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PubMed1987

Rat uterine progesterone receptor analyzed by [3H]R5020 photoaffinity labeling: evidence that the A and B subunits are not equimolar.

The hormone-binding components of the rat uterine progesterone receptor were investigated by the methods of [3H]R5020 photoaffinity labeling and sodium dodecyl sulfatepolyacrylamide gel electrophoresis analysis. Two specifically labeled peaks were observed at mol wt of 85,600 +/- 1,200 and 109,600 +/- 1,200 (n = 31), resembling the A and B progesterone receptor components previously described in other systems. However, in contrast to the equimolar ratio reported in other systems, the level of subunit A observed was consistently greater than that of B (A/B ratio = 3.2 +/- 0.3; n = 31). The unusual A/B ratio prompted a complete validation of the photolabeling procedure in this system. Although the levels of specific binding increased, there was no change in the A/B ratio with varying [3H]R5020 concentrations (5-80 nM) or with time of UV exposure (0.5 min to 3 h). Although adsorption to hydroxylapatite indicated that specific [3H]R5020 binding was reduced by 72.0 +/- 6.4% within 5 min of UV exposure, relabeling the irradiated preparations with [3H]R5020 resulted in little change in specific [3H]R5020 binding. TLC analysis of [3H]R5020 (Rf = 0.48 +/- 0.01; n = 4) after irradiation demonstrated rapid photolysis resulting in a 94.3 +/- 2.5% (n = 3) loss of authentic [3H]R5020 within 5 min. After photolysis, at least two new tritiated products were recovered with Rf values of 0.20 +/- 0.03 and 0.72 +/- 0.02. Analysis by adsorption to hydroxylapatite indicated that the photolysis products competed for specific [3H]R5020-binding sites in cytosol with only 10-fold lower relative binding activity than authentic R5020. Thus, these compounds probably account for the increase in specific photolabeling of the A and B peaks achieved when UV exposure is prolonged from 5 to 30 min. Further study indicated that the A/B subunit ratio in this system was not changed under a variety of in vitro conditions, including the absence or presence of molybdate, sulfhydryl protective reagents (dithiothreitol and thioglycerol), protease inhibitors (phenylmethylsulfonylfluoride and leupeptin), glycerol (0%, 10%, and 30%, vol/vol), or 1.5 mM EGTA, or after precipitation with 40% ammonium sulfate. This consistency of the A/B ratio under a wide variety of adverse in vitro conditions suggests that in vitro artefacts may not account for the ratio's deviation from unity. Estrogen withdrawal (48 h) enhanced by progesterone treatment (0.5 mg for 24 h) resulted in only a modest reduction in the A/B ratio to 1.9 +/- 0.1.(ABSTRACT TRUNCATED AT 400 WORDS)

Adsorption↗
PubMed1987

A nuclear binding assay to assess the biological activity of steroid receptors in isolated animal and human tissues.

This paper describes a nuclear binding assay (NB assay) which measures not only the presence of a steroid receptor in a tissue, but also the quantity of that receptor which is biologically active or functional, i.e. able to bind to nuclear acceptor sites. The assay involves the isolation viable cells from tissues and their incubation with an excess of radiolabeled steroid to encourage the activation and nuclear binding of all cellular receptors. The nuclei are isolated under conditions that remove unactivated (unbound) steroid-receptor complexes. This NB assay demonstrates, in both animal and human steroid target tissues, a saturable, tissue- and steroid-specific, and temperature- and time-dependent nuclear binding of radiolabeled steroids. These properties support a receptor-dependent nuclear binding of steroids. This assay is reproducible and requires relatively small amounts of tissue. The patterns of nuclear binding of the progesterone receptor, achieved with the assay in the avian oviduct model system, are shown to correlate with the nuclear binding of progesterone in vivo, the ability of the steroid to alter transcription, and the expression of a specific gene product, the protein avidin. The assay has been used to identify the existence of nonfunctional steroid receptors in endometrial and breast carcinomas. Therefore, this NB assay combined with the standard charcoal/hydroxylapatite methods of quantitating total cellular receptors should provide a means of assessing changes in the regulation of the biological activity of steroid receptors. Further, the assay should be useful to assess the ability of steroid analogs to properly activate their respective receptors for subsequent nuclear binding.

Animals↗
PubMed1988

Progesterone receptor regulation in T47D human breast cancer cells: analysis by density labeling of progesterone receptor synthesis and degradation and their modulation by progestin.

