On the regulation and turnover of progesterone metabolites in rat uterus

Recently the in vitro progesterone metabolism was shown to be inhibited in uterine tissue by association of the hormone with binding components. However, a dissociation of progesterone would impair the protection of the steroid hormone caused by complex formation. In order to study this effect, the influence of time was investigated on the metabolism of progesterone. Progesterone metabolites were analysed quantitatively from the recovered material of uteri and nutrient media by thin layer chromatography (TLC) at various time invervals. After finishing the incubation with the labelled steroid, the amount of progesterone metabolites produced increased continuously in the tissue during the following hour when the uteri were kept in nutrient medium. This indicated that the dissociation of progesterone from a hormone protein complex led to the subsequent metabolism of the unbound hormone. However, the metabolism was reduced markedly by an increase of the protein content in uterine tissue and with it by an increase of progesterone binding proteins in uterine cytosol as determined by charcoal adsorption technique. Additionally, the amount of progesterone metabolites was found to be much higher in uterine tissue than that released into nutrient medium during the time interval studied. Therefore, uterine tissue concentrates progesterone metabolites, and a rapid turnover of these substances does not occur.     

The in vivo regulation of the progesterone "receptor" in guinea pig uterus: dependence on estrogen and progesterone

An assay for quantifying the high affinity progesterone binding protein in guinea pig uterine cytosol was developed using Florisil to separate bound and free steroid. The activity of the progesterone binding protein increased between 4–12 hours following estrogen administration and by 4 days of treatment was 10-fold higher than castrate controls. When estrogen administration was discontinued the progesterone binding activity declined slowly with a half-life of 3 days. By contrast, progesterone treatment caused a rapid decline of binding activity within 3 hours. These studies suggest that the antagonistic actions of estrogen and progesterone determine the quantity of available progesterone binding sites in guinea pig uterine cytosol.     

Progesterone in the uterus. V. Correlation of the in vitro progesterone metabolism with the progesterone binding in rat uterus.

Incubations of rat uterine segments with varying 3H-progesterone concentrations were performed to study the hormone uptake by the tissue. The radioactivity of the uterus and the nutrient medium were plotted in form of a SCATCHARD plot. Additionally, the binding capacity of the uterine cytosol was measured. In both systems, the hormone was found to be associated with two components which differ from each other in their association constants. The progesterone metabolism occuring at a hormone concentration of 10(-6)M and more in the incubation medium is discussed with respect to the affinity and the capacity of the hormone binding components.     

Progesterone in the uterus. IV. Dependence of the in vitro progesterone metabolism in the rat uterus on the progesterone concentration.

After incubation of uterine segments of normal rats with various 3H-progesterone concentrations in nutrient medium, different patterns of radioactive steroids were obtained in uterine tissue. Using hormone concentrations of less than 5 X 10(-7)M progesterone metabolites could not be detected in the tissue. A series of metabolites appeared with progesterone concentrations of 10(-6)M and higher. Six radiometabolites were identified and two were characterized.     

Effect of estradiol and progesterone on phosphatidylinositol metabolism in the uterine epithelium of the mouse

The effect of estradiol and progesterone on uterine phosphatidylinositol (PtdIns) metabolism was examined in whole uteri and separated uterine luminal epithelium of ovariectomized mice. Incorporation of [3H]myo-inositol in vitro, into inositol-containing phospholipids extracted from whole uteri, increased in mice injected with estradiol, with maximal incorporation at 9-12 h. The breakdown of PtdIns to inositol polyphosphates was also stimulated in whole uteri by estrogen, with an abrupt increase between 6 and 9 h. Comparable increases in both processes occurred in the uterine epithelium after estrogen stimulation and were inhibited by progesterone pretreatment which by itself had little or no effect. These results suggest that PtdIns metabolism is involved in the stimulation of uterine epithelial cell proliferation by estrogens, and its inhibition by progesterone.     

