Effects of onapristone on postmenopausal endometrium

The progesterone antagonist mifepristone (RU486, Exelgyn) has been shown to exert a paradoxical agonist effect on postmenopausal endometrium. We conducted a study to investigate the effects of the 'pure' antiprogestin onapristone (ZK 98 299, Schering AG) on postmenopausal endometrium. Seventeen postmenopausal subjects (45-62 years), took 2 mg of oestradiol and either placebo, 1 mg onapristone or 10 mg of onapristone, daily for 56 days. An endometrial biopsy was performed during the final week of treatment and assessed for histology and immunohistochemistry for oestrogen receptors (ER), progesterone (PR), androgen receptors (AR) and the cell proliferation marker Ki 67. FSH fell in all 14 subjects who completed the study, consistent with the effect of oestradiol treatment. There was a dose-dependent additive effect of onapristone on suppression of gonadotrophins. All endometrial biopsies showed proliferative endometrium. A similar pattern and intensity of immunostaining of ER, PR and Ki 67 was observed in all groups, with positive immunoreactivity in both glands and stroma. AR immunostaining was observed in both glands and stroma from all subjects, but there was an increase in intensity of immunostaining within the glandular epithelium of women receiving 10 mg onapristone. The antiprogestin onapristone, in contrast to mifepristone, is not agonistic on postmenopausal endometrium and does not exert obvious antiproliferative effects. It does however cause a dose dependent suppression of FSH and LH release.     

The use of progesterone antagonists and progesterone receptor modulators in contraception

Progesterone antagonists (PAs) and progesterone receptor modulators (PRMs) have contraceptive potential by suppressing follicular development, delaying the surge of luteinizing hormone (LH), retarding endometrial maturation, and promoting endometrial bleeding. Mifepristone, in daily doses of 2-10 mg, blocks the LH surge and ovulation. Many of the studies were conducted in women not at risk of pregnancy, and thus the contraceptive efficacy is not yet known. Nevertheless, there is evidence that daily doses of 2 or 5 mg of mifepristone have contraceptive potential. Because of anovulation, there may be an unopposed estrogen effect on the endometrium, although this risk may be mitigated by the noncompetitive anti-estrogenic activity exhibited by both PAs and PRMs. Low doses of PAs and PRMs, which do not affect ovulation, retard endometrial maturation, indicating that the endometrium is exquisitely sensitive to these compounds. This raises the prospect of endometrial contraception, i.e. prevention of endometrial maturation without disturbing ovulation or producing alterations in bleeding patterns. This approach works well in monkeys but was not found to be very promising when given to women not using contraception. On the other hand, 200 mg mifepristone administered 48 h after the LH surge, which has minimal or no effect on ovulation and bleeding patterns, is an effective contraceptive; yet, it is not a practical approach to contraception. Late luteal phase administration of mifepristone produces menstrual bleeding. However, when mifepristone was administered every month at the end of the cycle either alone or together with prostaglandins, it was not very effective in preventing pregnancy. In contrast, a mifepristone-prostaglandin combination has been shown to be a very effective treatment for occasional menstrual regulation, with vaginal bleeding induced in 98% of pregnant women, with menses delay of 11 days or less. Mifepristone is an excellent agent for emergency contraception when used within 120 h of unprotected intercourse. It is also possible that PAs and PRMs may be used to reduce the occurrence of bleeding irregularities induced by progestin-only contraceptive methods. Both classes of progesterone receptor ligands may also have contraceptive efficacy by having a pharmacological effect on the embryo or altering tubal transport or other aspects of tubal physiology.     

Discovery of non-steroidal mifepristone mimetics: pyrazoline-based PR antagonists

Mifepristone is a non-selective antagonist of 3-oxosteroid receptors with both abortifacient and anti-endometriotic activities. Non-steroidal mimetics of mifepristone and progesterone are important templates for modulation of the progesterone receptor (PR). For our PR program, we sought an unexplored, synthetically accessible non-steroidal mimetic of mifepristone, suitable for parallel synthesis of analogues. Docking of compounds into a PR homology model identified 4-substituted pyrazolines, which, when synthesized and tested, exhibited functional antagonism of PR.     

