Estrogen receptors and androgen receptors in the mammalian liver

An estrogen receptor and an androgen receptor are present in the mammalian liver. In the liver of the rat, the estrogen receptor concentration increases markedly at puberty and this change correlates with enhanced estrogen stimulation of plasma renin substrate synthesis. High doses of estrogen are required for nuclear binding in liver when compared to doses for the uterus. The high dose requirement appears to be predominantly due to extensive metabolism in the hepatocyte of the estrogen to inactive derivatives. Furthermore, estradiol is much weaker than ethinyl estradiol for promoting nuclear binding in the liver. This is due to extremely rapid and extensive metabolism of estradiol. In human liver the concentration of estrogen receptor is low. An androgen receptor is present in high concentration in rabbit liver and is located predominantly in the nucleus after androgen administration. High concentrations of a putative androgen receptor are also present in human liver cytosol. Preliminary studies indicate that synthetic progestins can attach to the human liver androgen receptor. To date, a progesterone receptor has not been found in the mammalian liver. Thus, it appears that extensive steroid metabolism in liver preferentially diminishes sex steroid interaction with liver receptors and that androgen receptors may mediate progestin effects in liver. These observations provide a scientific basis for improved safety of oral contraceptives. Lowering the estrogen and progestin doses in oral contraceptives will decrease the major side-effects, which are liver mediated, and still maintain the desired effects at the hypothalamic-pituitary axis and uterus. Furthermore, it is likely that by selecting which estrogen, progestin or androgen is administered as well as by utilizing a parenteral route of administration that sex steroid effects on the liver could be minimized.     

Growth-related changes in histology and immunolocalization of steroid hormone receptors in gonads of the immature male green turtle (Chelonia mydas)

Studies on the population dynamics of sea turtles require histological evaluation of the ontogenetic development and the activity of the gonads for reproduction. To investigate the growth-related changes of gonads in the immature male green turtle (Chelonia mydas), the histological changes of testes and epididymides and the localization of the androgen receptor, estrogen receptor alpha, estrogen receptor beta, and progesterone receptor were examined. The testes were categorized histologically into six developmental stages, and a scarce relationship between straight carapace length and gonadal development was confirmed based on the histological analysis. Several kinds of steroid hormone receptors were examined to show distributions in both testes and epididymides, for which their immunoreactivities were enhanced according to the developmental stage of the testes. These results suggest that straight carapace length is not an adequate indicator of maturity determination, whereas histological and immunohistochemical evaluations are useful in identifying the growth stages of green turtles, owing to the higher sensitivity to steroid hormones that appear during growth.     

Arachidonic acid as a possible modulator of estrogen, progestin, androgen, and glucocorticoid receptors in the central and peripheral tissues

In an attempt to learn whether modulation of steroid hormone receptor by arachidonate is generalized or not, the arachidonate effect was examined in cytosol estrogen (ER), progestin (PR), androgen (AR) and glucocorticoid receptors (GCR) from the central and peripheral tissues of rats by sucrose density gradient centrifugation, and gel filtration on LH20 columns or dextran-coated charcoal absorption. Arachidonate and other long-chain fatty acids appear to inhibit the specific binding of estrogen ([3H]R2858), progestin ([3H]R5020), androgen ([3H]R1881) and glucocorticoid ([3H]dexamethasone) to the respective receptors in brain (neonatal cerebral cortex and hypothalamus-preoptic area, HPOA), uterus and prostate, with the exception of the potentiating effect on the brain estrogen receptors. The potency of the unsaturated fatty acids paralleled to some degree the number of cis double bonds and carbon, in that oleate (C18:1) arachidonate (C20:4) docosahexaenoate (C22:6). The arachidonate inhibition was dose-dependent in the tissue steroid hormone receptors, except for dose-dependent potentiation of the brain cortical estrogen receptors. Inhibitory potency as expressed by the concentration for 50% maximum inhibition (Ki) was in the range of 11-18 microM for the receptors other than the uterine estrogen receptors with the value of 44 microM, suggesting lower sensitivity for the estrogen receptor to the arachidonate effect in the uterus. Analysis on kinetics and Scatchard plot revealed the non-competitive type of the inhibition. In addition, arachidonate lowered dose-dependently the peak of labelled progestin or estrogen binding to the 8S receptor proteins, which were collected from fractions in the 8S region of the cytosols from intact or diethylstibestrol-primed rat uteri. These results suggest the generalized modulatory effect of arachidonate on the steroid hormone receptors in the central and peripheral tissues. Arachidonate could affect, negatively or positively, the estrogen receptors, and negatively the progestin, androgen and glucocorticoid receptors, through a possibly direct but weak binding at sites different from steroid binding sites on the receptor molecules. A potential messenger role of arachidonate itself has been implicated in the regulation or modulation of the steroid hormone receptors.     

