Regulation of estrogen receptor alpha and progesterone receptor (isoform A and B) expression in cultured human endometrial cells.

The effects of RU 486 together with estradiol and progesterone on estrogen receptor alpha and progesterone receptor (isoforms A and B) expression were studied in human endometrial long term cultures at the mRNA and protein level. We asked whether ligand induced receptor regulation, found in mammals in vivo, is also found in human cultured endometrial cells with special regard to the progesterone isoforms A and B. Endometrial cultures were maintained for 27 days. Media were supplemented with progesterone and/or estradiol alone or in combination with RU 486. Receptor expression (estrogen receptor alpha and progesterone receptor isoform A and B) was examined at the mRNA level by RT-PCR and at the protein level by western blot analysis. All receptor types examined were expressed in our culture model. Estradiol led to a general increase of receptor expression whereas treatment with estradiol in combination with progesterone down regulated receptor expression. The receptor down regulation was not found when RU 486 was additionally supplemented into the medium. Activation or inhibition of expression due to these treatments was similar for both PR isoforms. Our results (1) show that in our culture system estradiol induced up regulation of estrogen receptor and progesterone receptor A and B and suggest that the estrogen induced up regulation is prevented by progesterone (2) a clear cut antigestagenic effect of RU 486 and (3) suggest that both progesterone isoforms are analogously regulated in our culture model. We conclude that human endometrial cell cultures are suitable for the study of the dynamics of steroid receptor expression.     

The antiglucocorticoid and antiprogestin steroid RU 486 suppresses the adrenocorticotropin response to ovine corticotropin releasing hormone in man

The glucocorticoid and progesterone antagonist RU 486 normalizes the clinical and biochemical features of hypercortisolism in patients with nonpituitary Cushing's syndrome, presumably by antagonizing the action(s) of cortisol. Since RU 486 has progesterone agonist activity in addition to its progesterone antagonist action, the possibility that it might have some glucocorticoid agonist action did not seem unreasonable. To test this hypothesis we examined the effects of RU 486 on pituitary ACTH secretion in 10 patients with primary adrenal insufficiency in whom glucocorticoid replacement was withheld for 36 h. Each patient received, in randomized sequence 3-7 days apart, an oral dose of placebo, RU 486 (20 mg/kg), cortisol (0.1 mg/kg), or a combination of RU 486 and cortisol at 1800 h. Two hours later, an iv bolus dose of ovine CRH (1 microgram/kg) was administered, and plasma ACTH levels were measured serially for 3 h. RU 486 suppressed ovine CRH-stimulated ACTH secretion, albeit less than cortisol. Its glucocorticoid agonist effect was calculated to be approximately 1/250th that of cortisol on a weight basis. Additionally, RU 486 partially antagonized cortisol-induced suppression of ACTH secretion. These findings suggest that RU 486 is a partial glucocorticoid agonist and offer some insight as to its action in patients with Cushing's syndrome. Whether this degree of glucocorticoid agonist activity is adequate to support life, however, is not known.     

Progesterone but not estradiol 17beta potentiates local GH secretions by hormone-dependent breast cancer explants. An in vitro study.

The aim of this study was to evaluate the effects of treatment of breast cancer explants with tamoxifen (TMX) or RU486 on GH secretory dynamics in the presence of exogenous estrogen (E2), progesterone (P4) or both. Explants obtained during surgery were divided according to their sex steroid hormone receptor status. P4 (10(-7) M) or 17beta-estradiol (10(-5) M) or both were tested in vitro for their ability to induce hGH secretion and cell proliferation. TMX (10(-7) M) was added to E2, RU486 (10(-7) M) to P4, and both were applied to E2 plus P4-supplemented cultures. The stimulatory action of P4 on GH secretion was noted in hormone-dependent (ER+/PR+) but not in hormone-independent explants (ER-/PR-). RU486 did not abolish this effect. The stimulatory action of P4 on GH release was not parallel to the stimulation of cell proliferation. E2 alone was without effect on GH secretion by both types of breast cancer explants. Combined treatment with both steroids stimulated GH secretion and cell proliferation by (ER+/PR+) explants. Both TMX and RU486 reversed this effect on cell proliferation while only RU486 abolished augmentation of GH secretion. In none of the hormone-dependent breast cancers, the combined treatment with E2 and P4 had any effect on GH secretion and cell proliferation. Taken together, these results lead us to the hypothesis that P4 but not E2 potentiates local GH secretion by hormone-dependent breast cancer explants. The fact that RU486 reversed neither GH secretion nor cell proliferation in hormone-dependent explants indicates its non-receptor-mediated mechanism of action.     