Distribution of estrogen and progesteron receptors in the uterus: an immunohistochemical study in the immature and adult pseudopregnant rabbit.

In order to clarify the distribution and content of estrogen (ER) and progesteron receptors (PR) under changing hormonal influences within the various cell populations of the uterus (glandular and luminal endometrial epithelium, stroma, myometrium), immunohistochemical determinations using specific monoclonal antibodies were made. To correlate the immunohistochemical findings with peripheral hormone levels and specific tasks of the endometrium, 17 beta-estradiol and progesterone serum levels were measured and cell proliferation determined by use of BrdU-labelling-immunohistochemistry. At the subcellular level ER and PR were located exclusively in the cell nuclei of female rabbits, which were either immature and lacking any peripheral hormone levels or were pseudopregnant (d0-d8 p.hCG). In the immature rabbits a general faint ER and PR immunostaining was found. In addition to a general increase in ER and PR in all cell populations estrous rabbits (d0 p.hCG) showed a significant rise of ER in the epithelial cells and of PR in the myometrium. Within the epithelial cells and the myometrium the ER dropped heavily within a few days of pseudopregnancy. The PR, however, increased sharply during the first two days of pseudopregnancy and decreased gradually following d4 p.hCG. A close relationship was observed between the high PR content and the proliferation rate of the epithelial cells on d2 p.hCG. In spite of the more rapid decrease of ER compared with PR, the glandular epithelium retained positive immunostaining. In the stroma the ER and especially PR content did not change significantly during the course of pseudopregnancy suggesting that some of the well-known differentiation events in the luminal epithelium may be mediated by the stroma.     

Progesterone receptor involvement in independent tumor growth in MPA-induced murine mammary adenocarcinomas

We have developed a model of hormonal carcinogenesis in BALB/c female mice, in which MPA induced ductal mammary adenocarcinomas, expressing high levels of estrogen and progesterone receptors (ER and PR). A series of tumor lines, retaining both PR and ER expression, were obtained from selected tumors, which are maintained by syngeneic passages. In this model progesterone behaves as the growth-stimulating hormone (progesterone-dependent or PD tumors), whereas estrogens induce tumor regression. Through selective treatments we were able to derive a series of progesterone-independent (PI) variants. These lines do not require progesterone treatment to grow in ovariectomized female BALB/c mice, but retain, however, the expression of ER and PR. The aim of this paper is to investigate a possible regulatory role of the progesterone receptor (PR) on PI tumor growth. ER and PR were detected by immunocytochemistry in all lines studied. They were also characterized using biochemical assays and Scatchard plots. No differences in Kd of PR or ER were detected in PI variants. AR or GR were not detected in tumor samples using biochemical assays. Estradiol (5 mg silastic pellet) induced complete tumor regression in all tumors tested. We also evaluated the effects of different antiprogestins on tumor growth. Onapristone (10 mg/kg/day) and mifepristone (4.5 mg/kg/day) were able to induce complete tumor regression. The antiandrogen flutamide (5 mg silastic pellet) had no effect on tumor growth in agreement with the lack of androgen receptors. We used an in vitro approach to corroborate that the antiprogestin-induced inhibition was not attributable to an intrinsic effect. Cultures of a selected PI line were treated with PR antisense oligodeoxynucleotides (ASPR) to inhibit in vitro cell proliferation. A significant decrease of 3H-thymidine uptake was observed in cells of a PI line growing in the presence of 2.5% charcoalized fetal calf serum and 0.8-20 microg/ml ASPR. It can be concluded that the PR pathway is an essential path in the growth stimulation of PI tumors.     

Characterization and localization of estrogen and progesterone receptors of human fallopian tube

The cellular distribution of estrogen and progesterone receptors (ER and PR) in the human fallopian tube was investigated by immunohistochemical localization with specific monoclonal antibodies. Nuclear immunostaining was observed. Intense PR immunostaining was seen in tissues obtained at mid cycle and luteal stages of the normal menstrual cycle. On the other hand, enhanced staining for ER was seen in early follicular phase and mid cycle. Menopausal tissues showed negligible staining for both ER and PR. The ER and PR were characterized for their molecular size, anatomical distribution and levels during the menstrual cycle and in menopause. ER protein was present throughout the cycle and also during menopause. Western blot analysis revealed two forms of ER approximately 66 kDa and a truncated from approximately 49 kDa in hFT. Presence of A [approximately 90 kDa] and B [approximately 120 kDa] isoforms of human PR was detected. Follicular and early luteal tissue possessed relatively high concentration of immunoreactive PR whereas it was almost undetectable in menopausal tissues. These results suggests that ER and PR are regulated by the changing ovarian steroid hormones.     

