Transformation of androgen receptors from mouse submandibular glands by gel chromatography

[3H]Methyltrienolone-bound and unbound androgen receptors from mouse submandibular glands bound to DNA-cellulose to a similar extent after gel chromatography. The receptors sedimented at 6 S in a low-salt gradient whereas if receptors were transformed with KCl (0.4 mol/l) they sedimented at 4 S. On DEAE-anion exchange chromatography both were eluted at a concentration of 0.07 mol KCl/l. These results suggest that two states of transformed androgen receptors exist. A 6 S transformed receptor may well derive by partial dissociation of an 8 S non-transformed receptor and this is caused by gel chromatography. Molybdate ions completely prevent both the transformation induced by gel chromatography and the secondary transformation during DEAE-anion exchange chromatography.     

Binding of the glucocorticoid receptor complex to the nucleosomal core in the P1798 mouse lymphosarcoma

Binding of the glucocorticoid receptor complex to nucleosomes has been studied using the mouse P1798 lymphosarcoma. Cells were incubated with [3H]triamcinolone acetonide (TA), and nuclei prepared and digested with 3 different concentrations of micrococcal nuclease. After fractionation with EDTA and NaCl, it was observed that [3H]TA bound with similar specific radioactivity to mononucleosomes containing both core and linker DNA, of 183 +/- 5, and 168 +/- 4 base pair lengths, respectively, as well as to core size DNA, of 148 +/- 3 base pair length, suggesting that the glucocorticoid receptor bound to the core portion of the nucleosome. Steroid binding was found to be associated with regions of the nucleosome that were depleted in histone H1 and enriched in high mobility group (HMG) proteins 1 and 2; only negligible binding was noted in nucleosomes enriched in histone H1 and depleted in HMG proteins. In addition to binding to core nucleosomes, the glucocorticoid receptor complex was also shown to bind to a fraction sedimenting at 5-6 S on sucrose gradients characterized by subnucleosome and mononucleosome size DNA, as well as by core histones. While binding of the steroid receptor complex to linker regions of the nucleosome cannot be ruled out, this data would appear to present the first concrete evidence that glucocorticoid binding, at least in the P1798 lymphosarcoma, is to core nucleosomes. Some caution in interpretation of the results is indicated, however, on 2 points: (1) receptor redistribution during nuclease digestion cannot be ruled out; (2) only the binding of a small proportion of the steroid receptor complex may be physiologically relevant.     

Sex difference in the cytosolic and nuclear distribution of androgen receptor in mouse submandibular gland

Cytosol and nuclear androgen receptors in submandibular glands of male and female mice were measured by an exchange assay at 0 degree C. The binding of [3H]methyltrienolone to cytosol receptors in females was mostly saturated within a short period of incubation (3 h), whereas the saturation was much slower in males; suggesting that almost all of the cytosol receptors were unoccupied in females and the receptors were partially occupied in males. Nuclear receptors were extracted with pyridoxal 5'-phosphate (5 mmol/l) from nuclear fractions with 93-95% efficiency. The exchange of the bound steroids occurred by 24-48 h at 0 degree C, suggesting that most of the nuclear androgen receptor was occupied. The binding was low at higher temperatures, probably due to inactivation of the receptor. Scatchard analysis showed that the apparent dissociation constants of cytosol and nuclear receptors were similar (0.8 and 0.9 nmol/l respectively) in both sexes. On the other hand, the number of androgen-binding sites in the nucleus was much higher in males than in females (1052 fmol/mg DNA and 32 fmol/mg DNA respectively), while the number in the cytosol was higher in females than in males (512 fmol/mg DNA and 368 fmol/mg DNA respectively). These observations show that androgen receptors exist mainly (74%) in the nuclei of males, while they exist mostly (94%) in the cytosol of females.     

