Cytosolic Progesterone Research Articles

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)

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 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.

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.

Long-term effects of organophosphates in sheep dips?

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 (Ki = 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.

Uterine estradiol and progesterone receptor concentration, activities of certain antioxidant enzymes and dehydrogenases and histoarchitecture in relation to time of secretion of nidatory estrogen and high endometrial sensitivity in rat.

Alterations in uterine nuclear and cytosolic estradiol (ER) and progesterone (PR) receptor concentration, activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), glucose-6-phosphate dehydrogenase (G-6-PDH) and lactate dehydrogenase (LDH), surface and transmission electron microscopy and histology in relation to the time of secretion of nidatory estrogen and the onset of endometrial sensitivity in the rat were investigated. A significant increase in plasma estradiol (E2) concentration in control rats was observed at 22.00 h on day 4 post-coitum, whereas progesterone (P) concentration increased at 17.00 h on day 4 and was maintained until 17.00 h on day 5. The period of high endometrial sensitivity (10.00 h on day 5) was characterized by elevated uterine cytosolic ER and nuclear and cytosolic PR concentration and POD activity, low columnar luminal epithelium with undulating surface and intercellular membranes, covered with short microvilli and pinopods, and containing numerous electron-transparent apical vesicles, mitochondria, polyribosomes, rough (RER) and smooth (SER) endoplasmic reticulum, well developed Golgi, few lysosomes and lipid droplets and loose edematous antimesometrial stroma. Inhibition in endometrial sensitivity by post-coital centchroman was associated with a marked depletion in uterine cytosolic ER and an increase in nuclear ER concentration, a decrease in POD and G-6-PDH activities, compact fibroblastic stroma, an increase in luminal epithelial cell height with decreased RER, SER, polyribosomes, Golgi, straightening of intercellular membranes, reduced surface undulations and absence of pinopods. Electron-transparent vesicles appeared flattened and clumped in the apical portion of cells, tight junctions were more prominent and lipid droplets were translucent. Nuclear and cytosolic PR and the pattern of secretion or plasma E2 and P remained unaffected. CAT, SOD and LDH activities, although high throughout pre-implantation, did not vary in relation to the secretion of nidatory estrogen, endometrial sensitivity or centchroman treatment.

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.

Expression of steroid receptors in acoustic neuroma

The expression of steroid receptors has been investigated in an attempt to clarify the role of steroid hormones in the proliferation and progression of acoustic neuromas. Specimens of tumours taken during translabyrinthine surgery were tested for cytosolic (c) and nuclear (n) steroid receptors. Oestrogen and progesterone receptor levels were evaluated by enzymatic immuno-assay, while androgen receptor binding levels were detected by dextran-coated charcoal method in a single-step determination. In some cases, the six point Scatchard analysis of cytosolic and nuclear androgen receptor was also performed. Threshold values were: 3 fmol/mg of proteins for cytosolic steroid receptors and 20 fmol/mg DNA for nuclear steroids, which corresponded to approximate median values of cytosolic and nuclear oestrogen and progesterone, respectively. Oestrogen and progesterone appeared to be localized more frequently in the nuclei rather than in the cytosol (70% oestrogen and progesterone positivity in the nuclei; 30% oestrogen, 40% progesterone positivity in the cytosol), while androgen receptors were preferentially localized in the cytosol (80% positivity in the cytosol; 40% positivity in the nuclei). A negative non-linear correlation between cytosolic oestrogen and cytosolic androgen receptors was found. There was a direct linear correlation between cytosolic oestrogen and nuclear oestrogen levels. A strict correlation between nuclear oestrogen and nuclear progesterone incidence was shown. Preliminary analysis of clinical data and biochemical parameters showed that cytosolic progesterone levels inversely correlated with tumour size.

Modulation of natural killer cell-mediated cytotoxicity by tamoxifen and estradiol

