Cytosolic Progesterone Receptor Research Articles

Reproductive epidemiology of glial tumors may reveal novel treatments: high-dose progestins or progesterone antagonists as endocrino-immune modifiers against glioma.

Female gender, contraceptives, and menopausal hormone replacement treatments containing progesterone analogues associate with higher risk of meningiomas yet with lower risk of gliomas. Progesterone receptor (PR) expression and mifepristone treatment was highly discussed for meningiomas. However, much less is known in regard to progesterone actions in gliomas despite PR expression strongly correlates with their grade. Meningiomas and gliomas may grow faster during gestation; but paradoxically, parousity reduces lifetime risk of gliomas which can be explained with dichotomous cell growth-stimulating and inhibitory actions of progesterone at low versus high levels. Progesterone levels gradually increase in gestation up to 200-fold and the incidence of highly angiogenic brain tumors decreases in the last trimester. Indeed, progesterone stimulates glial tumor cell growth at low doses (10nM) while induces cell kill at higher doses. During gestation, some immune pathways are activated to protect the mother and the fetus against microbial pathogens. In parallel, high-dose medroxyprogesterone acetate (MPA) used in treatment of endometrial carcinoma decreases tumoral expression of PR-B and increases infiltration of cytotoxic T lymphocytes and natural killer cells. MPA also synergies with IL-2 in clinical treatment of renal cancer. In both glioma and meningioma, the dominant cytosolic PR is PR-B which increases cell growth, while PR-A limits cell growth. This seems also paradoxical at the first glance due to opposite behavior of these tumors in diverse endocrine conditions. High-dose progestins may inhibit brain tumor growth by downregulating PR-B, yet the dosage thresholds may differ between glial and meningeal tumors due to higher total PR expression in meningiomas. Supporting this proposal, certain progestins were reported to stimulate meningioma growth in anecdotal reports, but same agents at much higher doses reduced meningioma cell proliferation in pilot clinical studies. PR antagonist mifepristone reduced meningioma growth in some clinical studies, but lacked efficacy in others. In fact, mifepristone also has partial PR agonist efficacy and acts in synergy with MPA to block EC growth. Hence, a similar mechanism of receptor downregulation may also account for mifepristone. Both MPA and mifepristone also harbor myeloprotective features against chemotherapy. Ulipristal is another contraceptive PR antagonist and exerts promising anticancer activity on drug-resistant ovarian cancer and BRCA1-mutant breast cancer cells, which can be tested in animal glioblastoma models. We propose that progestins strongly deserve to be investigated in experimental models of glioblastoma alone and in combination with immunostimulating agents.

Cytosolic estrogen and progesterone receptor content in the endometriotic tissues and endometrium in women with and without endometriosis

In order to have a profile of cytosolic estrogen and progesterone receptors in either endometriotic tissue or endometrium in patients with and without endometriosis a cross-sectional study was  performed  involving  43  infertile women. They consisted of 31 (72.09%) endometriosis and 12 (27.91%) non-endometriosis cases; their average age was 32 ±4 years and 32  ±3 years respectively, with the average  length  of  menstrual  cycle  31  ±8  days  and  29  ±1  days  respectively.  The endometriotic tissue was obtained by excision during ope-rative laparoscopy procedure, while the endometrium was obtained  by  biopsy following hysteroscopy  procedure.  These procedures  were  conducted  within  the  periovulatory  period  (on  Day  13-18  of  the  cycle).The  sex  steroid  receptor  content  in  the cytosol  was  measured  quantitatively  using enzyme-immunoassaymethod,  and  calculated  as  sex  steroid  receptor/cytosol  protein (fmol/ml  cytosol).  It  was  found  that  the  average cytosolic estrogen  receptor concentration  in  the  respective  tissues  were 512.99fmol/ml  in  the  endometriotic  ovary compared  with  2369.17  in  normal  ovary and  632.18  fmol/ml  in  the  endometriotic  peritoneum compared  with  9607.61  fmol/ml  in  normal  peritoneum;while 99.28  fmol/ml and 608.33fmol/ml in the endometrium of women with endometriosis and those without endometriosis respectively. The average cytosolic progesterone receptor concentration found in the respective  tissues  were 50.64fmol/ml  in  the  endometriotic  ovary compared  with  6469.42  fmol/ml  in  normal  ovary and1631.40 fmol/ml  in  endometriotic  peritoneum compared with 12466.99 in normal peritoneum, while21.26 fmol/ml and 599.61fmol/ml in the endometrium  of  women  with  endometriosis  and  those  without  endometriosis  respectively.  There  is  no  significant difference in the  receptor concentration  between  each  tissue  according  to  its  topographic  origin. However,  this  result  may  assume  that  the  responsivity  on  hormonal treatment in endometriosis cases willdepend on the cytosolic sex steroid receptor content in the sick tissues, and the peritoneal lesions will possibly give better response than those in other sites. A further clinical trial is necessary. (Med J Indones 2005; 14: 133-41)

