The characteristics of the sex-differentiating action of sex steroids on the androgen receptors of the rat liver

A study was made of the specificities of polydifferentiating action of androgens (A) and estrogens (E) on the level of androgen receptors (AR) of the rat liver cytosol. Androgen receptors were shown to be detected in the neonatal period of ontogenesis, their level was progressively on the increase with the body development. Sex differences in their content were revealed in mature animals only. The AR level is a reversibly controlled, but unprogrammed sex-related sign. The effects of sex steroids are dose-related and modulated reciprocally by the action of endogenous A and E. The action of physiological doses of A and E is characterized by slow development of the effect. Correlation between the level of AR and the efficacy of A action on the content of these proteins was revealed.     

Studies of a thyroid hormone and androgen dependent protein in rat liver cytosol.

Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis of the liver cytosol of euthyroid male rats revealed a prominent band (molecular weight, 26 000 daltons), designated Protein II, which was virtually absent in the cytosol of hypothyroid animals. Injection of 500 mug triiodothyronine (T3) per 100 g body weight resulted in a maximal increase in the level of Protein II, reaching 90% of the euthyroidal level 3 days after hormone administration. Concomitant studies with the liver mitochondrial enzyme alpha-glycerophosphate dehydrogenase (alpha-GPD) indicated that this T3 dose also resulted in a maximal enzyme response in this time period. Since we have estimated that 500 mug of T3 will saturate nearly all nuclear T3 binding sites, these results support the concept that the synthesis of both proteins is limited by nuclear binding. Protein II was absent in the liver cytosol of female rats but could be induced in ovariectomized female rats by androgens. Treatment of male rats with oestradiol resulted in disappearance of Protein II. Since administration of testosterone to hypothyroid male rats caused only a minimal increase in the amount of Protein II, the absence of the protein in hypothyroid animals was not due to androgen deficiency. Similarities in the molecular weight and the response to hormonal manipulation of Protein II and of the urinary alpha2uglobulin, previously reported by Roy (1973) raise the possibility that these proteins are the same. The high concentration of Protein II in male rat cytosol and the relative ease in its identification by SDS polyacrylamide gel electrophoresis make it a potentially useful model protein for the study of thyroid hormone action at the cellular level.     

Regulation of the androgen and glucocorticoid receptors in rat and mouse skeletal muscle cytosol

Using a charcoal technique, we determined the relative binding affinity of some anabolic compounds for the androgen and glucocorticoid receptors in cytosol from rat skeletal muscle. Only a few of the compounds analyzed competed for the receptor-binding sites. The androgen and glucocorticoid receptors were analyzed in rat and mouse skeletal muscle cytosols by Scatchard analysis. In rats grouped according to sex and age, the cytosolic protein content was about the same in all groups, but the DNA content decreased with increased weight of the animal regardless of sex (male, female, or castrated male). The glucocorticoid receptor did not differ in concentration (2-3 pmol/g tissue) or ligand affinity (Kd, 10-40 nM) among the groups, but the androgen receptor concentration decreased with increased weight and age of the animals, more in the case of males than in the case of females or castrates. The Kd for the androgen receptor increased with age in males but was constantly about 0.2 nM for castrates or females. In adult intact rats, the androgen and glucocorticoid receptor concentrations in muscle cytosol from females were about 100 and 3000 fmol/g tissue, respectively, the corresponding values for males being about 50 and 2000 fmol/g tissue, respectively. Short term castration or adrenalectomy increased the concentration of and ligand affinity for the androgen and glucocorticoid receptors, respectively. After long term castration of male rats, the concentration of both receptors increased during 5 weeks to about the female level, only to decrease later. Neonatally castrated male rats had about the same androgen receptor concentrations and Kd values as female rats. Female mice had higher androgen receptor concentrations (approximately 700 fmol/g tissue) than rats. Intact male mice had about 200 fmol androgen receptor-binding sites/g tissue, and the same amount was found in mice bearing the testicular feminization (Tfm) mutant gene. In summary, the concentrations of androgen and glucocorticoid receptors in rat skeletal muscle are regulated at least by the testes. The presence of androgen receptors in skeletal muscle from Tfm mice is surprising and may motivate a reinvestigation of the regulation of androgen receptors in Tfm animals.     

