Flutamide counteracts the antiproliferative effects of antiprogestins in the primate endometrium

In addition to blocking progesterone (P) action, antiprogestins (APs) also inhibit estrogen-stimulated endometrial cell proliferation in nonhuman primates and women. This effect is paradoxical because APs do not bind to estrogen receptors (ER), and AP + estradiol (E(2)) treatment leads to elevation of ER levels, a recognized action of estrogen in the endometrium. Recently, we showed that APs (RU 486, ZK 137 316 and ZK 230 211) also elevate endometrial androgen receptor (AR) in macaques and women and we hypothesized that over expression of AR may play a role in the antiproliferative actions of APs. We now report that cotreatment with the specific antiandrogen flutamide (FLU) blocked the suppressive effects of APs on estrogen action in the endometrium. We treated ovariectomized rhesus macaques with either E(2) alone, E(2) + ZK 137 316 or E(2) + ZK 137 316 + FLU daily for 28 days. Endometrial wet weight (mean +/- SE) from one-half of the endometrium was as follows: E(2)-treated controls, 360 +/- 32 mg; E(2) + ZK 137 316, 64 +/- 10 mg; and E(2) + ZK 137 316 + FLU, 265 +/- 92 mg (P < 0.05). Mean mitoses/1000 epithelial cells +/- SE was: E(2) alone, 6.25 +/- 0.6; E(2) + ZK 137 316, 0.3 +/- 0.25; and E(2) + ZK 137 316 + FLU, 5.1 +/- 3.8 (P < 0.05). FLU also blocked the hyalinizing degradation of the spiral arteries typically induced by APs. These results indicate that many of the antiendometrial effects of APs in primates may involve the AR.     

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.     

The effect of the antiprogestins RU 486 and ZK 98734 on the synthesis and metabolism of prostaglandins F2 alpha and E2 in separated cells from early human decidua

Enriched preparations of glandular and stromal cells were obtained from early human decidua and incubated for 24 h in the presence of two progesterone antagonists, RU 486 (17 beta-hydroxy-11 beta-[4-dimethylaminophenyl]17 alpha-[1-propynyl]-estra-4,9-dien-3-one) and ZK 98734 (17 beta-hydroxy-11 beta-4[4-dimethylaminophenyl]17 alpha-[3-hydroxy-1-propynyl]estra-4,9-dien-3-one) to determine the effect of the antiprogestins on the release of prostaglandin F2 alpha (PGF2 alpha) and PGE2 and their subsequent conversion to 15-keto-13,14-dihydro-PGF2 alpha and 15-keto-13,14-dihydro-PGE2. In the presence of exogenous arachidonic acid (AA, 30 microM), both steroids stimulated PGF2 alpha release by glandular, but not stromal, cells (P less than 0.001) and inhibited the metabolism of PGF2 alpha by the glandular fraction (P less than 0.005 and P less than 0.001 respectively). In the absence of exogenous AA, RU 486 and ZK 98734 stimulated the release of PGF2 alpha from glandular, but not stromal, cells (P less than 0.001 and P less than 0.005, respectively). Neither steroid altered the release or metabolism of PGE2 when the cells were incubated with AA, but both RU 486 and ZK 98734 increased the release of PGE2 by glandular, but not stromal, cells when incubated without AA (P less than 0.005 and P less than 0.001, respectively). Both steroids inhibited the metabolism of PGE2 under these conditions (P less than 0.05). These results suggest that 1) antiprogestins stimulate the synthesis of PGs by glandular cells in early human decidua, but do not alter the synthesis of PGs by stromal cells; 2) this stimulation of PG synthesis involves an effect on cyclooxygenase activity and is not a consequence of increased availability of endogenous AA; 3) the metabolism of PGs by glandular cells is altered by RU 486 and ZK 98734; 4) as RU 486 has greater antiglucocorticoid activity than ZK 98734, these results suggest that both steroids act on decidua by antagonizing endogenous progesterone rather than glucocorticoid activity.     

Estrogenicity of isoflavones on human endometrial stromal and glandular cells

Endometrium consists of different cell populations such as epithelial and stromal cells and is mainly regulated by sex steroids. Isoflavones are plant-derived estrogenic compounds that have estrogenic and antiestrogenic properties in a cell-specific manner. We hypothesized that one of the potential health benefits of isoflavones may be their ability to regulate endometrial cell function. The present study was conducted to assess estrogenic and/or antiestrogenic effects of isoflavones (genistein, genistin, daidzein, and daidzin) in cultured human endometrial stromal and glandular (Ishikawa) cells by MTT colorimetric cell proliferation assay, proliferating cell nuclear antigen expression, and alkaline phosphatase activity assays. Experiments were performed in a time- (24-96 h) and concentration-dependent (10(-12) to 10(-5) M) manner. All isoflavones used in the present study induced endometrial stromal and Ishikawa cell proliferation when compared with control (vehicle) group in a time- (at 48 h and afterward) and concentration-dependent manner (at 10(-8) M and above) (P < 0.05). However, isoflavones (at 10(-8) and above concentrations) were also antiestrogenic when combined with estradiol (E(2)) (P < 0.05). The isoflavones revealed a weak estrogenic activity (39-67% less than E(2)) as assessed by alkaline phosphatase activity (P < 0.05), but when administered together with E(2), they antagonized estrogen induced alkaline phosphatase activity by 36-89% (P < 0.05). We conclude that, although isoflavones alone have weak estrogenic effects on endometrial stromal and glandular cells, in the presence of E(2) they act as antiestrogens.     