We have examined the effect of progestin on the regulation of cellular progesterone receptor (PR) levels and have used dense amino acid-density shift experiments to determine the mechanism by which progestin markedly decreases PR. We have utilized T47D human breast cancer cells which contain high levels of PR and are progestin responsive. When these cells are exposed to the progestin R5020, there is a time- and concentration-dependent decrease in PR levels. Experiments with different concentrations of R5020 reveal that the rate and extent of PR decrease reflect the time course of receptor occupancy and the fractional saturation of receptor. With a high concentration of ligand (20 nM) that labels all receptors rapidly, reductions in PR levels (processing) occur immediately and proceed rapidly to levels that are 15-20% of the initial; at lower concentrations (5 nM), where it takes several hours to achieve full saturation of receptors, there is a delay before the maximal rate of processing develops and then continues to achieve final receptor levels that are 15-20% of the initial; with a low concentration of ligand (0.5 nM), binding is even slower and never reaches full receptor saturation, with the consequence that processing is not only delayed but also less complete. Immunochemical detection of PR with a monoclonal antibody (B39) reveals a good correspondence between the loss of immunoreactive and hormone binding PR, and analysis of the A (Mr 85,000) and B (Mr 115,000) receptor forms on Western blots demonstrates that both A and B receptor forms are reduced after exposure to R5020. Density labeling of PR by biosynthetic incorporation of 2H, 13C, 15N (dense) amino acids reveals that PR turns over with a half-life of 21 h in control cells. In cells exposed to 20 nM R5020, PR levels decline and receptor half-life is reduced to 6 h. In addition, there is also a time-dependent decrease in the rate constant of PR synthesis, k8, which decreases to less than 10% of its initial value after 24 h of R5020 exposure. Thus, the R5020-evoked reduction in PR levels in this progestin-sensitive cell line is due both to a marked increase in the rate of receptor degradation as well as a dramatic decrease in the rate of receptor synthesis.

Antibodies, Monoclonal↗
PubMed1988

The ontogeny of sex differences in estrogen-induced progesterone receptors in rat brain.

The sex and age-related changes in the concentration of cytosol progesterone receptors (CPR) induced by estrogen (E) treatment in rat brain and pituitary were investigated by a modification of the Palkovits punch procedure using fresh tissue. Young male and female gonadectomized rats, 15, 21, 30, and 42 days of age, were treated for 44 h by a single sc injection of synthetic E. [Moxestrol (Ru2858)]. Adult gonadectomized animals were treated for 72 h by three injections of estradiol benzoate. Cytosol from pituitary and from punches of ventromedial nuclei (VMN), medial preoptic area (mPOA), arcuate nucleus (ARC), and cerebral cortex was labeled with 0.4 nM [3H]promegestrone (Ru5020) to maximize signal to noise and specificity of labeling of CPR. The developmental patterns of CPR differed across brain regions and between males and females. In VMN, females showed higher CPR levels after estrogen priming at 15, 21, and 42 days of age and in the adult; in ARC, females showed higher CPR levels after E priming at 15, 21, and 30 days of age, but not at 42 days or in the adult. In mPOA and pituitary, no consistent sex differences in CPR induction were found. Cortex showed no induction of CPR by E priming. Radioautography of [3H]Ru5020 uptake in VMN of E-primed 15-day-old male and female rats revealed significantly higher labeling in females, thus showing that the CPR levels in vitro reflect a difference in hormone retention in vivo. Female VMN contained more neurons with a higher labeling index than male VMN. Before puberty (approximately = 30 days), there was a decline in CPR levels induced by E priming in both sexes in pituitary, ARC, mPOA, and VMN. At 30 days, the female greater than male sex difference disappeared and tended to reverse in mPOA and VMN, only to be restored again by 42 days of age. Thus, the CPR induction by E priming may reflect underlying changes in E-sensitive brain regions associated with the preparation for puberty, as well as underlying sex differences in response to estrogen programmed by perinatal exposure to testosterone.

Aging↗
PubMed1988

Hepatic progesterone receptors: characterization in the turtle Chrysemys picta.