Demonstration of specific high affinity receptors for aldosterone in cytosol of rat colon

Since both aldosterone and glucocorticoids increase cation transport in rat distal colon, and a specific glucocorticoid high affinity cytosolic receptor has been identified in this tissue, it was possible that the action of aldosterone was dependent on interaction with the glucocorticoid receptor. Studies were, therefore, performed to determine whether a specific high affinity receptor for aldosterone was present in rat distal colon. At 4 C, aldosterone binding was saturable and exhibited a high affinity site with an apparent Kd of 6.2 +/- 0.9 X 10(-10) M and a calculated number of binding sites of 57.2 +/- 10.8 fmol/mg cytosol protein. Scatchard plot analysis also revealed a low affinity site with a Kd of 5.9 +/- 1.1 X 10(-8) M and 961 +/- 191 fmol/mg cytosol protein-binding sites. Competitive binding studies demonstrated that the high affinity binding protein was specific for aldosterone, compared to either dexamethasone or RU-28362. Since a specific high affinity receptor protein for aldosterone is present in rat distal colon, these data are consistent with a direct action of aldosterone that is independent of the glucocorticoid receptor system.     

Determination of the content of specific estrogen-binding protein in the rat liver cytosol

A method of quantitative determination of the areas of binding of a peculiar estrogen-binding protein (PEBP) in the cytosol of the rat liver by means of ion-exchange sorption on DEAE-cellulose is suggested. For differential determination of the PEBP contribution to the total estradiol (E2) binding with cytosol proteins a unique capacity of PEBP to form complexes with 3H-E2, which were rapidly decomposed on addition of an excess of unlabeled E2, was used. Theoretical prerequisites and experimental substantiation of the suggested method of the PEBP testing are considered. The content of the binding PEBP areas in the cytosol of the liver of male rats, measured by the mentioned method, constituted (5.8 +/- 0.7).10(-12) mol per 1 mg of protein (M +/- m, according to the data of 18 determinations). No analogous areas of E2 binding were revealed in the cytosol of the liver in female rats. It was also demonstrated that the cytosol of the liver of male rats contained specific areas of testosterone (T) binding, forming highly labile complexes with 3H-T, rapidly decomposing in the presence of an excess of T and E2.     

An unusual sex steroid-binding protein in mature male rat liver cytosol.

Mature male rat liver cytosol contains a moderate affinity and capacity estrogen-binding protein in at least a 200-fold higher level than mature female or immature male rat liver cytosol. Binding of estradiol to this protein is very rapid, is stabilized by EDTA, and is inhibited by divalent cations. This is the major binding protein for [3H]estradiol ([3H]E2) in mature male rat liver cytosol, and it has properties clearly distinguishing it from putative liver or uterine estrogen receptors. In addition to binding [3H]E2, this protein seems to rapidly bind a [3H]5alpha-dihydrotestosterone ([3H]DHT) metabolite at the same binding site. The binding of this androgen metabolite is stabilized by EDTA and is inhibited by divalent cations. The binding properties of the [3H]DHT metabolite suggest that these binding sites are not classical androgen receptors. Cytosol binding levels of both the [3H]E2 and the [3H]DHT metabolites change in a similar direction in resonse to endocrine manipulation. The putative liver estrogen receptor level, determined after partial purification (in a redissolved 30% ammonium sulfate-precipitated fraction), seems to change in an opposite direction in response to these same endocrine manipulations.     

Differences in the developmental patterns of somatotrophic and lactogenic receptors in rabbit liver cytosol