Immunocytochemical localization of estrogen receptors in the macaque endometrium during the luteal-follicular transition

We examined changes in estrogen receptors (ERs) in endometrial stromal and epithelial cells in cynomolgus macaques during artificially induced menstruation and repair. We used Silastic implants filled with either estradiol (E2) or progesterone (P) to treat spayed animals for 14 days with E2 followed by 14 days of E2 plus P. We then withdrew the P (but not the E2) implants and removed uteri 0, 0.5, 1, 2, 3, 4, 5, 7, and 14 days later. Uterine tissues were assayed biochemically for ER content, fixed for histology and frozen for immunocytochemistry of ER with monoclonal antiestrophilins. On day 0, ER levels in endometrium were low [1330 +/- 201 (n = 9) fmol/mg DNA]. An increase in total receptor was evident by 3-4 days of P withdrawal 2762 +/- 190 (n = 6) fmol/mg DNA; P less than 0.001]. Total receptor concentrations increased linearly with time from 0.5-7 days of P withdrawal (r = 0.88). On day 0, staining for nuclear ER in the glandular epithelium and stroma of zones I, II, and III of the endometrium was negative. Beginning at 12-24 h and continuing through 4 days of P withdrawal, nuclear staining became detectable and increased in intensity only in endometrial stromal fibroblasts and myometrial smooth muscle cells. The glandular epithelium of the endometrium did not develop nuclear staining until 5-7 days of P withdrawal, coincident with a 10-fold increase in the mitotic index of the epithelium of the upper zones. Thus, the increase in endometrial ER levels that occurred during the first 5 days of an induced luteal-follicular transition took place almost exclusively in stromal fibroblasts.     

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.     

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.     

Antiprogestins RU486 and ZK299 suppress basal and LHRH-stimulated FSH and LH secretion at pituitary level in the rat in an oestrous cycle stage-dependent manner

We have previously shown that administration of antiprogestin (AP) type II RU486 to ovariectomized (OVX) rats on the morning of pro-oestrus decreases the magnitude of preovulatory gonadotrophin surge. This suggests that the effect of RU486 on LHRH-dependent gonadotrophin release may be independent of its ability to block progesterone actions. The aim of the present research was to study the possible site of RU486 action and to determine whether the gonadotrophin suppressive effect of APs RU486 and ZK299 is dependent on the oestrogen background. Intact or OVX rats in the morning of pro-oestrus were injected s.c. with 4 mg of RU486 or ZK299 (AP type I) at 0900 h on pro-oestrus. At 1830 h, serum concentration of FSH and LH and median eminence (ME) content of LHRH were determined. In the second experiment, the effect of RU486 and ZK299 on pituitary responsiveness to LHRH (100 ng, i.p.) and ME content of LHRH at 1830 h pentobarbital-blocked intact or OVX rats was evaluated. In the last study, the anterior pituitary release of FSH and LH from pro-oestrus or metoestrus donors incubated with or without LHRH (1, 10 or 100 nM) in the presence or absence of APs (20 nM) was evaluated. Both APs reduced serum FSH and LH levels at 1830 h on pro-oestrus in intact and OVX rats. The suppressive effect on gonadotrophin release brought about by AP treatment was also evidenced in PB-blocked intact and OVX rats. This suggested that the inhibitory effect of APs occurred, at least in part, at pituitary level. Furthermore, in the absence of the natural ligand, APs significantly reduced basal and LHRH-stimulated FSH and LH release from pro-oestrous but not from metoestrus pituitaries. In conclusion, these experiments have shown, both 'in vivo' and 'in vitro', that APs RU486 and ZK299 have suppressive effects at pituitary level on basal and LHRH-stimulated FSH and LH secretion, regardless of their antiprogestagenic activity, in pro-oestrus but not in metoestrus.     

Inhibition of proliferation and differentiation by RU 486 in human endometrial stromal and epithelial cells in vitro.

