Progesterone receptor activation signals behavioral transitions across the reproductive cycle of the female rabbit

The female rabbit is an exceptional experimental model to define mechanisms by which progesterone (P) controls the expression of reproductive behaviors. In the rabbit, the rise in P levels during pregnancy inhibits estrous scent marking ("chinning"), stimulates the excavation of a nest burrow ("digging"), and primes behaviors later used for nest construction. The pre-parturient fall of P triggers the construction of a straw nest ("straw carrying") that is lined with hair that she pulls from her own body ("hair pulling"). These behaviors can be replicated in ovariectomized (ovx) females given a schedule of estradiol (E) and P that mimics hormone levels during pregnancy (E from days 0 to 4, E + P from days 5 to 17, E from days 18 to 27). We administered PR antagonists RU486 or CDB(VA)2914 to ovx female rabbits during either the initial (days 5-11) or late (days 12-17) phases of P treatment, to determine the role of PR activation in coordinating the expression of these behaviors. Both antiprogestins attenuated the P-mediated decline in chinning and increase in digging when administered during days 5-11. When given across days 12-17, both antiprogestins triggered an early decline in digging, the onset of nest building in some Ss, and the reinstatement of chinning. These results point to a central role of PR activation for establishing and maintaining the behavioral phenotype of pregnancy, and for the behavioral transition from pregnancy to estrus.     

Behavioral characteristics and pharmacological manipulations of a nicotine-entrainable circadian oscillator

Chronic daily administration of nicotine and other drugs of abuse has been found to entrain pre- and post-drug circadian locomotor activity episodes that oscillate on a 24-h schedule and persist for several days after administration ceases. This drug-entrainable oscillator system could conceivably lead to circadian rhythms of drug seeking and drug use in human drug addicts. The present study (1) characterizes the ability of daily nicotine administration to entrain circadian wheel-running activity episodes in rats across a range of doses, lighting schedules, and food access; and (2) tests whether pre- and post-nicotine episodes can be altered through pharmacological manipulations. Adult female rats were housed in wheel boxes for 35–60?d, and both wheel-running and feeding-related behaviors were measured continuously. Following acclimation, nicotine or saline was administered for 16–24?d, and the rats were left undisturbed for several test days to observe the persistence of nicotine-entrained activity. The results showed that nicotine dose-dependently entrains wheel-running activity, and the highest dose of 1.0?mg/kg produces robust pre- and post-nicotine circadian activity episodes under constant, fixed, and variable light/dark schedules. In the pharmacological manipulation experiment, nicotine-entrained rats were administered one of seven pharmacological treatments (varenicline, mecamylamine, acamprosate, topiramate, naltrexone, SB-334867, or bupropion) in place of the nicotine injection for 2?d, and the rats were not disturbed for four subsequent days. Most of the treatment drugs significantly reduced post-nicotine activity episodes, but only three treatments affected pre-nicotine episodes: the μ- and κ-opioid receptor antagonist naltrexone, the orexin-1 receptor antagonist SB-334867, and the AMPA (2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid)/kainate antagonist topiramate. These results show that chronic daily nicotine administration is a robust zeitgeber that dose-dependently entrains a nonphotic oscillator system that includes opioid, orexin, and glutamate pathways.     

Therapeutic administration of progesterone antagonist in a model of Charcot-Marie-Tooth disease (CMT-1A)

Charcot-Marie-Tooth disease (CMT) is the most common inherited neuropathy. The predominant subtype, CMT-1A, accounts for more than 50% of all cases and is associated with an interstitial chromosomal duplication of 17p12 (refs. 2,3). We have generated a model of CMT-1A by introducing extra copies of the responsible disease gene, Pmp22 (encoding the peripheral myelin protein of 22 kDa), into transgenic rats. Here, we used this model to test whether progesterone, a regulator of the myelin genes Pmp22 and myelin protein zero (Mpz) in cultured Schwann cells, can modulate the progressive neuropathy caused by moderate overexpression of Pmp22. Male transgenic rats (n = 84) were randomly assigned into three treatment groups: progesterone, progesterone antagonist (onapristone) and placebo control. Daily administration of progesterone elevated the steady-state levels of Pmp22 and Mpz mRNA in the sciatic nerve, resulting in enhanced Schwann cell pathology and a more progressive clinical neuropathy. In contrast, administration of the selective progesterone receptor antagonist reduced overexpression of Pmp22 and improved the CMT phenotype, without obvious side effects, in wild-type or transgenic rats. Taken together, these data provide proof of principle that the progesterone receptor of myelin-forming Schwann cells is a promising pharmacological target for therapy of CMT-1A.     

