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PubMed1987

Role of progesterone in mobility, fixation, orientation, and survival of the equine embryonic vesicle.

Luteal progesterone was removed by an injection of prostaglandin F2alpha or bilateral ovariectomy on Day 12 of pregnancy in pony mares. The embryonic vesicle remained mobile in the uterus until loss occurred on Days 13, 13, 15, or 19 in four prostaglandin-treated mares and Days 15, 17, 19, or 26 in four ovariectomized mares. Exogenous progesterone given daily, starting on Day 12, maintained pregnancy until Day 40 in five of five prostaglandin-treated and three of four ovariectomized mares. During two-hour mobility trials on Day 14, embryonic vesicles in mares without luteal or exogenous progesterone (n=9) moved to a different uterine segment less frequently (mean number of location changes per two-hour trial: 7.2+/-1.0 vs 10.4+/-1.1, P<0.05) and were observed more often in the uterine body (14.9+/-2.9 vs 8.9+/-1.3, P<0.10) compared to vesicles in mares with a progesterone influence (n=15). Of mares that still had a vesicle present on Day 18, fixation occurred by Day 17 in all (12/12) mares under the influence of luteal or exogenous progesterone but failed to occur in the three mares that were not under progesterone influence. Progesterone replacement was started on Day 16 in three mares that received prostaglandin F2alpha on Day 12 and still had a vesicle on Day 16. The vesicle was maintained and continued to develop in all three mares, indicating that the vesicles were viable four days after PGF2alpha treatment. However, fixation tended to be delayed (P<0.15) and orientation of the embryo proper was altered (P<0.005) compared to mares that were continuously under the influence of progesterone. The results demonstrated the importance of luteal progesterone to mobility, fixation, orientation, and survival of the embryonic vesicle.

Journal Article↗

PubMed1989

Comparisons of eight commercial on-farm milk progesterone tests.

To evaluate eight commercial on-farm milk progesterone kits, milk samples (50 ml each of foremilk and postmilk strippings) were collected during the estrous cycle from 10 cycling Holstein cows for 24 consecutive days. Relative concentrations of progesterone were classified as low or high by comparison with standard progesterone samples supplied with each kit. The concentration of progesterone in each milk sample was determined by radioimmunoassay (RIA). Accuracy of classification into low or high levels by commercial tests was determined by the percentage of similarity with RIA values using discriminant analysis. Accuracy of the eight tests ranged from 89.0 to 98.9% for low progesterone, 74.8 to 85.6% for high progesterone, and 80.3 to 87.3% for all samples (n = 238). The percentage of fat in milk or an interaction of the percentage of milkfat by day of estrous cycle influenced commercial test results for all tests except Accufirm and Calfcheck. Progesterone levels, estimated by the test-kits, were low from 1.5 +/- 0.5 to 2.8 +/- 0.9 days before estrus (X +/- SEM) and until 4.0 +/- 0.6 to 5.9 +/- 1.3 days after estrus. These data support the principle that a single low progesterone sample cannot be used to determine proper timing of insemination. All eight commercial kits can be used to determine accurately the relative concentrations of progesterone in milk samples.

Journal Article↗

PubMed1995

Secretion of progesterone during long and short days of the estrous cycle in goats that are continuous breeders.

This study was conducted 1) to determine if the secretion of progesterone, as an index of ovarian activity, during the estrous cycle of nonseasonal Shiba goats is affected by seasonal changes, and 2) to learn if the pulsatile secretion of ovarian progesterone can be estimated from samples obtained by cannulation into the caudal vena cava via the femoral vein. Progesterone concentrations in jugular venous plasma during the estrous cycle in spring (May) were similar to those in autumn (November). Plasma progesterone concentrations in the jugular vein and caudal vena cava monitored for 10 h on Day 12 of the estrous cycle in spring were similar to those in autumn. The mean concentration (21.9 to 28.9 ng/ml) and the pulse frequency (6.2 to 7.4 pulses/10 h) of plasma progesterone in the caudal vena cava during both seasons were 3.1- to 4.7-fold and 1.7- to 2.4-fold those in the jugular vein, respectively. The degree of change in the peak magnitude and the base-line concentration of progesterone were higher in the caudal vena cava than in the jugular vein. These results indicate that progesterone secretion during the estrous cycle in nonseasonal goats is not affected by seasonal changes, and suggest that the pulsatile secretion of ovarian progesterone can be evaluated better from samples obtained from the caudal vena cava, near where progesterone is released, than from those obtained from the jugular vein.

Journal Article↗

PubMed1995

Effects of sample handling temperatures on bovine skim milk progesterone concentrations.