The stimulation of prostaglandin production by two antiprogesterone steroids in human endometrial cells

Endometrial stromal cells and isolated endometrial glands obtained from women during days 6-26 of the ovarian cycle were cultured for 24 h in the presence of the progesterone antagonists 17 beta-hydroxy-11 beta-[4-dimethylaminophenyl]17 alpha-[1-propynyl] estra-4,9-dien-3-one (RU486) and 17 beta-hydroxy-11 beta-[4-dimethylaminophenyl] 17 alpha-[3-hydroxy-1-propenyl]estra-4,9-dien-3-one (ZK 98734). Both steroids stimulated prostaglandin F2 alpha (PGF2 alpha) production by stromal cells in a dose-dependent manner, in doses ranging from 10-1000 nM. Progesterone (100 nM) inhibited RU486 stimulation, except at the highest dose of antiprogestin. PGE2 was produced in smaller amounts than PGF2 alpha, but, when measurable, it also increased in the presence of RU486. In contrast, RU486 did not increase PG production by endometrial glands. In an experiment to determine the effect of pretreatment, stromal cells were incubated for 24 h with 1000 nM progesterone or RU486 (all with 100 nM 17 beta-estradiol) with either 30 or 6 microM arachidonic acid. These six batches of cells were incubated for a second 24 h with either progesterone or antiprogestin. Cells pretreated with the higher dose of arachidonic acid had a marked increase in PGF2 alpha production during the second 24 h only when also pretreated with progesterone. This finding suggests that progesterone allows an accumulation of PG precursor in a suitable accessible pool. Pretreatment with progesterone also allowed a greater conversion of PG to its 13,14-dihydro-15-keto metabolite. These results suggest that antiprogesterone steroids may act as menstrual regulators by: stimulating endogenous PG production within the endometrial stromal cells and inhibiting PG catabolism.     

Dickkopf-1, an inhibitor of Wnt signaling, is regulated by progesterone in human endometrial stromal cells

CONTEXT: Some members of the Wnt family, including ligands, receptors, inhibitors, and signaling components, are expressed in human endometrium. Dickkopf-1 (Dkk-1), a potent inhibitor of the Wnt signaling pathway, was recently found to be up-regulated in decidualizing endometrial stromal cells during the secretory phase of the menstrual cycle, suggesting regulation by progesterone. OBJECTIVES: To test the hypothesis that progesterone regulates Dkk-1 expression in human endometrial stromal cells, we investigated the following effects on stromal cell expression of Dkk-1 mRNA and protein: decidualizing stimuli (progesterone or cAMP), RU486 (an inhibitor of progesterone action), and withdrawal of progesterone. RESULTS: Short-term treatment (up to 72 h, which corresponds to the full decidualized phenotype in response to cAMP and an early response to progesterone) did not reveal regulation of Dkk-1 mRNA or protein by cAMP but did show induction of Dkk-1 expression when the cells were treated with progesterone, an effect that was blocked by RU486. In long-term cultures (from 14 to 23 d, which corresponds to the full decidualized phenotype in response to progesterone), a significant increase in Dkk-1 mRNA and protein production was observed. Addition of RU486 or withdrawal of progesterone after long-term decidualization resulted in a decrease of Dkk-1 mRNA and protein to control levels. Estradiol alone had no effect on stromal Dkk-1 expression. CONCLUSIONS: These data strongly support regulation by progesterone of Dkk-1 mRNA synthesis and protein expression in human endometrial stromal cells and that the response is specific for progesterone and independent of cAMP and estradiol.     

