Progesterone Antagonist RU486 Research Articles

Mouse Mammary Tumor Virus Chromatin in Human Breast Cancer Cells Is Constitutively Hypersensitive and Exhibits Steroid Hormone-Independent Loading of Transcription Factors In Vivo

We have stably introduced a reporter gene under the control of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) into human T47D breast cancer cells to study the action of the progesterone receptor (PR) on transcription from a chromatin template. Unexpectedly, the chromatin organization of the MMTV LTR in these human breast cancer cells differed markedly from what we have observed previously. The region adjacent to the transcription start site (-221 to -75) was found to be constitutively hypersensitive to restriction enzyme cleavage in the absence of hormone. This region is normally encompassed within the second nucleosome of a phased array of six nucleosomes that is assembled when the MMTV LTR is stably maintained in mouse cells. Characteristically, in these rodent cells, the identical DNA sequences show increased restriction enzyme cleavage only in the presence of glucocorticoid. The increased access of restriction enzymes observed in the human PR+ cells was not observed in adjacent nucleosomes and was unaffected by treatment with the progesterone antagonist RU486. In addition, exonuclease III-dependent stops corresponding to the binding sites for nuclear factor 1 and the PR were observed before and after hormone treatment. These results indicate that MMTV chromatin replicated in these cells is organized into a constitutively open architecture and that this open chromatin state is accompanied by hormone-independent loading of a transcription factor complex that is normally excluded from uninduced chromatin.

Progesterone and cell-cell adhesion interact to regulate rat granulosa cell apoptosis.

Ovarian follicles are composed of both small and large granulosa cells, but only the large granulosa cells undergo apoptosis within 24 h of culture in serum-free medium. The present study was designed to assess the relationship between cell-cell contact, progesterone treatment, and granulosa cell apoptosis. For this study, individual large granulosa cells were isolated from immature rat ovaries after sequential incubation with EGTA and EGTA-sucrose solutions. Granulosa cells were then cultured for 24 h in RPMI-1640 (control) supplemented with progesterone and (or) the progesterone antagonist RU 486. The cells were then fixed and assessed for apoptosis by either electron microscopy or in situ end labeling of DNA fragments. After 24 h of culture, the proportion of apoptotic granulosa cells was twofold lower for aggregated cells compared with single granulosa cells (p < 0.05). Aggregated granulosa cells were observed to be connected by gap junctions. Compared with controls, progesterone reduced and RU 486 increased the percentage of single and aggregated apoptotic granulosa cells present after culture. In the presence of RU 486, progesterone reduced the percentage of apoptotic single granulosa cells from 84 +/- 4% (RU 486 alone) to 66 +/- 8%. In granulosa cell aggregates, progesterone reduced the incidence of apoptosis from 86 +/- 3% to 44 +/- 7% (p < 0.05). Progesterone in the presence of RU 486 was more effective in inhibiting apoptosis of aggregated granulosa cells than in single granulosa cells (p < 0.05). Taken together, these data indicate that (i) progesterone acts through the progesterone receptor to inhibit granulosa cell apoptosis and (ii) cell-cell adhesion enhances progesterone's anti-apoptotic actions.

Hormone regulation and hormone antagonist effects on protein patterns of human endometrial secretion during receptivity.

Endometrial receptivity is a particular stage of maturation during the luteal phase to permit implantation. We have studied endometrial protein secretion and its patterns evaluated by SDS-PAGE, laser densitometry and Western blots. Uterine secretion electrophoresis (USE) permits highly sophisticated analyses of the intrauterine milieu and allows clinical determination of the receptive stage of the endometrium. This technique reveals direct parameters by patterns of numerous individual protein bands, mainly resolved between 68.0 and 6.5 kD. Characteristic bands appear during the typical functional states of the menstrual cycle presenting evidence on the diagnostic capacity of this method to identify stages of adequate (= normal) or inadequate (= defective) luteal phase maturation. Several individual protein bands appear as characteristic markers for the receptive stage of the luteal phase. We have isolated and molecularly identified several of these proteins: histones H2A, H2B, H3 and H4. In order to identify the endocrine dependency of the protein bands, which significantly contribute to the "receptive stage pattern," patients were treated with the progesterone antagonist RU 486 at day LH +2. The assessment 4 days later revealed deficient USE patterns, particularly diminished and missing bands of the H2A-, H2B-, and H3-histones. These results demonstrate progesterone-dependent components of the endometrium at the receptive stage, which can be used as useful markers for an improved precision in luteal phase diagnostics. On the other hand, essential parts of the protein pattern may serve as new targets for successful contraceptive interventions ("endometrial contraception").