The effects of incubation of whole milk at various temperatures and times on the concentration of progesterone in the skim milk fraction was determined. For the study, milk samples were collected from 10 pregnant Holstein cows. The milk from each cow was transferred to culture tubes to provide 32 replicates of 3 ml volume. To begin the incubation study, all samples were placed in a 37 degrees C water bath for 4 h. The end of this incubation was designated as time 0 and a sample from each cow was centrifuged to harvest skim milk. At time 0, samples from each cow were divided among incubation temperatures of 0 degrees, 4 degrees, 20 degrees and 37 degrees C. Samples were removed from each incubation group at 30, 60, 90 and 120 min. After 120 min, all remaining samples were returned to the 37 degrees C incubation and skim milk was collected at 30, 60 and 90 min. Progesterone was measured in skim milk by radioimmunoassay. The mean +/- SE concentration of progesterone in skim milk at time 0 was 10.9 +/- 1.1 nmol/L. The mean concentration of progesterone in skim milk was higher (P < 0.05) in all samples incubated at 0 degrees and 4 degrees C, with incremental increases ranging from 34% to 67% above time 0. Progesterone in skim milk returned to time 0 concentrations in milk samples transferred from 0 degrees or 4 degrees C to 37 degrees C. There was no change in skim milk progesterone in whole milk samples incubated at 20 degrees or 37 degrees C. From this study, it can be concluded that the concentration of progesterone in skim milk is temperature dependent. Inconsistency in handling whole milk samples can have a profound effect in the concentration of progesterone on skim milk. The temperature-dependent effect was reversible and may be related to solubility of progesterone in milk fat.

Journal Article↗

PubMed1995

Variable progesterone response and estradiol secretion in prepubertal beef heifers following treatment with norgestomet implants.

Two experiments were conducted to determine the effects of norgestomet ear implants on progesterone response and estradiol secretion in prepubertal beef heifers. In the first experiment, 47 beef heifers were treated with norgestomet. The implants were implanted subcutaneously for 9 d. After implant removal, blood samples were taken from heifers 2 to 4 d per week for 40 d. Following progesterone determination in jugular venous plasma, heifers were classified according to their progesterone response: 1) no response (Group 1); no rise in progesterone above 1 ng/ml throughout the sampling period; 2) one cycle (Group 2); one increase in progesterone above 1 ng/ml for at least 2 d followed by no further increase in progesterone during the sampling period; and 3) two cycles (Group 3); a rise in progesterone above 1 ng/ml for at least 2 d followed by another cycle of normal duration. Heifers treated with norgestomet were classified as 23 with no response, 9 with 1 cycle and 15 with 2 cycles. Concentrations of estradiol were measured in jugular venous samples on Day 2 after implant removal. Mean concentrations of estradiol were greater in Group 3 than in Group 1 (P < or = 0.01). In Experiment 2, 29 prepubertal beef heifers were assigned randomly to either a 9-d treatment with norgestomet (n = 14) or to serve as untreated controls (n = 15). Blood plasma samples were collected daily from Days 0 to 44 after implant removal. After progesterone determination, heifers were classified as 8 with no response, 4 with 1 cycle and 3 with 2 cycles in the control group, and 5 with no response, 3 with 1 cycle and 6 with 2 cycles in the norgestomet group (frequencies did not differ; P > 0.1). Jugular venous blood plasma was also collected at 4-h intervals from 0 h to 96 h after implant removal and concentrations of estradiol were measured. Patterns of estradiol secretion differed (P < or = 0.05) and overall mean concentrations of estradiol over the first 96 h following implant removal were greater (P < or = 0.01) in norgestomet-treated heifers versus the controls. We conclude that norgestomet can produce a variable progesterone response with heifers with 2 cycles secreting more estradiol. Implants of norgestomet also causes more acute secretion of estradiol in prepubertal beef heifers.

Journal Article↗

PubMed2003

Effect of progesterone prior to GnRH-PGF2alpha treatment on induction of oestrus and pregnancy in anoestrous Awassi ewes.

An experiment was conducted to examine the effect of progesterone prior to a GnRH-PGF2alpha treatment on oestrus and pregnancy in seasonally anoestrous Awassi ewes. Twenty-four ewes were randomly assigned to three groups to be pre-treated with 60 mg medroxyprogesterone acetate sponges (group A), 600 mg progesterone sponges (group B) or blank sponges (group C) for 4 days. All ewes were injected with 100 microg of GnRH 24 h after sponge removal followed, 5 days later, by 20 mg PGF2alpha injection. Ewes were exposed to three fertile rams at the time of PGF2alpha injection (day 0, 0 h) and were checked for breeding marks at 6-h intervals for 5 days. Blood samples were collected from all ewes 1 day (day -10) prior to sponge insertion, at the time of sponge removal (day -6), 1 day following sponge removal (day -5, at the time of GnRH injection) and at the time of PGF2alpha injection (day 0) for analysis of progesterone. Progesterone concentrations on days -10 and -5 were basal and averaged 0.2 +/- 0.04 and 0.2 +/- 0.2 ng/ml, respectively. Progesterone concentrations on day -6 were elevated only in group B ewes and were higher (p < 0.0001) than those of groups A and C. Progesterone concentrations on day 0 were higher (p = 0.002) in groups A and B than group C. Oestrous responses occurred only in ewes of groups A and B (p > 0.05). Induced oestrus conception rate was greater (p < 0.01) in group A than groups B and C. Ewes returned to oestrus 17-20 days following day 0 were two of eight, six of eight and three of eight of groups A, B and C, respectively, all of which eventually lambed. The overall lambing rate was 82% in progesterone-primed ewes compared with only 38% non-progesterone-primed ewes (p < 0.05). Progesterone priming apparently sensitizes GnRH-PGF2alpha-treated seasonally anoestrous ewes and increases their response in oestrus and pregnancy rates.