Effects of RU486 on progesterone secretion by human preovulatory granulosa cells in culture

The effects of RU486 on progesterone synthesis were studied in human preovulatory granulosa cells in culture. No effect was observed at 1 and 10 micrograms/mL, but at 100 micrograms/mL, RU486 inhibited the simulation of progesterone secretion induced by LH and cAMP. It is suggested that the main target of RU486 is the cytochrome P450scc function [catalyzing the formation of pregnenolone (D5P) from cholesterol], since no accumulation of D5P or hydroxy derivatives of progesterone was observed. As RU486 is an antiglucocorticosteroid and antiprogesterone agent, the effects of dexamethasone and progesterone were also investigated. Dexamethasone did not modify progesterone secretion, but progesterone inhibited its own synthesis in both the presence and absence of LH. Thus, under these experimental conditions RU486 displayed a progesterone-like effect. However, since the effect of RU486 was observed only at a concentration around 10(-4) M, the mechanism of action may not involve a receptor pathway and may not apply to most clinical circumstances.     

Endometrial and pituitary responses to the steroidal antiprogestin RU 486 in postmenopausal women

The effects of the antiprogestin RU 486 on the human endometrium were investigated. Seventeen postmenopausal women were injected with estradiol (E2) benzoate (0.625 mg/day) for 15 days. Progesterone (P) (25 mg/day) and/or RU 486 (100 or 200 mg/day) were given to groups of 2-3 women during the last 6 days of E2 benzoate treatment. Serial blood samples were drawn for the measurement of plasma E2, P, and LH and FSH. An endometrial biopsy was performed on the last day of treatment, and processed for histology or for assays of DNA polymerase alpha, E2-dehydrogenase (E2DH), and P receptor (PR). Treatment with E2 benzoate alone resulted in a marked decrease of plasma gonadotropins; in those patients who received either P, RU 486, or both, in addition to E2 benzoate, the concentrations of plasma LH and FSH were further decreased to premenopausal levels. In absence of glycerol, the affinity of RU 486 for the endometrial PR (Kd = 0.8 nM) was higher than that of P (Kd = 1.2 nM). Glycerol decreased markedly the affinity of RU 486, whereas the affinity of P for the PR was unchanged. RU 486 had negligible affinity for plasma transcortin. Either P or RU 486, but not both together, induced secretory changes in the endometrium as determined from histologic sections of tissue biopsies. Either P or RU 486 decreased DNA polymerase alpha and increased E2-DH activities in the endometrium. Unexpectedly, when P and RU 486 were given together. E2-DH activity remained at the level found in E2-treated women. In vitro cultures of proliferative endometrium treated with the synthetic progestagen R 5020 or with RU 486 also had increased E2-DH activity; RU 486 counteracted R 5020 effects. We conclude that, contrary to previous results with experimental animals, the anti-P RU 486 has some progestomimetic activity in humans under specific conditions. Paradoxically, when given together with P, RU 486 lost most of its progestomimetic activity in the endometrium and behaved as a pure antagonist.     

Divergent effects of antiprogesterone, RU 486, on progesterone, relaxin, and prolactin secretion in pregnant and hysterectomized pigs with aging corpora lutea

Porcine corpora lutea produce progesterone and relaxin during pregnancy and after hysterectomy. Peak amounts of relaxin are released into peripheral blood in both pregnant and hysterectomized animals on about day 113 (estrus = day 0 and term = 114), and this release coincides with an abrupt decrease in the progesterone concentration. RU 486, a progesterone receptor antagonist, was used to investigate the effects of interruption of progesterone binding to its receptor on luteal function and gonadotropin secretion of pigs with aging corpora lutea. RU 486 was administered orally to hysterectomized gilts (surgery on day 8) once a day (0800 h) on days 111-115 at two dosages (group 1, 2 mg/kg BW; group 2, 4 mg/kg BW). During 5 days of RU 486 treatment, plasma progesterone concentrations in both treated groups were markedly elevated (32 and 37 ng/ml for groups 1 and 2) compared with 22 ng/ml in the controls (group 3; P less than 0.01). PRL concentrations increased in both groups (9 and 13 ng/ml) and differed significantly from those of the controls (3 ng/ml) (P less than 0.04). RU 486 treatment delayed the time of relaxin peak to days 116.1 and 117.0 in groups 1 and 2 compared with day 114.1 in the controls (P less than 0.01). Pregnant gilts received RU 486 orally once a day (0800 h) at 4 mg/kg BW beginning on day 111 until parturition occurred. Parturition was induced on day 112.7 after only two RU 486 treatments compared with day 114.7 in the control group (P less than 0.01). Progesterone decreased abruptly from a pretreatment mean of 11 to less than 0.6 ng/ml during the 2 days that RU 486 was given compared with a shift from 12 to 6 ng/ml during the same period in the controls (P less than 0.01). The time of the relaxin peak was advanced to day 112.1 in RU 486-treated gilts compared with day 113.9 in the controls (P less than 0.01). Results from this study provide strong evidence that the antagonistic effect of RU 486 on progesterone receptor results in an abrupt increase in PRL and progesterone secretion in hysterectomized gilts with aging corpora lutea. In marked contrast with hysterectomized animals, the acute luteolytic effects of RU 486 depend on the presence of the uterus and/or conceptuses in the pig. Disruption of the regulatory loop of progesterone secretion by RU 486 alters the ability of corpora lutea to produce and release peak quantities of relaxin.     