Distribution of estrogen and progesteron receptors in the uterus: an immunohistochemical study in the immature and adult pseudopregnant rabbit

Summary In order to clarify the distribution and content of estrogen (ER) and progesteron receptors (PR) under changing hormonal influences within the various cell populations of the uterus (glandular and luminal endometrial epithelium, stroma, myometrium), immunohistochemical determinations using specific monoclonal antibodies were made. To correlate the immunohistochemical findings with peripheral hormone levels and specific tasks of the endometrium, 17-estradiol and progesterone serum levels were measured and cell proliferation determined by use of BrdU-labelling-immunohistochemistry. At the subcellular level ER and PR were located exclusively in the cell nuclei of female rabbits, which were either immature and lacking any peripheral hormone levels or were pseudopregnant (d0–d8 p.hCG). In the immature rabbits a general faint ER and PR immunostaining was found. In addition to a general increase in ER and PR in all cell populations estrous rabbits (d0 p.hCG) showed a significant rise of ER in the epithelial cells and of PR in the myometrium. Within the epithelial cells and the myometrium the ER dropped heavily within a few days of pseudopregnancy. The PR, however, increased sharply during the first two days of pseudopregnancy and decreased gradually following d4 p.hCG. A close relationship was observed between the high PR content and the proliferation rate of the epithelial cells on d2 p.hCG. In spite of the more rapid decrease of ER compared with PR, the glandular epithelium retained positive immunostaining. In the stroma the ER and especially PR content did not change significantly during the course of pseudopregnancy suggesting that some of the well-known differentiation events in the luminal epithelium may be mediated by the stroma.     

Tissue- and hormone- dependent progesterone receptor distribution in the rat uterus

Background  

Estradiol (E2) and progesterone (P) are well known regulators of progesterone receptor (PR) expression in the rat uterus. However, it is not known which receptor subtypes are involved. Little knowledge exist about possible differences in PR regulation through ERalpha or ERbeta, and whether the PR subtypes are differently regulated depending on ER type bound. Thus, in the present study PR immunostaining has been examined in uteri of ovariectomized (ovx) rats after different treatments of estrogen and P, in comparison with that in immature, cycling, and pregnant animals.     

Termination of pregnancy and induction of premature luteolysis by the antiprogestagen, mifepristone, in dogs

Five pregnant beagle bitches were treated with 2.5 mg mifepristone/kg body weight, twice a day, for 4.5 days starting at Day 32 of gestation. Results of fetal ultrasonography and assay of serum progesterone concentrations every 2-4 days were compared to those in 5 control bitches. Mifepristone resulted in a premature (P less than 0.01) termination of pregnancy (36 +/- 1 vs 65 +/- 1 days), without side effects. The antiprogestagen also caused progesterone to decline to less than 1 ng/ml by Day 40-45 after the preovulatory LH peak (vs 64-67 days in controls) and reduced (P less than 0.05) mean concentrations on Days 34-50 (2.2 +/- 0.5 vs 6.3 +/- 0.3 ng/ml). The results suggest that antiprogestagen therapy is a safe means to terminate unwanted pregnancy in dogs, and that luteal function in pregnant bitches is dependent on luteotrophic support that is blocked by antiprogestagen treatment, directly or indirectly, due to termination of pregnancy.     