The synergistic effect of selective sigma receptor agonists and uncompetitive NMDA receptor antagonists in the forced swim test in rats.

Data acquired to date show that some sigma receptor ligands reveal "antidepressant-like" activity in the forced swim test in mice and rats. Moreover, our preliminary results suggested that joint administration of sigma receptor ligands and amantadine (AMA, a glutamatergic/NMDA receptor antagonist) caused a positive interaction in the Porsolt test in rats, as had already been observed in the case of co-treatment with clinically active antidepressants and AMA. The aim of the present study was to examine the effect of combined administration of sigma1 or sigma2 receptor agonists, SA4503 or siramesine, respectively, and AMA or memantine (MEM) (uncompetitive NMDA receptor antagonist). SA4503 or siramesine given jointly with MEM (as well as with AMA) decreased the immobility time in rats. The effect of SA4503 and AMA co-administration was antagonized by progesterone, a sigma1 receptor antagonistic neurosteroid. Combined treatment with siramesine and AMA was modified by neither progesterone nor BD1047 (a novel sigma antagonist with preferential affinity for sigma1 sites); but it was counteracted by sulpiride and prazosin (a dopamine D2- and an alpha1-adrenergic receptor antagonist, respectively). The "antidepressant-like" effect induced by siramesine and MEM was not antagonized by progesterone, but was attenuated by BD1047, sulpiride and prazosin. The obtained results give support to the hypothesis that sigma (particularly sigma1) receptors may be one of the possible mechanisms by which drugs induce antidepressant-like activity in the forced swim test, and that this effect may be enhanced by NMDA receptor antagonists. Combined treatment with sigma ligands and AMA or MEM (applied in the clinic) may be an alternative to the treatment of antidepressant-resistant depressive patients in the future.     

Development of the selective progesterone receptor modulator CDB-2914 for clinical indications

CDB-2914 (17 alpha-acetoxy-11 beta-[4-N,N-dimethylaminophenyl]-19-norpregna-4,9-diene-3,20-dione) is a 19-norprogesterone derivative that acts as an antagonist in progesterone-responsive tissues. It binds to progesterone receptors A and B with high affinity. After oral dosing in humans, CDB-2914 serum levels peak at 60-90 min. CDB-2914 binds to serum proteins and is cleared slowly. Doses of 1, 10 and 50 mg exhibit proportional increases in peak serum levels, but serum levels from higher doses, 100 and 200 mg, are not dose-dependent, suggesting saturation of carrier sites. The biological effects of CDB-2914 vary according to time of the menstrual cycle that the drug is given. In the mid-follicular phase, CDB-2914 (50 mg) inhibits follicular development and delays ovulation and menses. At 100 mg, in some cases the original follicle ceases development and a new follicle is recruited. Endometrial maturation is delayed at all doses tested (10, 50, 100 mg). Given at mid-luteal phase, there was a dose-dependent effect on menses, with higher doses (100-200 mg) resulting in earlier menses. On average, CDB-2914 tends to lengthen the menstrual cycle by approximately 1-2 days although the amount of delay varies with timing in the menstrual cycle and dose.     