Progesterone effects on cell growth of U373 and D54 human astrocytoma cell lines

Astrocytomas are the most frequent primary brain tumors and constitute a leading cause of cancer-related deaths. We studied the effects of progesterone and its antagonist, RU486, on cell growth of two human astrocytoma cell lines with different evolution grade (U373, grade III; and D54, grade IV). Progesterone receptor expression was determined by Western blot. The effects of different doses of progesterone and RU486 on cell number, cell cycle, and apoptosis were analyzed for five consecutive days. Progesterone (10 nM) significantly increased the number of D54 cells from the second day of culture, and the number of U373 cells on days 3–5. RU486 (10 μM) blocked progesterone effects in both astrocytoma cell lines. Interestingly, RU486 administered without progesterone significantly reduced the number of cells from the second day of culture in both cell lines. Progesterone increased S phase of cell cycle in U373 cells (61%, on day 5). RU486 blocked the effects of progesterone on cell cycle but administered alone did not significantly change cell cycle profile. DNA fragmentation (TUNEL) assay showed that the diminution in the number of astrocytoma cells produced by RU486 was not by apoptosis. Progesterone receptor isoforms were detected in both cell lines. Our data suggest that progesterone induces cell growth of human astrocytoma cell lines through the interaction with its nuclear receptor.     

Progesterone antagonists increase androgen receptor expression in the rhesus macaque and human endometrium

Antiprogestins (APs) inhibit estradiol (E(2))-stimulated endometrial growth in women and nonhuman primates, but the mechanism of this "antiestrogenic" action is unknown. Here, we report that APs up-regulate endometrial androgen receptor (AR) in both women and macaques, an effect that might play a role in the antiproliferative effects of APs on the primate endometrium. In addition, because there are discrepancies in the literature on the regulation and localization of AR in the primate endometrium, we used both in situ hybridization and immunocytochemistry to evaluate hormonal influences on endometrial AR in women and macaques. In ovariectomized macaques, the following treatments were given for 4 weeks each: E(2) alone, E(2) + progesterone (P), E(2) + mifepristone (RU 486), and E(2) + P + RU 486. In women, samples were obtained during the normal menstrual cycle and after treatment with either RU 486 for 30 days at 2 mg/day, or after a single oral administration of 200 mg RU 486 on cycle day LH + 2. In macaques, E(2) significantly increased AR expression above vehicle controls; E(2) + RU 486 increased binding further; E(2) + P decreased AR binding; and E(2) + P + RU 486 treatment caused an intermediate elevation in AR binding. In macaques treated with E(2) alone, stromal AR staining was predominant, and P treatment suppressed that staining. E(2) + RU 486 or E(2) + P + RU 486 treatment produced a striking up-regulation of glandular epithelial AR staining and enhanced the stromal AR signal. In situ hybridization analyses confirmed the immunocytochemistry data. Similar induction of glandular AR staining and enhanced stromal AR staining were obtained in macaques treated with ZK 137 316 and ZK 230 211. During the natural cycle in women, stromal AR staining predominated and was greater in the proliferative than the late secretory phase. RU 486 treatment of women up-regulated glandular epithelial AR staining after either daily treatment for 30 days with 2 mg/day or after a single oral dose of 200 mg. In summary, endometrial AR was highest in the stroma during the human proliferative phase (or during E(2) treatment in macaques) and lowest during the late secretory phase in women (or after E(2) + P treatment in macaques). In both species, RU 486 induced AR expression in the glands and enhanced AR expression in stromal cells. Because androgens can antagonize E(2) action, enhanced endometrial AR expression induced by APs could play a role in the antiproliferative, "antiestrogenic" effects of APs in primates.     

