Effect of RU486 on development and implantation of rat embryos

This study evaluated the effects of postcoital treatment with the antiprogestin RU486 on transport, development and implantation of rat embryos. Doses of 0.1, 0.5, 1.0, 2.0, or 3.0 mg/rat of RU486 were injected subcutaneously on days 1, 1 + 2, or 4 of pregnancy. Autopsies were carried out on days 5 or 12 of pregnancy. RU486 provoked a significant dose-related reduction in the number of recovered embryos and inhibited their development (day 5) and decreased the number and size of implanted embryos (day 12). Treatment on day 4 was the least effective. Blastocysts recovered from RU486-treated rats exhibited comparable rate of trophoblastic outgrowth in vitro as the controls. Blastocysts transferred from RU486-treated rats to synchronous untreated pseudopregnant recipients yielded implanted embryos 12 days later in all recipients, although at a significantly lower rate than the controls. Blastocysts transferred from control pregnant rats to RU486-treated pseudopregnant recipients failed to implant completely when the dose was greater than or equal to 1.0 mg. The interceptive mechanism of postcoital treatment with RU486 in the rat involves loss of embryos from the reproductive tract and altered development prior to implantation. Endometrial receptivity or the ability of the uterus to retain the embryos until the time of implantation are also impaired by RU486. The embryos that survive these effects may experience delayed implantation in their mothers.     

A genomic approach to identify novel progesterone receptor regulated pathways in the uterus during implantation

The cellular actions of steroid hormone progesterone (P) are mediated via its nuclear receptors, which regulate the expression of specific target genes. The identity of gene networks that are regulated by the P receptors (PRs) in the uterus at various stages of the reproductive cycle and pregnancy, however, remain largely unknown. In this study, we have used oligonucleotide microarrays to identify mRNAs whose expression in the pregnant mouse uterus is modulated by RU486, a well-characterized PR antagonist, which is also an effective inhibitor of implantation. We found that, in response to RU486, expression of mRNAs corresponding to 78 known genes was down-regulated at least 2-fold in the preimplantation mouse uterus. The PR regulation of several of these genes was ascertained by administering P to ovariectomized wild-type and PR knockout (PRKO) mice. Detailed spatio-temporal analysis of these genes in the pregnant uterus indicated that their expression in the epithelium and stroma could be correlated with the expression of PR in those cell types. Furthermore, time-course studies suggested that many of these genes are likely primary targets of PR regulation. We also identified 70 known genes that were up-regulated at least 2-fold in the pregnant uterus in response to RU486. Interestingly, initial examination of a number of RU486-inducible genes reveals that their uterine expression is also regulated by estrogen. The identification of several novel PR-regulated gene pathways in the reproductive tract is an important step toward understanding how P regulates the physiological events leading to implantation.     

RU486 suppresses progesterone-induced acrosome reaction in boar spermatozoa

The effects of progesterone on the acrosome reaction, as well as the effects of RU486 on the progesterone-induced acrosome reaction in capacitated boar spermatozoa, were investigated. Progesterone, a major steroid that is secreted by the cumulus cells of oocyte, clearly induced the acrosome reaction in a dose-dependent manner in capacitated boar spermatozoa, even though it failed to show similar effects in non-capacitated spermatozoa. RU486, a potent antiprogestin, significantly reduced the effects of progesterone on the progesterone-induced acrosome reaction; however, when treated alone, it showed no inhibitory effects on the acrosome reaction. The inhibitory effects of RU486 were also shown to be dose dependent. These results imply that in addition to the wellknown inducer of the acrosome reaction, zona pellucida, progesterone can also induce the acrosome reaction through its specific receptors on spermatozoa after the spermatozoa undergo capacitation.     

Relationship between uterine expression of matrix metalloproteinases and their inhibitors and endometrial receptivity

In order to investigate the relationship between the endometrial receptivity and matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-1,-3 (TIMP-1,-3) in the endometrium, we used early pregnant mice as the animal model and studied the expression of MMP-2, TIMP-1,-3 in the endometrium in relation to the number of implantation sites after RU486 treatment. The results indicated that RU486 could significantly inhibit embryo implantation and change the expression of MMP-2 and TIMP-1,-3 in a dose-dependent pattern. When the mice were treated with 12 mg/kg RU486, there were a few embryos implanted as compared with the control. The expression of matrix metalloproteinase MMP-2 was low during the period of "implantation window", while the tissue inhibitor of metalloproteinase in the endometrial cells was high, suggesting that the activity of the proteolytic enzyme was strictly controlled by its inhibitors. After RU486 treatment, the generation of TIMP-1,3 was decreased while the MMP-2 was significantly increased, indicating that the normal balance between the activators and their inhibitors in the tissue was broken and the extracellular matrix was excessively degraded, subsequently the embryo implantation was inhibited. Therefore, it is suggested that the anti-implantation effect of RU486 may be mediated by MMPs and their inhibitors TIMPs.     