Regulation of uterine progesterone receptors by the nonsteroidal anti-androgen hydroxyflutamide

We have recently reported that the anti-androgen hydroxyflutamide causes delayed implantation and exhibits antideciduogenic activity in the rat. The present experiments were conducted to examine whether hydroxyflutamide binds to the uterine progesterone receptors and/or alters the progesterone binding sites in the uterus. Cytosol and nuclear fractions from decidualized rat uterus were incubated with [3H]-R5020 without or with increasing concentrations of radioinert R5020, RU486, dihydrotestosterone, or hydroxyflutamide. From the log-dose inhibition curves, the relative binding affinity of both hydroxyflutamide and dihydrotestosterone was less than 0.1% and 2%, compared with R5020 (100%) for displacing [3H]-R5020 bound to uterine cytosol and nuclear fractions, respectively. Injection of estradiol-17 beta (1 microgram/rat) to ovariectomized prepubertal rats induced a 1.85-fold increase in uterine weight by 24 h. Hydroxyflutamide at 2.5 or 5.0 mg did not significantly alter the estrogen-induced increase in uterine weight. Compared to vehicle alone, estrogen induced an approximately 5-fold increase in uterine cytosolic progesterone binding sites. Hydroxyflutamide at both 2.5- and 5.0-mg doses significantly attenuated the estrogen-induced elevation in uterine progesterone binding sites. These studies demonstrate that hydroxyflutamide does not bind with high affinity to progesterone receptors, but suppresses the estrogen-induced elevation in progesterone receptor levels in the uterus.     

Metabolism of progesterone in rat uterus

The in vitro metabolism of progesterone was studied in uteri of untreated and estrogen stimulated immature rats. In intact uteri the rate of metabolism varied with the hormonal status of the animal in a concentration dependent manner. At a low (3 × 10^?9M) progesterone concentration the rate of ring A reduction was decreased in estrogen stimulated uteri. At a high progesterone concentration (3 × 10^?6M) the rate of ring A reduction was increased after estrogen treatment. The rate of reduction of the C20 ketone was increased after estrogen treatment at all concentrations of incubated progesterone. In dilute homogenates of uterus, estrogen stimulation always increased the rate of progesterone metabolism.Estrogen stimulation results in increased concentration of progesterone receptor in the uterus. It is proposed that increased activity of ring A reductases also occurs. The relative influence of these two factors on the metabolism of progesterone is dependent on the progesterone concentration in the incubation medium.     

Uterine glutathione reductase activity: modulation by estrogens and progesterone

The aim of this study was to determine whether glutathione reductase activity in uterine tissue is regulated by sex hormones. In spayed rats uterine glutathione reductase was significantly increased by exogenous estrogen (P< 0.01), progesterone (P< 0.01) or estrogen plus progesterone (P<0.01). When enzyme activity is expressed per mg protein, daily administration of estrogen or progesterone induces a progressive increase of this enzyme between 24 to 48 h or 24 to 72 h of treatment, respectively. Whereas the combination of both steroids causes an earlier and higher increase in glutathione reductase activity at 24 h of treatment. Estradiol singly or in combination with progesterone induced the highest protein concentration in the uterus. Whereas uterine DNA concentration is only significantly affected by estradiol. Our results suggest that uterine glutathione reductase is regulated by estradiol and progesterone and may be involved in maintaining levels of reduced glutathione in the uterus. This compound may be required for control of the redox state of thiol groups and in detoxification reactions involving H2O2 and electrophylic substances. The antioxidant action of estrogens is partially due to the stimulation of glutathione reductase.     

Changes of progesterone content of rat uterine flushings in relation to serum concentrations of progesterone during the oestrous cycle.