From the structure of steroid receptors to their assessment by immunocytochemistry in target cells

Immunohistochemical studies with antibodies to steroid hormone receptors provide new insight in the mechanism of action of steroid hormones. Immunologically reactive estrogen and progesterone receptors are found exclusively in cell nuclei of target cells even in the absence of the hormonal ligand. A hormonal treatment inducing receptor transformation and "translocation" to the nucleus does not modify the intracellular distribution of the receptor. This result is in contradiction with most biochemical studies which show a displacement of receptor from the cytosolic fraction to the nuclear fraction after hormone-receptor complex formation. We propose that different affinity levels of the non-transformed and hormone-complexed receptor molecules for nuclear structure produce unequal losses of nuclear receptor during homogenization. A lesser loss appears as an increase in nuclear binding sites or immunologically reactive receptor. The glucocorticosteroid receptor differs from the others in that it shows an increase of nuclear immunoreactive receptor after hormone administration. This result was accepted as evidence for a nuclear translocation in the sense initially proposed for all steroid hormones. Alternatively, one may propose another explanation based on the same experimental artefact as invoked for the estrogen and progesterone cytosol receptors. A higher affinity of the hormone-complexed receptor entails a lesser loss from the nucleus during tissue processing, and consequently an apparent increase in nuclear staining. Such a possibility is currently tested in parallel with the progesterone receptor.     

Spatial and molecular aspects of estrogen and progesterone receptor expression in human uteri and uterine carcinomas

The expression of steroid hormone receptors as molecules reflecting processes of development and differentiation in the human uterine tissue was analysed in a spatial distinct fashion: tissue samples were excised at the fundus and at different, spatially distinct positions of the uterus. They were analysed for concentrations of cytosolic estrogen and progesterone receptors in supernatants from frozen sections using an isoelectric focusing technique. The spatial and molecular distinct, qualitative and quantitative pattern of their expression in the human uterus and uterine adenocarcinomas were studied by sectioning tissue sample from the functionalis through the basalis of the endometrium until reaching deep myometrial parts of the tissue: (1) Specific spatial patterns of estrogen and progesterone receptor levels were detectable throughout the menstrual cycle. (2) For proliferative endometrium from the functionalis to the basalis of the endometrium, the content of both cytosolic receptor species increased up to 6-fold. (3) Differences detectable were less pronounced in the myometrial part of the tissue. (4) Differences of steroid receptor concentrations measured in the endometrium at different uterine positions were highest between fundus and corpus of the endometrium. (5) Maximal differences were detectable around ovulation. (6) After secretory transformation of the organ, specific patterns were still detectable, however quantitative differences were less pronounced. (7) Additionally, quantitative differences measurable were accompanied by variations of molecular properties of the progesterone receptor as demonstrated in an isoelectric focusing gel. (8) In endometrial adenocarcinomas, not only significant quantitative alterations in steroid receptor content were measured, but also a significantly changed spatial pattern of receptor concentrations, also a change of the molecular properties of the progesterone receptor was resolved if these tumor parameters were compared to those detected in the normal tissue of the same organ surrounding the tumor.     

Characterization of steroid hormone receptors with ion-exchange fast protein liquid chromatography

Androgen, estrogen and progesterone receptors have been characterized with anion exchange Fast Protein Liquid Chromatography (FPLC) on a Mono Q column (Pharmacia). In the presence of sodium molybdate androgen receptors in cytosols from rat prostate, rat epididymis and calf uterus eluted as a single sharp peak at 0.32 M NaCl with recoveries of approx 90%. The molybdate-stabilized form of the androgen receptor from rat prostate was purified about 75-fold. The receptor containing FPLC-peak fractions sedimented in high salt (0.4 M KCl) linear sucrose gradients at 3.6 S (prostate) and at 4.6 S (epididymis and calf uterus) respectively. Multiple forms of the androgen receptor were present in cytosols from rat prostate prepared in the absence of sodium molybdate, probably due to proteolytic breakdown of the native form. Calf uterine estradiol and progesterone receptors prepared in the presence of sodium molybdate (20 mM) eluted from the Mono Q column at 0.32 M NaCl. The molybdate-stabilized forms of the oestradiol and progesterone receptors were purified approx 70-fold and 30-fold respectively. In the absence of molybdate the estradiol receptor dissociated into two major forms eluting at 0.23 M NaCl and 0.37 M NaCl. After heat induced transformation (30 min at 25 degrees C) of the estradiol receptor one major peak was eluted at 0.42 M NaCl, indicating a change in the surface charge of the estradiol receptor as a result of the 4 S to 5 S transformation. It is concluded that the FPLC anion exchange system is a powerful, fast tool for characterization and partial purification of steroid receptors. In addition this technique could be applied as a rapid procedure for the quantitative estimation of steroid receptors in small biological samples.     