Relationship between uterine expression of matrix metalloproteinases and their inhibitors and endometrial receptivity

In order to investigate the relationship between the endometrial receptivity and matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-1 ,-3 (TIMP-1,-3) in the en-dometrium, we used early pregnant mice as the animal model and studied the expression of MMP-2, TIMP-1 ,-3 in the endometrium in relation to the number of implantation sites after RU486 treatment. The results indicated that RU486 could significantly inhibit embryo implantation and change the expression of MMP-2 and TIMP-1,-3 in a dose-dependent pattern. When the mice were treated with 12 mg/kg RU486, there were a few embryos implanted as compared with the control. The expression of matrix metalloproteinase MMP-2 was low during the period of "implantation window", while the tissue inhibitor of metalloproteinase in the endometrial cells was high, suggesting that the activity of the proteolytic enzyme was strictly controlled by its inhibitors. After RU486 treatment, the generation of TIMP-1,3 was decreased while the MMP-2 wa     

Steroid RU 486 inducible myogenesis by 10T1/2 fibroblastic mouse cells

For reconstruction or repair of damaged tissues, an artificially regulated switch from proliferation to differentiation would be of great advantage. To achieve conditional myogenesis, we expressed MyoD in mouse C3H 10T1/2 fibroblastic cells, using a gene regulation system based on the synthetic steroid RU 486. By stable co-transfection of a plasmid construct with the RU 486 dependent activator and an integrating inducible MyoD construct, a cell clone, designated 10T-RM, was obtained in which MyoD expression was stringently controlled by RU 486. 12 h after addition of 10 nM RU 486 to 10T-RM cells, saturation levels of MyoD mRNA were observed and >/=2 days later, mRNA for embryonal myosin heavy chain (MyHC(emb)) was abundant and mononucleated cells fused into myotubes.     

In vitro effects of RU486 on proliferation and differentiation capabilities of human bone marrow mesenchymal stromal cells

Although exogenous glucocorticoids (GC) play a role in the regulation of bone marrow mesenchymal stem/stromal cells (MSCs) proliferation and differentiation, the function of endogenous GC is not well understood. The purpose of this study was to investigate the effect of the blockage of endogenous GC using RU486, an antagonist of the glucocorticoid receptor, on the in vitro proliferation and differentiation capabilities of human MSCs. We quantitatively measured cell proliferation of human MSCs after treatment with increasing concentrations of RU486. We also evaluated multiple MSC differentiation capabilities, as well as the expression of stemness and senescence genes after proliferation of these human cells in vitro in the presence of RU486 at 10(-8)M. It was observed that RU486 treatment significantly increases the proliferation of human MSCs, although the optimal dose of RU486 for this increase in proliferation differs depending on the gender of the MSC donor. This improvement in MSC proliferation with RU486 treatment was higher in MSCs from male donors than that from females. No effect of RU486 on MSC proliferation was observed in a steroid-free medium. RU486 pretreatment significantly increased the expression of mRNA for alkaline phosphatase in human MSCs and the mRNA expression of osteocalcin of these cells up-regulated earlier after their exposure to osteogenic differentiation medium. Although no statistical significance in terms of chondrogenic differentiation markers was detected, mRNA expression for aggrecan and collagen type 2 were higher in a majority of the RU486-pretreated donor MSCs than their untreated controls. No significant difference in terms of MSC adipogenic differentiation capabilities were observed after RU486 treatment. RU486 treatment up-regulated the expressions of FGF-2 and Sox-11 in human MSCs. These results indicate that blockage of endogenous GCs may be developed as a novel approach to effectively improve the proliferation and osteochondral differentiation capabilities of human MSCs for potential clinical applications. Additional studies will be required to determine the potential long-term effects of RU486 treatment on these bone marrow cells.     