High and low affinity progesterone (P)-binding proteins similar to those described in the chick oviduct were characterized for the first time in liver cytosol in an oviparous turtle, Chrysemys picta. Two forms of high affinity P receptor were separated by diethyl aminoethyl (DEAE)-Sepharose anion exchange chromatography; the A form eluted in low salt buffer, and the B form at a KCl concentration of 0.20 M in a linear gradient. After photoaffinity labeling of crude cytosol with [3H] R5020, two overlapping peaks of radioactivity were detected with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After separation with DEAE-Sepharose and photoaffinity labeling, the A form migrated as a single peak with a mol wt of 90,000, and the B form as a single peak with a mol wt of 123,000. The affinity (Kd, 2.9 X 10(-9) M), capacity (1 pmol/mg protein), and binding specificity were characteristic of a P receptor. P competed most effectively for [3H]P binding. R5020 and deoxycorticosterone were good competitors; 5 alpha-pregnenolone, 17 alpha-hydroxyprogesterone, and testosterone were quite effective; cortisol, corticosterone, aldosterone, and 5 alpha-dihydrotestosterone were poor competitors. A lower affinity P-binding component (Kd, 1 X 10(-7) M; maximum binding, 15 pmol/mg protein) co-eluted from DEAE-Sepharose columns with the A and B receptor forms. This is the first description of a hepatic P receptor in any vertebrate.

Affinity Labels↗
PubMed1988

The distribution of progesterone receptor in the 20-day-old fetal mouse: an autoradiographic study with [125I]progestin.

The distribution of progestin target sites in 20-day-old fetuses of estrogen-primed pregnant mice was investigated by thaw-mount autoradiography. Pregnant mice received a Silastic estradiol implant on day 17 and were ovariectomized on day 19 of pregnancy. Twenty-four hours after ovariectomy 10 prematurely delivered fetuses were each injected with 0.33 microgram/100 g BW [125I]progestin (SA, 2200 Ci/mM). To show specificity of progestin localization two additional fetuses were each injected sc with 20 micrograms R5020, a synthetic progestin, 1 h before the injection of [125I]progestin. The fetuses were frozen 2 h after injection of [125I]progestin, sectioned, and processed for thaw-mount autoradiography. Cells with nuclear uptake and retention of radioactivity were observed in numerous tissues, including certain regions of the oral mucosa and developing teeth, esophagus, larynx, skin, mammary gland, skeletal muscle, kidney, and reproductive glands and ducts. Injection of unlabeled R5020 1 h before [125I]progestin prevented nuclear concentration of radioactivity in all target tissues. The results indicate that progesterone receptors are expressed with a regional, cellular, and subcellular distribution in term fetal mouse tissues and suggest that progesterone is important to the growth and development of certain fetal tissues.

Affinity Labels↗
PubMed1989

Anatomical localization of the effects of 17 beta-estradiol on oxytocin receptor binding in the ventromedial hypothalamic nucleus.

Oxytocin (OT) neurotransmission plays a role in the facilitation of steroid-dependent sexual receptivity in the rat. One way in which the ovarian steroid 17 beta-estradiol (E2) has been shown to modulate OT transmission is by increasing OT receptor binding in certain brain areas involved in the regulation of female sexual behavior such as the ventromedial hypothalamic nucleus (VMN). This study was undertaken to describe the distribution of OT receptors within the VMN that are regulated by physiological levels of E2. With quantitative autoradiographic methods, we measured [3H]OT binding in ovariectomized female rats implanted with Silastic capsules containing cholesterol, 5% E2, or 100% E2. In addition, plasma E2 levels, pituitary progestin receptor binding, and uterine weights were measured in animals from each treatment group. Results of this study showed that physiological levels of E2 increased [3H]OT binding in caudal regions of the ventrolateral VMN and stimulated maximal uterine growth and pituitary progestin receptor binding. However, in more rostral VMN sections, E2 induced a dose-dependent increase in [3H]OT binding. These data suggest that ovarian steroids sensitize the brain to OT by increasing OT receptor binding in certain brain areas involved in the regulation of sexual receptivity.

Animals↗
PubMed1989

Autologous down-regulation of messenger ribonucleic acid and protein levels for estrogen receptors in MCF-7 cells: an inverse correlation to progesterone receptor levels.