Administration, Intravaginal↗

PubMed1984

Potent inhibition of the hypothalamic progesterone 5 alpha-reductase by a 5 alpha-dihydroprogesterone analog.

The ability of the 5 alpha-dihydroprogesterone analog, 4-aza-4-methyl-5 alpha-pregnane-3,20-dione (AMPD), to inhibit the progesterone 5 alpha-reductase and the two 5 alpha-dihydroprogesterone 3 alpha-hydroxysteroid oxidoreductase activities (NADH- and NADPH-linked) from female rat hypothalamus has been studied. Dose response experiments indicate that AMPD is a potent antagonist of hypothalamic progesterone 5 alpha-reduction but is an ineffective inhibitor of the NADPH- and NADH-linked 3 alpha-hydroxysteroid oxidoreductase activities, even at concentrations up to 10 microM. Kinetic analyses of the interaction of AMPD with the progesterone 5 alpha-reductase show that it is a competitive inhibitor versus progesterone (Ki(slope) = 6.2 +/- 0.5 nM; apparent Km (progesterone) = 130 +/- 12 nM) and an uncompetitive inhibitor versus NADPH (Ki(intercept) = 11.8 +/- 0.8 nM). These inhibition patterns are consistent with the concept that NADPH binding precedes that of either AMPD or progesterone. The inhibition of the progesterone 5 alpha-reductase by AMPD does not appear irreversible since preincubation of the enzymatic activity (at 37 degrees C) with inhibitor and NADPH, for periods of time up to 60 min, does not lead to a time-dependent loss of activity. Furthermore, this inhibition can be easily removed via dilution, even following a 60-min preincubation with AMPD and NADPH. It is postulated that the specific and powerful inhibition of the progesterone 5 alpha-reductase by AMPD may be due to this compound functioning as a transition state analog. This inhibitor should prove valuable in studying the characteristics of the progesterone 5 alpha-reductase and the function of hypothalamic progestin metabolism.

Animals↗

PubMed1984

The postovulatory cat oviduct and uterus: correlation of morphological features with progesterone receptor levels.

Oviducts and uteri were obtained from cats at estrus and up to 14 days after coitus. Progesterone binding sites were estimated by Scatchard analysis, serum levels of progesterone were determined by radioimmunoassay, nd key morphological parameters of the lining of the epithelium of the oviduct and the deep uterine glands were quantitated. Progesterone cytosol binding was highest at estrus, significantly depleted by day 4, and continued a gradual decline out to day 14 in both tissues. Nuclear binding was markedly elevated at day 4; with time the binding decreased until by day 14 it was equal to or lower than that detected at estrus. The initial depletion of cytosol binding and rise in nuclear binding coincided with the initial rise in serum progesterone levels. Within the oviduct, the increase in nuclear binding coincided with the onset of regressive changes within the epithelium. These changes included atrophy, deciliation, and apoptosis. Within the uterine glands, the increase in nuclear binding coincided with the onset of the progestational response. This response included cell hypertrophy, glycogen synthesis and deposition, and with longer exposure to elevated levels of progesterone, the disappearance of these glycogen deposits. These results clearly illustrate the immediate effect of low levels of progesterone on the subcellular compartmentalization of the progesterone receptor system, and the down regulation of the total receptor system when target tissues are exposed to a continuous elevation of serum progesterone. These results also illustrate that similar changes in receptor compartmentalization induce different morphological responses within target tissues from the same animal.

Animals↗

PubMed1998

Plasma membrane mediated action of progesterone in amphibian (Rana dybowskii) oocyte maturation.