The nonsteroidal antiestrogenic drug, tamoxifen, inhibits the growth of estrogen receptor-positive tumors by interfering with the growth-stimulatory effect of estradiol. However, there is compelling evidence that tamoxifen treatment also is beneficial for patients with estrogen receptor-negative tumors. The hypothesis that tamoxifen is capable of enhancing the immunologic defense of tumor-bearing hosts was been investigated as a possible method for targeting receptor-negative neoplasms. Natural killer (NK) cells in the spleen of female Fisher and Wistar-Furth rats were used against the YAC-1 murine lymphoma target in 51Cr-release assays. The effect of various concentrations of estradiol and tamoxifen (1 nM-1 microM) and of the metabolic inhibitors actinomycin D and cyclohexamide on target-cell killing was investigated. Tamoxifen enhanced and estradiol inhibited killing if applied for the entire 5 hours of the cytotoxic reaction. When applied jointly in this experimental setup, estradiol interfered with the enhancing effect of tamoxifen. Pretreatment of target cells with tamoxifen led to highly significant enhancement of cytotoxicity; estradiol also enhanced target cell killing, but to a lesser extent. After joint treatment, the level of cytotoxicity was comparable with that obtained with tamoxifen alone. Both pharmacologic (100 nM and 1 microM) and physiologic (1 or 10 nM) concentrations of estradiol and equimolar tamoxifen enhanced target cell lysis. However, pharmacological levels of estradiol inhibited effector cells when applied alone or in combination with tamoxifen. Highly significant enhancement of target-cell destruction occurred if both target and effector cells were pretreated with tamoxifen, whereas estradiol treatment of both cell types led to slight enhancement or no effect on cytotoxicity. Treatment of the target cells with actinomycin D or cycloheximide inhibited the lysis of untreated and tamoxifen- or estradiol-exposed cells. Treatment of YAC-1 target cells with tamoxifen or estradiol also enhanced the NK cell-mediated release of the nuclear label, 3H-thymidine, indicating DNA degradation. Similarly treated P815 cells resisted lysis by NK cells, but showed sensitization when the NK cells were stimulated by interleukin-2 for 48 hours before the lytic reaction. Estradiol and tamoxifen changed the kinetics of 3H-thymidine incorporation by YAC-1 cells, but the cells were capable of growing with the highest drug concentrations (1 microM) used in the cytotoxicity experiments. YAC-1 cells have no cytosolic estradiol receptors and are weakly positive for cytosolic progesterone receptors. These experiments indicate that NK cell-mediated target-cell destruction can be enhanced by tamoxifen primarily through sensitizing the target for lysis. Estradiol also sensitizes the target but inhibits the effector cells simultaneously so that little or no change results in cytolysis. Target-cell sensitization is not mediated by classical estrogen receptors and requires the active metabolic participation of the cells treated. A likely mechanism of this phenomenon is priming the target cell for apoptosis.

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.

Uterine estradiol and progesterone receptor concentration in relation to circulating hormone levels and histoarchitecture during high endometrial sensitivity and induced decidualization in guinea pigs

Alterations in nuclear and cytosolic estradiol (ER) and progesterone (PR) receptor concentration in the antimesometrial (AM) and mesometrial (M) segments of the uterus in relation to circulating hormone levels, histology and surface topography during the period of high endometrial sensitivity and development of trauma-induced decidualization in cyclic guinea pigs were investigated. The period of high endometrial sensitivity (i.e. day 5 of the estrous cycle) was characterized by elevated plasma estradiol and progesterone and their receptors in the nuclear and cytosolic fractions of the uterus. There was, however, no difference in the concentration of these receptors or the surface ultrastructure in the AM and M segments. Unilateral traumatization by scissor cut along the AM length of the uterus on day 5 of the estrous cycle induced decidual cell reaction resulting in a marked increase in weight of the decidualized (traumatized) uterine horn with advancing decidualization to reach maximum levels (926% of the contralateral nontraumatized uterine horn) 7 days after traumatization. This was associated with decidual transformation and a marked increase in nuclear and cytosolic ER and PR concentration in the AM segment of the traumatized uterine horn. An increase in receptor concentration in the M segment of the traumatized uterine horn or the AM segment of the nontraumatized uterine horn was transitory and of a low order. Receptor concentration in the M segment of the nontraumatized uterine horn remained low throughout days 8–12 of the cycle. Findings indicate a possible role of both estradiol and progesterone in induction of endometrial sensitivity and development and maintenance of decidua in the guinea pig.

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.

Bovine Uterine, Cervical and Ovarian Androgen Receptor Concentrations

Bovine cytosol androgen receptor (ARC) concentrations were examined simultaneously in various regions of the uterus and in ovarian tissues of cows, and were related to cytosol estrogen (ERC) and progesterone receptor (PRC) concentrations and circulating steroid levels. ERC concentrations were 3-7-fold and PRC concentrations 13-29-fold those of ARC in bovine endometrial and myometrial tissues. When serum progesterone levels were low, both endometrial and myometrial ARC, endometrial ERC, and endometrial and myometrial PRC concentrations were higher (p < 0.05) than those observed during higher progesterone concentrations. Because serum 5 alpha-dihydrotestosterone (5 alpha-DHT) concentrations were higher during the luteal phase, it is possible that ARC was down-regulated by this natural ligand at this phase of the cycle. There were no differences between uterine horns in endometrial or myometrial ARC concentrations. Bovine cervical and ovarian stromal tissue also contained ARC, and the concentrations were about the same as in the endometrium and the myometrium. The relative binding affinities (RBAs) of some steroid hormones towards ARC in vitro were: the synthetic compound R1881 (146%), 5 alpha-dihydrotestosterone (100%), testosterone (75%) while estradiol-17 beta, progesterone and dexamethasone had lower RBAs (2, < 1, < 1% respectively). Cytosol androgen receptor concentrations correlated significantly with cytosol progesterone (PRC) and estrogen receptor (ERC) concentrations, both in the endometrium and myometrium. These data show that androgens, such as 5 alpha-DHT, may participate the endocrine regulation of bovine reproductive tissues.