Modulation of estrogen action during preimplantation period and in immature estradiol-primed rat uterus by anti-implantation agent, ormeloxifene

Studies were undertaken to evaluate the influence of estrogen antagonist-cum anti-implantation agent, ormeloxifene, on 17β-hydroxydsteroid dehydrogenase (17β-HSD) activity and estrogen action in rat uterus during preimplantation period and to examine its ability to induce progesterone receptor (PR) in immature rat model. A group of female rats received orally a contraceptive dose of 1.25 mg/kg of ormeloxifene on Day 1 postcoitum (pc). Rats were sacrificed on Days 3, 4 and 5 pc, and uterine tissues were processed for enzymatic, estrogen receptor and estradiol (E 2) estimations. Immature ovariectomized rats received ormeloxifene, subcutaneously for 3 days at various doses in the absence or presence of estradiol, and uterine PR levels were measured using 3H-R5020 as radioligand. Results revealed that ormeloxifene treatment caused a marked increase in enzyme activity of 17β-HSD on Days 3, 4 and 5 pc as compared to respective controls. Further, total uterine estrogen receptors as estimated by exchange assay showed a noticeable decrease on Days 4 (35%) and 5 (>80%) pc in ormeloxifene-treated groups. The results correlated well with a decrease in tissue E 2 levels. In immature rats, ormeloxifene caused a dose-dependent increase in cytosolic PR levels; ormeloxifene given along with E 2 (0.1 μg) for 3 days caused a significant reduction in concentration of PRs at 10 μg and higher doses. Ormeloxifene also induced 3H-progesterone (P) uptake by immature rat uterus. However, in the presence of E 2, it significantly reduced 3H-P uptake. The in vitro competitive binding experiments did not reveal any displacement of 3H-R5020 either by ormeloxifene or by its hydroxy derivative from PR. The results suggest that in addition to its competitive antagonism at estrogen receptor level, ormeloxifene enhances the inactivation of intracellular E 2 to estrone, a biologically less active form, thus declining estrogen receptor pool. Moreover, it causes indirect anti-progestational effects in the uterus by virtue of its anti-estrogenic profile rather than by blocking the PRs.

Specific interactions of steroids, arylhydrocarbons and flavonoids with progesterone receptors from the cytosol of the fungus Rhizopus nigricans

Rhizopus nigricans ( R. nigricans) transforms fungitoxic progesterone into the less toxic 11α-hydroxyprogesterone which is then able to exit the mycelia into the surrounding water. Hydroxylation of progesterone is an inducible process in which cytosolic progesterone receptors could be involved. In the present study, we characterised receptors with respect to ligand specificity and to their involvement in progesterone induction of hydroxylase. EC 50 values of different ligands (steroids, xenobiotic arylhydrocarbons and natural flavonoids) were determined by competition studies using 40 nM ( 3H)progesterone. C21 and C19 3-oxo-4-ene steroids were good competitors (EC 50 of progesterone 2.3 ± 0.1 × 10 −7 M, EC 50 of androsten-3,17-dione 24 ± 2 × 10 −7 M). The presence of hydroxyl groups in steroids significantly decreased the affinity for receptors. The arylhydrocarbons α-naphthoflavone and ketoconazole exhibited EC 50 values of 0.3 ± 0.01 × 10 −7 M and 27 ± 5 × 10 −7 M, respectively, whereas β-naphthoflavone and benzo(a)pyrene were not able to displace labelled progesterone completely. The competition curves obtained by natural flavonoids also did not reach the bottom level of non-labelled progesterone, indicating the interaction at some allosteric binding site(s) of progesterone receptors. All ligands were examined for their involvement in progesterone-hydroxylase induction. Steroid agonists induced the enzyme in a dose-dependent manner in accordance with their affinity for receptors, whereas arylhydrocarbons and natural flavonoids did not induce the enzyme. The agonistic action of steroids, together with the antagonistic action of α-naphthoflavone, strongly suggests the involvement of progesterone receptors in progesterone signalling resulting in the induction of progesterone-hydroxylase.