Mifepristone (RU486) protects Purkinje cells from cell death in organotypic slice cultures of postnatal rat and mouse cerebellum

Mifepristone (RU486), which binds with high affinity to both progesterone and glucocorticosteroid receptors (PR and GR), is well known for its use in the termination of unwanted pregnancy, but other activities including neuroprotection have been suggested. Cerebellar organotypic cultures from 3 to 7 postnatal day rat (P3-P7) were studied to examine the neuroprotective potential of RU486. In such cultures, Purkinje cells enter a process of apoptosis with a maximum at P3. This study shows that RU486 (20 microM) can protect Purkinje cells from this apoptotic process. The neuroprotective effect did involve neither PR nor GR, because it could not be mimicked or inhibited by other ligands of these receptors, and because it still took place in PR mutant (PR-KO) mice and in brain-specific GR mutant mice (GRNes/Cre). Potent antioxidant agents did not prevent Purkinje cells from this developmental cell death. The neuroprotective effect of RU486 could also be observed in pathological Purkinje cell death. Indeed, this steroid is able to prevent Purkinje cells from death in organotypic cultures of cerebellar slices from Purkinje cell degeneration (pcd) mutant mice, a murine model of hereditary neurodegenerative ataxia. In P0 cerebellar slices treated with RU486 for 6 days and further kept in culture up to 21 days, the synthetic steroid increased by 16.2-fold the survival of pcd/pcd Purkinje cells. Our results show that RU486 may act through a new mechanism, not yet elucidated, to protect Purkinje cells from death.     

Involvement of the adrenergic system on the release of prolactin and lactogenesis at the end of pregnancy in the rat

The part played by the adrenergic system on the release of prolactin and lactogenesis induced by prostaglandin F2 alpha and the antiprogesterone RU 486 was studied in pregnant rats. Two doses of prostaglandin F2 alpha (150 micrograms) administered at 08.00 and 12.00 h on day 19 of pregnancy induced, at 12.00 h on day 20 (24 h after administration), a significant increase in the serum concentration of prolactin, with a significant decrease in serum progesterone levels. These hormonal changes significantly augmented casein and lactose levels in the mammary gland. Treatment with RU 486 (2 mg/kg) at 08.00 h on day 19 augmented casein and lactose concentrations in the mammary gland at 12.00 h on day 20 without modifying serum concentrations of prolactin and progesterone. The adrenergic antagonists, propranolol (3 mg/kg), metoprolol (10 mg/kg), ICI 118,551 (200 micrograms/kg), idazoxan (100 micrograms/kg) and prazosin (10 mg/kg), were administered s.c. at 12.00 and 20.00 h on day 19 and 08.00 h on day 20 of pregnancy to intact rats or to rats previously treated with RU 486 or prostaglandin F2 alpha. These adrenergic antagonists did not modify serum prolactin or progesterone levels in intact or RU 486-treated rats, but serum prolactin levels in the prostaglandin F2 alpha-treated group were significantly reduced by treatment with propranolol, metoprolol or prazosin. In addition, propranolol and ICI 118,551 also decreased the casein and lactose concentrations in the mammary glands of RU 486- and prostaglandin F2 alpha-treated rats, while the other compounds had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Progesterone, but not estrogen, stimulates vessel maturation in the mouse endometrium

The human endometrium undergoes regular periods of growth and regression, including concomitant changes in the vasculature, and is one of the few adult tissues where significant angiogenesis and vascular maturation occurs on a routine, physiological basis. The aim of this study was to investigate the effects of estrogen and progesterone on endometrial vascular maturation in mice. Endometrial tissues were collected from early pregnant mice (d 1-4) and ovariectomized mice given a single 17beta-estradiol (100 ng) injection 24 h before dissection (short-term estrogen regime) or three consecutive daily injections of progesterone (1 mg) with/without estrogen priming (progesterone regime). Experiments were then repeated with the inclusion of mice treated concurrently with progesterone and either RU486 or a vascular endothelial growth factor-A antiserum. Proliferating vascular mural cells (PVMC) were observed on d 3-4 of pregnancy, corresponding with an increase in circulating progesterone. A significant increase in PVMC and alpha-smooth muscle actin (labels mural cells) coverage of vessel profiles were observed in mice treated with progesterone in comparison to controls; no significant change was noted in mice treated with estrogen or with vascular endothelial growth factor antiserum. RU486 treatment did not inhibit the progesterone-induced increases in PVMC and mural cell coverage, although progesterone-induced changes in endothelial and epithelial cell proliferation were inhibited. These results show that progesterone, but not estrogen, stimulates vessel maturation in the mouse endometrium. The work illustrates the relevancy of the mouse model for understanding endometrial vascular remodeling during the menstrual cycle and in response to the clinically important progesterone receptor antagonist RU486.     