Mifepristone: a novel estrogen-free daily contraceptive pill

When the first synthetic progesterone antagonist (mifepristone) was synthesized over 20 years ago, it was clear that it had a potential as an antifertility agent. Research into the use of antiprogestogens for contraception have concentrated on three general approaches: (1) inhibition of ovulation, (2) inhibition of implantation and (3) disruption of implantation or "menstrual induction". The effect of mifepristone on the ovarian and endometrial cycle depends on dose, timing and frequency of administration. Doses of 10 mg per day or more suppress follicular development and estradiol levels. Ovulatory cycles are maintained in the dose of less than 2 mg although there is increased variability in cycle length. The endometrium shows some minor asynchronous changes, although these are not sufficient to prevent pregnancy. We have chosen to investigate daily doses between 2 and 5 mg which inhibit ovulation and menstruation in over 90% of cycles while still maintaining follicular development and levels of estradiol within the range found during the follicular phase. The endometrium shows proliferative or cystic changes lined by a layer of inactive glandular epithelium set in densely packed stroma. There is, however, an absence of proliferative activity as reflected by a reduced mitotic index and Ki67 staining. These unusual histological appearances are associated with downregulation of PR but a massive upregulation of AR in particularly glandular epithelium. The antiproliferative effect of mifepristone is reassuring suggesting that the risk of atypical hyperplasia due to the effect of prolonged exposure to estrogen unopposed by progesterone is low. In a pilot study, there were no pregnancies in 200 months of exposure in 50 women who used this method as their sole method of contraception. Daily mifepristone could provide a novel contraceptive method which should be devoid of the risks associated with estrogen containing combined oral contraceptive (COC), e.g. venous thromboembolism. The health benefits of avoiding the morbidity associated with menstruation are considerable. Recent surveys suggest that amenorrhoea would be popular with many women.     

Steroid receptors in canine endometrial cells can be regulated by estrogen and progesterone under in vitro conditions

The expression of estrogen (ER) and progesterone receptors (PR) in the endometrium is regulated by steroid hormones. An increase in plasma estrogen leads to upregulation of the number of both steroid receptors, whereas a decrease in both receptors population is due to high concentration of plasma progesterone. To study the exact effect of different concentrations of beta-estradiol and progesterone on canine epithelial and stromal endometrial cells an in vitro model from dog uterus was developed and kept for 20 days. Material was obtained from healthy dogs, undergoing ovariohysterectomy. Endometrial epithelial and stromal cells were gained after collagenase treatment, followed by filtration steps. Electron microscopy and immunolabeling were used to study cell morphology and differentiation. Immunocytochemistry was used to determine proliferation rate (Ki-67), ER and PR status on Days 3, 8, 10, 13, and 20. Mitotic activity of both cells was stimulated with different concentrations of steroids and revealed high values until cells reached confluency. ER and PR expression in confluent layer from epithelial and stromal cells was upregulated with beta-estradiol. In addition progesterone significant downregulated both receptors population in stromal cells, whereas the reduction was less pronounced in epithelial cells. Results showed that our in vitro system is a useful tool to study the influence of beta-estradiol and progesterone on cell proliferation rate, ER and PR expression. The primary cell culture model helps to avoid experiments on living animals.     

Endocrine pharmacological characterization of progesterone antagonists and progesterone receptor modulators with respect to PR-agonistic and antagonistic activity

Studies were conducted to assess progesterone antagonists (PAs) and progesterone receptor modulators (PRMs) with respect to PR agonistic and antagonistic activities in vivo. These properties are not always adequately reflected in transactivation in vitro models. Studies were performed in pregnant rats, estrogen-primed rabbits (McPhail -Test), and cycling and pregnant guinea pigs. Tested compounds included mifepristone (RU486), onapristone, J867, J956, J1042, and ZK137316. J-compounds induced sub-maximum endometrial transformation and, paradoxically, inhibited effects of progesterone in rabbits. Mifepristone, onapristone, and ZK137316 behaved as 'pure' antagonists in this species. Inhibition of uterine PGF(2alpha) secretion and inhibition of luteolysis in cycling guinea pigs were more sensitive parameters of PR-agonistic and antagonistic properties. 'Pure' PAs inhibited uterine PGF(2alpha) secretion and luteal regression completely. The PR agonist R5020 reversed both effects which demonstrates a PR mediation. Agonistic PRMs (J-substances and mifepristone) showed no or blunted antiluteolytic effects compared to the 'pure' PR antagonist onapristone. When tested in pregnant guinea pigs for their labor-inducing potential, PR agonistic PRMs had much reduced or abolished abortifacient activity compared to mifepristone (mifepristone > J956 > J867/J912 > J1042). However, in cycling animals, superior antiovulatory and antiproliferative properties of the J-substances were seen. Antiovulatory effects of 'pure' and agonistic PRMs are probably due to different mechanisms. The relevance of rodent studies for antiovulatory and uterine antiproliferative effects for the human is still uncertain. The non-abortifacient PRM J1042 induced stromal compaction and inhibition of endometrial proliferation in monkeys, but this effect was not stronger than that of the 'purer' PAs. 'Pure' PAs are important pharmacological tools analogous to PRKO models to study the role of PR in the menstrual cycle and in pregnancy.     