Progesterone suppresses tyrosine hydroxylase messenger ribonucleic acid levels in the arcuate nucleus on proestrus.

This study examined the intracellular mechanisms for the regulation of tyrosine hydroxylase in the tuberoinfundibular dopaminergic neurons of cycling female rats. It also evaluated the hormonal influences that contribute to the control of this enzyme on proestrus. Tyrosine hydroxylase messenger RNA (mRNA) levels in the arcuate nucleus of the hypothalamus were assessed by in situ hybridization. Tyrosine hydroxylase activity in the stalk-median eminence was determined from the in vitro or in vivo rate of 3,4-dihydroxyphenylalanine (DOPA) accumulation after inhibiting DOPA decarboxylase with brocresine or m-hydroxybenzylhydrazine, respectively. Tyrosine hydroxylase mRNA levels and in vitro DOPA accumulation were similar on diestrous day 2 and proestrous mornings, but were reduced by 50% on estrus. Although circulating PRL concentrations were similar on the morning of each day of the estrous cycle, a broad preovulatory PRL surge was observed on the afternoon of proestrus. In vitro DOPA accumulation was similar at 1000 h before the PRL surge and at 1330 h during the peak phase of the PRL surge, but was reduced during the plateau phase of the PRL surge (1700 and 2200 h) coincident with the preovulatory progesterone rise and remained low on estrus. However, in vivo DOPA accumulation was transiently decreased only at 1700 h on proestrus. Tyrosine hydroxylase mRNA levels were similar at 1000, 1330, and 1700 h on proestrus, were reduced by 50% at 2200 h on proestrus subsequent to the decrease in enzyme activity, and remained low on the morning of estrus. Okadaic acid, a protein phosphatase-1 and -2A inhibitor, induced a similar increase in tyrosine hydroxylase activity in vitro at 1330 and 2200 h on proestrus and at 1100 h on estrus, indicating that tyrosine hydroxylase was capable of being activated in spite of decreased mRNA levels. Ovariectomy between 1100-1200 h on proestrus prevented the decrease in tyrosine hydroxylase mRNA levels and in vitro DOPA accumulation at 2200 h. The effects of ovariectomy were completely reversed by progesterone, whereas estradiol had no effect. Circulating PRL levels at 2200 h were suppressed to basal levels after ovariectomy, but were increased by progesterone treatment at 1530 h to levels similar to those in the plateau phase of the PRL surge in control rats. Administration of the progesterone antagonist RU486 at 1200 h on proestrus did not alter tyrosine hydroxylase activity, tyrosine hydroxylase mRNA levels, or circulating PRL concentrations at 2200 h.(ABSTRACT TRUNCATED AT 400 WORDS)

Progesterone stimulates fibronectin production by chicken granulosa cells in vitro.

Experiments were conducted in vitro to examine the effect of progesterone on fibronectin production by chicken ovarian granulosa cells. Granulosa cells isolated from the largest (F1; mature) and third-largest (F3; developing) preovulatory follicles as well as form a pool of immature small yellow follicles (SYF) of the domestic chicken ovary were incubated in serum-free Medium-199 and the amounts of fibronectin and progesterone produced were quantified by enzyme-linked immunosorbent assay and radioimmunoassay respectively. The amounts of basal fibronectin and progesterone produced by granulosa cells from F1, F3 and SYF follicles increased with advancing stages of follicular development. Thus, the quantity of basal fibronectin secreted by granulosa cells was directly proportional to the amount of progesterone produced by them. Exogenously supplied progesterone increased the amount of fibronectin secreted by F1 and F3 cells in a dose-dependent manner, but its effect on SYF cells was marginal. Cyanoketone (an inhibitor of progesterone synthesis) suppressed basal fibronectin production by F1 and F3 granulosa cells and its inhibitory action was reversed by exogenous progesterone. The progesterone antagonist RU 486 also attenuated basal fibronectin production by F1 and F3 granulosa cells, but only the highest concentration affected SYF cells. The inhibitory effect of RU 486 was diminished in the presence of exogenous progesterone. These data show that progesterone regulates fibronectin production by chicken granulosa cells. They suggest that in avian granulosa cells, endogenous progesterone can stimulate fibronectin synthesis in an intracrine or autocrine manner.