In the present study we have examined the effects of estradiol on mRNA levels for estrogen (ER) and progesterone receptors (PR) in the estrogen-dependent mammary carcinoma MCF-7 cell line. The changes in ER immunoactivity and specific binding of [3H]R5020 were also assessed. Estradiol (10(-7) M) caused a transient and time-dependent reduction of the level of mRNA for ER, with a maximal effect (30-40% of control; n = 3) after 72 h. This was associated with a similar decrease in ER immunoactivity. Further treatment (96 and 120 h) revealed a return of ER mRNA to control values, whereas the ER immunoactivity remained depressed. The effect on the mRNA level for PR gave almost the inverse curve. Initially (24-72 h), we observed a pronounced increase in this mRNA, with a maximal effect (6-7 times the control value; n = 3) after 72 h. Treatment beyond 72 h was associated with a gradual return of mRNA for PR toward the control level. The variation in specific binding of [3H]R5020 revealed similar changes, except that changes in specific receptor binding were delayed 24 h compared to the levels of mRNA. Incubation with low concentrations (10(-11) and 10(-10) M) of estradiol for 72 h was associated with slightly elevated levels of mRNA for ER, whereas higher concentrations gave a dose-dependent decrease. The mRNA for PR was biphasically stimulated, with a maximal effect at 10(-10)-10(-8) M, where a 10- to 13-fold stimulation was observed. The highest concentration (10(-7) M) gave a lower response. Assessment of concentration-induced variations in protein receptor levels of ER and PR reflected the effects of estradiol on their mRNAs. Low concentrations of estradiol slightly enhanced the ER level, whereas high concentrations clearly reduced ER immunoactivity. The PR level was stimulated by all concentrations used, and 10(-8) M estradiol raised the PR level more than 11-fold. Our results indicate autologous regulation of estrogen receptor gene transcripts and proteins and a clear induction of PR mRNA and receptor proteins by estradiol.

Adenocarcinoma↗
PubMed1990

Regulation and localization of estrogen and progestin receptors in the pituitary of steroid-treated monkeys.

PRL increases during pregnancy in primates with rising levels of placental estradiol (E) and progesterone (P). However, while E will increase PRL secretion in monkey pituitary cell cultures, P has no effect. We recently localized progestin receptors (PR) to gonadotropes, but not lactotropes, with an immunocytochemical technique to double stain monkey pituitary cell cultures. The following studies were performed to confirm the immunocytochemical localization of PR in intact pituitary tissue and to determine the effect of E and P on the levels of estrogen receptors (ER) and PR in the pituitary. ER and PR levels were determined in the endometrium of the same animals for an internal comparison. Thirteen adult cycling female cynomolgus monkeys were ovariectomized and treated for 28 days with 1) an empty Silastic capsule (Spay), 2) a 2-cm E-filled capsule (E), or 3) a 2-cm E-filled capsule for 14 days plus a 6-cm P-filled capsule implanted for an additional 14 days (E + P). Blood samples were drawn daily for assay of serum E, P, and PRL levels. Serum PRL was not significantly affected by E, but the sequential addition of P significantly increased serum PRL levels over those observed in Spray animals. The anterior pituitary and endometrium were removed for measurement of ER and PR levels by a sucrose gradient shift assay incorporating monoclonal antibodies against ER and PR. Pituitary ER levels did not vary significantly with steroid treatment (158.2 +/- 33.6, 135.5 +/- 24.9, 104.3 +/- 13.4 fmol/mg DNA in Spay, E, and E + P animals, respectively). Pituitary PR levels were undetectable in Spay animals, were induced by E (393.3 +/- 53.4 fmol/mg DNA), and were suppressed to undetectable levels by the addition of P. A portion of the pituitary was frozen for immunocytochemical single staining for ER, PR, PRL, and LH and double staining for PRL + PR and LH + PR. ER staining was observed in many parenchymal cells, but there was no apparent change with steroid treatment. PR staining was absent in the Spay animals; many PR-positive cells were observed in E-treated females, and only a small number of faintly staining cells were detected in the E + P animals. Double staining for PRL + PR and LH + PR revealed PR in gonadotropes, but not lactotropes. In conclusion, PR, but not ER, are regulated by E and P in the monkey pituitary. Importantly, PR is regulated within gonadotropes, but not lactotropes. Therefore, P probably increases PRL secretion through a hypothalamic action.

Animals↗
PubMed1990

Multihormonal regulation of the progesterone receptor in MCF-7 human breast cancer cells: interrelationships among insulin/insulin-like growth factor-I, serum, and estrogen.