Insulin-like growth factor I receptors in human corpora lutea

To determine the presence and binding characteristics of insulin-like growth factor (IGF) receptors in corpora lutea (CL) of spontaneous and clomiphene citrate (CC)-induced cycles and to identify any relationship between IGF receptors and cytosol progesterone (P) and 17 alpha-hydroxyprogesterone (17 alpha-OHP) levels. Women undergoing bilateral tubal ligation were divided into two groups. One group received no medication (controls) and the other took 50 mg of CC. Midluteal phase CL were recovered at tubal ligation for hormone and receptor analysis. Patients were recruited from a university hospital setting. Eleven fertile women 26 to 37 years of age requesting bilateral tubal ligation were studied. Four women were given 50 mg/d of CC from days 5 through 9 of study cycle. Seven women did not take any medication. Minilaparotomy bilateral tubal ligation and luteectomy were performed 7 to 9 days after midcycle urinary (LH) surge. Insulin-like growth factor receptor concentrations and binding characteristics and cytosol P and 17 alpha-OHP levels in individual CL. Optimal binding for 125I-IGF-I with membrane fractions of homogenized CL was obtained with incubation at 4 degrees C for 16 hours. Specific binding (mean +/- SEM) was significantly higher in CC-treated (53.6% +/- 4.8%) than in control cycles (25.9% +/- 5.5%, P less than 0.001). Receptor concentrations were also significantly higher in CL from CC-induced (145.6 +/- 21.8 pmol/mg protein) than from control cycles (74.8 +/- 15.2 pmol/mg protein, P less than 0.02). Insulin-like growth factor receptor levels correlated with neither serum nor cytosol P and 17 alpha-OHP in CL from either cycles. Specific IGF-I receptors are present in human CL of the menstrual cycle with higher concentrations present in CC-induced cycles. Thus IGF may express its action on luteal function through its receptors in CL.

Enhancement of Progesterone Receptor Bindings by Estradiol in Pituitary Ceils of the Hen

Isolated cells from pituitaries of nonlaying hens were incubated in vitro with and without estradiol-17β (E2), and the amount of progesterone receptor binding in the cytosolic fraction of the cells was measured by an exchange assay. When the cells were incubated with 1.0 ng E2 for 1, 2, or 4 h, the progesterone receptor bindings were increased. The bindings were not increased when incubated together with cycloheximide, actinomycin-D, or α-amanitin. The results suggested that the cytosolic progesterone receptor binding of pituitary cells was enhanced by the action of estrogen through a protein-synthesizing pathway.

Progesterone regulation of progesterone receptor in mesometrial and antimesometrial deciduoma in pseudopregnant hamster

Experiments were performed to examine the thropic effects of progesterone (P) on the progesterone receptor (PR) and the maintenance of the deciduoma. Deciduomal reactions in response to artificial stimuli were induced in hamsters' uteri on the 4th (D 4) day of pseudopregnancy (PSP). On and after the 8th day, PSP hamsters received P supplement (2mg; s.c.) daily. Histological observation revealed that the life span of the deciduoma was partially prolonged. The maintenance and regression of the deciduoma was heterogeneous. P was able to maintain the morphology of the cells at the mesometrial site (MS) on D 12, whereas cells at the antimesometrial site (AMS) regressed. The cytosol progesterone receptor (cPR) concentration in cells of the MS was higher than that of the AMS on D 10. The cPR in MS and AMS decreased drastically on D 12. The nuclear PR (nPR) remained at a higher concentration in both tissues on D 10 followed by a precipitous decrease. The deciduomal nPR in the AMS decreased at a faster rate. By day 12, the nPR in the MS was much higher than that in the AMS. These data show that P acts as a trophic hormone of the deciduoma. The maintenance of deciduoma is closely related to the presence of PR. Other mechanisms may exist for the heterogeneous responses observed at the MS and AMS.