Progesterone receptors: their localization, binding activity and expression in the pig oviduct during follicular and luteal phases

Estrogens (E) and progesterone (P) are known to require their respective steroid receptors in order to exert structural and functional effects on the oviduct. Cyclic changes in progesterone receptor (PR) localization in the oviductal tissue of female pigs were determined using an immunohistochemical technique with mouse monoclonal antibody mPRI against PR. The variations observed during the estrous cycle in the progesterone receptor (PR) intensity and proportion between ampulla and isthmus probably reflect different response of these regions to progesterone. Immediately before ovulation, during follicular phase, no staining was observed in either the ampulla or the isthmus stroma. However, a low expression of PR in the epithelium of the ampulla was observed. After ovulation, during luteal phase, PR immunostaining was more intense in the whole oviduct. According to immunohistochemical assays, the binding assays for nuclear and cytosolic PR (PRn and PRc, respectively), by using [ 3 H ] R5020 at 4 °C for 15 h, also showed a higher specific binding during luteal phase. However, the PR mRNA in the oviduct, analyzed by RT-PCR, showed similar levels at both stages of the estrous cycle. Although this methods could not be quantitative, indicate the possibility that a post-transcriptional control could differentially regulate the PR in the pig oviduct.

Progesterone inhibits human infragenicular arterial smooth muscle cell proliferation induced by high glucose and insulin concentrations

Diabetes mellitus is a significant risk factor for atherosclerotic peripheral vascular disease. Hyperglycemia and hyperinsulinemia, as encountered in patients with type II diabetes, have been shown to stimulate vascular smooth muscle cell (VSMC) proliferation, a paramount feature in atherosclerosis. Female sex hormones, such as estrogen, have been suggested to inhibit VSMC proliferation. However, the role of progesterone, particularly in patients with diabetes mellitus, has not been examined. Therefore, we studied the effect of progesterone on VSMCs exposed to various concentrations of glucose and insulin. Human infragenicular VSMCs isolated from the tibial arteries of five male patients with diabetes undergoing lower extremity amputation were used. Immunocytochemical studies with confocal microscopy were performed for progesterone receptor identification in these VSMCs. Cells were grown to subconfluence, followed by exposure to deprived media with various glucose (100 and 200 mg/dL) and insulin (no insulin and 100 ng/mL) concentrations. Cells were then additionally exposed to physiologic progesterone (10 ng/mL, progesterone group) and compared with a no-progesterone group. Cell count and methyl-(3)H-thymidine incorporation were used to determine cellular proliferation. Cell count with hemocytometry was performed on day 6. DNA synthesis as reflected through methyl-(3)H-thymidine incorporation was measured at 24 hours. Immunocytochemical studies with confocal microscopy showed cytosolic progesterone receptors. The no-progesterone group showed a significant rise in cell count (P <.05) at all concentrations of glucose or insulin compared with the control group containing 100 mg/dL glucose concentration. The no-progesterone group also showed a significant rise in thymidine incorporation (P <.05) in the 100 mg/dL glucose-100 ng/mL insulin group and the 200 mg/dL glucose-100 ng/mL insulin group compared with the 100 mg/dL glucose group. In the cell count studies, progesterone significantly inhibited cellular proliferation in several settings. All cell groups cultured with insulin or an elevated glucose concentration showed a significant (P <.05) antiproliferative effect when exposed to progesterone. With thymidine incorporation, progesterone showed a similar antiproliferative effect in cells stimulated with glucose or insulin. Significant reductions in cell proliferation as determined with both cell count and thymidine incorporation suggest that progesterone is an inhibitor of VSMC proliferation induced by our in vitro models of hyperglycemia and hyperinsulinemia. Therefore, progesterone may have a protective role against the atherosclerotic changes associated with type II diabetes.