These studies have examined the ontogeny of specific GH- and PRL-binding proteins in rabbit liver cytosol across the fetal, early neonatal, and adult periods. The precise hormonal specificity of binding of the somatotropic/lactogenic ligand, [125I]human GH, was determined by displacement with monoclonal antibodies against either rabbit mammary gland PRL receptors or liver GH receptors. The developmental changes were evaluated by gel filtration chromatography, which allowed a measure of both the extent of binding and the molecular size (Mr). Scatchard analysis indicated that fetal liver cytosol contained mostly high affinity PRL receptors (Ka, 13.78 +/- 0.98 nM-1; capacity, 127 +/- 24.0 fmol/g tissue; mean +/- SEM; n = 5) and was low in GH-specific binding. This contrasts markedly with adult liver cytosol, which is a rich source of GH receptors (Ka, 2.44 nM-1; capacity, 20,765 fmol/g tissue), but is low in PRL-specific binding. Despite the deficiency of GH receptors in fetal liver, an abundant receptor-like GH-binding protein was present in fetal serum. Of several fetal tissue cytosols examined, only the placenta contained significant GH-specific binding; no tissues other than liver contained PRL-specific binding. After parturition PRL receptors in liver cytosol increased and predominated up until day 3 (Ka, 27.97 +/- 6.27 nM-1; capacity, 495 +/- 95.09 fmol/g tissue; n = 7), a period during which GH-specific binding was increasing only slightly. By day 6 a striking switch-over occurred, and GH receptors, which had a 4-fold lower affinity, became predominant (Ka, 6.89 +/- 0.63 nM-1; capacity, 600 +/- 47 fmol/g tissue; n = 5), while PRL-specific binding fell dramatically to levels observed in adult liver cytosol. After day 6 GH receptors increased steadily, reaching the high levels observed in adulthood by 2 months (Ka, 3.95 nM-1; capacity, 16,300 fmol/g tissue; n = 2), while PRL-specific binding appeared to change little. Structural and immunological analyses of the cytosolic GH and PRL receptors in the fetal/early neonatal period revealed similarities with the adult liver cytosolic GH receptor and mammary gland cytosolic PRL receptor, respectively. The increase in GH receptors on day 6 was clearly illustrated by cross-linking studies which showed the emergence of a GH-specific binding protein structurally distinct from PRL-specific binding proteins. These studies have demonstrated that in the rabbit major changes occur in the GH/PRL receptor profile during the early period of growth and suggest that important developmental changes occur in the requirement for GH and/or PRL action during this period.     

Role of hormones in maintaining the sex-differentiated level of estrogen receptors in rat liver cytosol

A study was made of the endocrine mechanisms of the formation and maintenance of a sex-differentiated level of estrogen receptors (ER) in rat liver cytosol. The administration of testosterone-propionate (TP) at a dose of 3 mg for 3 days was shown to cause a significant decrease in the concentration of ER in the liver of gonadectomized animals to the level in intact male rats. In a week after the discontinuation of TP, a complete restoration of the basal level of receptors was observed. Neonatal and prepubertal administration of TP to gonadectomized male rats at early stages of ontogenesis made no effect on the level of ER in the liver cytosol of these animals at the age of 12-14 weeks. The removal of the adrenal and thyroid glands produced no changes in the level of ER in the liver of rats of both sexes. Hypophysectomy in rats resulted even on the 1st day in a decrease in ER concentration in the liver of male and female animals to the same basal level which later on remained unchanged. Ectopic transplantation of a homologous hypophysis and human STH administration led to a significant rise of the level of ER in hypophysectomized animals. TP inhibited a stimulating effect of STH in rats with the removed hypophysis.     

Specificity of androgen receptors of hepatocellular carcinoma and liver in humans

The specificity of androgen receptor in hepatocellular carcinoma and the liver was investigated using auto-radiographic techniques. Partial hepatectomy was carried out on 11 patients with hepatocellular carcinoma and associated parenchymal disease of the liver. Androgen receptors were assayed biochemically for hepatocellular carcinoma and the surrounding liver in all cases. Estrogen receptor was also measured in five patients. In eight patients, fresh resected specimens as thick as 3 mm were first incubated for 15 minutes in a medium containing estradiol and hydrocortisone, and then radio-labeled testosterones were added to the medium. After another 60 minutes incubation, macro-autoradiographic studies were carried out. With the same medium and chemicals, and using the same principle, micro-autoradiographic studies were performed using fresh hepatocellular carcinoma and liver cell suspensions in six cases. The radio-labeled testosterones were incorporated into hepatocellular carcinoma and the liver to a parallel extent with the androgen receptor titers biochemically assayed. The current results seem to indicate that androgen receptors present in hepatocellular carcinoma and the liver of humans specifically bind androgens. Further studies are needed to elucidate the role of AR in hepatocarcinogenesis in humans.     