Clinical use of RU 486: control of the menstrual cycle and effect on the hypophyseal-adrenal axis

RU 486 is a synthetic 19 norsteroid with a great affinity for progesterone and glucocorticosteroid receptors. The antiprogesterone activity of RU 486 permits interruption of the luteal phase of the menstrual cycle and of early pregnancy, while the antiglucocorticosteroid activity interrupts the feedback mechanisms of cortisol at the hypothalamic-pituitary axis. 11 women aged 18-34 years who were 6-8 weeks pregnant were given daily doses of 200 mg of sensation of lipothymia. Blood pressure and laboratory test results remained normal. The daily increase of beta human chorionic gonadotropin (HCG) and progesterone was stopped by the antiprogesterone. It has been demonstrated in vitro that RU 486 inhibits secretion of beta HCG by cells of trophoblastic origin. 2 weeks after expulsion of the products of conception, the blood levels of beta HCG, progesterone, and estradiol had returned to nonpregnant values and ovarian activity was normal in women not using oral contraceptives. The reason for failure of expulsion in 2 cases was not known. RU 486 was also used to interrupt the luteal phase of normal menstrual cycles in 3 young women. 50 mg/day of RU 486 starting on the 22nd day was sufficient to induce bleeding within 48 hours. Studies using RU 486 50 mg/day beginning on the 10th cycle day indicate that progesterone plays a central and follicular role during the preovulatory period. The antiglucocorticosteroid activity of RU 486 was observed in pregnant women who received the abortifacient dose of 200 mg/day. The 8 a.m. plasma level of ACTH and beta-LPH cortisol remained elevated for thee 4 days of treatment. Nonpregnant women receiving 50 mg of RU 486 for 4 days beginning on the 22nd cycle day had no change in the level of hypophyseal-adrenal hormones.dd Tests in young male volunteers showed that RU 486 amplified circadian rhythms of the hypophysealadrenal axis, affecting specifically the morning hormonal levels without influencing afternoon levels. A test in which RU 486 was used to antagonize the inhibition producedd by dexamethasone during a test of suppression of the hypophyseal-adrenal axis confirmed that the antiglucocorticosteroid effect of RU 486 is produced at the level of the glucocorticosteroid receptors. The antiglucocorticosteroid effect is independent of the antiprogesterone effect. RU 486 may be utilizable in fertility control, to control undesirable effects of steroids in cases of hypercorticism, and perhaps eventually to test the hypophyseal-adrenal axis.     

Effects of an antiprogesterone (RU486) on the hypothalamic-hypophyseal-ovarian-endometrial axis during the luteal phase of the menstrual cycle