Progesterone receptor Localization and Isoforms in Myometrium, Decidua, and Fetal Membranes From Rhesus Macaques: Evidence for Function Progresterone Withdrawal at Parturtion

It is not known whether withdrawal of progesterone (P) action is a prerequisite for parturition in women or in nonhuman primates because concentrations of circulating progesterone or progesterone receptors (PR) in myometrium and decidua do not decrease before delivery. To examine this potentially important regulatory mechanism, we determined PR isoforms, PR localization, and mRNA in myometrium, decidua, and fetal membranes from rhesus monkeys during pregnancy and in spontaneous labor at term. Gestational tissues were obtained midpregnancy (day 80-100), late pregnancy (day 130-145), and during spontaneous labor at term (day 161-167). Samples of rhesus monkey myometrium, decidua, chorion-decidua, and amnion were collected and analyzed for total nuclear and cytosolic PR by competitive binding assay. Progesterone receptor isoforms were identified and quantified by Western blot analysis, and PR mRNA was determined by a specific ribonuclease protection assay. Nuclear PR was localized by immunohistochemistry with monoclonal anti-PR (JZB39) after microwave stabilization. Myometrium and decidua showed no change in total PR during pregnancy and labor. Nuclear PR was not detected in fetal membranes by binding assay but was localized in amnion epithelial and mesenchymal cells and in chorion laeve cytotrophoblasts by immunohistochemistry. Staining for PR was substantially less by serial antibody dilution in fetal membranes than in decidua. Message for PR was confirmed in all tissues analyzed. A significant (P <.05) shift in the ratio of PR isoforms (from PR-B dominance at midpregnancy to PR-A dominance in labor) was observed in myometrium but not in decidua. Both PR-A and PR-B isoforms and PR nuclear staining were nearly undetectable in amnion obtained during labor. A shift to PR-A dominance in myometrium at term together with a loss of PR in fetal membranes provides evidence for a functional progesterone withdrawal mechanism, which may facilitate the initiation of parturition in primates.

Relative binding activity of new antigestagens with progesterone receptors in human hyperplastic endometrium.

We determined the content and binding capacity of progesterone receptors in the endometrium of patients with adenomatous and fibroid polyps, adenocystic hyperplasia, and atypical hyperplasia before and after gestagen therapy. Hyperplasia of the endometrium was accompanied by changes in affinity of cytosolic progesterone receptors for antigestagens, which provides the possibility of individual correction of hormone therapy.

Estrogenic Activity and Metabolism of N-Butyl Benzyl Phthalate in Vitro: Identification of the Active Molecule(s)

Some phthalates are suspected to disrupt the endocrine system, especially by mimicking estrogens. N-butyl benzyl phthalate (BBP) has estrogenic effects in vitro but not in vivo. The aim of this study was to identify the active molecule(s) (parent compound and/or metabolite(s)) involved in the estrogenic activities of BBP. The estrogenic effects of BBP and its in vivo metabolites were assessed using the following tests: E-Screen, ER binding, and PR induction tests on the human breast cancer cell line MCF-7 (ER+). BBP, the parent compound, was a partial agonist. It stimulated MCF-7 proliferation in the E-Screen assay and increased cytosolic progesterone receptors (PR) levels in a concentration-dependent manner. No BBP metabolites were active except hippuric acid (HA), which had a weak effect at very high concentrations. BBP and HA stimulatory effects on MCF-7 proliferation were antagonized by tamoxifen. However, no competition was observed between BBP or HA and 17β-estradiol for binding to the estrogen receptor (ER). BBP metabolism by MCF-7 cells was also investigated. After a 48-h incubation, only 10% of the initial BBP remained in the culture medium, demonstrating that BBP was extensively metabolized by the MCF-7 cells. The radioactivity recovered in the medium was represented by: mono-n-butyl phthalate (MBuP, 25%) and mono-n-benzyl phthalate (MBeP, 48%), phthalic acid (6%), and benzoic acid (3%). Since none of these metabolites had estrogenic activities, this study demonstrates that the parent compound was the active molecule involved in the in vitro estrogenic effects of BBP.