Epidermal growth factor receptors in human breast and endometrial carcinomas

The incidence and levels of epidermal growth factor receptor (EGFR) were studied in 67 breast tumors and 22 endometrial carcinomas. Estrogen receptors (ER) were also measured in all samples and progesterone receptors (PR) were analyzed in 57 breast samples and 21 endometrial tumors. A high level of EGFR expression is found in both breast and endometrial carcinomas, although the incidence of EGFR content is greater in breast carcinomas. 36% of breast tumors had EGFR at levels 3-49.5 fmol/mg membrane protein, whereas this percentage of positivity was 27% for endometrial tumors. In 51% of breast carcinoma and 73% of endometrial tumors, there was a positive ER content, whereas 53% of breast tumors and 62% of endometrial carcinomas were positive. A clear inverse relationship between EGFR content and ER and PR status has been observed in breast tumors. Our data confirm the previously described inverse correlation between expression of EGFR and estrogen receptors in human breast cancer. We also show here that there is a similar inverse relationship between EGFR content and ER levels in endometrial tumors.     

Suckling-induced prolactin release potentiates mifepristone-induced lactogenesis in pregnant rats

Suckling, starting at 19:00 h on Day 18 of pregnancy, induced a significant increase in serum prolactin concentration at 20:00 h on Day 19 of pregnancy, but no increase in mammary gland casein or lactose content. Mifepristone (2 mg/kg) injection at 08:00 h on Day 19 of pregnancy induced significant increases in casein, but not in lactose, 24 h after administration. Mifepristone alone did not induce prolactin secretion, indicating that lactogenesis was induced by placental lactogen in the absence of progesterone action. When mifepristone was injected into suckling rats, serum prolactin concentrations were higher than in the untreated suckling rats. Casein in these rats increased significantly 12 h after mifepristone administration and lactose at 24 h after. If the suckling mifepristone-treated rats were given two injections of bromocriptine (1.5 mg/kg) at 12:00 h on Days 18 and 19 of pregnancy, serum prolactin concentrations were not increased by suckling, but casein and lactose concentrations in the mammary gland showed values similar to those obtained in the mifepristone-treated non-suckling rats. Mifepristone can therefore potentiate suckling-induced prolactin release in pregnant rats, demonstrating a direct central inhibitory action of progesterone on prolactin secretion. This suckling-induced prolactin secretion, unable to induce casein or lactose synthesis in the presence of progesterone, enhanced significantly synthesis of these milk components in the absence of progesterone action (rats treated with mifepristone). Fatty acid synthase, which is stimulated by the suckling stimulus in lactating rats, was not modified by mifepristone or suckling in pregnant rats.     

Monoclonal antibody characterization of progesterone receptors, estrogen receptors and the stress-responsive protein of 27 kDa (SRP27) in human uterine leiomyoma