Effect of RU 486 on luteal function in the early pregnant rat

A dose of 30 mg RU 486/kg, an antiprogesterone, was administered to pregnant rats on Day 2 (Group 1) or Day 4 (Group 2) of pregnancy. RU 486 significantly changed serum progesterone and oestradiol concentrations and luteal 3 beta-HSD and 20 alpha-HSD activities in Group 1, and implantation was significantly inhibited. The luteal 3 beta-HSD activity in Group 2 rats on Day 6 was significantly (P less than 0.01) lower than the control value (7.5 +/- 0.6 and 10.1 +/- 0.6 mU/mg protein respectively). This decline in the 3 beta-HSD activity was followed by a marked decrease in the serum progesterone concentration, resulting in a significant decrease of the progesterone/oestradiol ratio and implantation was completely inhibited. The 20 alpha-HSD activity, which could not be detected on Day 6 in the control rats, was twice as great in Group 2 than in Group 1 rats (17.5 +/- 1.2 and 7.4 +/- 3.1 mU/mg protein respectively). Ultrastructural examination of corpora lutea of Group 2 rats confirmed luteolysis. These results suggest that RU 486 has a luteolytic effect and its anti-implantation effect is concomitant with luteolysis of the corpora lutea of pregnancy.     

Inhibition of glucocorticoid-induced cataracts in chick embryos by RU486: a model for studies on the role of glucocorticoids in development

Cataract formation can be induced by glucocorticoid treatment of developing chick embryos. We show here that this response can be blocked very effectively by use of the antiglucocorticoid RU486. When dexamethasone (0.02 micromol/egg) was administered from day 13 to 16 chick embryos, their lenses (over 80%) became cataract (GC-induced cataract; stage IV-V) within 48 hrs. These GC-induced cataract formations were prevented by administration of RU486 (0.2 micromol/egg) on day 9. However, RU486 also inhibited hatching even though the embryos showed normal growth and appearance. In control embryos, more than 90% live chicks (39/42 chicks) were hatched on day 22. Chick embryos treated with RU486 on day 9 appeared to grow normally until 21, but could not hatch. When chick embryos were treated with RU486 (0.2 micromol/egg) on day 15, more than 80% live embryos (34/42 chicks) were hatched on day 23 with normal appearance, which was one day delay comparing to the control. These observations indicate that endogenous glucocorticoids are involved in the ability to hatch and that RU486 is able to block the actions of endogenous glucocorticoids. Thus, RU486 should be a very useful tool for studies on other biochemical and physiological aspects of chick embryo development that are under glucocorticoid control.     

Luteolytic effect of the antiprogestin and antiglucocorticoid agent RU486 in rats

Ovarian cells of pregnant rats were cultured with synthetic progestins (R5020, R2323), dexamethasone and RU486. Progesterone and 20 alpha-hydroxy-pregn-4-en-3-one (20 alpha-dihydroprogesterone) in the medium were measured by specific radioimmunoassay. Both R5020 and R2323 increased concentrations of these intrinsic progestins. RU486 decreased concentrations of progesterone, however, the addition of R5020 or R2323 counteracted this action. Immature hypophysectomized rats treated with pregnant mare serum gonadotropin (PMS) and human chorionic gonadotropin (hCG) were administered with RU486; the serum levels of progesterone and 20 alpha-dihydroprogesterone tended to decrease. R5020 and R2323 inhibited the effect of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), whereas RU486 did not. Inhibition of the cholesterol side chain cleavage enzyme (CSCC) by RU486 was more marked than that by R5020 or R2323. These results show that RU486 decreases progesterone synthesis in cultured ovarian cells. A part of the mechanism may involve an inhibition of CSCC.     

Inhibitory effects of the antiprogestin, RU 486, on progesterone actions and luteinizing hormone secretion in pituitary gonadotrophs