Impaired estrogen priming of progesterone receptors in uterus of MRL/MP- lpr/lpr mice, a model of systemic lupus erythematosus (SLE)

Estrogens exacerbate the autoimmune disease SLE and progesterone is immunoprotective. Estrogens increase synthesis of progesterone receptors (PR) and it is hypothesized that this physiological balance may be impaired in SLE. To test this, cytosolic PR were measured in hypothalamus, thymus and uterus from 6-week-old female ovariectomized BALB/c and MRL/MP- lpr/ lpr mice 48 h after s.c. injection of estradiol benzoate (3.2 μg/0.1 ml; OB) in peanut oil or 0.1 ml peanut oil alone. PR were measured using [ 3H]ORG 2058, which does not bind to corticosteroid-binding globulin (CBG), and bound and free ligand were separated using minicolumns of Sephadex LH20 at 0°C. PR were measured in cytosols from hypothalamus and uterus of oil-treated BALB/c mice, but were undetectable in thymus, whereas receptors were measurable in all three tissues of MRL mice. There was a significantly greater priming effect of OB on PR in uterus of BALB/c mice, but not in hypothalamus, and PR became detectable in thymus cytosols from BALB/c mice. Also, the apparent affinity of the binding reaction between [ 3H]ORG 2058 and PR was significantly higher than those measured in other tissues in hypothalamic cytosols of both strains. These results suggest that there is an impairment of estrogen priming of progesterone receptors in uterus and perhaps thymus of MRL mice.

Gene expression of cytokines relevant to natural killer cell differentiation in mouse uterus during pregnancy

The effects of fetuses or placentas (with fetuses) removal on progesterone receptors (PR) in deciduomal tissues were investigated in unilateral pregnant (ULP) hamsters. Deciduomal reactions in response to artificial stimuli were induced on the tube-ligated uterine horns of ULP hamsters. Fetuses or placentas (with fetuses) were removed on day 8 of ULP, and the cytosolic and nuclear PR (cPR, and nPR, respectively) levels in deciduomal tissues at mesometrial (MS) and antimesometrial (AMS) sites were separately studied on day 10 and 12 of ULP. In the sham control group, cPR at the MS decreased with time, but at a slower rate than that at the AMS. The MS deciduoma contained higher cPR than the AMS deciduoma on day 12. Removal of fetuses did not cause any significant change of the decline rate, and the PR levels were similar to that of the control. The nPR at the MS and AMS also declined with time in all three groups. Removal of placentas induced drastic losses of cPR and nPR at the MS and AMS on day 10 and 12. Supplement of progesterone (P; 2 mg/animal/day) to the placenta-removed group restored the cPR somewhat at the MS site, but not back to the level of the control. The effect of P to restore nPR levels could be observed at the MS and AMS on day 10, but only at the MS on day 12. These data suggest that placental factor(s) other than P may play an important role on the maintenance of PR in deciduoma. Other cellular mechanisms may exist at the MS and AMS sites for the heterogeneous responses.

Vinclozolin does not alter progesterone receptor (PR) function in vivo despite inhibition of PR binding by its metabolites in vitro