Progesterone and RU486: opposing effects on human sperm.

Progesterone induced a rapid influx of calcium in capacitated human sperm, followed by a long-lasting, dose-dependent increase of intracellular free calcium. Thereafter, progesterone increased the fraction of hyperactivated sperm and the acrosome reaction. On the contrary, the progesterone antagonist RU486 (mifepristone) induced an immediate and transient, dose-dependent decrease of intracellular free calcium and a drop in the values of sperm movement parameters related to hyperactivation. Moreover, RU486 counteracted the effects of progesterone on calcium influx, lateral sperm head displacement, and the acrosome reaction. Therefore, RU486 effects were opposite to those of progesterone. The nature of the membrane receptor(s) involved is unknown. Several steroids bearing 11 beta-phenyl substitutions, with different pharmacological profiles, were also investigated. It was concluded that the steroid structure and chemical groups added to the 11 beta-phenyl influence effects on calcium influx.

Immunohistochemical localization of estradiol and progesterone receptors in human uterus throughout pregnancy: expression in endometrial blood vessels.

Although progesterone and estrogens are essential to maintain human pregnancy after implantation, the localization of their specific receptors in different uterine cell types during pregnancy has not been investigated. We studied uteri (n = 40) obtained during the first 3 months of pregnancy (n = 21) and in late pregnancy (n = 9) as well as from women 5-14 weeks pregnant (n = 10) who had received the antiprogestagen RU 38486 (Roussel-UCLAF) to induce cervical dilation. Frozen tissues were processed for indirect immunocytochemical staining with specific monoclonal antibodies against estrogen receptors (ER; Abbott Laboratories) and progesterone receptors (PR; Li 417). Specific staining for steroid receptors was only detected in the nucleus. In the endometrium, PR staining remained fairly constant throughout pregnancy, whereas ER staining was initially weak and then undetectable. PR was widely expressed in stromal cells and in spiral arterial wall cells, whereas ER was expressed in scattered stromal cells and arterial cells. Both PR and ER were absent from glandular epithelium, contrasting with the secretory activity during the first trimester. Spiral arteries of the endometrium and myometrial smooth muscle cells showed intense PR and moderate ER staining in early pregnancy. The progesterone antagonist RU 38486 (mifepristone), given in early pregnancy at a dose of 200 mg, caused a marked increase in ER staining and a smaller increase in PR staining in stromal cells, whereas the glandular epithelium remained negative for both ER and PR (except for one and two specimens, respectively). We conclude the following. 1) Stromal cells retain PR despite the high progesterone levels during pregnancy, in keeping with the role of progesterone in stromal decidualization. The absence of PR from the secretory glandular epithelium suggests a paracrine link between decidualized stromal cells and epithelial cells. 2) Significant PR down-regulation by progesterone during pregnancy occurs only in epithelial cells of the endometrium. 3) In contrast, the absence or low level of ER staining in the various cell types of the endometrium during gestation concurs with the known effect (down-regulation) of steroid hormones on ER mRNA or protein levels. The increase in ER in human decidua after RU 38486 treatment indicates that the main cause of the low ER levels is progesterone secretion. 4) The intense PR staining in smooth muscle cells of spiral arteries during early pregnancy suggests that progesterone is essential for modulating blood flow during pregnancy.