Uterine fluid was collected from four-day cyclic rats at each stage of the oestrous cycle and assayed for progesterone and protein content. Progesterone was determined by radioimmunoassay either after ethanol (or 2.5% NaOH) denaturation of proteins from uterine flushings ('total' progesterone) or without protein denaturation ('ether-extractable' progesterone). The amount of 'ether-extractable' progesterone in the lumen was constant from metoestrus to pro-oestrus (340 pg per uterus) but lower in oestrus (200 pg per uterus). However, 'total' progesterone content of uterine fluid was subject to cyclic variations and was highest in dioestrus (890 pg per uterus) and lowest in oestrus (350 pg per uterus), in contrast to serum progesterone which is lowest in dioestrus and highest in oestrus. Protein content of uterine flushings peaked to 780 micrograms per uterus in pro-oestrus then fell to about 140 micrograms per uterus until the end of the oestrous cycle. Changes in protein content of the lumen were followed by qualitative variations since the mean amount of 'bound' progesterone ('total' progesterone minus 'ether-extractable' progesterone) released per milligram of denatured lumen protein rose from 1.8 pmol in pro-oestrus to 18.2 pmol in dioestrus. The changes of luminal 'bound' progesterone during the oestrous cycle suggest that progesterone binding to luminal proteins could be an important modulator of progesterone action in rat uterus. Moreover, the variations in progesterone content of the lumen, irrespective of serum progesterone concentrations, are consistent with the hypothesis that progesterone synthesis occurs in the uterus.     

Binding of radiolabelled luteinizing hormone to intact and ovariectomised rat uterus.

Binding of ovine LH to uterine tissue preparation from intact and ovariectomised rat clearly indicates that uterus possesses specific binding sites for LH. Binding characteristics of LH to uterine tissue preparation from intact rat showed saturability with high affinity and low capacity. Scatchard plot analysis showed dissociation constant of the specific binding site to be 0.12 x 10(-9) mol/l and the number of binding sites was 2.31 +/- 0.05 f mol/mg protein. Ovariectomy did not change the binding affinity but effected a decrease in the number of binding sites (1.7 +/- 0.08 f mol/mg protein). LH treatment of ovariectomized (ovx) rat had no effect on binding affinity but significantly increased the number of binding sites (3.23 +/- 0.1 f mol/mg protein). Reduction of uterine weight due to ovariectomy and marked increase of ovx rat uterine weight by LH administration indicate a source of estrogen in ovx rat. An in vitro uterine tissue slice (from intact and ovx rat) incubation showed depletion of 17 beta-estradiol (E2) content in ovx rat which significantly elevated on LH addition. Data suggest that LH binding to rat uterine tissue has biological relevance.     

Rapid recovery of nuclear estrogen receptor and oxytocin receptor in the ovine uterus following progesterone withdrawal

We previously showed that progesterone rapidly down regulates nuclear estrogen receptor (Re) in the estrogen-primed rodent uterus. We have now extended these studies to test the response of the Re system in sheep uterus to progesterone withdrawal. Since the estrogen-Re complex is believed to regulate hormone-dependent gene expression, it was of interest to determine whether withdrawal of progesterone under constant estrogen stimulation would lead to the recovery of nuclear Re levels and estrogen action, i.e. oxytocin receptor (ROT) synthesis. Ovariectomized ewes were primed with estradiol-17 beta and serum steroid levels were maintained by constant infusion of estradiol (0.5 microgram/h) and progesterone (500 micrograms/h) for 5 days. The animals were anesthetized with fluothane/O2, and uterine samples were excised 1 h before and 3, 6 and 12 h after progesterone withdrawal. Estradiol infusion was continued during the experiment in order to maintain estrogen levels at a steady state (14 pg/ml plasma). Re, ROT and progesterone receptor (Rp) were measured in endometrium and myometrium using standard 3H-hormone binding assays. Following progesterone withdrawal, the nuclear Re concentration increased in both uterine compartments, and the nuclear Re level was correlated significantly with the ROT concentration in the membrane fraction of both uterine tissues (endometrium, r = 0.79; myometrium, r = 0.86). Although cytosol Re rose between 6 and 12 h in the endometrium, cytosol Re levels remained unchanged in myometrium. Cytosol Rp appeared to increase in endometrium but not in myometrium. Uterine tissue sampled from a control animal before stopping the progesterone infusion revealed that the observed changes in receptor concentration following progesterone withdrawal were not due to regional differences in receptor levels. These results demonstrate that the recovery of nuclear Re in the ovine endometrium and myometrium following progesterone withdrawal represents a selective effect on Re retention in the nucleus rather than on cytosol Re availability or Re activation which was controlled by constant estrogen infusion. Thus, these results are consistent with the hypothesis that progesterone induces an Re regulatory factor which acts to down regulate nuclear Re, and that the activity of this factor diminishes rapidly after progesterone withdrawal.     