The effects of progesterone and RU 486 on cellular proliferation and differentiation in long term cultures of mixed human endometrial cells were studied. The endometrial tissue was obtained from women with normal menstrual cycles who were undergoing hysterectomy for benign growths. Estradiol supplemented cultures were treated with progesterone and/or RU 486 for 27 days. Cell number was measured by crystal violet assay, and prolactin secretion was used as a marker of differentiation. Progesterone doubled the rate of proliferation, but the addition of RU 486 reduced it to baseline again. The gestagen increased prolactin secretion up to 30 times, while the addition of RU 486 suppressed it to baseline levels. When administered to cells that were pretreated with progesterone for 15 days RU 486 abolished the progesterone effects. RU 486 alone was without any effect. Our results indicate that (1) in vitro progesterone is essential for the initiation and maintenance of proliferation and differentiation of endometrial cells and (2) RU 486 acts as a pure progesterone antagonist in our culture model.     

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.     

Disruption of follicular maturation and delay of ovulation after administration of the antiprogesterone RU486

To investigate the role of progesterone in the follicular phase, we examined the effects of RU486 in eight normal cycling women studied with daily and frequent blood sampling (every 10 min for 10 h) during three menstrual cycles (control-treatment-recovery). RU486 (3 mg/kg, orally) was administered for 3 consecutive days after ultrasound documentation of a dominant follicle. In six of the eight women, RU486 was given after emergence of the dominant follicle, while in two women, RU486 was initiated during the preovulatory period when estradiol levels had exceeded 917 pmol/L. In the six women given RU486 after emergence of the dominant follicle, RU486 significantly prolonged the follicular phase duration from 15.6 +/- 1.9 (+/- SD) to 28.6 +/- 9.3 days (P less than 0.01) and extended the treatment cycle length to 42.3 +/- 9.1 (+/- SD) days (P less than 0.01). During RU486 treatment, mean serum estradiol levels decreased from 385 +/- 43 to 228 +/- 28 pmol/L (P less than 0.01), while LH, FSH, ACTH, cortisol, and progesterone levels changed little. LH pulse frequency and amplitude on the last day of RU486 administration did not differ from control values. Collapse of the dominant follicle was evident on ultrasound after RU486 administration and was not accompanied by uterine bleeding. In the two women treated during the preovulatory period, the follicular phase was not prolonged, and RU486 failed to delay the onset of the LH surge. Our findings indicate that RU486 treatment during the follicular phase interrupts normal follicular development, resulting in a delay of ovulation and a reinitiation of follicular recruitment.     

Progesterone withdrawal up-regulates vascular endothelial growth factor receptor type 2 in the superficial zone stroma of the human and macaque endometrium: potential relevance to menstruation

Several reports indicate that vascular endothelial growth factor (VEGF) expression is increased in endometrial glands and stroma during the menstrual phase in the human endometrium. Here we report that VEGF receptor type 2 (KDR), normally expressed only in the vascular endothelium, was dramatically up-regulated in the stromal cells of the superficial endometrial zones during the premenstrual phase in both human and macaque endometrium. This increase was detectable by Northern analysis, in situ hybridization, and immunocytochemistry and was cell specific, zone specific, cycle phase specific, and VEGF receptor type specific. That is, it only occurred during the premenstrual/menstrual phase, did not occur in glandular epithelium, endothelium, or stromal cells of the deepest endometrial zones, and was not observed for VEGF receptor type 1. The upregulation of stromal KDR was induced by progesterone (P) withdrawal in both women and macaques, and adding back P 24 h after P withdrawal in macaques blocked stromal, but not vascular, endothelial KDR expression. Promatrix metalloproteinase-1 (MMP-1) was coordinately up-regulated in the same stromal cell population by P withdrawal. Because of reports that VEGF can enhance MMP expression, we hypothesize that VEGF-KDR interactions may influence MMP expression in the superficial zones of the primate endometrium during the premenstrual phase, and that these interactions play a role in the induction of menstruation.     

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.     

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 the antiprogesterone steroid RU 486 during midluteal phase in normal women

The antiprogesterone steroid RU 486 (17 beta-hydroxy-11 beta-4-dimethyl-aminophenyl)17 alpha(1-propynyl)estra-4,9-dien-3-one) was given orally to 32 normally cycling women for 4 days, starting on the fourth day of the luteal phase. Uterine bleeding occurred on the third day of RU 486 administration in all 14 women treated with 100 mg/day, in 7 of the 8 women treated with 50 mg, and in 8 of 10 women receiving 25 mg/day. Premature luteal regression induced by RU 486 occurred in 8 women treated with 100 mg/day, in 3 treated with 50 mg, and in 2 receiving 25 mg/day. Plasma LH was measured every 15 min from 0800-1200 h for 5 days in 17 women. Mean LH levels decreased and pulsatile release disappeared in 7 of the 8 women treated with 100 mg, in 2 of 4 receiving 50 mg, and in 1 of 5 treated with 25 mg. RU 486 had no effect when given to 5 women with anovulatory cycles for 4 days starting on day 18 of the cycle. In conclusion: 1) RU 486, given to normally cycling women at midluteal phase, provokes uterine bleeding. 2) This effect occurs whether or not luteal regression is induced by the compound, indicating that RU 486 acts directly upon the endometrial tissue, very likely at the progesterone receptor level. 3) The drug may impair simultaneously or separately luteal function and gonadotropin secretion in a dose-dependent manner. 4) The lack of antiglucocorticosteroid activity, at the dosage of 100 mg/day, suggests that RU 486 may be useful for fertility control.     