Vinclozolin, a dicarboximide fungicide, alters morphological sex differentiation in male rats following perinatal exposure. The occurrence of these abnormalities correlates with the in vivo formation of two antiandrogenic metabolites of vinclozolin, (i.e. 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid (Ml) and 3′, 5′-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2)), which are potent inhibitors of rat androgen receptor binding. As steroid hormone receptors exhibit promiscuity in their ability to bind different ligands, the present study evaluated the ability of these vinclozolin metabolites to bind to the estrogen (ER) and progesterone (PR) receptors in vitro, and to alter ER and PR function following in vivo exposure. To this end, in vitro ligand binding assays demonstrated that both M1 and M2 can compete with endogenous ligand for binding to the PR ( K i = 400 and 60 μM, respectively). In contrast, neither metabolite exhibited the ability to bind ER. Subsequent in vivo studies to evaluate the potential of vinclozolin to alter ER or PR function demonstrate that, (1) the estrogen-dependent increases in uterine weight and PR induction were not altered by vinclozolin; (2) the distribution of nuclear and cytosolic PR was not altered following short-term vinclozolin exposure; and (3) vinclozolin did not disrupt ovulation in cycling female rats. These studies indicate that although vinclozolin metabolites can compete for binding to the PR in vitro, concentrations of these metabolites do not reach sufficient levels to disrupt female reproductive function following short-term in vivo exposure to vinclozolin. In addition, these studies demonstrate the importance of correlating in vitro receptor binding data with in vivo studies in order to understand the physiological consequences of exposure to environmental toxicants.

Interaction of nistranol with estradiol and progesterone receptors in the rat uterus cytosol in vitro and in vivo

Interactions between nistranol and estradiol and progesterone receptors in the cytosol fraction of the uterine tissue of oophorectomized rats and the relative competitive capacity of nistranol have been studied 24 h after a single injection of the drug. The results demonstrate the effects of nistranol on estradiol and progesterone binding. Nistranol boosting of uterine growth in rats is explained by its capacity to accelerate the translocation of hor- mone-receptor complexes into the nucleus. Investigations of the capacity of new estrogens to compete with estradiol for binding in the tissues of target organs in vitro and affect estradiol and progesterone binding in vivo permit a more effective screening of estrogens than use of only the classical in vitro method.

Failure of Chloro-S-triazine-Derived Compounds to Induce Estrogen Receptor-Mediated Responsesin Vivoandin Vitro

The potential estrogenic activities of atrazine and simazine were investigatedin vivousing the immature female Sprague–Dawley rat uterus andin vitrousing the estrogen-responsive MCF-7 human breast cancer cell line and the estrogen-dependent recombinant yeast strain PL3. Animals that were dosed with 50, 150, or 300 mg/kg of atrazine or simazine alone for 3 consecutive days did not exhibit any significant increases in uterine wet weight while decreases in cytosolic progesterone receptor (PR) binding levels and uterine peroxidase activity were observed. 17β-estradiol (E2)-induced increases in uterine wet weight were not significantly affected by cotreatment with either chemical; however, some dose-independent decreases in E2-induced cytosolic PR binding and uterine peroxidase activity were observed.In vitro,atrazine and simazine did not affect basal or E2-induced MCF-7 cell proliferation or the formation of nuclear PR–DNA complexes as determined by gel electrophoretic mobility shift assays. In addition, these chloro-S-triazines did not display agonist activity or antagonize E2-induced luciferase activity in MCF-7 cells transiently transfected with a Gal4-human estrogen receptor chimera (Gal4-HEGO) and a Gal4-regulated luciferase reporter gene (17m5-G-Luc). Moreover, the estrogen-dependent PL3 yeast strain was not capable of growth on minimal media supplemented with atrazine or simazine in place of E2. Collectively, these results indicate that the reported estrogenic and antiestrogenic effects elicited by these chemicals are not mediated by the estrogen receptor.

Effects of Progesterone, Estrogen, and Androgen on Progesterone Receptor Binding in Hen Oviduct Uterus (Shell Gland)

The specific [3H]progesterone binding per milligram of protein of the cytosolic fraction of the hen uterus was found to have decreased at 1 and 4 h after the injection of progesterone (P4), but increased at 4 and 8 h after the injection of estradiol-17β (E2), and at 1 and 4 h after the injection of testosterone (T). The equilibrium dissociation constant obtained by Scatchard analysis was not significantly different between the hens receiving steroid injections [P4, E2, T, and dihydrotestosterone (DHT)] and controls. The maximum binding capacity was lower in hens injected with P4 and greater in hens injected with E2, T, or DHT than in uninjected hens. The results suggest that the cytosolic progesterone receptor binding in the hen uterus may be modulated by the sex steroid hormones.