Anxiolytic-like effects of nociceptin/orphanin FQ in the elevated plus maze and in the conditioned defensive burying test in rats

Different reports suggest that nociceptin/orphanin FQ (N/OFQ) may have either anxiolytic- or anxiogenic-like effect in rodents. Since N/OFQ elicits hypolocomotion, which undergoes rapid tolerance, and hypolocomotion may be associated to emotional consequences, the present study was designed to investigate the effect of N/OFQ on anxiety after development of tolerance to its hypolocomotor effect. The effect of single or double intracerebroventricular (i.c.v.) injection of N/OFQ was evaluated on anxiety-related behaviors in rats, in the elevated plus maze (EPM) and conditioned defensive burying (CDB) tests. After single administration, N/OFQ displayed an anxiogenic-like pattern of response on the elevated plus maze but hypolocomotion was also observed. Conversely, in the CDB test, N/OFQ induced a clear-cut anxiolytic pattern. To produce tolerance to N/OFQ-induced hypolocomotion the peptide was administered by two i.c.v. injections separated by 120 min; in these conditions it decreased the expression of anxiety-related behaviors in both tests without affecting locomotor activity. The nociceptin/orphanin FQ peptide (NOP) receptor antagonist UFP-101 significantly reduced the effects of N/OFQ to control values in either tests. Corticosterone levels were significantly increased after a single N/OFQ administration (not in a dose-dependent manner) but this increase did not reach significance after double administration (1 nmol/rat). Our results support the idea that N/OFQ may act as an anxiolytic-like agent in the rat; the apparent anxiogenic-like effect observed following its single administration in the EPM may be consequent to its effect on locomotion.     

Progesterone Receptor Expression Declines in the Guinea Pig Uterus during Functional Progesterone Withdrawal and in Response to Prostaglandins

Progesterone withdrawal is essential for parturition, but the mechanism of this pivotal hormonal change is unclear in women and other mammals that give birth without a pre-labor drop in maternal progesterone levels. One possibility suggested by uterine tissue analyses and cell culture models is that progesterone receptor levels change at term decreasing the progesterone responsiveness of the myometrium, which causes progesterone withdrawal at the functional level and results in estrogen dominance enhancing uterine contractility. In this investigation we have explored whether receptor mediated functional progesterone withdrawal occurs during late pregnancy and labor in vivo. We have also determined whether prostaglandins that induce labor cause functional progesterone withdrawal by altering myometrial progesterone receptor expression. Pregnant guinea pigs were used, since this animal loses progesterone responsiveness at term and gives birth in the presence of high maternal progesterone level similarly to primates. We found that progesterone receptor mRNA and protein A and B expression decreased in the guinea pig uterus during the last third of gestation and in labor. Prostaglandin administration reduced while prostaglandin synthesis inhibitor treatment increased progesterone receptor A protein abundance. Estrogen receptor-1 protein levels remained unchanged during late gestation, in labor and after prostaglandin or prostaglandin synthesis inhibitor administration. Steroid receptor levels were higher in the non-pregnant than in the pregnant uterine horns. We conclude that the decreasing expression of both progesterone receptors A and B is a physiological mechanism of functional progesterone withdrawal in the guinea pig during late pregnancy and in labor. Further, prostaglandins administered exogenously or produced endogenously stimulate labor in part by suppressing uterine progesterone receptor A expression, which may cause functional progesterone withdrawal, promote estrogen dominance and foster myometrial contractions.     

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.     