The effects of RU 486 on the modulation of LH release by progesterone were investigated in cultured anterior pituitary cells from ovariectomized adult female rats. The inhibitory effect of progesterone on LH secretion was demonstrable in estrogen-treated pituitary cells, in which addition of 10(-6) M progesterone to cells cultured in the presence of 10(-9) M estradiol for 52 h reduced the LH response to GnRH (10(-11) to 10(-7) M). When RU 486 was superimposed upon such combined treatment with estradiol and progesterone, the suppressive effect of progesterone on GnRH-induced LH release was completely abolished. The converse (facilitatory) effect of progesterone on LH secretion was observed in pituitary cells pretreated with 10(-9) M estradiol for 48 h and then with 10(-6) M progesterone for 4 h. When RU 486 was added together with progesterone during the 4 h treatment period, the facilitatory effect of progesterone was blocked and LH release fell to below the corresponding control value. The direct effect of RU 486 on LH secretion in the absence of exogenous progesterone was evaluated in cells cultured in the absence or presence of 10(-9) M estradiol and then treated for 4 to 24 h with increasing concentrations of RU 486 (10(-12) to 10(-5) M) and stimulated with GnRH (10(-9) M) during the last 3 h of incubation. In estrogen-deficient cultures, 4 h exposure to RU 486 concentrations of 10(-6) M and above decreased the LH response to GnRH by up to 50%. In cultures pretreated with 10(-9) M estradiol, GnRH-stimulated LH responses was inhibited by much lower RU 486 concentrations, of 10(-9) M and above. After 24 h of incubation the effects of RU 486 were similar in control and estradiol-pretreated pituitary cell cultures. Thus, RU 486 alone has a significant inhibitory effect on LH secretion that is enhanced in the presence of estrogen. The antiprogestin is also a potent antagonist of both the inhibitory and the facilitatory actions of progesterone upon pituitary gonadotropin release in vitro.     

Expression of galectin-1 mRNA in the mouse uterus is under the control of ovarian steroids during blastocyst implantation

Galectin-1 is a member of β-galactoside-binding lectins expressed in a variety of mammalian tissues. We report here that galectin-1 mRNA is abundantly expressed in the mouse reproductive organs such as the uterus and ovary. Uterine expression of galectin-1 mRNA is specifically regulated in the embryonic implantation process. Its expression increased at a high level on the fifth day post coitum (dpc 5) when embryos hatched into the endometrial epithelial cells. In the absence of embryos, however, galectin-1 expression in the mouse uterus decreased on dpc 5. In the delayed implantation mice, galectin-1 mRNA level was augmented by the termination of the delay of implantation. Ovarian steroids progesterone and estrogen differentially regulated galectin-1 mRNA level in uterine tissues. Treatment with RU486, a progesterone receptor antagonist, blocked progesterone-induced galectin-1 mRNA level in uterine tissues of ovariectomized mouse. ICI182780, a pure estrogen receptor antagonist, clearly blocked the estrogen effect. Taken together, galectin-1 gene expression in the uterine tissues was regulated by ovarian steroids and this regulation correlated with the implantation process. Mol. Reprod. Dev. 48:261–266, 1997. © 1997 Wiley-Liss, Inc.     

Agonist and antagonist-induced qualitative and quantitative alterations of progesterone receptor from breast cancer cells

T47D cells, cultured in medium containing serum stripped of endogenous steroids, proliferate in response to treatment with the progesterone receptor (PR) agonist, R5020 or the PR agonist/antagonist, RU486, whereas the full PR antagonist, ZK98299 has no proliferative effects. Under estrogenized conditions, all of the PR ligands tested inhibit cell growth [23]. In order to determine whether the levels or phosphorylation state of PR are reflected in the growth patterns of T47D cells, we monitored the effects of these PR ligands on the immunoblotted PR band intensities, the relative intensities, of PR-A and PR-B, and their phosphorylation states that are reflected in their altered mobility during SDS-PAGE. Under conditions where the PR ligands inhibit cell proliferation, each ligand had distinctively different qualitative and quantitative effects on PR. Short term treatment of the cells with R5020 or RU486 induced a characteristic phosphorylation-dependent upshift of both PR-A and PR-B. The phosphorylated PR was stable for up to 4 days after treatment of the cells with RU486, but was down regulated between 6-24 h after treatment with R5020. No replenishment of PR in cells treated with R5020 was detected. ZK98299, at concentrations tested, had no qualitative or quantitative effects on PR. Culturing cells for 8 days in medium containing steroid-depleted serum caused a significant reduction in the PR band intensity without causing a change in the ratio of PR-A and PR-B or their phosphorylation states. This decrease in the PR band intensity was reversed by maintaining the cells in 1 nM estrogen, but was potentiated by RU486 or ZK98299. These observations support the view that decreased PR levels may play a role in the stimulatory effects of R5020 and RU486 when cells are cultured under non-estrogenized conditions.     