Influence of androgen on translatable renin mRNA in the mouse submandibular gland

In mature outbred Swiss male mice, submandibular gland renin enzyme activity is 4- and 10-fold higher than in glands of prepubescent males and mature females, respectively. Levels of translatable renin mRNA have been studied in mouse submandibular gland during postnatal development and following administration of testosterone. The [35S]methionine-labeled cell-free translation products directed by male glandular mRNA contain a 47 +/- 2kd renin precursor that is not detected in products coded by prepubescent male or female gland mRNA. This cell-free synthesized precursor is detected immunochemically only in the translation products of gland mRNA from males of 33 days or older and from females receiving testosterone administration, a pattern consistent with the measurements of renin enzyme activity. This increase in biologically active renin mRNA is a selective one, since unfractionated male and female mRNAs have similar overall nucleotide sequence complexity corresponding to 1% of mouse single copy DNA. The cDNA transcribed from male gland mRNA reacts 5- and 10-fold faster with the template mRNA than with female or prepubescent male gland mRNA, respectively, which indicates that the male gland contains abundant nucleotide sequences that exist at low concentration in the female or prepubescent male. Selective hybrid arrested translation confirms that the levels of renin mRNA are lower in the glands of prepubescent males than in those of the mature males. These data indicate that the regulation of renin enzymatic activity by androgens is mediated by an increase in the levels of translatable renin mRNA both during postnatal development and after testosterone administration.     

Developmental study of androgen responsiveness in the submandibular gland of the mouse.

The development of androgen responsiveness in the submandibular gland of normal and androgen-resistant (Tfm) mice of different ages was studied after varying hormonal treatments. Total esteroproteolytic (tamase) activity of submandibular gland homogenates was used as a marker for androgen action. Newborn mice of all four genotypes (normal male, normal female, carrier Tfm females, and Tfm males) were resistant to androgen. However, at 3 weeks of age the capacity to develop a tamase response appears in normal and carrier Tfm animals given androgen and rapidly rise to maximal levels. The level in the normal animal is regulated thereafter primarly by the level of circulating androgen. In contrast, the tamase response in the Tfm male of all ages and under all androgen regimens was minimal.     

Binding of the partially purified glucocorticoid receptor of rat liver to chromatin and DNA

The binding of the glucocorticoid receptor of rat liver to chromatin and DNA has been studied with crude and partially purified preparations of cytosol receptor labelled with [³H]-triamcinolone acetonide in vitro. The use of crude preparations of receptor and increasing protein concentrations leads to an apparent saturation of chromatin and DNA, suggesting a limited number of high affinity nuclear acceptor sites for the receptor. Appropriate controls indicate that the observed saturability of chromatin acceptor sites is due to the presence in crude receptor preparations of heat-stable protein factors which interfere with the binding of the receptor to the genome; whereas the apparent saturation of DNA is due to contamination with deoxyribonucleases.If the activated complex of receptor and triamcinolone acetonide (R-TA) is partially purified to a step where it is free from nucleases and inhibitors, its binding to both chromatin and DNA is linearly dependent on the concentration of free (R-TA) in the incubation medium. There is no absolute specificity with respect to the source of DNA or chromatin, although liver chromatin has considerably higher receptor binding capacity than chromatin from avian erythrocytes.The rate kinetics of association and dissociation for the binding of (R-TA) to DNA and chromatin are very similar, but DNA exhibits a 10-fold higher receptor binding capacity than chromatin. These data, in conjunction with the effect of poly-(D)-lysine and NaCl on the binding of (R-TA) to chromatin and DNA, suggest that most of the receptor molecules bound to chromatin in vitro interact with the ‘accessible’ DNA stretches. Although a small population of receptor molecules may bind specifically to target tissue genome, the detection of these specific sites against the background of unspecific binding is not possible with unfractionated chromatin or DNA preparations.     

Binding of hormone accelerates the kinetics of glucocorticoid and progesterone receptor binding to DNA.

Steroid hormone receptors induce genes by virtue of their interaction with DNA regulatory sequences. The hormonal response of a particular gene in vivo correlates with binding of the hormone to the receptor and supposedly reflects the degree of occupancy of the corresponding DNA regulatory sequences. However, in vitro the steroid-free glucocorticoid and progesterone receptors bind specifically to the regulatory sequences of mouse mammary tumor virus, thus raising questions on the role of the hormone in DNA binding in vivo. By using monoclonal antibodies, gel retardation assays, and filter binding techniques we show here that binding of a functional steroid to either the glucocorticoid or the progesterone receptors influences the kinetics of the protein-DNA interaction in vitro. In the presence of hormone the on rate of receptor binding to DNA fragments with or without regulatory sequences is accelerated 2- to 5-fold, and the off rate is accelerated 10- to 20-fold. The receptors complexed to an antihormone bind to DNA with kinetics intermediate between those of the steroid-free and the hormone-bound protein. Thus, ligand binding accelerates the kinetics of receptor binding to DNA and this could partly account for the behavior of the hormone receptor observed in vivo.     