Estrogen (E) is well known to be an important stimulator of progesterone receptor (PR) synthesis in target cells. We have observed that E stimulation of PR in MCF-7 human breast cancer cells (as monitored by progestin binding or Western blotting with anti-PR antibodies) increases as a function of serum concentration in the cell culture medium; PR stimulation by E is greatest in high serum medium (5% or 10% charcoal dextran-treated calf serum) and is not observed when cells are in medium containing serum concentrations below 1%, although estrogen receptor levels are well maintained. This suggests that some serum factor(s) may be essential for E to be able to stimulate PR. To better understand such factors, we have grown cells in serum-free medium and in serum-free medium supplemented with insulin (6.25 micrograms/ml) [corrected], transferrin (6.25 micrograms/ml), selenium (6.25 ng/ml), albumin (1.25 mg/ml) [corrected], and linoleic acid (5.35 micrograms/ml; ITS+). Unexpectedly, we found that addition of ITS+ (without E) increases PR levels in these cells, especially in the absence of serum and under low serum conditions where E stimulation of PR is poor. Analyses of the individual components in ITS+ reveal that insulin is the major active component. Dose-response studies indicate that high superphysiological (greater than 1 microgram/ml) concentrations of insulin are required. In contrast, low physiological levels of insulin-like growth factor-I (IGF-I; 10 or 40 ng/ml) are active, suggesting mediation by the IGF type I receptor system. At all serum concentrations (0-10%), the effects of ITS+ and E in increasing PR are synergistic. The fact that anti-E are able to suppress the insulin/IGF-I stimulation as well as the E stimulation of PR suggests that the anti-E can actively interfere with the action of the growth factor as well as the action of E. These results indicate that regulation of PR is multifactor and raise the possibility that PR may be regulated in vivo by both E and growth factors such as IGF-I that are known to be increased in these breast cancer cells by E.

Blood↗
PubMed1990

Estrogen binding is a noncooperative process in primary rat uterine cells.

Estrogen binding in a primary rat uterine cell culture system did not exhibit positive cooperativity as judged by Hill coefficients. Under defined culture conditions, total specific [3H]estradiol (E2) binding in intact cells attained apparent equilibrium within 3 h after E2 had been varied from 5 to 250 pM. Thereafter, at each concentration of E2 studied, estrogen receptor (ER) levels were constant for up to 6 additional h. We have characterized a single high affinity E2 binding site with a dissociation constant (Kd alpha) of 0.01-0.05 nM E2. The binding capacity of the alpha-site is approximately 5-8 fmol/micrograms DNA, corresponding to approximately 20,000 receptors per cell. In extensive studies, we have seen no evidence for positive cooperative E2 binding. The Hill coefficient was never significantly (95% confidence level) greater than 1. Data are also presented which indicate that [3H]E2 exogenously added to the extracellular medium is in simple equilibrium with the intracellular compartment and the ER. When saturation analyses were conducted using medium that contained BSA, increased concentration of serum protein increased the apparent Kd of E2 for the receptor. No effect was observed on either the shapes of the binding curves or on the calculated Hill coefficients. Furthermore, dose correlations between E2 binding and stimulated response(s) indicated that the ER in these cells was biologically active, which was a major advantage of this culture system over freshly dispersed cells or cytosolic extracts.

Animals↗
PubMed1990

Progesterone action in normal mouse mammary gland.

Previously it has been shown that progesterone, as well as estrogen, plays an important role in the growth of the mammary gland. Eighty percent of mammary progesterone receptors (PgR) are estrogen-inducible and are localized in the epithelium; the remaining 20% of PgR are estrogen-independent and appear to be localized in the mammary stroma. The purpose of the present study was to investigate how progestins promote mammary growth in relation to their interactions with epithelial and stromal components of the gland and to assess the role of estrogen in these interactions. Progestins [progesterone, [17 beta-methyl-3H]promogestone (R5020), and medroxy progesterone acetate] alone or in combination with estrogen were combined with Elvax 40P and implanted directly into mammary glands. The effect of hormones on cell proliferation was determined by observing changes in mammary gland morphology and by quantitating DNA synthesis in both epithelial and stromal cells by DNA histoautoradiography. The results demonstrate that in mammary epithelial cells the effects of progestins on mammary gland morphology and DNA synthesis are locally mediated such that proliferative changes in the hormone-implanted glands were greater than in contralateral control glands. Dose-response studies with estrogen and R5020 revealed that the extent of progestin activity was only partially dependent upon the R5020 dose with the major determining factor being the dose of estrogen. Analysis of the effect of estrogen on mammary PgR concentration indicates that the degree and pattern of the morphological response of ductal sidebranching and increases in DNA synthesis are largely due to the increase in estrogen-dependent PgR. The antiprogestin, 11 beta-(4-dimethylamino-phenyl)1-17 beta-hydroxy-17 alpha-(prop-1ynyl)-estra-4,9-diene-3-one (RU486), blocks the proliferation in the epithelium that is mediated through estrogen-dependent PgR. In contrast, in stromal cells progestin activity is not estrogen-dependent, and stimulation of DNA synthesis was not confined to the hormone-implanted glands. Furthermore, RU486 stimulates stromal cell DNA synthesis, and this response is augmented by estrogen. While progestin effects in epithelial cells appear to be mediated by estrogen-dependent PgR, the mechanism operative in stromal cells appears to be different and remains to be elucidated.