The effect of the administration of different estrogens on the content of estrogen receptors in the cytosol and nuclear subfractions of liver cells and on the level of angiotensinogen in the blood plasma of rats

The level of estrogen receptors (ER) in the cytosol and nuclear subfractions of female rat hepatocytes was studied 1 h and plasma angiotensinogen (AG) concentration 24 h after single and multiple administration of different doses of estradiol (E2) and synthetic estrogens. Synthetic weakly metabolized estrogens, used at doses corresponding to physiological concentrations of the natural female sex steroid, were shown to be much more effective than E2 in relation to ER redistribution between the cytosol and nuclear fractions of hepatocytes as well as in relation to the stimulation of AG production by the liver. Differences in the ER level in hepatocytic nuclei 1 h after single or multiple administration of the same estrogen were undetectable. An increase in a plasma AG level after a single injection of estrogens was noted after achieving a certain threshold (more than 3-fold as compared to the normal level) level of ER accumulation in hepatocytic nuclei. The sensitivity of AG production by the liver to a stimulating effect of low doses of estrogens was on the increase as a result of their repeated effect in prolonged administration and combined administration of E2 and glucocorticoids.     

Progestin receptors in human uterine cytosol

Progestin(norethindrone and norethindrone acetate)-binding protein, exhibiting characteristics similar to uterine progesterone receptor, has been identified in human uterine cytosol. The progestin receptor was characterized by sedimentation coefficient 4.2 S; Stokes radius, 39 Å; frictional ratio 1.29; isoelectric pH 4.6; molecular radius 2.7 nm; and molecualr weight in the range 67 000–74 000. The ammonium-sulfate-precipitated progestin-receptor complex was eluted from a DEAE-cellulose column at 0.18 M KC1. The progestin binding was saturable and stereospecific. The sequential variation in receptor concentration (early proliferative, 3800–4300 sites/cell; late proliferative, 9500–11200 sites/cell; early secretory, 4900–6200 sites/cell; late secretory, 1800–2300 sites/cell) was in conformity for progesterone and the progestins, when concurrently measured. Oral administration of norethindrone significantly reduced the cytoplasmic and nuclear receptor concentration for estradiol and progesterone. A significant observation was that the progestins stabilized the progestin receptor by forming a slowly dissociating complex with a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{～ 110?130}\text{min}$ as compared with the progesteronereceptor complex dissociating with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{～41}\text{min}$. Thus, the uterine progestin receptor recognizes progestins in general, although with a varying degree of affinity, and the altered rate constants could be of putative importance in determining the biological potency of the progestins.     

Mechanisms of sex differentiation of the level of corticosteroid-binding globulin in rats

The role of sex steroids was studied at different stages of ontogenesis in the regulation and programming of the level of corticosteroid binding globulin (CBG) in the blood serum of female and male rats. The blood concentration of CBG was shown to be a sex determined feature: its level in female rats was a 2.5-fold higher than that in male rats; sex differences of this function of the liver were preserved in a primary culture of hepatocytes. Androgens (A) in adult animals were shown to decrease the level of CBG, and estrogens (E) did not influence the blood level of this protein. Castration of adult males leading to an increase of the level of CBG by 1.5-fold did not eliminate sex differences. However gonadectomy of males on the first day of the life or in prepubertal period (at the age of 3-4 weeks) resulted in complete "feminization" of the CBG content in these animals at the age of 10-12 weeks. Ovariectomy in female rats in the prepubertal period did not influence the level of CBG in adult female rats. The administration of A to females or castrated males did not prevent the development of a high level of CBG at older age. Irreversible suppression of the level of CBG in adult animals could be artificially induced by the administration of A to gonadectomized female and male rats in the prepubertal period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prolactin influence on cytosol and nuclear androgen receptors in the ventral, dorsal, and lateral lobes of the rat prostate

PRL augments testosterone-mediated growth of the prostate in a permissive manner. To elucidate the mechanism of this hormonal interaction, the present study examined the effect of PRL on cytosol and nuclear androgen receptors in the three prostate lobes. Adult male Sprague-Dawley rats were castrated, given 5-mm Silastic implants of testosterone, and either grafted with two anterior pituitary glands under the kidney capsule or sham operated. Three weeks later, animals were killed, serum was collected for PRL and testosterone RIA, and the ventral, dorsal, and lateral prostate lobes were processed for either cytosol or nuclear androgen receptor quantitation. Pituitary grafts significantly elevated the serum PRL concentration and increased the weight and the content of protein and DNA of the lateral prostate lobe compared to control values. There was no effect on these parameters in the ventral or dorsal lobes. Androgen receptor levels and apparent distributions were different in the ventral, dorsal, and lateral lobes of control animals. Unoccupied and total cytosolic androgen receptors in the three separate prostate lobes were not significantly affected by the presence of the grafts. However, an elevated PRL concentration was associated with an increase (P less than 0.005) in nuclear androgen receptor content in the lateral lobe exclusively. The binding affinity was not altered by pituitary grafts in any of the lobes. These findings suggest that PRL promotes lateral prostatic growth by increasing nuclear androgen receptor levels in that tissue and, thus, optimizes its response to circulating testosterone.     