Regulation of anterior pituitary gonadotropin subunit mRNA levels during the preovulatory gonadotropin surge: A physiological role of progesterone in regulating LH-β and FSH-β mRNA levels

In a previous study we demonstrated that in the ovariectomized estrogen-primed immature rat, progesterone induced a gonadotropin surge while the gonadotropin mRNA subunit levels were either suppressed or unaltered. This observation has now been confirmed using more frequent time points. Progesterone administered at 0900 h was found to suppress LH-β mRNA levels at 1300, 1400, and 0800 h the next day, with no subsequent effects at 1000, 1200 or 1600 h. FSH-β mRNA levels were unaffected by progesterone except for a slight elevation at 1400 h and a suppression at 0800 h. Progesterone was either suppressive or had no effect on alpha mRNA levels. Since elevations in LH-β and FSH-β mRNA levels were observed in the cycling rat, the observed differences in the ovariectomized estrogen-primed rat could be due to a higher basal synthesis occurring due to ovariectomy. This was indeed the case because LH-β and FSH-β mRNA levels were 3.7- and 42.7-fold higher in such animals as compared to intact estrogen-primed rats. In contrast to the ovariectomized estrogen-primed rats, in intact estrogen-primed rats LH-β mRNA levels were increased at 1000 h and FSH-β mRNA levels were increased at 1000, 1200 and 1300 h after the administration of progesterone. In pregnant mare's serum gonadotropin-primed immature rats, LH-β, FSH-β and α-subunit mRNA levels were significantly elevated at 1800 and 2000 h, paralleling the serum LH and FSH surge. The progesterone antagonist RU486 (0.2 and 1.0 mg) significantly reduced serum LH and FSH levels at 2000 h. The lower dose reduced LH-β and α-subunit mRNA levels at 2000 h and FSH-β mRNA levels at 1800 h. The higher dose caused an increase in LH-β mRNA levels at 1200 and 1800 h and a decrease in FSH-β mRNA levels at 1800 and 2000 h. In conclusion, the present study provides evidence that preovulatory progesterone plays an important role in the increase in FSH-β mRNA levels as well as the release of LH and FSH during the normal preovulatory gonadotropin surge. This relationship appears to be dependent on the ongoing rate of synthesis because this does not occur in the ovariectomized estrogen-primed rat in which synthesis is at a high basal level. Furthermore, the correlation with FSH appears to be tighter as compared to LH.

Regulation of transcripts encoding the myometrial gap junction protein, connexin-43, by estrogen and progesterone.

In the rat, transcripts encoding the myometrial gap junction protein, connexin-43 (Cx-43), increase dramatically with the onset of labor in association with an increase in the ratio of estrogen to progesterone in plasma. We examined whether the level of Cx-43 transcripts might be regulated by these steroids in the rat myometrium. Administration of progesterone to late pregnant rats abolished the more than 12-fold increase in transcripts seen in control rats at term and blocked delivery of the fetuses. Treatment of rats on day 15 of gestation (when Cx-43 mRNA levels are low) with the progesterone antagonist RU486 resulted in a significant (2.5-fold) increase in transcripts within 9 h, with the maximal (5.6-fold) increase occurring between 24-48 h, and preterm delivery occurring between 48-72 h. Administration of a single dose of 17 beta-estradiol (5 micrograms, sc) to nonpregnant rats resulted in a significant increase in Cx-43 transcripts within 3 h; these levels were increased 3-fold between 6-24 h, before falling to lower levels by 48 h. Progesterone (4 mg, sc) administration at the same time as estradiol significantly attenuated the estradiol response. Chronic estradiol administration (5 micrograms/12 h for 36 h) failed to maintain elevated levels of Cx-43 transcripts beyond 36 h. Administration of progesterone 12 h after estradiol prematurely reduced the level of Cx-43 transcripts. These data demonstrate that steroid hormones can modulate the level of steady state transcripts of Cx-43 during pregnancy in association with changes in uterine contractile activity. Furthermore, the data from the nonpregnant studies suggest that the levels of transcripts are regulated positively by estradiol and negatively by progesterone.

Recurrent fever associated with progesterone action and persistently elevated serum levels of immunoreactive tumor necrosis factor-alpha and interleukin-6.

We describe two women who suffer from recurrent fever up to 40 C in association with progesterone action and who have continuously elevated serum levels of immunoreactive tumor necrosis factor-alpha (TNF alpha) and interleukin-6 (IL-6). In patient 1, recurrent fever began at age 17 yr and has now continued for 11 yr. The patient has had three early pregnancy terminations because of continuous fever and, thereafter, three early pregnancy losses associated with fever. In patient 2, fever first appeared at age 18 yr, and the attacks have now continued for 3 yr. The association between fever and progesterone action is supported by the following facts. 1) The episodes of fever appear in the midluteal phase of the menstrual cycle concomitantly with the highest concentration of serum progesterone. 2) Fever is further exaggerated in early pregnancy. 3) Synthetic progestins induce fever regardless of the day of the menstrual cycle. 4) The progesterone antagonist RU 486 and an agonist of GnRH, nafarelin, are capable of preventing the fever, with no effect on serum cytokine levels. Although the underlying mechanism of elevated TNF alpha and IL-6 levels in our patients remains unknown, the data suggest that these cytokines cooperate with progesterone in exerting a pyrogenic response in the hypothalamic thermoregulatory center.