Animals↗
PubMed1991

Progestin receptor cells in the 8-day-old male and female mouse cerebral cortex: autoradiographic evidence for a sexual dimorphism in target cell number.

The present study examined the number and distribution of progestin receptor cells in the 8-day-old male and female cortex and compared cortical labeling with that in the preoptic area and central hypothalamus. Eight-day postnatal mice (four males and four females), treated with estradiol, were each sc injected with 0.32 micrograms/100 g BW [125I]progestin (SA, 2200 Ci/mM). Brains were frozen 2 h after injection of [125I]progestin, sectioned, and processed for thaw-mount autoradiography. Cells with a nuclear concentration of radioactivity were localized in lamina VI of the lateral cortical regions of the male and female brain, while only a few cortical cells were seen in laminae II, III, and V of the suprarhinal, lateral, and cingulate/paracingulate regions. Comparison of the number of labeled cells revealed that the female cortex contained significantly more labeled cells than the male at three of the four levels investigated. Similarly, the number of target cells was higher in the female medial preoptic nucleus, but not in the arcuate nucleus and ventromedial hypothalamic nucleus, while the distributions of labeled cells in the male and female preoptic/hypothalamic regions were comparable. Injection of unlabeled progesterone or R5020 1 h before [125I]progestin reduced the nuclear concentration of radioactivity in all target regions and verified the specificity of [125I]progestin for the progestin receptor. The results of these studies indicate that mouse 8-day-old cortex and preoptic area in the female animal have more progestin receptor cells than those in the male and demonstrate that progestin receptor cells are localized in a region of the cortex known to contain few estrogen target cells. These results further suggest that a sexual dimorphism in progestin cell number may result in a differential effect of progestin on the cortex and preoptic area of the mouse, perhaps establishing a dimorphism in development and function.

Animals↗
PubMed1991

Progesterone receptor regulation in uterine cells: stimulation by estrogen, cyclic adenosine 3',5'-monophosphate, and insulin-like growth factor I and suppression by antiestrogens and protein kinase inhibitors.

Primary uterine cell cultures were used to study multifactor regulation of progesterone receptor (PR) and the signal transduction pathways which may serve to mediate that regulation. Increases in intracellular cAMP, brought about by treatment with cholera toxin plus isobutyl methyl xanthine or by addition of 8-bromo-cAMP, result in 6- to 7-fold increases in the intracellular content of PR as monitored by [3H]R5020 binding and by Western immunoblot using anti-PR antibodies. In these primary cultures of uterine cells isolated from 19-day-old immature rats, 8-bromo-cAMP evokes significant increases in PR by 8 h with maximal increases by 24 h. This time course and magnitude of PR stimulation are similar to those evoked by maximally effective concentrations of estradiol (3 x 10(-9) M) or IGF-I (20 ng/ml). Dose-response studies reveal that 10(-6) to 10(-4) M concentrations of 8-bromo-cAMP (8-Br-cAMP) elicit a maximal response. In contrast, 8-bromo-cGMP over a wide concentration range was unable to elevate cellular PR levels. Under these culture conditions, cell proliferation was not altered by treatment with any of these agents. Although estrogen, cAMP, and insulin-like growth factor I (IGF-I) may act via different pathways to increase PR, the effects evoked by maximally effective concentrations of these agents are not additive implying involvement of a common component. The increases in PR evoked by estradiol, cAMP, or IGF-I are markedly suppressed by treatment with antiestrogen (ICI 164,384) or the cyclic nucleotide-dependent protein kinase inhibitor H8 or the protein kinase A inhibitor PKI, indicating the involvement of the estrogen receptor and phosphorylation pathways in PR regulation by these three agents. The present studies identify cAMP, as well as estrogen and IGF-I, as important regulators of the level of PR in uterine cells and suggest that multiple factors, including those affecting intracellular cAMP levels, might influence responsiveness to progestins via regulation of the intracellular PR content.