Medroxyprogesterone acetate: receptor binding and correlated effects on steroidogenesis in rat granulosa cells

Medroxyprogesterone acetate (MPA), a widely used synthetic steroid, was studied to determine both its effects on steroid receptors and steroidogenesis in the well-characterized rat ovarian granulosa cell model. Initial receptor binding studies showed MPA was as potent as progesterone and 10-fold less potent than R-5020 (an active synthetic progestin) in binding to progesterone cytosolic receptors in rat ovarian granulosa cells. MPA was 20-fold less potent than testosterone, and 10-fold less potent than dexamethasone in binding to the androgen and glucocorticoid cytosolic receptors, respectively. The binding of MPA to progestrone, androgen and glucocorticoid receptors predicted direct effects of MPA on FSH-stimulated estrogen (E), progesterone (P), and 20 alpha-dihydroprogesterone (DHP) production by cultured rat ovarian granulosa cells. MPA at 10(-7) to 10(-6) M significantly augmented FSH-stimulated P and DHP production (a previously documented progestin, androgen and glucocorticoid effect). This augmentation was blocked by the concurrent addition to cell culture of 10-fold excess RU-486 (a potent anti-progestin and anti-glucocorticoid). At concentrations greater than 10(-6) M, MPA inhibited the production of P and DHP (a progestin effect), and the production of E (a progestin and glucocorticoid effect). MPA, structurally a progestin, has complex steroid hormone effects predicted by its interaction with progesterone, androgen and glucocorticoid receptors.     

Neurotransmitter-controlled steroid hormone receptors in the central nervous system

Results are discussed indicating that neurotransmitters affect steroid hormone activity not only by controlling via neuroendocrine events the hypophysial-gonadal and hypophysial-adrenal axes, but also by modulating cell responsiveness to steroids in target cells. Hyper- or hypoactivity of pineal nerves result in enhancement or impairment of estradiol and testosterone effects on pineal metabolism in vivo and in vitro. Pineal cytoplasmic and nuclear estrogen and androgen receptors are modulated by norepinephrine released from nerve endings at the pinealocyte level. Neural activity affects the cycle of depletion-replenishment of pineal estrogen receptors following estradiol administration. Another site of modulation of steroid effects on the pinealocytes is the intracellular metabolism of testosterone and progesterone; nerve activity has a positive effect on testosterone aromatization and a negative effect on testosterone and progesterone 5α-reduction. NE activity on the pineal cells is mediated via β-adrenoceptors and cAMP. In the central nervous system information on the neurotransmitter modulation of steroid hormone action includes the following observations: (a) hypothalamic deafferentation depresses estrogen receptor levels in rat medial basal hypothalamus; (b) changes in noradrenergic transmission affect, via α-adrenoceptors, the estradiol-induced increase of cytosol progestin receptor concentration in guinea pig hypothalamus; (c) cAMP increases testosterone aromatization in cultured neurons from turtle brain; (d) electrical stimulation of dorsal hippocampus augments, and reserpine or 6-hydroxydopamine treatment decrease, corticoid binding in cat hypothalamus. In the adenohypophysis changes in dopaminergic input after median eminence lesions or bromocriptine treatment of rats result in opposite modifications of pituitary estrogen receptor levels. Therefore all these observations support the view that neurotransmitters can modulate the attachment of steroid hormones to their receptors in target cells.     

Influence of neonatal diethylstilbestrol treatment on androgen and estrogen receptor levels in the mouse anterior prostate, ventral prostate and seminal vesicle

Perinatal exposure to the synthetic estrogen, diethylstilbestrol (DES), affects the structure of both male and female reproductive systems. Changes may also occur in the levels of steroid hormone receptors. Cytosolic and nuclear androgen and estrogen receptor levels (expressed per mg DNA) from the sex accessory glands of male BALB/c mice exposed neonatally to DES were analyzed by exchange assays. Neonatal DES exposure caused significant decreases in: (1) cytosolic androgen and cytosolic and nuclear estrogen receptor levels in the anterior prostate and (2) cytosolic estrogen receptor levels in the ventral prostate. A significant increase was seen in the cytosolic estrogen receptor levels in the seminal vesicle. Significant decreases in cytosolic protein levels occurred in all DES-exposed glands.     