The progesterone antagonist RU486 acquires agonist activity upon stimulation of cAMP signaling pathways.

The protein kinase A stimulator cAMP can potentiate the ability of progestins to induce the transactivation function of the human progesterone receptor (hPR). We questioned in the present study whether cAMP could functionally cooperate with the progestin antagonist RU486. In T47D human breast cancer cells, RU486 behaves as a pure antagonist with respect to induction of the progesterone-responsive mouse mammary tumor virus chloramphenicol acetyltransferase (MMTV-CAT) reporter gene. It fails to stimulate MMTV-CAT expression and completely inhibits induction by the synthetic progestin R5020. However, when RU486 is combined with 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), MMTV-CAT is induced to levels approaching that stimulated by R5020 alone. Also, RU486 in the presence of 8-Br-cAMP is only partially effective in antagonizing R5020 action. The agonist activity exhibited under these conditions appears to be due to RU486 acting through hPR as evidenced by the fact that 8-Br-cAMP alone has no effect on MMTV-CAT, whereas induction by the combination of 8-Br-cAMP and RU486 is dose responsive to RU486 in a saturable manner and can be inhibited by the type I antiprogestin (prevents hPR-DNA binding) ZK98299, which does not exhibit positive functional cooperation with cAMP. Acquisition of agonist activity in the presence of 8-Br-cAMP also extends to the type II antiprogestin (permits hPR-DNA binding) ZK112993. Since RU486 is also a type II antagonist, these results suggest that detection of functional synergism between cAMP and antiprogestins may require binding of the hPR-antagonist complex to DNA. We propose that cross-talk between second messenger and steroid receptor signal transduction pathways may be one mechanism for resistance to steroid antagonists that frequently develops in breast cancer.

Progesterone antagonist RU 486 accommodates but does not induce labour and delivery in primates.

The hormonal mechanisms of parturition in primates remain controversial. Even so, the well-known decrease of plasma progesterone concentration near term is considered by many as the 'labour inducer'. The progesterone antagonist RU 486, which blocks progesterone activity at the cellular receptor level, appears to be a useful hormonal tool by which to study this tissue. Here, we tested its capacity to induce labour and delivery. A total of 23 Cynomolgus monkeys (Macaca fascicularis), within 9-17 days of expected term, were assigned to four different protocols to study various doses, routes and regimens of RU 486 administration. Observations included uterine contractile patterns, pharmacokinetics of RU 486 in plasma and passage of RU 486 into breast milk. None of the protocols tested successfully induced labour resulting in vaginal delivery within 24 h. Instead, the data demonstrate that blockade of progesterone activity by the progesterone antagonist was not sufficient by itself to achieve parturition in these primates. Uterine myometrial contractile activity under RU 486 exposure was not sufficient to induce labour and delivery. Moreover, the progesterone antagonist concentration in breast milk was very low, indicating little passage to suckling newborn infants.

Estrogen-induced calbindin-D 9k gene expression in the rat uterus during the estrous cycle: late antagonistic effect of progesterone.

