated with (1) various sensory pathways and (2) ventricular recess organs, constituting the periventricular gland. Androgens, in addition, stimulate selectively neurons of the somatomotor system and circuits of aggression. In general, the wide existence of hormone target cells in the central nervous system reflects the multiple actions of the steroid hormones on endocrine regulation, autonomic functions and behavior.

Distribution of androgen target cells in rat forebrain and pituitary after [3h]-dihydrotestosterone administration

The distribution of androgen concentrating cells in male rat forebrain and pituitary was studied by autoradiography. One h after injection of a non-aromatizable androgen [3H]-dihydrotestosterone (17β-hydroxy-5α-androstan-3-one) (DHT), nuclear concentration of radioactivity is found in neurons at specific hypothalamic and extrahypothalamic sites as well as in certain anterior pituitary cells. In the forebrain radioactively labeled cells are found in certain nuclei of the septum, preoptic region, anterior and central hypothalamus, mammillary body, epithalamus, amygdala and hippocampus. Furthermore, nuclear labeling is observed in ventricular recess organs, including the pituitary, the pineal and the subfornical organ. Competition studies with unlabeled estrad-17β at a dose similar to the labeled DHT has no or little effect on the nuclear uptake of radioactivity in neurons, whereas a dose of estradiol-17β ten times higher than the dose of the labeled DHT reduces the nuclear uptake in certain neuronal groups by 20–25%. Unlabeled DHT inhibits nuclear uptake of radioactivity. The autoradiographic results suggest that DHT is one of the major active metabolites responsible for the central action of testosterone.