Studies of a plasma membrane steroid receptor in Xenopus oocytes using the synthetic progestin RU 486

A steroid binding protein (Mr = 110,000) has previously been identified in the plasma membrane of Xenopus laevis oocytes by photoaffinity labeling with [3H]R5020. In order to further characterize this steroid receptor, the photoaffinity labeled receptor protein was solubilized with 0.1% Brij 35. The solubilized labeled receptor yielded an approximate mol. wt of 102,000 +/- 2,000 by sucrose density gradient centrifugation, suggesting that the solubilized receptor exists as a monomer. RU 486, a synthetic progestin antagonist for mammalian cytosolic receptor systems, inhibited up to 70% of [3H] R5020 photoaffinity binding to the 110,000-Dalton receptor with an IC50 of 5 microM and induced germinal vesicle breakdown (GVBD) with an EC50 of 9.0 +/- 0.6 microM. GVBD induced by RU 486 was slower than with progesterone, and RU 486 was less powerful than progesterone. Micromolar concentrations of RU 486 also potentiated GVBD induced by sub-optimal concentrations of progesterone or R5020. Furthermore, RU 486 inhibited oocyte plasma membrane adenylate cyclase with an apparent IC50 of 7.5 +/- 2.5 microM. The close correlation of the EC50 value for RU 486 induction of GVBD with the IC50 values for inhibition of [3H]R5020 photoaffinity labeling of the 110,000-Dalton receptor and inhibition of adenylate cyclase activity further supports the physiological significance of the oocyte plasma membrane steroid receptor.     

RU486 inhibits induction of aromatase by dexamethasone via glucocorticoid receptor in cultured human skin fibroblasts

Effects of RU486 on the induction of aromatase by dexamethasone via glucocorticoid receptor were determined using cultured human skin fibroblasts. Competition of [3H]dexamethasone binding to the cytosol receptor was 7 times stronger with RU486 than with dexamethasone. The order of the strength of competition was RU486 greater than dexamethasone greater than betamethasone greater than prednisolone greater than hydrocortisone. RU486 abolished a specific 8.6 S [3H]dexamethasone binding peak in the cytosol, determined using a sucrose density gradient analysis. Dexamethasone markedly induced aromatase and this event was strongly suppressed by RU486, in a dose-dependent manner, in the cultured skin fibroblasts. A linear correlation between the strength of competition and the induction of aromatase of various glucocorticoids was observed. RU486 non-competitively inhibited aromatase induction by dexamethasone determined from a double reciprocal plot of aromatase activity, with respect to [3H]androstenedione concentration in the presence of RU486. These results show that RU486 is a peripheral noncompetitive antiglucocorticoid on aromatase induction by glucocorticoid in human skin fibroblasts and that aromatase induction is a good marker for the biological function of glucocorticoid receptor in human skin fibroblasts.     

Non-classical androgenic actions of RU38486 in androgen-responsive Shionogi carcinoma 115 cells in serum-free culture

Antiglucocorticoid and antiprogestin RU38486 (RU486) stimulated the growth of highly androgen- and moderately glucocorticoid-sensitive SC-3 cells (a cloned cell line from Shionogi mouse mammary carcinoma 115) in a dose-dependent manner. A maximal 8-fold stimulation of growth by RU486 has been observed at 10(-7) M in a serum-free medium and its potency has been found to be almost the same as that of dexamethasone (Dex). The growth rate of SC-3 cells treated by triamcinolone acetonide (TA) or Dex combined with RU486 at 10(-9)-10(-7) M was enhanced compared to cells treated by TA or Dex alone, indicating that RU486 had additive rather than antagonistic effects. Our previous study revealed that RU486 could compete with the specific uptake of [3H]testosterone in intact SC-3 cells at relatively low affinity and the present study showed that the stimulatory effect of RU486 on the growth of SC-3 cells was significantly inhibited by pure antiandrogen flutamine and that half-maximal inhibition by flutamine was achieved at 10(-6) M. Moreover, we demonstrated that the conditioned medium from RU486-stimulated SC-3 cells contained growth-promoting activity which caused a 3.5-fold increase in DNA synthesis by SC-3 cells in the absence of RU486 and which was abolished by treatment with heparin-Sepharose. These results indicate that RU486-induced growth of SC-3 cells may be expressed as an androgenic activity through androgen receptor and mediated by a heparin-binding growth factor.