Regulation of prolactin production by progestin, estrogen, and relaxin in human endometrial stromal cells

Human decidua synthesizes and secretes PRL. We identified the PRL synthesized in endometrial stromal cells and investigated the effect of medroxyprogesterone acetate (MPA), estradiol (E2), porcine relaxin (RLX), and RU486, an antiprogestin, on PRL production by stromal cells from non-pregnant endometrium in primary culture. Stromal cells were isolated from proliferative and secretory endometria and individually cultured in nutrient medium or medium supplemented with different hormone(s). The immunoreactive PRL isolated from culture medium of hormone-stimulated stromal cells was identified and compared to pituitary PRL. Bio-Gel elution pattern and mol wt analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting showed that PRL produced by stromal cells had properties identical to those of pituitary PRL. In addition, PRL mRNA was identified in hormone-stimulated stromal cells using human pituitary PRL cDNA as a hybridization probe. Analysis of mRNA by Northern blotting showed that the size of PRL mRNA isolated from stromal cells was indistinguishable from that of PRL mRNA in human decidua and pituitary tissue. These results indicated that PRL measured in culture medium was synthesized de novo by stromal cells. The PRL content in culture medium was quantitated by RIA. The PRL production rate in stromal cells cultured without hormones ranged from 6-10 ng/day.mg cell protein. After 4-5 days of incubation with RLX or MPA alone, the PRL production rate increased about 2- to 3-fold over the control value. E2 alone had either no effect or slightly decreased the stromal cell PRL production rate. Stromal cells responded to 0.02 microM MPA, and the maximal response was at 0.1-1 microM MPA. A further increase in PRL production was found when stromal cells were treated with a combination of MPA and E2 and MPA, E2 and RLX. In the presence of MPA or MPA and E2, 0.1 ng/ml relaxin increased the PRL production rate. A potent progestin antagonist, RU486, inhibited PRL production in stromal cells treated with MPA, MPA and E2, or MPA and RLX. These results indicate that endometrial PRL production is regulated by the combined effects of steroid hormones (progestin and estrogen) and a peptide hormone (relaxin).     

Blockade of the spontaneous midcycle gonadotropin surge in monkeys by RU 486: a progesterone antagonist or agonist?

Preovulatory ovarian secretion of progesterone (P4), several hours before the onset of the typical midcycle gonadotropin surge, occurs in humans and monkeys. We investigated the potentially obligatory role of preovulatory P4 secretion in stimulating the midcycle LH surge by administering a potent P4 antagonist, RU 486(17 beta-hydroxy-11 beta-[4-dimethylaminophenyl-1]17 alpha-[prop-1-ynyl]estra-4,9-dien-3-one), to sexually mature, normally ovulatory cynomolgus monkeys on days 10-12 of the menstrual cycle (n = 18). Monkeys were randomized to receive RU 486 alone (5 mg/day, im; group I); RU 486 plus dexamethasone (1 mg/day, im; group II); dexamethasone alone (group III); or vehicle (ethanol; 0.5 ml; group IV). Before drug treatment, the follicular phases were quite similar among groups. The administration of RU 486 blocked (delayed) the expected gonadotropin surge, despite rising estrogen concentrations (greater than 250 pg/ml). The expected LH surge was delayed by RU 486 (n = 5) or RU 486 with dexamethasone (n = 3) until 36 +/- 7 (+/- SEM) and 27 +/- 8 days in groups I and II, respectively. In contrast, groups III (n = 3) and IV (n = 5) had timely midcycle surges after the administration of dexamethasone or vehicle alone (4 +/- 2 and 6 +/- 2 days, respectively). The intermenstrual interval was lengthened by RU 486 administration in both group I and II animals (61 +/- 6 and 54 +/- 6 days) compared to controls (30 +/- 2; P less than 0.0001). In summary, RU 486 effectively blocked imminent midcycle gonadotropin surges, delayed subsequent folliculogenesis, and significantly extended the menstrual cycle length. If RU 486 acted as a pure P4 antagonist, then P4 is necessary for timely midcycle gonadotropin surges to occur. However, recent evidence showing agonistic properties of RU 486 (in the virtual absence of P4) at both endometrial and pituitary levels may favor a P4-like (agonistic) blockade of the estrogen-induced FSH/LH surges by RU 486.