Nestorone: a progestin with a unique pharmacological profile

Nestorone(R) (Nestorone 16-methylene-17alpha-acetoxy-19-norpregn-4-ene-3,20-dione), formerly referred to as ST 1435, is a potent progestin when given parenterally via sustained release formulations. The pharmacological profile of Nestorone was compared with that of levonorgestrel and 3-keto-desogestrel by steroid receptor binding studies and by in vivo bioassays in rats and rabbits. 3-Keto-desogestrel showed the highest binding affinity to progesterone receptors (PR) followed by Nestorone, levonorgestrel, and progesterone. The binding affinity of Nestorone to androgen receptors (AR) was 500- to 600-fold less than that of testosterone. However, both levonorgestrel and 3-keto-desogestrel showed significant binding (40 to 70% of testosterone) to AR. None of the progestins bound to estrogen receptors (ER). The progestational activity of Nestorone, levonorgestrel, and progesterone was compared using McPhail index in immature rabbits and pregnancy maintenance and ovulation inhibition tests in rats after subcutaneous (s.c.) administration. In all three tests, Nestorone was the most potent progestin. The progestational activity of Nestorone was also compared after oral and s.c. administration in rabbits. The potency of Nestorone was over 100-fold higher upon s.c. administration than via the oral route. The androgenic activity of progestins, based on the stimulation of ventral prostate (androgenic target) and levator ani (anabolic target) growth in castrated immature rats, showed good correlation with their binding affinity to AR. Nestorone showed no androgenic or anabolic activity. Nestorone did not bind to sex hormone binding globulin (SHBG), whereas both levonorgestrel and 3-keto-desogestrel showed significant binding to SHBG. The estrogenic/antiestrogenic activity of Nestorone was investigated in immature ovariectomized rats. In contrast to estradiol and levonorgestrel, Nestorone showed no uterotropic activity in ovariectomized rats. Despite significant binding to glucocorticoid receptors (GR), Nestorone showed no glucocorticoid activity in vivo. It is concluded that a strong progestational activity, combined with lack of androgenic, estrogenic, and glucocorticoid-like activities, confer special advantages to Nestorone for use in contraception and hormone replacement therapy.     

Antiprogestins as a model for progesterone withdrawal

The key physiological function of the endometrium is preparation for implantation; and in the absence of pregnancy, menstruation and repair. The withdrawal of progesterone is the initiating factor for breakdown of the endometrium. The modulation of sex steroid expression and function with pharmacological agents has provided an invaluable tool for studying the functional responses of the endometrium to sex steroids and their withdrawal. By administration of the antiprogestin mifepristone, it is possible to mimic progesterone withdrawal and study local events in early pregnancy decidua that may play a role in the process of early pregnancy failure. Our data indicate that antagonism of progesterone action at the receptor level results in an up-regulation of key local inflammatory mediators, including NF-kappaB, interleukin-8 (IL-8), monocyte chemotactic peptide-1 (MCP-1), cyclooxygenase 2 (COX-2) and others in decidua. Bleeding induced by mifepristone in the mid-luteal phase of the cycle is associated with changes in the endometrium similar to those that precede spontaneous menstruation including up-regulation of COX-2 and down-regulation of PGDH. Administration of antagonists of progesterone provide an excellent model to study the mechanisms involved in spontaneous and induced abortion as well as providing information which may help devise strategies for treating breakthrough bleeding associated with hormonal contraception.     

A novel pathway involving progesterone receptor, endothelin-2, and endothelin receptor B controls ovulation in mice

The steroid hormone progesterone (P) plays a pivotal role during ovulation. Mice lacking P receptor (Pgr) gene fail to ovulate due to a defect in follicular rupture. The P receptor (PGR)-regulated pathways that modulate ovulation, however, remain poorly understood. To identify these pathways, we performed gene expression profiling using ovaries from mice subjected to gonadotropin-induced superovulation in the presence and in the absence of CDB-2914, a synthetic PGR antagonist. Prominent among the genes that were down-regulated in response to CDB-2914 was endothelin (ET)-2, a potent vasoactive molecule. ET-2 mRNA was transiently induced in mural granulosa cells of the preovulatory follicles immediately preceding ovulation. This induction was absent in the ovaries of PGR null mice, indicating a critical role of this receptor in ET-2 expression. To investigate the functional role of ET-2 during ovulation, we employed selective antagonists of endothelin receptors, ETR-A and ETR-B. Mice treated with an ETR-B antagonist exhibited a dramatic (>85%) decline in the number of released oocytes. Strong expression of ETR-B was observed in the mural and cumulus granulosa cells of the preovulatory follicles as well as in the capillaries lining the inner border of the theca interna. We also identified cGMP-dependent protein kinase II, a previously reported PGR-regulated gene, as a downstream target of ET-2 during ovulation. Collectively, our studies uncovered a unique pathway in which ET-2, produced by PGR in mural granulosa cells, acts in a paracrine or autocrine manner on multiple cell types within the preovulatory follicle to control the final events leading to its rupture.     