Failure of Chloro-S-triazine-Derived Compounds to Induce Estrogen Receptor-Mediated Responses in Vivo and in Vitro

The potential estrogenic activities of atrazine and simazine were investigated in vivo using the immature female Sprague-Dawley rat uterus and in vitro using the estrogen-responsive MCF-7 human breast cancer cell line and the estrogen-dependent recombinant yeast strain PL3. Animals that were dosed with 50, 150, or 300 mg/kg of atrazine or simazine alone for 3 consecutive days did not exhibit any significant increases in uterine wet weight while decreases in cytosolic progesterone receptor (PR) binding levels and uterine peroxidase activity were observed. 17 beta-estradiol (E2)-induced increases in uterine wet weight were not significantly affected by cotreatment with either chemical; however, some dose-independent decreases in E2-induced cytosolic PR binding and uterine peroxidase activity were observed. In vitro, atrazine and simazine did not affect basal or E2-induced MCF-7 cell proliferation or the formation of nuclear PR-DNA complexes as determined by gel electrophoretic mobility shift assays. In addition, these chloro-S-triazines did not display agonist activity or antagonize E2-induced luciferase activity in MCF-7 cells transiently transfected with a Gal4-human estrogen receptor chimera (Gal4-HEGO) and a Gal4-regulated luciferase reporter gene (17m5-G-Luc). Moreover, the estrogen-dependent PL3 yeast strain was not capable of growth on minimal media supplemented with atrazine or simazine in place of E2. Collectively, these results indicate that the reported estrogenic and antiestrogenic effects elicited by these chemicals are not mediated by the estrogen receptor.

Inhibition of rat sexual behavior by antisense oligonucleotides to the progesterone receptor.

To test further the idea that sexual behavior in rodents is mediated via the progesterone receptor (PR) in the ventromedial nucleus of the hypothalamus, antisense and sense oligonucleotides to progesterone receptor were administered intracerebroventricularly into the third cerebral ventricle of ovariectomized estrogen-primed animals. Progesterone-facilitated sexual behavior was inhibited in animals treated with antisense oligonucleotides, with proceptive and receptive responses being minimal or completely suppressed. Sexual behavior was not altered by control sense oligonucleotides. In vitro binding assays of the cytosol progesterone receptors demonstrated a 52.2% reduction of PRs in the hypothalamus of animals that received antisense oligonucleotides, suggesting a reduction in PR synthesis. These data suggest that a threshold level of estrogen-induced hypothalamic PR is critical in the regulation of progesterone-facilitated sexual behavior in female rats.

Changes of progesterone receptors in mesometrial and antimesometrial deciduoma after removal of fetus or placenta in unilateral pregnant hamster

The effects of fetuses or placentas (with fetuses) removal on progesterone receptors (PR) in deciduomal tissues were investigated in unilateral pregnant (ULP) hamsters. Deciduomal reactions in response to artificial stimuli were induced on the tube-ligated uterine horns of ULP hamsters. Fetuses or placentas (with fetuses) were removed on day 8 of ULP, and the cytosolic and nuclear PR (cPR, and nPR, respectively) levels in deciduomal tissues at mesometrial (MS) and antimesometrial (AMS) sites were separately studied on day 10 and 12 of ULP. In the sham control group, cPR at the MS decreased with time, but at a slower rate than that at the AMS. The MS deciduoma contained higher cPR than the AMS deciduoma on day 12. Removal of fetuses did not cause any significant change of the decline rate, and the PR levels were similar to that of the control. The nPR at the MS and AMS also declined with time in all three groups. Removal of placentas induced drastic losses of cPR and nPR at the MS and AMS on day 10 and 12. Supplement of progesterone (P; 2 mg/animal/day) to the placenta-removed group restored the cPR somewhat at the MS site, but not back to the level of the control. The effect of P to restore nPR levels could be observed at the MS and AMS on day 10, but only at the MS on day 12. These data suggest that placental factor(s) other than P may play an important role on the maintenance of PR in deciduoma. Other cellular mechanisms may exist at the MS and AMS sites for the heterogeneous responses.