Regulation of calcitonin gene-related peptide receptors in the rat uterus during pregnancy and labor and by progesterone.

Calcitonin gene-related peptide (CGRP) is a potent smooth muscle relaxant in a variety of tissues. We recently demonstrated that CGRP relaxes uterine tissue during pregnancy but not during labor. In the present study we examined whether uterine (125)I-CGRP binding and immunoreactive CGRP receptors are regulated by pregnancy and labor and by sex steroid hormones. We found that (125)I-CGRP binding to membrane preparations from uteri was elevated during pregnancy and decreased during labor and postpartum. Changes in immunoreactive CGRP receptors were similar to the changes in (125)I-CGRP binding in these tissues, suggesting pregnancy-dependent regulation of CGRP receptor protein. CGRP receptors were elevated by Day 7 of gestation, and a precipitous decrease in these receptors occurred on Day 22 of gestation prior to the onset of labor. Both (125)I-CGRP-binding and immunofluorescence studies indicated that CGRP receptors were localized to myometrial cells. Hormonal control of uterine CGRP receptors was assessed by the use of antiprogesterone RU-486, progesterone, and estradiol-17beta. RU-486 induced a decrease in uterine CGRP receptors during pregnancy (Day 19). On the other hand, progesterone prevented the fall in uterine CGRP receptors at term (Day 22). In addition, progesterone also increased uterine CGRP receptors in nonpregnant, ovariectomized rats, while estradiol had no effects. These hormone-induced changes in uterine CGRP receptors were demonstrated by (125)I-CGRP-binding, Western immunoblotting, and immunolocalization methods. These results indicate that CGRP receptors and CGRP binding in the rat uterus are increased with pregnancy and decreased at term. These receptors are localized to the myometrial cells, and progesterone is required for maintaining CGRP receptors in the rat uterus. Thus, the inhibitory effects of CGRP on uterine contractility are mediated through the changes in CGRP receptors and may play a role in uterine quiescence during pregnancy.     

Phospholipase A2 activity in the rat uterus: Modulation by steroid hormones

Phospholipase A2 (PLA2), an enzyme which provides free arachidonic acid for the synthesis of prostaglandins (PG), has been studied in the rat uterus under various experimental conditions. Uterine PLA2 activity increased 14 fold in hypophysectomized rats implanted with Silastic capsules containing estradiol-17β as compared to those treated with oil vehicle. Dexamethasone treatment reduced the PLA2 activity induced by estrogen by 78%. Hypophysectomized animals treated with progesterone (2mg/day) for 5 days had low levels of uterine PLA2 activity but a single injection of estradiol (10ug/rat) given 24 h after the last injection of progesterone increased activity 5 fold within 12 h. Administration of the protein synthesis inhibitor cycloheximide in the rats treated with progesterone, before and after injection of estradiol, prevented the stimulating action of the estrogen on PLA2 activity. If the estrogen was given at the time of the last injection of progesterone, PLA2 activity did not increase until 24 h later and the level was much less than when progesterone was absent. The results are consistent with the view that estrogen stimulates uterine prostaglandin production because of its effect upon PLA2; this effect can be greatly reduced by a glucocorticoid. Progesterone may modulate the PLA2 stimulating effect of estrogen in order to direct the production of specific PGs by regulating the amount of arachidonic acid available for PG synthetase.     