The impact of the antiprogesterone RU486 [17 beta-hydroxy-11 beta-(4-dimethylaminophenyl) 17 alpha-(1-propynyl)estra- 4,9-dien-3-one] on the hypothalamic-pituitary-ovarian-endometrial axis was examined in normal cycling women during the mid (MLP)- and late (LLP) luteal phases. During the MLP, 10 women received 3 mg/kg RU486 for 3 days. During the LLP, a single dose of 600 mg RU486 was administered to 4 women, and in another 4 women a single dose of 3 mg/kg was given during corpus luteum rescue by hCG. Longitudinal studies with daily and frequent blood samples (every 10 min for 10 h) were conducted during 3 consecutive cycles (control-treatment-recovery). During the MLP, RU486-induced uterine bleeding occurred in all 10 women 36-72 h after the first dose. No histological evidence of endometrial breakdown was found in endometrial biopsies taken 12-24 h before the onset of bleeding. Significant decreases in LH secretion (P less than 0.001) and LH pulse amplitude (P less than 0.006) and blunted pituitary responses to GnRH (P less than 0.01) were evident by the last treatment day, but LH pulse frequency did not change. Complete luteolysis occurred in 2 of the 10 women. Incomplete luteolysis occurred in 8 women and was associated with an initial decline of serum estradiol (P less than 0.001), but not progesterone levels, followed by rebound increases (P less than 0.001) in LH, estradiol, and progesterone levels 3 days later, which may have reversed the luteolytic processes and prolonged corpus luteum function. Spontaneous luteolysis ensued 3-5 days later with the onset of second episodes of uterine bleeding. For serum FSH, an early rise occurred during the luteal phase in advance of the onset of the second episodes of uterine bleeding. This rise may have resulted in early follicle recruitment and accounted for the shorter duration of the follicular phase during recovery cycles. During the LLP, the single RU486 dose resulted in significant decreases in LH pulse amplitude (P less than 0.03), frequency (P less than 0.05), and secretion (not significant) within 12 h. The recovery cycle was entirely normal. Corpus luteum rescue with incremental doses of hCG did not prevent uterine bleeding after RU486 treatment. These findings indicate that RU486 operates at multiple sites and implies that progesterone is important in the control of luteal function. Further, our data provide a basis for exploring the potential use of RU486 as a once a month birth control agent.     

The effects of RU 486 on immune function and steroid-induced immunosuppression in vitro

The effect of RU 486 [17 beta-hydroxy-11 beta-(4-dimethylamino-phenol)17 alpha-(prop-1-ynyl)estra- 4,9diene-3-one] on [3H]thymidine incorporation into Concanavalin-A-stimulated human peripheral blood mononuclear cells and its influence on the suppressive effects of cortisol and progesterone were investigated. Cortisol suppressed lymphocyte thymidine incorporation at 10(-5), 10(-6), and 10(-7) M (17.6%, 20%, and 38% of control, respectively; P less than 0.01). Cortisol-induced suppression was reversed when low concentrations of RU 486 (10(-7) and 10(-6) M) were added. RU 486 at 10(-5) M further suppressed lymphocyte thymidine incorporation when added to cultures with cortisol. Progesterone significantly inhibited lymphocyte thymidine incorporation at 10(-5) M (8.2% of control; P less than 0.01). No reversal of progesterone-induced suppression of thymidine incorporation was seen when RU 486 was added to cultures; rather, further suppression of thymidine incorporation was seen. RU 486 alone in culture at concentrations achieved therapeutically (10(-5) M) significantly inhibited thymidine incorporation (7.2% of control; P less than 0.01). These findings suggest that RU 486 may have dose-dependent mixed agonist/antagonist effects on cortisol-induced immunosuppression. The lack of an antagonist effect of RU 486 on progesterone suggests that progesterone's immunosuppressive effects may not be receptor mediated. Finally, our findings would suggest that some immunosuppression may be seen at currently used doses of RU 486.     

Different effects of the antiprogesterone RU486 on progesterone secretion by the corpus luteum of rats with 4- and 5-day oestrous cycles.

Administration of the antiprogesterone RU486 (2 mg/day) for 14 days to rats with a 5-day reproductive cycle resulted in an increase in both ovarian and pituitary weight in contrast with rats with a 4-day oestrous cycle. Luteal progesterone production decreased earlier in 4-day than in 5-day cyclic rats. Treatment of 5-day cyclic rats with antiprogesterone from the day of metoestrus onwards resulted in the advancement of the preovulatory prolactin surge by 24 h. Progesterone production by the corpus luteum was, however, not affected, indicating that in 5-day cyclic rats the corpora lutea are still functionally active at the time of the preovulatory surge of prolactin. They become, therefore, stimulated both in size and progesterone production. In contrast, the corpora lutea in 4-day cyclic rats are functionally inactive at the time of the preovulatory surge of prolactin, and prolactin acts luteolytically. In conclusion, the advancement of the preovulatory surge of prolactin by 24 h accounts, at least in part, for the increase in ovarian weight in 5-day cyclic rats after treatment with antiprogesterone. The results of these experiments do not agree with a direct effect of the antiprogesterone RU486 on progesterone secretion by the corpus luteum.     

Antiprogesterone RU486 increases serum immunoreactive inhibin levels and LH:FSH and testosterone:oestradiol ratios in cyclic rats.

