Progesterone antagonists and progesterone receptor modulators in the treatment of breast cancer

Progesterone antagonists (PAs) (antiprogestins) or progesterone receptor modulators (PRMs) form an interesting category of new hormonal agents in the treatment of breast cancer. In vitro, antiproliferative effects of different PAs are mainly observed in estrogen-stimulated growth of PR-positive tumor cell lines. Both progestin agonist/antagonist actions on mammary tumor cells are dependent on the type of cell line, culture medium and concentrations of the PAs used, and type of biologic response measured. In various experimental animal tumor models, different PAs showed a greater antitumor activity than tamoxifen or high-dose progestins. Most interestingly, combination treatment of different PAs (mifepristone, ORG 31710, onapristone) or PRMs with different antiestrogens (tamoxifen, droloxifen, ICI 164384) or with an aromatase inhibitor (atemestane) showed greater antitumor efficacy than treatment with each single type of drug alone. These additive antiproliferative effects were demonstrated in various experimental in vitro and in vivo models. In some studies, these effects were accompanied by additive effects on several cell biologic parameters. In pretreated postmenopausal patients with metastatic breast cancer, objective responses have been observed in 10-12%, and stable disease in 42-46% of the patients; in previously untreated patients objective response rates of 11 and 56% have been reported. The clinical development of onapristone was stopped because of liver toxicity. At the present time, apart from development of new pure potent PAs, clinical investigation of combined therapy of PAs with antiestrogens are urgently needed.     

Regulation of cell growth of a progestin-dependent murine mammary carcinoma in vitro: progesterone receptor involvement in serum or growth factor-induced cell proliferation

Primary cultures of the medroxyprogesterone acetate-induced mouse mammary tumor line C4-HD are stimulated by medroxyprogesterone acetate (MPA) or progesterone. Serum obtained from ovariectomized, MPA-treated animals (OVX-MPA) exerts a stimulatory effect that is significantly higher than that induced by serum obtained from OVX mice with the exogenous addition of MPA, suggesting the involvement of MPA-induced serum factors potentiating the proliferative effect of MPA. The object of this paper is to further explore the stimulatory effect of mouse serum and to investigate the role of aFGF and bFGF on cell proliferation. The role of PR as possible mediators was tested using two different antiprogestins and antisense oligodeoxynucleotides of PR A isoform. Serum was obtained from OVX untreated or MPA-treated mice and was charcoalized and/or heat-inactivated. The effect of MPA or mifepristone at 10 nM concentrations was tested. Charcoalization and heat inactivation exerted a stimulatory effect (P<0.01) when OVX-serum was used. This effect was potentiated by MPA. Charcoalized OVX-MPA serum induced a significant inhibition of cell proliferation that was restored by the exogenous addition of MPA or by heat inactivation. Mifepristone induced an inhibition of ³H-thymidine uptake when OVX-MPA serum was used. These results suggest that serum factors activated by different manipulations may replace the stimulatory effect of MPA. When charcoalized fetal calf serum (chFCS) was used, a higher proliferative activity was obtained using higher serum concentrations. Mifepristone and onapristone 10 nM also inhibited this effect. aFGF and bFGF 100 ng/ml were both able to stimulate ³H-thymidine uptake. MPA exerted an additive effect. Mifepristone 10 nM inhibited bFGF and MPA+bFGF induced cell proliferation. Antisense oligodeoxynucleotides of PR (ASPR) were used to further confirm the participation of PR in the proliferative pathway of these cells. They inhibited serum and bFGF-induced cell proliferation in a specific dose-dependent manner. Our results suggest that PR play a central role in proliferation and suggest the existence of a cross-talk between steroid and growth factor signaling pathways.     