Progesterone modulates estrogen-stimulated responses in the uterus. Calbindin-D 9k (CaBP9k), a 17 beta-estradiol-responsive gene expressed in the uterus, was used as a marker to examine the interactions between endogenous progesterone and estradiol in the rat. The variations in uterine CaBP9k messenger RNAs (mRNAs) during the rat estrous cycle indicated that CaBP9k gene expression was greatest during the estrogen-dominated phases (proestrus and estrus) and became totally repressed during diestrus, when progesterone predominates. Estradiol was found to be the major controlling factor of CaBP9k gene expression in vivo, progesterone antagonizing estrogen-induced CaBP9k gene expression. The inhibitory role of progesterone was further examined in two experiments. Mature cyclic rats were injected with the progesterone antagonist RU486 before the progesterone surge of proestrus, and the estrous cycle was mimicked in ovariectomized rats by sequential injections of estrogen and progestin. Progesterone did not appear to be involved in the rapid decrease in CaBP9k mRNA during estrus but was implicated in the down-regulation of the estrogen-stimulated CaBP9k gene expression at the end of estrus and during diestrus. This delayed effect of progesterone was confirmed in the ovariectomized rat model. CaBP9k mRNA accumulation in estrogen-primed ovariectomized rats was suppressed by estrogen followed 1 h later by the progesterone agonist R5020. This effect occurred more than 24 h after progestin treatment. The inhibition of the estrogen-induced CaBP9k gene expression in the rat uterus by progesterone is certainly mediated by the progesterone receptor, because progesterone had no effect without estrogen priming or when the antagonist RU486 was used. The delayed progesterone effect probably does not involve depletion of nuclear estrogen receptors, the major rapid mechanism proposed for estrogen inhibition by progesterone in the rodent uterus, or control of estrogen receptor synthesis, as shown by Northern blot analysis of estrogen receptor mRNA.

Time-related effects of RU486 treatment in experimentally induced endometriosis in the rat

To evaluate the time-dependent effect of the progesterone antagonist RU486 on experimentally induced endometriosis in the rat. Endometriosis was induced by implanting endometrium onto the peritoneal side wall. Size of the implants was measured before and after treatment. Fifty-two adult female Sprague-Dawley female rats weighing 200 to 225 g. Departmental research laboratory of university hospital. Animals treated with RU486 or vehicle only for 2, 4, 6, or 8 weeks. Size of the implant was measured before and after treatment; relative change in size of the implants was calculated. There was not a statistically significant difference between the treatment groups and the control groups. There was no regression of endometriosis in any of the treatment groups. RU486 administered for 8 weeks in the rat model was not effective therapy for endometriosis.

Modulators of cellular protein phosphorylation alter the trans-activation function of human progesterone receptor and the biological activity of progesterone antagonists.

Addition of progesterone to breast cancer cells in vivo increases phosphorylation of human progesterone receptor (PR), suggesting that phosphorylation has a regulatory role in producing the activated form of receptor. Kinetic analysis indicates that hormone-dependent phosphorylation is sequential and that early stages of phosphorylation(s) are closely associated with enhancement of PR-DNA binding while later stages are associated with a trans-activation function. Various agents that stimulate cellular protein phosphorylation (8-Br cAMP, okadaic acid, TPA) functionally synergize with progesterone to enhance progesterone-dependent PR trans-activation in intact cells. These results suggest that protein phosphorylation does have a role in modulating the trans-activation function of PR in vivo. They also demonstrate cross-talk between second messenger signal transduction pathways and nuclear steroid receptors. Whether the phosphorylated target that provides the link between these two signal transduction pathways is PR itself or another protein involved in PR-mediated gene transcription is not known. Positive cooperative interactions were also observed between cAMP signaling pathways and the progesterone antagonist RU486, that resulted in RU486 exerting substantial agonist activities. This ability of cross-talk between second messenger and steroid receptor signal transduction pathways to override the antagonistic effects of RU486 suggests a novel mechanism to explain the problem of resistance to clinically important steroid antagonists.

Clinical use of mifepristone (RU 486).

Because progesterone is essential for the establishment and maintenance of pregnancy, it has long been recognized that a substance which antagonized the action of progesterone would have potential as an antifertility agent. Within 2 years of the synthesis of the progesterone antagonist RU 486 (mifepristone) it was demonstrated that bleeding and uterine contractions occurred following its administration in non-pregnant and pregnant women. Extensive trials over the last 10 years have established that a single dose of mifepristone followed 36-48 hours later by a prostaglandin, is an effective, safe alternative to vacuum aspiration for the termination of early pregnancy. Although this combination is licensed in France, China and the United Kingdom for induction of abortion, research is continuing to determine the minimum effective dose of mifepristone and type of prostaglandin which is associated with minimum side effects without loss of efficacy. In addition, studies to determine the acceptability of this type of medical abortion to women in different cultures and societies are required. The facilities necessary for medical termination differ from those for surgical abortion, although the requirements for access to emergency resuscitation and treatment of (rare) complications are similar for the two methods. Mifepristone is very effective in the management of prostaglandin-induced midtrimester abortion. By sensitizing the uterus to prostaglandin, the dose of prostaglandin can be reduced with a shortened prostaglandin-abortion interval. Administration of mifepristone in the early luteal phase of the cycle delays the development of a secretory endometrium. Preliminary studies suggest that it may be highly effective when given at this time as a post-coital contraceptive or 'once a month' pill. Although antigestagens offer great promise as agents to help regulate human fertility, their widespread use may be constrained more by religious and political factors, rather than by demonstration of clinical efficacy.