Since administration of the antiprogesterone RU486 to cyclic rats results in a dissociation of basal LH and FSH secretion we studied its effects on peripheral levels of inhibin, oestradiol and testosterone throughout the oestrous cycle. Cyclic rats were given RU486 (2 mg) twice daily (09.00 and 17.00 h) on metoestrus, dioestrus and pro-oestrus. Oil-treated rats were used as controls. Serum concentrations of immunoreactive inhibin in oil-treated rats increased from metoestrus to pro-oestrus and decreased at oestrus. RU486-treated rats had serum inhibin concentrations significantly increased over oil-treated rats at dioestrus and pro-oestrus, but not at oestrus. At both pro-oestrus and oestrus serum concentrations of LH, testosterone and oestradiol were significantly raised in RU486-treated rats compared with oil-treated controls. In contrast, serum FSH concentrations in RU486-treated rats were decreased on both days. Ovaries from RU486-treated rats showed an increased testosterone content at pro-oestrus, mainly in the interstitial tissue. The results of the present study demonstrate that RU486 has a stimulatory effect on inhibin secretion, and offer an explanation for the decrease in basal serum FSH levels. The low FSH secretion on the morning of oestrus in spite of the low levels of inhibin suggests that progesterone is involved in FSH secretion at this time.     

Lack of synergy between estrogen and progesterone on local IGF-I, IGFBP-3 and IGFBP-2 secretion by both hormone-dependent and hormone-independent breast cancer explants in vitro. Effect of tamoxifen and mifepristone (RU 486).

The aim of the present study was to investigate direct effects of estrogen (E2) or progesterone (P4) given separately vs. estrogen+progesterone on local IGF-I, IGFBP-3 and IGFBP-2 secretion. Explants obtained from estrogen receptor positive plus progesterone receptor positive (ER+/PR+) and hormone receptors negative (ER-/PR-) tumors were incubated with E2, P4 or both. Tamoxifen was added to E2-exposed cultures; mifepristone (RU 486) was added to P4, and both were given to E2+P4-supplemented cultures. In hormone-dependent and hormone-independent tissues, treatment with estrogen+progesterone increased IGF-I and IGFBP-2 secretion with concomitant decrease in IGFBP-3, in the same manner as E2 or P4 given alone. Tamoxifen decreased the E2- and E2+P4-stimulated IGF-I secretion by hormone-dependent breast cancer explants. RU 486 decreased the P4- and E2+P4-stimulated IGF-I secretion with parallel stimulation of IGFBP-3 secretion by ER+/PR+ explants. Estradiol and progesterone had a synergistic action on IGFBP-2 secretion by hormone-dependent breast cancer explants. In conclusion, the presented data suggest that there is no synergistic action of E2 and P4 in influencing IGF/IGFBPs ratio and, additionally, suggest a protective action of antiestrogen and antiprogestagen against excessive IGF-I secretion.     

Effects of an antiprogesterone agent, RU-486, on the menstrual cycle of the rhesus monkey

RU-486 (RU), a synthetic steroid with antiprogesterone receptor activity, was used to study the role of progesterone in the normal menstrual cycle of rhesus monkeys. The drug, at dosages of 2, 4, or 12 mg/kg, was administered as a single im injection during the luteal phase (days 5-8) in 16 experiments in 8 monkeys. RU had acute effects on the endometrium, as it induced menstruation within 3 days in spite of persistant progesterone elevations, thereby shortening the cycle length by about 5 days. Long term effects on menstrual cyclicity were also found with the higher RU doses. The intermenstrual interval after RU treatment increased from 31.8 +/- 2.4 (+/- SE) days after a 2 mg/kg dose of RU to 82.4 +/- 6.8 days after a 12 mg/kg dose of RU, with a control cycle length of 29.4 +/- 0.7 days. This prolonged interval was related to a delay in the completion of the follicular maturation process and, therefore, a delay in ovulation, as judged by estrogen and progesterone concentrations. Subsequent menstrual cycles were regular and ovulatory. Our results suggest that RU binds to the endometrial progesterone receptor, thereby inducing premature menstruation in the presence of elevated progesterone levels. RU also exerts long term effects on the hypothalamo-hypophyseal axis, since it significantly alters menstrual cyclicity.