1-Methyl-3-isobutylxanthine↗
PubMed1992

Progestin target cell distribution in forebrain and midbrain regions of the 8-day postnatal mouse brain.

The present study investigated the anatomical distribution of progestin target cells throughout the forebrain and midbrain regions of the 8-day postnatal female mouse. Female ICR mice were sc injected with 100 micrograms/100 g BW estradiol valerate on postnatal day 5 (birth = day 0). On postnatal day 8, treated mice were sc injected with 0.32 micrograms/100 g BW (Z)-17 beta-hydroxy-17 alpha-(2-[125I]iodovinyl)4-estren-3-one ([125I] progestin). For competition, additional estrogen-treated mice were each injected with 320 micrograms R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione; a potent synthetic progestin), 320 micrograms dihydrotestosterone, or 32, 160, or 320 micrograms corticosterone 1 h before [125I]progestin to show the specificity of [125I]progestin for the progestin receptor. Two hours after injection of [125I]progestin, the brains were removed, frozen, and processed for high resolution thaw-mount autoradiography. After 8-60 days of exposure, nuclear uptake and retention of [125I]progestin were detected in many brain regions, including the septum; bed nucleus of the stria terminalis; and preoptic area, periventricular nucleus, ventromedial nucleus, arcuate nucleus, and dorsomedial nucleus of the hypothalamus. In addition, labeling was seen in the cerebral cortex, caudate putamen, hippocampus, amygdala, and substantia nigra. Competition studies showed that excess R5020 prevented nuclear concentration of ligand, while dihydrotestosterone and corticosterone did not. The results indicate that the distribution of progestin target cells in extrahypothalamic regions of the developing brain is more extensive than that in the adult, while a similar topography was seen in the preoptic area and hypothalamus. The results further suggest that progestin action during brain development may influence the growth and development of target cells not only in the hypothalamus but also in regions of the brain previously not considered to be sites of hormone action.

Animals↗
PubMed1993

Androgens augment vasoactive intestinal peptide- and growth hormone-releasing hormone-stimulated progestin production by rat granulosa cells.

Vasoactive intestinal peptide (VIP) has been shown to stimulate steroid production by cultured rat granulosa cells independently of FSH. In the present study, we have examined the modulatory effects of various steroids on this response. Rat granulosa cells cultured for 2 days with only VIP showed small but significant increases in progesterone and 20 alpha-dihydroprogesterone (20 alpha-OH-P) production. Concomitant treatment with either a synthetic estrogen (diethylstilbestrol), a synthetic progestin (R5020), or cortisol did not augment VIP-stimulated progesterone production; however, the latter two steroids slightly, but significantly, augmented VIP-stimulated 20 alpha-OH-P production. In contrast, concomitant treatment with a synthetic androgen (R1881) dramatically augmented both VIP-stimulated progesterone and 20 alpha-OH-P production. These effects were dose dependent for both VIP and R1881 and could be blocked by the androgen antagonist cyproterone acetate. Time course studies revealed that progesterone content of the culture media rapidly increased over the first 24 h of culture then remained fairly constant for the next 48 h; 20 alpha-OH-P content, on the other hand, was low for the first 12 h of culture and steadily increased thereafter. Dose-response analysis for R1881 revealed an ED50 of approximately 2 x 10(-8) M for the synthetic androgen, and comparison with other naturally occurring androgens provided the rank order of potency R1881 > androstenedione > testosterone = dihydrotestosterone. Additional studies with another member of the VIP peptide family, GH-releasing hormone, showed dose-dependent stimulation of progesterone and 20 alpha-OH-P production by this peptide. These effects were also augmented by R1881 but not by diethylstilbestrol, R5020, or cortisol. These studies demonstrate that androgens, but not estrogens, progestins, or glucocorticoids, augment VIP- and GH-releasing hormone-stimulated progestin production by cultured rat granulosa cells.

20-alpha-Dihydroprogesterone↗
PubMed1993

Differential regulation of human progesterone receptor A and B form-mediated trans-activation by phosphorylation.