Prolactin receptor in rat liver: sex difference in estrogenic stimulation and imprinting of the responsiveness to estrogen by neonatal androgen in male rats

Rat hepatic prolactin receptor is regulated by sex steroids. A high level of the receptor was found in female rats but the level was nearly undetectable in males. Gonadectomy reduced the receptor level in females but increased the level in males. Administration of estradiol benzoate (0.05 μmoles/kg on alternate days subcutaneously for 9 days) to adult gonadectomized females increased the receptor level by 473% whereas the same treatment in adult gonadectomized males produced a more modest 276% increase. This sexually dimorphic pattern in the responsiveness to estrogen stimulation in adult rats appeared to be determined neonatally. Neonatal gonadectomy of male rats changed the hepatic response system to a more female pattern in adulthood. Replacement of testosterone (1.45 μmoles at days 1 and 3 after birth) to these neonatally gonadectomized male rats restored the male pattern. Diethylstilbestrol replacement (1.45μmoles at days 1 and 3 after birth) to the neonatally gonadectomized male rats showed the same effect as neonatally administered testosterone. Scatchard analysis revealed that the observed changes in binding are related to changes in binding capacity but not affinity. Desaturation by 4 M MgCl₂ indicated that the amount of endogenously bound hormone was negligible in our membrane preparations.     

Identification of an androgen receptor in the cytosol of the female Mastomys prostate

An in vivo and in vitro study was carried out on the prostate from the female Praomys (Mastomys) natalensis to identify and characterize the binding of androgens within the cytoplasm. The labelled cytosol was prepared and subjected to gel exclusion chromatography and density gradient centrifugation. A macromolecular protein associated with the radioactivity was isolated on Sephadex G-200. Subsequent analysis of the steroid receptor complex showed that the major part of the radioactive steroid (64 percent) was dihydrotestosterone. This binding was inhibited by unlabelled testosterone and could not be demonstrated in liver cytosol. Characterization of this dihydrotestosterone receptor complex revealed a sedimentation coefficient of 4.6 s in the presence of a high salt solution (0.4 M KCl). The complex aggregated in the absence of 0.4 M KCl and sedimented preferentially from 5.6-7.4 s together with polydisperse aggregates of higher sedimentation coefficients. The use of this animal as an experimental model for hormonal studies on the prostate is suggested.     

Corticosteroid receptors in liver cytosol of the clawed toad, Xenopus laevis: daily and seasonal variations

In postmetamorphic Xenopus laevis liver cytosol the glucocorticoid binding capacity R0 and the dissociation constant Kd were determined. The receptor assay included an incubation period of 24 hr at 0-4 degrees with sodium molybdate to stabilize the receptor. Dexamethasone, triamcinolone acetonide, and corticosterone as tritiated ligands were compared regarding the R0 (67.6, 57.2, and 30.7 fmol/mg protein), the Kd (3.54, 0.56, and 9.03 nM), and the rate of dissociation in young specimens of X. laevis. In adult toads the [3H]dexamethasone receptor binding capacity was threefold higher in females than in males (153.86 +/- 12.19, 54.29 +/- 4.5 fmol/mg protein)--with about the same Kd (3.97 +/- 0.57, 4.08 +/- 0.28 nM). Young toads were kept under an artificial light regime (light from 600 to 1800 hr) and dexamethasone binding was measured every 3 hr. Unlike Kd, R0 showed a significant diurnal variation with maximal values at 600 and 1800 hr, which occurred about 9 hr after a maximal level of corticosterone in serum was reached (900, 2100). Seasonal variations of the [3H]dexamethasone and [3H]corticosterone binding capacity were different in both sexes of adult X. laevis. Maximal values in males were found in June/July and October/November. In females, the R0 was increased in the second half of the year with the maximum in August (275.5 +/- 45.02 fmol/mg protein). No correlation between R0 and the concentrations of corticosterone or aldosterone existed.