Synergistic effects of antiprogestins and iNOS or aromatase inhibitors on establishment and maintenance of pregnancy

Progesterone is known to be involved in many steps in female reproduction including control of implantation and uterine-cervical function during pregnancy. Our studies in rats and guinea pigs indicate that progesterone inhibits uterine contractility and cervical softening during pregnancy. Progesterone levels or actions decline near the end of pregnancy leading to the onset of labor. Treatment with progestin agonists prolongs pregnancy and inhibits cervical softening, whereas treatment with antiprogestins (mifepristone or onapristone) stimulates uterine contractility, cervical softening and premature delivery. Thus the effect of progesterone receptor modulators in the uterus and cervix depend up on the degree of intrinsic agonistic/antagonistic activities. Our recent studies show that progesterone interacts with nitric oxide (NO) to maintain pregnancy and that administration of progesterone antagonists with NO synthase inhibitors act synergistically to stimulate labor. In addition our studies show that combinations of progesterone antagonists with aromatase inhibitors act synergistically to induce labor. Similarly antiprogestins interact with NO synthase or aromatase inhibitors to block implantation through action on the endometrium. These studies suggest new applications for combined therapies of progestin receptor modulators with aromatase inhibitors or agents that modify NO production for contraception, stimulation of labor, estrogen-dependent diseases and improved outcomes in pregnancy.     

Progesterone antagonists: Tumor-inhibiting potential and mechanism of action

A new approach for the treatment of breast cancer could be the use of progesterone antagonists. These compounds were originally developed for the inhibition of progesterone-dependent processes and have been shown to be effective in inhibition of nidation and interruption of pregnancy. Although the roles of progesterone and the progesterone receptor in control of cell growth remain unclear, it was found in progesterone receptor positive mammary carcinoma cell lines that the antiprogestin, Mifepristone, had an inhibitory effect on cell growth-and a growth-inhibiting action on the DMBA-induced mammary carcinoma of the rat. We have shown that the progesterone antagonists, Onapristone and ZK 112993, which possess a reduced antiglucocorticoid activity compared to Mifepristone, exert a strong tumor-inhibiting effect in a panel of hormone-dependent mammary tumor models. The effects of these compounds were in some systems superior to those of tamoxifen or high dose progestins and comparable to ovariectomy. Although prerequisites for their antiproliferative potency are an affinity to the progesterone receptor as well as a sufficient number of available receptors in the tumors, the strong tumor inhibiting potential of the antiprogestins cannot be explained by a classical antihormonal mechanism. Surprisingly, the antitumor activity is evident in spite of elevated serum levels of ovarian and pituitary hormones. It was established by morphometric procedures that treatment with Onapristone triggers differentiation of the mitotically active polygonal tumor epithelial cell towards secretory active glandular structures and acini. All our quantitative light and electron microscopic data indicate that the antitumor action of antiprogestins is accompanied by the initiation of terminal differentiation leading to (apoptotic) cell death. Finally, our flow cytometry studies revealed an accumulation of the tumor cells in the G₀G₁ phase of the cell cycle, which may result from induction of differentiation since a differentiation-specific G₁ arrest has already been proposed for other stem cell systems. It can be concluded from these data that the progesterone receptor antagonists differ in their mode of action from compounds used in established endocrine treatment strategies for mammary carcinoma. The ability of progesterone antagonists like Onapristone to reduce the number of cells in S-phase may offer a significant clinical advantage, since it is established that the S-phase fraction is a highly significant predictor of disease-free survival among axillary node-negative patients with diploid mammary tumors.     

Loss of Progesterone Receptor-Mediated Actions Induce Preterm Cellular and Structural Remodeling of the Cervix and Premature Birth