Hormone-dependent phosphorylation of progesterone receptors (PRs) plays a functional role in their transcriptional activity. However, hormone-independent phosphorylation has also been shown to modulate the chicken PR-mediated trans-activation in the presence of phosphorylating agents. The present study was designed to investigate the effects of protein kinase A- and protein kinase C-mediated signal transduction pathways on the regulation of the activity of the two forms of human PR (hPRA and hPRB). Similar to chicken PR, hPR was activated by 8-bromo-cAMP (8-Br-cAMP) in the absence of ligand, whereas 8-Br-cAMP synergized with the progestin agonist R5020 to amplify hPRA- and hPRB-mediated reporter activity. Interestingly, the effect of 8-Br-cAMP was much more pronounced on hPRA-induced trans-activation than on hPRB. This differential regulation by 8-Br-cAMP could also be mimicked by okadaic acid. Both mouse mammary tumor virus-thymidine kinase-chloramphenicol acetyl transferase and progesterone response element-thymidine kinase-chloramphenicol acetyl transferase showed a similar response to 8-Br-cAMP in the presence of R5020. Protein kinase C, on the other hand, did not discriminate between hPRA- and hPRB-mediated trans-activation. Unlike 8-Br-cAMP, phorbol 12-myristate 13-acetate did not cause marked ligand-independent trans-activation through either of the two receptor forms. RU486, an antagonist of progestin, preferentially blocked R5020-induced trans-activation compared to R5020 + 8-Br-cAMP synergism. As expected, H-89, a specific inhibitor of protein kinase A was more effective in inhibiting ligand-independent activity. Western analysis of transfected receptors suggested that 8-Br-cAMP and 8-Br-cAMP + R5020 but not R5020 alone down-regulated the level of hPRB in COS-1 cells. Only marginal modulation of hPRA levels was observed with R5020 treatment in the presence and absence of 8-Br-cAMP. These data suggest that R5020 and 8-Br-cAMP mediate PR-dependent transactivation through distinct pathways, and that phosphorylation can differentially regulate the activity of hPRA and hPRB forms, an observation which may be important for selective target gene activation in vivo by progestins.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

Intracrine role of progesterone in follicle-stimulating hormone- and cyclic adenosine 3',5'-monophosphate-induced fibronectin production and deposition by chicken granulosa cells: influence of follicular development.

The role of progesterone in FSH- and 8-bromo-cAMP (8-Br-cAMP)-induced fibronectin production by chicken ovarian granulosa cells was examined. Granulosa cells isolated from the third largest (F3; developing; 15-20 mm in diameter) preovulatory follicle and a pool of immature small yellow follicles (SYF; 6-8 mm in diameter) were incubated in serum-free medium 199, and the total amount of fibronectin produced (deposited, secreted into the medium, and associated with cells) was measured by enzyme-linked immunosorbent assay. Unstimulated F3 cells deposited greater amounts of fibronectin than unstimulated SYF cells. FSH or 8-Br-cAMP significantly increased fibronectin deposition. Similarly, both agents increased the quantity of fibronectin secreted into the medium and that associated with cells. The magnitude of FSH- and 8-Br-cAMP-enhanced fibronectin deposition or secretion into medium by SYF cells was greater than that by F3 cells. Cyanoketone (an inhibitor of progesterone synthesis) significantly suppressed basal fibronectin production by F3 cells, but not that by SYF cells. Cyanoketone completely blocked FSH- or 8-Br-cAMP-induced fibronectin production by F3 cells, but caused only a modest inhibition (nonsignificant) of agonist-induced fibronectin production by SYF cells. Exogenous progesterone completely reversed the inhibitory effects of cyanoketone on agonist-induced fibronectin production. The nondegradable synthetic progestin R5020 also reversed the inhibitory effects of cyanoketone on agonist-induced fibronectin production. The antiprogestin, ZK 98.299, inhibited basal and FSH-stimulated fibronectin production. The data demonstrate that FSH- and cAMP-stimulated fibronectin production by chicken granulosa cells is dependent (at least in part) on de novo progesterone synthesis. Furthermore, they indicate that fibronectin production and deposition by these cells are stimulated by progesterone, perhaps in an intracrine/autocrine manner, and that the role of progesterone increases with advancing stages of follicular development.

8-Bromo Cyclic Adenosine Monophosphate↗