Uterine leiomyoma occurs in one of every four or five women during their reproductive life. Its origin is unknown but it is accepted that estrogens play a significant role in its development. In order to learn more about the estrogen dependency of leiomyoma, the biochemical and immunological properties of two markers of estrogen response in target cells (the progesterone receptor (PR) and the stress-responsive protein of 27 kDa (SRP27)) were studied in leiomyoma. The ER (estrogen receptor) and PR content were determined by conventional DCC exchange assays. Specific anti-ER, anti-PR and anti-SRP27 monoclonal antibodies were used in immunoblots and immunohistochemical (IHC) studies. The binding properties of PR from cytosol of leiomyoma showed a Kd of 0.8-1.3 nM, which is in the range described for other human tissues. 80% of all studied leiomyoma contained PR, in a range of 805-2000 fmol/mg protein. The Kd for leiomyoma ER was 0.1-0.9 nM, and 84% of the samples were positive for ER. The PR of leiomyoma has the two A and B forms of 120 and 94 kDa, as shown in the immunoblot using the AB52 anti-PR monoclonal antibody. The IHC study revealed that the PR is concentrated in the cell nuclei, in the form of perinuclear bodies, with a homogeneous staining pattern from cell to cell. The leiomyoma fibres contain SRP27 in a higher concentration than the healthy myometrium. The leiomyoma SRP27 shows a typical doublet of 24 kDa and 27 kDa in immunoblot, the same as in MCF-7 cells. The IHC study revealed a high degree of organization of SRP27 in leiomyoma cells, suggesting that this protein may be part of the cytoskeleton. The results obtained show that human leiomyomas contain ER, PR and RSP27 with similar immunological and biochemical properties to those of other human tissues, including the MCF-7 breast cancer cell line.     

Estradiol and progesterone receptor concentrations and 17β-hydroxysteroid-dehydrogenase activity in estrogenprogestin stimulated endometrium of women with gonadal dysgenesis

Four hypergonadotrophic women between 25 and 37 years of age with gonadal dysgenesis were treated sequentially with estrogens and a progestin. The hormonal environment induced by this therapy was similar to that of ovulating women, as demonstrated by serum levels of estradiol, endometrial histology and pituitary gonadotropin secretion before and after LH-RH double stimulation.The concentrations of estradiol and progesterone receptors (ER and PR) and the activity of the 17β-hydroxysteroid-dehydrogenase (17β-HSD) were determined in endometrial curettings obtained from the above patients at 5 different days of their 28-day artificial cycles. The results were correlated to the histology of the endometrium and the serum concentrations of estradiol at the corresponding days of the cycle.The cytoplasmic ER and PR concentrations in the endometrium were 3–6 times higher during the estrogen than progestin phase of the induced endometrial cycle. For the activity of the 17β-HSD, the contrary was the case, being 6–10 fold higher during the progestin phase. A 22-day administration of estrogens only did not lead to a rise in enzyme activity or decrease in receptor content as observed under progestin influence after day 14. Since ER and PR concentrations and 17β-HSD activities were similar to those in the endometrium of normally ovulating women, these results confirm experimentally the present concept concerning the dependence of the cytoplasmic ER and PR content and 17β-HSD activity on female sex hormone action.     

Myometrial effects of selective estrogen receptor modulators on estradiol-responsive gene expression are gene and cell-specific

We examined in vivo effects of selective estrogen receptor modulators (SERMs) 4-OH-tamoxifen (Tam), GW 5638 (GW) and EM-800 (EM) on myometrial gene expression. The uteri of ovariectomized ewes were infused with 10⁻⁷ M of one SERM via indwelling catheters for 24 h preceding hysterectomy. Half of the ewes in each SERM group received an intramuscular injection of 50 μg 17β-estradiol (E2) 18 h prior to hysterectomy. Northern blot analysis and in situ hybridization demonstrated that E2 increased estrogen receptor (ER), progesterone receptor (PR) and cyclophilin (CYC) gene expression in the cells of both inner layer of myometrium (IM) and outer layer of myometrium (OM) as well as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression in OM. Tam also increased ER mRNA levels in OM. EM appeared to increase ER gene expression, but antagonized E2’s up-regulation of PR and CYC gene expression in both IM and OM. Tam and GW also antagonized E2 up-regulation of PR gene expression in OM but not IM. No SERM affected GAPDH gene expression with or without E2. Immunohistochemistry indicated that E2 increased nuclear ER and PR protein levels in both IM and OM. EM was unique in up-regulating ER protein levels, opposite to its effects in endometrial cells. All SERMs tested antagonized this increase in PR immunostaining preferentially in OM compared to the IM layer. These results illustrate gene and cell layer-specific effects of SERMs in sheep myometrium.