Receptor progesterone complex in the nuclear fraction of the rat uterus: demonstration by 3H-progesterone exchange

We have previously shown that ³H-estradiol exchange can be used to measure the quantity of estrogen receptor complex in the nuclear fraction of target tissue cells. This method has been modified for the measurement of the progesterone receptor complex (R_n·P) in the nuclear fraction of uterine cells. Nuclear fractions were incubated for 5 hrs. at 15°C in the presence of varying concentrations of ³H-progesterone (³H-P) with or without a 250-fold excess of non-labeled progesterone (P). R_n·P was determined by subtracting the ³H-P bound in the presence of excess P (non-specific binding) from ³H-P bound in the absence of excess P (total binding). All R_n·P studies were done in adult castrate female rats that had received estradiol benzoate (0.4 mg) one week before use. The quantity of R_n·P increased in the uterine nuclear fractions by 280% 30 min. after injection of 5 mg of P. R_n·P was not increased in muscle or fat pad by this treatment. Injections of corticosterone (B), cortisol (F), dexamethasone (Dx) or testosterone (T) failed to increase R_n·P. The exchange reaction was specific for P; B, F, Dx or T did not compete. These results demonstrate the existence of a low capacity, high affinity, stereospecific progesterone binding site in the nuclear fraction of the uterus.     

Inhibition of uterine contractility by progesterone and progesterone metabolites: mediation by progesterone and gamma amino butyric acidA receptor systems.

Progesterone and several progesterone metabolites are capable of inhibiting uterine contractility. Some progesterone metabolites have shown little or no affinity for the progesterone receptor but have been found to be potent modulators of the GABAA receptor system. This study examined whether the inhibition of uterine contraction by progesterone and its metabolites was progesterone receptor-mediated or gamma amino butyric acidA (GABAA) receptor-mediated. Uterine contractions were measured in annular rings of uterine tissue, 5 mm in length, from diestrous II rats, under a fixed tension of 1 gram. The steroids tested were 3 beta-hydroxy-5 beta-pregnan-20-one (6 micrograms/ml), 5 beta-pregnane-3,20-dione (10 micrograms/ml), 3 alpha-hydroxy-5 alpha-pregnan- 20-one (3 alpha,5 alpha-THP, 27.5 micrograms/ml), and progesterone (40 micrograms/ml). All compounds significantly inhibited spontaneous uterine contractions when compared to controls. No effect was seen by either 16 micrograms/ml of the progesterone antagonist, RU486, or 32 micrograms/ml of the GABAA antagonist, pictrotoxin, when administered alone. However, when uterine tissues were exposed to a combination of the steroid and the antagonist, the effect of 3 beta-hydroxy-5 beta-pregnan-20-one and 3 alpha,5 alpha-THP was blocked by picrotoxin but not by RU486, indicating that the action of these steroids was mediated through the GABAA system. The effect of 5 beta-pregnane-3,20-dione and progesterone was effectively blocked by RU486 but not by picrotoxin, suggesting that their actions were mediated through the progesterone receptor system. These results indicate that multiple mechanisms exist in the uterus for inhibiting uterine contractility by progesterone and its metabolites.     

A biphasic action of estradiol on estrogen and progesterone receptor expression in the lamb uterus

Regulation of the uterine expression of estrogen and progesterone receptors was studied in 20 three-month-old lambs that were not treated or treated with estradiol- 17beta. Determinations of receptors were performed by binding assays in the nuclear and cytosolic fractions, receptor mRNAs by solution hybridization, and estrogen receptor protein by an enzyme-immunoassay. Estradiol treatment decreased the receptor binding capacity of both receptors and the levels of immunoreactive estrogen receptor 12 h after injection in the absence of decreased receptor mRNAs, suggesting that the initial decrease is due to degradation of the proteins or that mRNAs are translated into new receptor proteins at a reduced rate. The mRNA levels increased after estradiol treatment suggesting that the replenishment phase consists of synthesis of new receptors rather than recycling of inactivated receptors.     

Histochemical demonstration of estrogen and progesterone binding in endometriotic tissue and in uterine endometrium: A comparative study