Asoprisnil (J867): a selective progesterone receptor modulator for gynecological therapy

Asoprisnil is a novel selective steroid receptor modulator that shows unique pharmacodynamic effects in animal models and humans. Asoprisnil, its major metabolite J912, and structurally related compounds represent a new class of progesterone receptor (PR) ligands that exhibit partial agonist and antagonist activities in vivo. Asoprisnil demonstrates a high degree of receptor and tissue selectivity, with high-binding affinity for PR, moderate affinity for glucocorticoid receptor (GR), low affinity for androgen receptor (AR), and no binding affinity for estrogen or mineralocorticoid receptors. In the rabbit endometrium, both asoprisnil and J912 induce partial agonist and antagonist effects. Asoprisnil induces mucification of the guinea pig vagina and has pronounced anti-uterotrophic effects in normal and ovariectomized guinea pigs. Unlike antiprogestins, asoprisnil shows only marginal labor-inducing activity during mid-pregnancy and is completely ineffective in inducing preterm parturition in the guinea pig. Asoprisnil exhibits only marginal antiglucocorticoid activity in transactivation in vitro assays and animal models. In male rats, asoprisnil showed weak androgenic and anti-androgenic properties. In toxicological studies in female cynomolgus monkeys, asoprisnil treatment abolished menstrual cyclicity and endometrial atrophy. Early clinical studies of asoprisnil in normal volunteers demonstrated a dose-dependent suppression of menstruation irrespective of the effects on ovulation, with no change in basal estrogen concentrations and no antiglucocorticoid effects. Unlike progestins, asoprisnil does not induce breakthrough bleeding. With favorable safety and tolerability profiles thus far, asoprisnil appears promising as a novel treatment of gynecological disorders, such as uterine fibroids and endometriosis.     

Valproate is an anti-androgen and anti-progestin

Anti-convulsant treatment is associated with a high prevalence of reproductive dysfunction compared with age-matched non-epileptics. We examined the widely used anti-convulsants valproate (VPA) and carbamazepine (CBZ) for steroidal bioactivity using a yeast-based steroid receptor-beta-galactosidase reporter assay for the androgen receptor (AR), progesterone receptor (PR) or estrogen receptor (ER). Bioassays were performed (a) to detect agonist activity by exposing yeast to 100 microM CBZ or VPA or (b) to detect antagonist activity by exposing yeast stimulated with testosterone (5 x 10(-9) M, AR), progesterone (1.6 x 10(-9) M, PR) or estradiol (2.6 x 10(-11) M, ER) together with either VPA or CBZ for 4 (PR) or 16 (AR, ER) hours. VPA showed dose-dependent (1-800 microM) inhibition of progesterone-induced PR- and testosterone-induced AR activity but had no ER antagonist bioactivity and no significant PR, AR or ER agonist bioactivity. VPA also showed a dose-dependent (1-200 microM) blockade of DHT's suppression of AR-mediated NF-kappaB activation in human mammalian cells. By contrast, CBZ had no significant PR, AR or ER agonist or AR and ER antagonist bioactivity but at the highest concentration tested (800 microM) it did antagonize PR activity. We conclude that VPA is a non-steroidal antagonist for human AR and PR but not ER. VPA's androgen and progesterone antagonism at concentrations within therapeutic blood levels (350-700 microM) seems likely to contribute to the frequency of reproductive endocrine disturbances among patients treated with VPA.     

Nonsteroidal progesterone receptor ligands (I): synthesis and SAR of new tetrahydronaphthofuranone derivatives

We have synthesized a series of nonsteroidal progesterone receptor (PR) ligands, tetrahydronaphthofuranones, structurally based on the fungal metabolite PF1092C. Structure-activity relationship studies revealed that substituents at the 6- and 7-positions were critical for PR binding affinity and for agonist or antagonist activity. Compounds in this series, exemplified by 19i, exhibited high affinity and high specificity for PR over other steroid hormone receptors and acted as selective PR antagonists. Further modification of PF1092C may generate compounds of potential pharmacological interest.     