A decline in serum progesterone or antagonism of progesterone receptor function results in preterm labor and birth. Whether characteristics of premature remodeling of the cervix after antiprogestins or ovariectomy are similar to that at term was the focus of the present study. Groups of pregnant rats were treated with vehicle, a progesterone receptor antagonist (onapristone or mifepristone), or ovariectomized on day 17 postbreeding. As expected, controls given vehicle delivered at term while rats delivered preterm after progesterone receptor antagonist treatment or ovariectomy. Similar to the cervix before term, the preterm cervix of progesterone receptor antagonist-treated rats was characterized by reduced cell nuclei density, decreased collagen content and structure, as well as a greater presence of macrophages per unit area. Thus, loss of nuclear progesterone receptor-mediated actions promoted structural remodeling of the cervix, increased census of resident macrophages, and preterm birth much like that found in the cervix at term. In contrast to the progesterone receptor antagonist-induced advance in characteristics associated with remodeling, ovariectomy-induced loss of systemic progesterone did not affect hypertrophy, extracellular collagen, or macrophage numbers in the cervix. Thus, the structure and macrophage census in the cervix appear sufficient for premature ripening and birth to occur well before term. With progesterone receptors predominantly localized on cells other than macrophages, the findings suggest that interactions between cells may facilitate the loss of progesterone receptor-mediated actions as part of a final common mechanism that remodels the cervix in certain etiologies of preterm and with parturition at term.     

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.     

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.     

Failure of two progesterone antagonists, mifepristone and onapristone, to affect luteal activity in lactating rats

The progesterone antagonists, mifepristone (RU-38,486) and onapristone (ZK-98,299), given as 2 mg daily, did not markedly affect lactation in rats. Both litter growth and time spent by 10-pup litters attached to their mothers were similar in antagonist-treated mothers and in solvent-treated controls. The progesterone antagonists did not affect the steroid content in corpora lutea remaining from the preceding pregnancy. Corpora lutea formed after post-partum ovulation also showed nearly normal function throughout the first 17 days of lactation. It is concluded that progesterone itself plays no role in the initiation or maintenance of luteal function when prolactin secretion is governed through an action independent of the ovaries, as through suckling. Antagonist-treated rats ovulated around Day 13 of lactation despite suckling. This ovulation was not associated with a decrease of progesterone production by the corpora lutea formed after post-partum ovulation. Apparently, elimination of progesterone action may protect corpora lutea from luteolysis. The latter finding indicates a possible role of progesterone in luteolysis and deserves further analysis.     

Progestins and breast cancer

In the last years there has been an extraordinary development in the synthesis of new progestins. These compounds are classified, in agreement with their structure, in various groups which include progesterone, retroprogesterones, 17-hydroxyprogesterones, 19-norprogesterones, 17-hydroxyprogesterone derivatives, androstane and estrane derivatives. The action of progestins is a function of many factors: its structure, affinity to the progesterone receptor or to other steroid receptors, the target tissue considered, the biological response, the experimental conditions, dose, and metabolic transformation. The information on the action of progestins in breast cancer patients is very limited. Positive response with the progestins: medroxyprogesterone acetate and megestrol acetate was obtained in post-menopausal patients with advanced breast cancer. However, extensive information on the effect of progestins was obtained in in vitro studies using hormone-dependent and hormone-independent human mammary cancer cell lines. It was demonstrated that in the hormone-dependent breast cancer cells, various progestins (nomegestrol acetate, tibolone, medrogestone, promegestone) are potent sulfatase inhibitory agents. The progestins can also involve the inhibition of mRNA of this enzyme. In another series of studies it was also demonstrated that various progestins are very active in inhibiting the 17β-hydroxysteroid dehydrogenase for the conversion of estrone to estradiol. More recently it was observed that the progestins promegestone or medrogestone stimulate the sulfotransferase for the formation of estrogen sulfates. Consequently, the blockage in the formation of estradiol via sulfatase, or the stimulatory effect on sulfotransferase activity, by progestins can open interesting and new possibilities in clinical applications in breast cancer.     