Progesterone reverses the estradiol-induced decrease in tyrosine hydroxylase mRNA levels in the arcuate nucleus.

Estradiol (E2) and progesterone (P4) interact to influence tuberoinfundibular dopaminergic neuronal activity and contribute to the control of prolactin (PRL) release. This study examined tyrosine hydroxylase mRNA signal levels in the arcuate nucleus of the hypothalamus and tyrosine hydroxylase activity in the stalk-median eminence after 1 week of steroid treatment and related these to circulating PRL levels. Ovariectomized rats were untreated (control) or were implanted with E2, P4 or both E2 and P4 pellets and were sacrificed after 7 days at either 10:00 or 18:00 h. Some E2 + P4-treated rats were injected with either RU 486 or its vehicle at 12-hour intervals for the last 3 of the 7 days of steroid treatment. Tyrosine hydroxylase mRNA signal levels in the arcuate nucleus were decreased by 70% at both 10:00 and 18:00 h in the E2-treated rats compared to control rats. P4 alone had no effect on tyrosine hydroxylase mRNA levels, but reversed the E2-induced decrease so that mRNA levels in the E2 + P4-treated rats were similar to control levels. The progesterone antagonist RU 486 blocked this effect of P4, supporting the observation of decreased mRNA levels in E2-treated rats. Steroid treatment had no effect on tyrosine hydroxylase mRNA levels in the medial zona incerta. Tyrosine hydroxylase activity in the stalk-median eminence was similar at 10:00 and 18:00 h in control rats, and was decreased by 25 and 36% at 10:00 and 18:00 h, respectively, in E2-treated rats. P4 alone had no effect on tyrosine hydroxylase activity, but reversed the E2-induced decrease in enzyme activity to control levels at both 10:00 and 18:00 h. In contrast to the effect of RU 486 on tyrosine hydroxylase mRNA, tyrosine hydroxylase activity in E2 + P4-treated rats was not significantly altered by RU 486 at either 10:00 or 18:00 h. Circulating PRL levels were elevated in the E2-treated and E2 + P4-treated rats. A diurnal PRL rise was evident at 18:00 h in E2-treated rats, but was abolished by concomitant treatment with P4. The diurnal PRL surge was re-established in E2 + P4-treated rats after administration of RU 486, whereas basal circulating PRL levels were not altered by RU 486. These data indicate that P4 antagonizes the profound inhibitory effect or E2 on tyrosine hydroxylase mRNA content in the tuberoinfundibular dopaminergic neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of the progesterone antagonist RU486 on human myometrial spontaneous contractility and PGI 2 release

We studied the effect of antiprogesterone RU 486 on spontaneous uterine contractility and PGI 2 release with human myometrial strips superfused “in vitro”. A decrease of PGI 2 release into the superfusion medium was observed after 20 min superfusion. The inhibition was dose-dependent and reversible. After 20 min washing with tyrode medium without RU 486, the uterine strips recovered their initial rate of release. R5020, a progesterone agonist, did not affect PGI 2 release nor dexamethasone and testosterone. Parallel to the decrease of PGI 2 observed during RU 486 superfusion, the uterine spontaneous contraction frequency decreased, while the amplitude and duration of contractions increased. The alteration of uterine contractility was also rapid, dose-dependent and reversible. Modifications of uterine strip spontaneous contractility, similar to those induced by RU 486, were also observed with superfusions of R5020 at concentrations as low as 10 −9M, dexamethasone (10 −8M), but not with superfusions of testosterone. These observations are not in favour of a progesterone-receptor mediated effect of RU 486 in our model. The mechanism of action may be related to the antiprogesterone specific structure i.e. the bulky substituent at the C-11 position. The RU 486 effect on uterine strip contractility, mimicked by other steroids, could point to a non-specific lipid/membrane interaction. However, the fact that testosterone did not affect motility, may indicate a possible specificity of steroids having a 3 oxo pregnene structure.