Effects of immunosuppressants FK506 and rapamycin on the heterooligomeric form of the progesterone receptor

The non-DNA binding form of the rabbit uterus cytosol progesterone receptor (PR) contains, in addition to the hormone binding unit and heat shock protein M r 90kDa (hsp90), a H eat shock protein B inding I mmunophilin (p59/HBI) which interacts with hsp90. P59/HBI binds the immunosuppressants FK506 and Rapamycin (RAP) and belongs to the FK506 binding protein family. A recombinant p59/HBI-glutathione- S-transferase fusion protein, purified by Sephadex LH-20 filtration of tritiated drug-p59/HBI complexes, binds FK506 and RAP with apparent K d values of 75±40 and 40±15 nM, respectively. Immunopurification from cytosol of [ 3H]steroid-labeled tungstate-stabilized PR with anti-PR immunoadsorbent yielded “9S”-PR species in which hsp90, hsp70 and p59/HBI were present. In the absence of tungstate ions, only the 4–6S Pr was eluted, and Western blot analysis demonstrated the absence of hsps and p59/HBI. In contrast 30 to 50% of the original 9S-PR species containing hsps and p59/HBI, was eluted in the absence of tungstate ions but after exposure of cytosol to 5 μM FK506 or RAP. Other experiments showed that cytosol fractions incubated for 2 h at 25°C with 0.05 to 10 μM FK506 or RAP, then with [ 3H]steroids (the agonist [ 3H]Org 2058 or the anti-progestin [ 3H]RU486), contains greater amounts of 9S-PR species than that detected in non-immunosuppressant exposed control cytosol. Scatchard analysis showed an up to 2-fold decrease of the K d values for both hormones following exposure to drugs, without modification of the number of steroid binding sites. Purification of cytosol PR on immobilized FK506 yields a 9S form still containing hsp90, hsp70 and p59/HBI associated to PR units. Altogether, these results suggest that binding of immunosuppressants to p59/HBI does not promote hsps dissociation from the receptor and, as a consequence, that inhibition of peptidyl-prolyl isomerase activity of p59/HBI by immunosuppressants binding does not transform (activate) PR in vitro. However, given the assumption that hsp90 binds to receptor and that p59/HBI binds hsp90 but not directly to receptor, immunosuppressants affect hormone binding by an unknown mechanism involving receptor associated proteins. In addition, we show that the chick oviduct cytosol 9S-PR, not displaced with the EC1 antibody specific for several mammalian p59/HBI, also binds to FK506 columns and can be eluted by exchange with either FK506 or RAP, suggesting that there is an avian HBI homolog. These findings extend the notion of immunophilin-steroid receptor association and suggest that, besides the already described molecular chaperone hsp90, other associated proteins might be involved in steroid receptor function.

Effect of tamoxifen alone and in combination with RU 486 on the endometrium in the mid-luteal phase.

The effects of RU 486 combined with tamoxifen and tamoxifen alone on hormonal parameters and endometrial development at the time of implantation were studied. Measurements of cytosolic oestrogen and progesterone receptors in endometrium and placental protein 14 (PP14) in plasma were also included. Three dosage schedules were used: single oral dose of 40 mg tamoxifen alone and in combination with 200 mg RU 486, and 40 mg tamoxifen for three consecutive days starting on the first day after the luteinizing hormone (LH) surge. The combined treatment prolonged the luteal phase (P < 0.05) and increased the plasma levels of progesterone. A single dose of tamoxifen did not affect the bleeding pattern and plasma hormone levels, but raised plasma oestradiol and LH with the 3-day treatment. The endometrium was retarded after the combined and the 3-day treatment with tamoxifen. Concentrations of cytosolic progesterone receptors were higher after the combined therapy, but were unaffected after tamoxifen only. PP14 levels were higher (P < 0.05) after repeated tamoxifen doses than in controls, but were lower with combined treatment. Progesterone and oestrogen are evidently necessary for endometrial maturation during the secretory phase of the menstrual cycle. PP14 levels in plasma cannot be used for clinical assessments of endometrial function because high levels coincide with disturbed endometrial development.