Estrogen and progesterone receptors in the ovine cervix during the postpartum period

Cervical estrogen (E) and progesterone (P) receptors were characterized and quantified during the postpartum period in Corriedale ewes lambing in the late breeding season. Cervices and uteri were collected after ovariohysterectomy at 1 d (n = 2), 5 d (n = 4), 17 d (n = 2) or 30 d (n = 2) post partum. The estrogen and progesterone receptors were measured using binding assays with tritiated hormones, dextran charcoal separation and inverse Scatchard analysis. Similar kinetic parameters in cytosolic binding sites for both hormones were found in all cervical and uterine samples, indicating that the binding protein in both tissues is of the same nature. Receptor concentrations (fmol/mg cytosolic protein) in the cervix of early (1 to 5 d, n = 6) and late (17 to 30 d, n = 4) postpartum ewes were 348 +/- 66 vs 994 +/- 145 (P < 0.05) for E and 618 +/- 126 vs 1170 +/- 201 (P < 0.05) for P, respectively. These data suggest an increased synthesis of receptors, probably due to the presence of ovarian estrogen-active follicles. Cervical E and P receptor concentrations were similar or higher than those in the uterus (1.40 +/- 0.15, n = 10 and 1.51 +/- 0.19, n = 10; for E and P respectively), and these receptor ratios did not differ between the early and late postpartum period. The high ratio between cervical/uterine receptors suggests that the ovine cervix may be a very sensitive to steroid action. In conclusion, it was shown that restoration of steroid receptors during the postpartum period in the ovine cervix is similar to receptor dynamics in the uterus, and is probably associated with the recovery of ovarian cyclicity.     

Effect of antibodies against the specific estrogen-binding protein in the rat liver on the hormone-binding activity of this protein and steroid hormone receptors

The effects of rabbit polyclonal antibodies (AB) against an unusual estrogen-binding protein (UEBP) isolated from rat liver by immunoadsorption on the interaction of [3H]estradiol with UEBP, estrogen receptors of uterus and other tissues, of [3H]dihydroxytestosterone with prostate androgen receptors, of [3H]progesterone with uterine progesterone receptors and of [3H]dexamethazone with rat thymus glucocorticoid receptors were investigated. It was shown that preincubation of cytosol of the above tissues with AB decreases the ability of UEBP, estrogen and androgen receptors to bind the corresponding ligands. The hormone-binding activity of progesterone and glucocorticoid receptors in the presence of AB remains thereby unchanged. The binding activity of UEBP in the presence of ABUEBP diminishes due to the decrease in the concentration of protein binding sites, whereas that of estrogen and androgen receptors decreases due to the diminution of affinity for their ligands which, in turn, is the result of reduction of the association rate constant. A cross influence of AB on the activity of uterine estrogen receptors of rabbit, guinea pig and mouse was observed. It was assumed that the structure of UEBP and sex steroid receptors has some similar elements.     

A comparative study of estrogen receptors alpha and beta in the rat uterus.

The uterus is an important target organ for steroid hormones. The effects of these hormones are mediated via specific receptors. The aim of this study was to compare the expression, distribution, and regulation of estrogen receptor (ER) alpha and beta in the rat uterus in order to establish possible different biological roles for the two receptor forms. Ovariectomized rats were treated with either estradiol (E(2)), progesterone (P(4)), or combinations of these for 24 or 48 h. The mRNA levels were measured by solution hybridization. Distribution of the mRNAs and receptor proteins was detected by in situ hybridization and immunohistochemistry. The results showed that ERalpha is the dominating subtype in the rat uterus. E(2) seemed to increase the ERalpha mRNA level in the glandular and luminal epithelium, but it caused a decrease of the immunostaining intensity in the glandular epithelium. P(4) reduced ERalpha expression in luminal epithelium whereas no effect was seen in the glandular epithelium. E(2) or P(4) did not alter the expression of ERbeta, on either the mRNA or protein level. In conclusion, the distribution and regulation of ERalpha and ERbeta differ in the different compartments of the rat uterus. The complex uterine responses to E(2) and P(4) are directly or indirectly mediated by differential cell-specific expression of their receptors. The low expression in the uterus and the limited regulation by gonadal steroids in this study suggest that ERbeta probably plays a minor role in the regulation of uterine physiology.     