Role of progesterone in the modulation of the preovulatory surge of gonadotropins and ovulation in the pregnant mare's serum gonadotropin-primed immature rat and the adult rat

The role of progesterone in the regulation of the preovulatory surge in gonadotropins and ovulation was examined in this study by use of a potent antagonist of progesterone, RU 486 (17 beta-hydroxy-11 beta-[4-dimethyl-aminophenyl]-17 alpha- [prop-1-ynyl]estra-4,9-diene-3-one). The immature rat primed with pregnant mare's serum gonadotropin (PMSG) and the cycling adult animal were the models used to verify the role of progesterone. When RU 486 (200 micrograms/rat) was given as a single dose on the morning of proestrus, there was a significant reduction in the preovulatory surge levels of gonadotropins and ovulation in both animal models. Serum progesterone levels in both models at the time of death on the evening of proestrus were unaltered upon treatment with RU 486. RU 486 did not have any effect on gonadotropin levels in immature rats 7 days after castration. These results show that the actin of RU 486 on the preovulatory gonadotropin surge is due to an antagonism of the action of progesterone on the hypothalamic-pituitary axis. Thus, a role for progesterone in modulating the preovulatory surge of gonadotropins and, consequently, ovulation is strongly suggested.     

In vitro and in vivo characterization of novel nonsteroidal progesterone receptor antagonists derived from the fungal metabolite PF1092C

We studied the pharmacological effects of novel nonsteroidal progesterone receptor antagonists CP8661 and CP8754, which were synthesized from the fungal metabolite PF1092C. CP8661 possess a tetrahydrobenzindolone skeleton and CP8754 possess a tetrahydronaphthofuranone skeleton. In binding assays for steroid receptors, CP8661 and CP8754 inhibited [(3)H]-progesterone binding to human progesterone receptors (hPR), though they are less potent than RU486. CP8661 also showed moderate affinity to rat androgen receptors (rAR), although CP8754 did not. Neither compound showed affinity to human glucocorticoid receptors (hGR) or human estrogen receptors (hER). In exogeneous and endogeneous PR-dependent enzyme expression assays using human mammary carcinoma T47D, CP8661 and CP8754 showed pure antagonistic activity. In a rabbit endometrial transformation test, CP8661 and CP8754 showed anti-progestational activity by s.c. administration in a dose-dependent manner; meanwhile, these compounds showed no progestational activity at the same dose. These results suggested that CP8661 and CP8754 are in vivo effective pure progesterone receptor antagonists and presented the possibility of synthesizing pure progesterone receptor antagonists from both tetrahydronaphthofuranone and tetrahydrobenzindolone skeletons.     

Estrogen downregulates the number of caveolae and the level of caveolin in uterine smooth muscle

Estrogen and progesterone, while regulating uterine functions, also regulate the number of caveolae and the level of caveolin. Large numbers of caveolae, as well as elevated expression of caveolin-1 and caveolin-2 isoforms in the myometrium of ovariectomised (OVX) rats were detected. 17beta-estradiol (E2) has a downregulating effect: the treatment of OVX rats with E2 (5 microg/animal) reduced the formation of caveolae by approx. 90%. Western blots clearly demonstrated the reduction of membrane caveolin-1 and -2 content. Progesterone treatment (2.5 mg/animal) alone did not cause any substantial change, but prevented the effect of estrogen. Control experiments showed that the quantity of Na+/K+-ATPase, a plasma membrane protein excluded from caveolae, was not downregulated by E2. The administration of the pure estrogen receptor (ERalpha) antagonist ICI 182,780 (1 mg/animal) not only compensated for the inhibitory effect of E2, but further increased the level of caveolin-1 in the myometrium of OVX rats and facilitated the formation of caveolae by approximately 70%. In contrast, the partial antagonist tamoxifen (1 mg/animal) mimicked the effect of estrogen. The amount of caveolin also changed during pregnancy. During the first half of pregnancy the expression of caveolin was suppressed, but it gradually increased until delivery. Our results indicate that the formation and number of caveolae are influenced by the physiological state of the uterus in a hormone dependent manner.