Estrogen and progesterone binding to endometriotic and endometrial tissue was studied histochemically using estradiol and progesterone fluorochrome derivatives (E2-bovine serum albumin-fluorescein isothiocyanate and progesterone-bovine serum albumin-tetramethylrhodamine isothiocyanate). Thirty endometriotic samples from 21 women were studied, together with endometrial specimens obtained simultaneously from 14 of the women. In 77% of the endometriotic samples binding of the estrogen conjugate was indicated by specific fluorescence in more than half of the epithelial cell population, and in 20% in less than half. The corresponding figures for the progesterone conjugate binding were 75 and 18%, respectively. Blocking studies indicated a reasonable degree of ligand specificity. In endometrial tissue the corresponding figures were 64 and 29%, respectively, for binding of the estrogen conjugate and 54 and 38%, respectively, for binding of the progesterone conjugate. In 7 of 13 cases where evaluable samples of both tissues had been obtained, the relative proportion of fluorescent cells, with either reagent, was similar in the two tissue types. Our results suggest that the cytoplasm of epithelial cells in endometriotic tissue and in uterine endometrium contains specific binding sites for both estrogen and progesterone. The binding pattern of the two conjugates in endometriotic tissue was unrelated to the menstrual phase.     

Cytosol type II sites in the rat uterus: interaction with an endogenous ligand

Previous studies from our laboratory demonstrated that normal, but not malignant tissues, contain a ligand which competes for [3H]estradiol binding to nuclear type II sites in the rat uterus. Since elevated nuclear levels of type II sites are correlated with estrogen stimulation of uterine growth and DNA synthesis, we believe this ligand may regulate cell growth. The present studies show that the ligand for nuclear type II sites also interacts with type II sites in uterine cytosol. This was demonstrated by dilution experiments which show that greater quantities of type II sites are measured in dilute (10 mg/ml) than in concentrated (40 mg/ml) uterine cytosol. Furthermore, stripping of uterine cytosol with 1% dextrancoated charcoal, or pre-binding cytosol type II sites to hydroxylapetite (HAP) prior to binding analysis, removed the ligand from these preparations such that high levels of type II sites were measured. Following charcoal stripping, cytosol type II sites demonstrated good specificity for estrogenic hormones but not progesterone, corticosterone, or the triphenylethylene anti-estrogen, nafoxidine. Since the level of type II sites in the cytosol always preceded and exceeded the level of this site measured in uterine nuclei at all times following estrogen treatment (0-96 h), we believe cytosol type II sites may function as an type II-ligand binding protein (LBP) which regulates the availability of the ligand for interaction with nuclear type II sites. This is consistent with our observation that type II sites are not depleted from uterine cytosol by estrogen treatment and nuclear type II sites are very tightly associated with the nuclear matrix.     

Role of 17 beta-hydroxysteroid dehydrogenase in the modulation of nuclear estradiol receptor binding by progesterone in the rat anterior pituitary gland and the uterus

Progesterone has been shown to decrease occupied pituitary and uterine nuclear estradiol receptor (E2R) binding in mature and immature estrogen-primed rats. Progesterone has also been shown to stimulate pituitary but not uterine 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) in the rat. The conversion of estradiol to its less active metabolite estrone by 17 beta-HSD and activation of phosphatase are among mechanisms considered to be involved in the reduction of E2R. To determine if 17 beta-HSD stimulation was a mechanism by which progesterone induced nuclear E2R decrease, the synthetic estrogen ethinylestradiol, which is not oxidized by 17 beta-HSD, was used instead of estradiol to prime adult ovariectomized rats. When ethinylestradiol-primed rats received 0.8, 2.0 or 4.0 mg/kg body wt of progesterone 2 h before sacrifice, the total and occupied nuclear E2R accumulation in the anterior pituitary by a subsequent ethinylestradiol injection 1 h later did not show any decrease. This response was different from that observed previously in estradiol-primed animals in which progesterone showed a multiphasic decrease of occupied form of nuclear E2R. However, in the uterus of ethinylestradiol-primed rats, a partial decrease of total and occupied nuclear E2R accumulation was observed in the presence of the three doses of progesterone used. The decrease of uterine nuclear E2R with the three progesterone doses was different from the dose-dependent effect of progesterone observed in the uterus of estradiol-primed rats. Affinity constants of the interaction between [3H]estradiol and the nuclear E2R were similar among groups treated with ethinylestradiol, estradiol and progesterone. These results demonstrate the involvement of 17 beta-HSD in the reduction of anterior pituitary gland E2R by progesterone in the estradiol-treated animals. Furthermore, the mechanism of decrease of E2R by progesterone in the uterus appears to be different from the pituitary gland.