Transcriptional regulation of the mouse calbindin-D9k gene by the ovarian sex hormone

Calbindin-D9k levels in the rat uterus are under the control of estrogen. We found that the putative estrogen response element (ERE) failed to bind to the estrogen receptor from the mouse uterus. We therefore isolated mouse genomic clones of the calbindin-D9K gene and analyzed their expression in the mouse uterus. The promoter region of the gene contained several putative steroid hormone receptor binding sites. To characterize these elements, we constructed several promoter-reporter plasmids, and transiently transfected them into T47D breast cancer cells that express both estrogen and progesterone receptors. Luciferase activity was expressed from a promoter region containing the putative progesterone response element (PRE) and expression was stimulated by progesterone. In the uterus of oophorectomized mice, the calbindin-D9k gene was up-regulated by progesterone, but not by estrogen. These results suggest that the mouse uterine calbindin-D9k gene is expressed under the control of a PRE.     

A study of cytoplasmic and nuclear estrogen and progestin receptors in gynecologic neoplasms

A rapid method for simultaneous preparation of cytosol and nuclear estrogen (E) and progestin (P) receptors and their in vitro determination is described. The method was applied to several uterine or ovarian surgical specimens to evaluate their steroid hormone "dependence". The results suggest that low cytoplasmic E receptor levels (ERc) are associated with higher nuclear E receptor (ERn) levels but no apparent correlation was observed between PRc and ERn levels. The method appeared to be suitable for screening steroid hormone receptor content in tumor tissues and may provide better estimation of steroid dependence since both cytoplasmic and nuclear compartments can be studied simultaneously.     

Preferential nuclear binding of estrogen in the formalin-fixed rat uterus

Rat uterus fixed overnight in buffered formalin retains the ability to specifically bind estradiol. However, the estrogen binding property of fixed tissue appears preferentially localized in the nuclear fraction regardless of hormonal status. Furthermore, the quantity of the nuclear estrogen receptor in fresh or fixed uterus is virtually identical in the presence or absence of estrogenic hormone. Yet, while both tissue preparations exhibit equivalent increases in the total nuclear receptor occupancy after hormone exposure, only the fresh uterus contains a major cytosolic estrogen binder which decreases in availability upon the estrogen-induced elevation of the nuclear bound steroid. However, the cytosolic estrogen receptor exhibits a significant loss in its ligand binding property after formalin exposure. Thus, the preferential localization of estrogen binding in the nuclear fraction of fixed whole tissue may just reflect that only the tightly bound nuclear estrogen receptor's functional and/or structural integrity survives long-term formation fixation. Our observation of estrogen binding in preserved tissue may also be a clinically useful tool in therapy analysis.     

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.     

Photoaffinity labelling of androgen receptors with 17 beta-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one

The synthetic androgen 17 beta-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one (R1881) has been used as photoaffinity label to characterize androgen receptors in calf uterus and rat prostate. Polyacrylamide gel electrophoresis under denaturing conditions showed that the DNA-binding form of the androgen receptor in calf uterus cytosol is a protein with a molecular mass of 98 kD. In rat prostate cytosol an androgen receptor with a molecular mass of 46 kD could be photoaffinity labelled with R1881. The photoaffinity labelling procedure described here provides a method for studying the hormone binding domain of androgen receptors in partial purified preparations.     

Estrogen and progesterone receptors in the oviduct during egg transport in cyclic and pregnant rats

We investigated the temporal relationships between ovum transport and changes in the concentration of nuclear steroid receptors in the oviduct of cyclic and pregnant rats. A lack of parallelism between estrogen and progesterone fluctuations in plasma and their respective nuclear receptor concentrations in the oviduct predominated during egg transport. In pregnant animals, oviductal egg transport took 24 h longer than in nonpregnant animals. In both conditions, transport was initiated while the action of estrogen and progesterone on the oviduct--measured as nuclear receptor accumulation--was decreasing. Three or four days later, depending on whether the animal was pregnant, the eggs entered the uterus shortly after an increase in the nuclear receptor accumulation of both hormones. Treatment with RU486, a progesterone receptor-blocking agent known to cause premature arrival of eggs in the uterus, advanced estrogen receptor accumulation in the oviduct of pregnant rats. These data suggest that the arrival of eggs in the uterus is timed by a transitory increase in nuclear estrogen receptor in the oviduct that does not necessarily reflect a similar change of circulating estradiol. Moreover, in pregnant rats, the onset of this estrogenic action is delayed by a progesterone receptor-mediated effect that hinders nuclear estrogen receptor accumulation.