Immunofluorescent and immunoenzymatic analysis of an insulin-like protein of the mouse submandibular glands using monoclonal antibodies to insulin

As has been reported earlier, submandibular glands of animals and man contain insulin-like protein (ILP), similar in some of its properties to pancreatic insulin. An immunocytochemical method with polyclonal anti-insulin serum has shown that ILP is contained in cells of the submandibular gland granular ducts. This paper is concerned with the determination of localization of ILP and estimation of its structural identity with insulin by using monoclonal antibodies to pig insulin. ILP and insulin were extracted from mouse submandibular glands and pancreas and partially purified. Solid-phase immunoenzymatic analysis was used to determine the capacity of mouse ILP and pancreatic insulin to bind specifically with monoclonal antibodies to pig insulin indicating the structural identity of ILP and insulin. The absence of cross-reaction of antibodies with the nerve growth factor and kallikrein (biologically active substances contained in the submandibular gland granular ducts) was established. An immunofluorescent indirect method with the use of monoclonal antibodies to insulin has shown that ILP is localized in cells of the submandibular gland granular ducts. It confirms the previously reported results.     

Inhibitory effect of androgen on the synthesis of proteinase F in the male mouse submandibular gland

Androgenic regulation of one of the esteroproteinases (proteinase F) in the mouse submandibular gland was studied using specific antiserum. In contrast to esteroproteinases such as proteinases A, D or P-esterase, proteinase F content in male but not in female mice was increased by gonadectomy and decreased by the injection of various androgens. In-vivo incorporation of [3H]leucine into proteinase F in males was increased after castration and decreased by the injection of testosterone propionate; androgens inhibited the de-novo synthesis of proteinase F in male mice. The dose-response curves for testosterone propionate and time-courses following castration or after the injection of testosterone propionate were reciprocal between proteinase F and total esteroproteinase activity. Proteinase F, like other esteroproteinases in the submandibular gland of the mouse, was localized in granular convoluted tubular cells. These data indicate that granular convoluted tubular cells of the male mouse submandibular gland synthesize both androgen-inducible proteinases and androgen-inhibitory proteinase (proteinase F).     

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.     

Interaction of androgen and glucocorticoid receptor DNA-binding domains with their response elements

Fusion proteins containing the glucocorticoid and the androgen receptor DNA-binding domain (ARF1 and GRF1) were produced in Escherichia coli. DNAse I footprinting was used to compare the interaction of these proteins with responsive elements (REs) in a typically glucocorticoid-responsive gene (mouse mammary tumour virus (MMTV)) and in an androgen-responsive gene (the C3(l) gene of rat prostatic binding protein). It is demonstrated that response elements which most closely resemble the consensus sequence show identical footprinting patterns for ARF1 and GRF1. The protected regions suggest that these sequences are occupied by two DNA-binding domains (DBDs) forming a dimer. Regions that constitute imperfect RE sequences, however, are apparently recognized by only one DBD, which mainly protects the TGTTCT motif. At these REs, the protection patterns produced by ARF1 and GRF1 are not identical. In the long terminal repeat (LTR) of MMTV but not in C3(1), a mechanism other than classical dimer formation seems to increase the affinity of ARF1 and GFR1 for these imperfect REs.     