Influence of progestins on serum hormone levels in postmenopausal women with advanced breast cancer--II. A differential effect of megestrol acetate and medroxyprogesterone acetate on serum estrone sulfate and sex hormone binding globulin

Serum estradiol, estrone, estrone sulfate and sex hormone binding globulin were measured in 10 postmenopausal patients with advanced breast cancer receiving sequential treatment with medroxyprogesterone acetate and megestrol acetate. Treatment with megestrol acetate caused a non-significant reduction in serum estradiol (mean reduction of 19%, 0.05 less than P less than 0.1) but significant reductions in serum estrone (mean reduction of 20%, P less than 0.02) and serum estrone sulfate (mean reduction of 54%, P less than 0.005) compared to treatment with medroxyprogesterone acetate. In contrast, treatment with medroxyprogesterone acetate reduced serum sex hormone binding globulin more compared to treatment with megestrol acetate (mean reduction of 69%, P less than 0.01). These findings suggest that the two progestins have differential effects on serum hormone levels. The finding that treatment with megestrol acetate causes a significant reduction in serum estrone sulfate level warrants further investigations of this potentially important mechanism of action of this drug in advanced breast cancer.     

Effect of steroids and antisteroids on human meningioma cells in primary culture

Human meningiomas are rich in progestin receptors but virtually devoid of oestrogen receptors. We have studied the hormonal sensitivity of meningioma cells in vitro during 8 days of primary culture in the presence of different steroids and antisteroids. On day 8 the thymidine labelling index (TLI) was determined as a measure of cell growth. To date 30 cultures have been established from 39 tissue specimens. 13 cultures had a TLI below 1.0 and their growth were not affected by hormones. The TLI of the other 17 cultures was 3.0 +/- 1.7 (mean +/- SD; range 1.2-7.7). Following culture in the presence of 1 and 10 nM progesterone TLI was 83 +/- 28% (n = 9) and 61 +/- 29% (n = 3) of that of the control cultures respectively. Although in individual cultures occasional differences were found, the overall values are not statistically different from 100. Similarly, 1 nM of oestradiol and testosterone had no effect on the TLI (n = 3). Tamoxifen at 1 nM increased the TLI to 138% in one culture and decreased it to 66% of the control in another. The antiprogestin mifepristone (RU 486) in concentrations of 0.1, 1.0, 10, 100 and 1000 nM decreased the TLI to 72 +/- 30; 54 +/- 20; 55 +/- 20; 59 +/- 18 and 65 +/- 10 respectively (n = 6-15; P less than 0.05 vs control). It is concluded that although a growth promoting effect of progestins on meningioma could not be shown, the therapeutic possibilities of antiprogestins warrant further investigation.     

Characterization of ligand binding,DNA binding and phosphorylation of progesterone receptor by two novel progesterone receptor antagonist ligands

In order to gain a better understanding of the distinctive mechanisms of the various types of antiprogestins, we have characterized in vitro ligand binding, specific DNA binding and phosphorylation of progesterone receptor (PR) from T47D cells after treatment of cells with progestins (progesterone, R5020) and antiprogestins (RU486, ZK98299, Org 31806 and Org 31710). Treatment of the cells with R5020 or PR antagonists, with the exception of ZK98299, resulted in a quantitative upshift of PR-A and PR-B indicative of ligand/DNA-induced phosphorylation of PR. Treatment of cells with RU486, Org 31710 or Org 31806, but not R5020 or ZK98299 resulted in detectable PR-progesterone response element complexes (PR-PREc) as assessed by gel mobility shift assay. Although treatment of cells with ZK98299, a type I PR antagonist, did not induce phosphorylation, the antiprogestins, Org 31806 and Org 31710, in a manner identical to RU486, did. Our data suggest that Org 31806 and Org 31710 affect propertie s of PR from T47D cells that are similar to RU486. (Mol Cell Biochem 175: 205–212, 1997)     

Molecular properties and preclinical pharmacology of JNJ-1250132, a steroidal progesterone receptor modulator that inhibits binding of the receptor to DNA in vitro