Effects of the steroid antagonist RU486 on dimerization of the human progesterone receptor.

We previously reported, using a coimmunoprecipitation assay, that the B form (PR-B) of the human progesterone receptor from T47D human breast cancer cells dimerizes in solution with the A receptor (PR-A) and that the extent of dimerization correlates with receptor binding activity for specific DNA sequences [DeMarzo, A.M., Beck, C.A., Oñate, S.A., & Edwards, D.P. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 72-76]. This suggested that solution dimerization is an intermediate step in the receptor activation process. The present study has tested the effects of the progesterone antagonist RU486 on solution dimerization of progesterone receptors (PR). As determined by the coimmunoprecipitation assay, RU486 binding did not impair dimerization of receptors; rather, the antagonist promoted more efficient solution dimerization than the progestin agonist R5020. This enhanced receptor dimerization correlated with a higher DNA binding activity for transformed receptors bound with RU486. RU486 has been shown previously to produce two other alterations in the human PR when compared with R5020. PR-RU486 complexes in solution exhibit a faster sedimentation rate (6 S) on salt-containing sucrose density gradients than PR-R5020 complexes (4 S), and PR-DNA complexes have a faster electrophoretic mobility on gel-shift assays in the presence of RU486. We presently show that the 6 S PR-RU486 complex is a receptor monomer, not a dimer. The increased sedimentation rate and increased mobility on gel-shift assays promoted by RU486 were also observed with recombinant PR-A and PR-B separately expressed in insect cells from baculovirus vectors. These results suggest that RU486 induces a distinct conformational change both in PR monomers in solution and in dimers bound to DNA. We also examined whether conformational changes in PR induced by RU486 would prevent a PR polypeptide bound to RU486 from heterodimerization with another PR polypeptide bound to R5020. To evaluate this, PR-A and PR-B that were separately bound to R5020 or RU486 in whole cells were mixed in vitro. PR-A-RU486 was capable of dimerization with PR-B-R5020, and this was demonstrated for heterodimers both formed in solution and bound to specific DNA. The capability to form heterodimers in vitro raises the possibility that the antagonist action of RU486 in vivo could in part be imposed in a dominant negative fashion through heterodimerization between one receptor subunit bound to an agonist and another bound to RU486.

Decreased luteinizing hormone-stimulated progesterone secretion by preovulatory follicles isolated from cyclic rats treated with the progesterone antagonist RU486.

Since administration of the antiprogesterone RU486 to cyclic female rats at metestrus and diestrus results in increased serum levels of LH, estradiol, and testosterone at proestrus, we investigated whether RU486 affects follicular steroidogenesis. Female rats with a 4-day estrous cycle, induced experimentally by a single injection of bromocriptine on the morning of estrus, were given RU486 (2 mg) twice daily (0900 and 1700 h) on metestrus and diestrus. At proestrus the preovulatory follicles were isolated and incubated for 4 h in the absence and presence of LH. In the absence of LH, accumulation of estradiol, testosterone, and progesterone in the medium was not different for RU486-treated rats and oil-treated controls. In contrast, LH-stimulated estradiol, testosterone, and progesterone secretions were significantly lower in RU486-treated rats compared with controls. Addition of pregnenolone to the incubation medium resulted in a significantly lower increase of progesterone in follicles from RU486-treated rats compared with those from oil-treated controls. This suggests that 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity is decreased by administration of RU486 in vivo. Aromatase and 17 alpha-hydroxylase/C17-20 lyase activities were not affected: addition of substrate (androstenedione and progesterone respectively) did not affect differently the amount of product formed (estradiol and testosterone) in RU486- and oil-treated rats. However, LH-stimulated pregnenolone secretion was lower in follicles from RU486-treated rats compared with follicles from oil-treated controls, suggesting that either cholesterol side-chain cleavage activity or LH responsiveness is decreased. At proestrus the preovulatory follicles from RU486- and oil-treated rats were not morphologically different.(ABSTRACT TRUNCATED AT 250 WORDS)