Melatonin inhibits glucocorticoid receptor nuclear translocation in mouse thymocytes

The antiapoptotic effect of melatonin (MEL) has been described in several systems. In particular, MEL inhibits glucocorticoid-mediated apoptosis. Our group previously demonstrated that in the thymus, MEL inhibits the release of Cytochrome C from mitochondria and the dexamethasone-dependent increase of bax mRNA levels. In this study we analyzed the ability of MEL to regulate the activation of the glucocorticoid receptor (GR) in mouse thymocytes. We found that even though the methoxyindole does not affect the ligand binding capacity of the receptor, it impairs the steroid-dependent nuclear translocation of the GR and also prevents transformation by blocking the dissociation of the 90-kDa heat shock protein. Coincubation of the methoxyindole with dexamethasone did not affect the expression of a reporter gene in GR-transfected Cos-7 cells or HC11 and L929 mouse cell lines that express Mel-1a and retinoid-related orphan receptor-alpha (RORalpha) receptors. Therefore, the antagonistic effect of MEL seems to be specific for thymocytes, in a Mel 1a- and RORalpha-independent manner. In summary, the present results suggest a novel mechanism for the antagonistic action of MEL on GR-mediated effects, which involves the inhibition of 90-kDa heat shock protein dissociation and the cytoplasmic retention of the GR.     

Differential distribution of an estrogen receptor in the submandibular and parotid salivary glands of female rats

The purpose of this study was to measure the availability of the estrogen receptor in submandibular and parotid salivary glands in female rats. The presence of a specific, competitive, and saturable estrogen binder in rat salivary gland tissue was determined by saturation analysis and steroid competition in cell-free homogenates of salivary gland tissue from adult ovariectomized females. Scatchard analysis of the data indicated an estrogen receptor content of 1971.1 +/- 651.4 femtomoles/gm of tissue in submandibular salivary gland. This was significantly (p less than 0.01) greater than the number of estrogen binding sites in the parotid gland (457.1 +/- 123.4 femtomoles/gm tissue). Thus, there is a differential distribution in estrogen receptor content between parotid and submandibular salivary glands. The presence of an estrogen receptor in salivary gland tissue may serve to promote gender differences in submandibular salivary gland EGF content, to mediate changes in saliva composition during the female reproductive cycle and to regulate EGF release for cyclic uterine growth.     

Enhanced sexual behaviors and androgen receptor immunoreactivity in the male progesterone receptor knockout mouse

Reproductive and behavioral functions of progesterone receptors (PRs) in males were assessed by examining consequences of PR gene deletion. Basal hormone levels were measured in male progesterone receptor knockout (PRKO) mice and compared to wild-type (WT) counterparts. RIA of serum LH, testosterone, and progesterone levels revealed no significant differences. Levels of FSH were moderately but significantly lower and inhibin levels were higher in PRKOs; these differences were not accompanied by gross differences in testicular weight or morphology. PRKOs exhibited significant alterations in sexual behavior. In initial tests PRKOs exhibited reduced latency to mount, compared with WT. In second sessions, PRKOs again showed a significantly reduced latency to mount and increased likelihood of achieving ejaculation. RU486 treatment in WT produced increased mount and intromission frequency and decreased latency to intromission. In anxiety-related behavior tests, PRKO mice exhibited intermediate anxiety levels, compared with WT, suggesting that enhanced sexual behavior in PRKOs is not secondary to reduced anxiety. Immunohistochemical analysis revealed significantly enhanced androgen receptor expression in the medial preoptic nucleus and bed nucleus of the stria terminalis of PRKO. We conclude that testicular development and function and homeostatic regulation of the hypothalamic-pituitary testicular axis are altered to a lesser extent by PR gene deletion. In contrast, PR appears to play a substantial role in inhibiting the anticipatory/motivational components of male sexual behavior in the mouse. The biological significance of this inhibitory mechanism and the extent to which it is mediated by reduced androgen receptor expression remain to be clarified.     

米非司酮与孕激素受体及糖皮质激素受体结合力的研究

目的探讨米非司酮的抗孕激素及抗糖皮质激素作用。方法用放射配体结合实验测定米非司酮与猕猴子宫胞浆孕激素受体及大鼠肝胞浆糖皮质激素受体的结合力(BA)。结果米非司酮和孕酮与孕激素受体的BA分别为175.58%±19.20%、100%,50%抑制浓度(IC50)分别为(17.20±1.20)nm o l/L、(30.20±2.31)nm o l/L,两者相比,P均<0.01;米非司酮和地塞米松与糖皮质激素受体的BA分别为344.41%±57.41%、100%,IC50分别为(4.21±1.02)nm o l/L、(14.50±1.89)nm o l/L,两者相比,P均<0.01。结论米非司酮具有强的抗孕激素作用和一定的抗糖皮质激素作用。