Progesterone receptor modulators have diverse potential therapeutic uses, including the treatment of endometriosis, uterine fibroids and breast cancer. Here we describe the molecular properties and preclinical pharmacology of a new steroidal progestin antagonist, JNJ-1250132. The compound is a high affinity ligand for the progesterone receptor, possessing cross-reactivity with other steroid receptors comparable to that of steroidal antagonists such as mifepristone. It inhibits progestin-inducible alkaline phosphatase gene expression in T47D human breast cancer cells, and also inhibits their in vitro proliferation. It inhibits gestation in rats and progesterone-dependent endometrial transformation in rabbits with efficacies comparable to mifepristone. Like mifepristone, it is a glucocorticoid antagonist in vivo. In cell-free DNA binding assays, the compound inhibits binding of the human progesterone receptor to a progesterone response element, and thus is similar to onapristone in this regard. In contrast, as judged by proteolytic analysis, JNJ-1250132 induces a receptor conformation more similar to that induced by mifepristone, which promotes receptor binding to DNA. Therefore, JNJ-1250132 has unique effects on the progesterone receptor that may translate into a novel clinical profile.     

Interaction of progestins with steroid receptors in human uterus

Norethindrone (17β-hydroxy-19-nor-17α-pregn-4-en-20-yn-3-one) and norethindrone acetate (17β-acetoxy-19-nor-17α-pregn-4-en-20-yn-3-one) interfered to a varying degree, by competitive inhibition, with the binding of progesterone and oestradiol to respective cytoplasmic receptors in the human uterus. Progesterone binding to 4S macromolecule was saturable and co-specific for progestins. Competitors like norgestrel (17β-hydroxy-18-methyl-19-nor-17α-pregn-4-en-20-yn-3-one), 19-norprogesterone, medroxyprogesterone acetate (17α-acetoxy-6α-methylpregn-4-ene-3,20-dione) and compound R₅₀₂₀ (17,21-dimethyl-19-norpregna-4,9-diene-3,20-dione) possessed higher binding affinities for the progestin receptor. The dissociation constant (K_d) for the progesterone–receptor interaction was 0.6–1.6nm and the receptor concentration ranged between 6600 and 8200 sites/cell. Norethindrone and norethindrone acetate competed for the progesterone receptor with inhibition constants (K_i) of 6.8 and 72nm respectively. Gradient displacement and competitive-receptor assays indicated that norethindrone acetate-binding affinity for progestin receptor was approximately one-tenth that of norethindrone and progesterone. The progestins also inhibited oestradiol binding to 4.6S oestrogenic receptor by 8–12%, involving interaction at the oestradiol-binding site with a calculated K_i value of 0.5–0.8μm. The competitive interaction of progestins with steroid receptors may be of putative importance in explaining the progestin action at the target site.     

Characterization of a putative membrane receptor for progesterone in rat granulosa cells.

Progesterone (P(4)) inhibits granulosa cell apoptosis in a steroid-specific, dose-dependent manner, but these cells do not express the classic nuclear P(4) receptor. It has been proposed that P(4) mediates its action through a 60-kDa protein that functions as a membrane receptor. The present studies were designed to determine the P(4) binding characteristics of this protein. Western blot analysis using an antibody that recognizes the P(4) binding site of the nuclear P(4) receptor (C-262) confirmed that the 60-kDa protein was localized to the plasma membrane of both granulosa cells and spontaneously immortalized granulosa cells (SIGCs). To determine whether this protein binds P(4), proteins were immunoprecipitated with the C-262 antibody, electrophoresed, transferred to nitrocellulose, and probed with a horseradish peroxidase-labeled P(4) in the presence or absence of nonlabeled P(4). This study demonstrated that the 60-kDa protein specifically binds P(4). Scatchard plot analysis revealed that (3)H-P(4) binds to a single site (i.e., single protein), which is relatively abundant (200 pmol/mg) with a K(d) of 360 nM. (3)H-P(4) binding was not reduced by dexamethasone, mifepristone (RU 486), or onapristone (ZK98299). Further studies with SIGCs showed that P(4) inhibited apoptosis and mitogen-activated protein kinase kinase (MEK) activity, and maintained calcium homeostasis. These studies taken together support the concept that the 60-kDa P(4) binding protein functions as a low-affinity, high-capacity membrane receptor for P(4).     

Novel antiprogestins Org 31806 and 31710: interaction with mammalian progesterone receptor and DNA binding of antisteroid receptor complexes.

We have examined steroid binding parameters and transformation of calf uterine progesterone receptor (PR) liganded with progestins (progesterone and R5020) and the newly synthesized antiprogestins (Org 31806 and 31710). Species specificity analysis indicated that [3H]R5020 binding in the chicken oviduct cytosol could be eliminated in the presence of 100-fold excess radioinert progesterone and R5020 but not Org 31806 and 31710. In the calf uterine cytosol, the progestins and the antiprogestins appeared to interact with the same PR as revealed by the displacement of [3H]R5020 by all of the above steroids. When the extent of [3H]R5020 binding was examined in the presence of different concentrations of radioinert steroids, the relative affinity with which these compounds interacted with the uterine PR was found to be comparable. A 23 degrees C incubation of cytosol transformed the progestin-bound PR complexes increasing their binding to DNA-cellulose from 5 (0 degrees C, nontransformed) to 35%. Under these conditions, 20% Org 31710- and RU486-occupied PR complexes bound to DNA-cellulose whereas only 10% Org 31806-receptor complexes were retained by the resin. Transformation (23 degrees C) of cytosol receptor caused a loss of the larger 8 S form and an increase in the smaller 4 S form. In its unliganded state or when it was complexed with R5020 or the antiprogestins, incubation of PR at 23 degrees C led to dissociation of the receptor-associated 90 kDa heat-shock protein (hsp90). The PR-hsp90 association was stabilized in the presence of 10 mM iodoacetamide when the ligand binding site was occupied by Org 31806 and 31710. The R5020-receptor complexes, however, allowed release of hsp90 under the above transforming conditions. Our results indicate that although Org 31806 and 31710 show no affinity for the avian PR, these steroids interact with the mammalian PR. We propose that the reported antiprogestational effects of Org 31806 and 31710 are mediated via their interaction with PR which appears similar to one that exists between PR and RU486.     

Cell surface-binding sites for progesterone mediate calcium uptake in human sperm.

Recent studies (e.g. Blackmore, P. F., Beebe, S. J., Danforth, D. R., and Alexander, N.) (1990) J. Biol. Chem. 265, 1376-1380) have shown that in human sperm, progesterone produces a rapid increase in intracellular free calcium ([Ca2+]i) and an induction of the acrosome reaction (e.g. Osman, R. A., Andria, M. L., Jones, A. D., and Meizel, S. (1989) Biochem, Biophys. Res. Commun. 160, 828-833). In this study, the location of progesterone receptors on the cell surface of human sperm was identified using progesterone immobilized on bovine serum albumin (BSA) (progesterone 3-(O-carboxymethyl)oxime:BSA) as well as progesterone and its 3-O-carboxymethyloxime derivative. Using fluorescence microscopy, BSA-fluorescein isothiocyanate was shown to be excluded from intact sperm, thus validating the use of progesterone 3-(O-carboxymethyl)oxime:BSA to identify cell surface-binding sites for progesterone. The immobilized progesterone and the 3-O-carboxymethyloxime derivative rapidly increased [Ca2+]i and were full agonists, although they were approximately 1.5 orders of magnitude less potent than progesterone. They also displayed an identical time course to increase [Ca2+]i as free progesterone, and the entire increase in [Ca2+]i was due to the influx of Ca2+. This progesterone-mediated response displayed different steroid receptor characteristics since the very potent inhibitors of genomic progesterone responses, RU38486 and ZK98.299, were very ineffective at inhibiting the progesterone-mediated increase in [Ca2+]i. Also the synthetic progestins megestrol, medroxyprogesterone acetate, norgestrel, norethynodrel, norethindrone, R5020, and cyproterone acetate did not mimic the effects of progesterone to increase [Ca2+]i. It is proposed that a distinct nongenomic cell surface receptor for progesterone exists in human sperm.