The mechanism of progesterone action within the ovarian follicle was investigated in Rana dybowskii, by using immobilized progesterone. Fluorescein isothiocyanate-labeled progesterone 3-O-carboxymethyloxime-BSA (P-BSA) was localized on the outside surface of the denuded oocyte, which indicated that P-BSA did not cross the barrier of cell surface. Progesterone-BSA induced germinal vesicle breakdown (GVBD) of denuded oocytes in a dose-dependent manner but failed to induce GVBD of follicle wall-enclosed oocytes. The time course of P-BSA-induced GVBD in denuded oocytes was similar to that observed with progesterone. Furthermore, both P-BSA and progesterone induced oocyte maturation in the presence of RU486, a well-known nuclear progesterone receptor antagonist. Treatment of denuded oocytes with P-BSA resulted in a threefold increase in inositol triphosphate (IP3) and a fourfold increase in diacylglycerol levels within 10 min. Additionally protein kinase C (PKC) activity was markedly increased by 30 min of incubation following exposure to P-BSA. Such changes were not observed in denuded oocytes exposed to beta-estradiol-6-O-carboxymethyloxime-BSA, which failed to induce GVBD. These results suggest that progesterone acts initially at the oocyte surface where it triggers generation of membrane-mediated second messengers during oocyte maturation in amphibians.

Animals↗

PubMed1985

Gap junctions and myometrial steroid hormone receptors in pregnant and postpartum rats: a possible cellular basis for the progesterone withdrawal hypothesis.

Myometrial gap junctions, levels of cytosol and nuclear estradiol and progesterone receptors, and serum estradiol and progesterone levels were determined simultaneously in pregnant and postpartum rats. Between days 15 to 20 after conception, levels of progesterone nuclear receptors decreased (776.8 +/- 88.5 versus 241.1 +/- 56.5 fmol/mg of DNA, p less than 0.05). The mean serum progesterone also fell by about 50% during this period, but variation in individual levels between days 15 to 20 did not allow for this group to achieve statistical significance until day 21. As the progesterone nuclear receptors decreased, estradiol remained stable, but estradiol nuclear receptors increased (day 21 versus 22: 1710.6 +/- 61.1 versus 3254.8 +/- 203.8 fmol/mg of DNA, p less than 0.05), preceding the increase in gap junctions observed at parturition (day 21 versus 22: 2.0 +/- 0.2 versus 8.0 +/- 3.2 [per 1000 micron plasma membrane], p less than 0.05). Gap junctions fell to prepartum levels by 12 hours, when progesterone nuclear receptors were markedly increasing (6 versus 12 hours post partum: 637.7 +/- 324.8 versus 1509.6 +/- 283.2 fmol/mg of DNA, p less than 0.05). Relationships between gap junctions and cellular estradiol and progesterone nuclear receptors are clearer than could be forecast by circulating hormone measurements alone, and may offer a cellular basis for the role of progesterone in controlling labor.

Animals↗

PubMed1989

Preventing ruptured ectopic pregnancy with a single serum progesterone.

Retrospective data on patients with documented ectopic pregnancies have demonstrated the potential usefulness of serum progesterone in decreasing the time to diagnosis. We report a prospective trial utilizing serum progesterone in the emergency department of a large city-county hospital. Sixty-seven ectopic pregnancies were document in the 582 patients screened; 54 (80.6%) had a progesterone level less than 15 ng/ml. Only one (1.5%) ruptured ectopic pregnancy was associated with a progesterone level greater than 25 ng/ml. There were 387 documented or presumed normal intrauterine pregnancies, 41 (10.6%) had progesterone concentrations of less than 15 ng/ml and 236 (61%) had a progesterone level greater than 25 ng/ml. We decreased minimum time of diagnosis from 48 to 72 hours to less than 24 hours by use of a progesterone screening protocol. During this time the percentage of ruptured ectopic pregnancies decreased from 79.2% to 38.8% (p less than 0.05). Because of its simple interpretation and 24-hour maximum delay, we conclude that the determination of serum progesterone should be used for ectopic pregnancy screening in all patients at risk for ectopic pregnancy, or at any time when the diagnosis is in question.

Algorithms↗

PubMed1987

Cellular progesterone receptor phosphorylation in response to ligands activating protein kinases.

Progesterone receptors were immunoprecipitated with monoclonal antibodies KD68 from lysates of human breast carcinoma T47D cells labelled to steady state specific activity with 32Pi. The 120 kDa 32P-labelled progesterone receptor band was resolved by polyacrylamide gel electrophoresis and identified by autoradiography. Phosphoamino acid analysis revealed serine phosphorylation, but no threonine or tyrosine phosphorylation. Treatment of the 32Pi-labelled cells with EGF, TPA or dibutyryl cAMP had no significant quantitative effect on progesterone receptor phosphorylation, though the EGF receptor and the cAMP-dependent protein kinases have been reported to catalyze phosphorylation of purified avian progesterone receptor preparations in cell free systems. Progesterone receptor phosphorylation on serine residues was increased by 2-fold in cells treated with 10 nM progesterone; EGF had no effect on progesterone-mediated progesterone receptor phosphorylation.

Antibodies, Monoclonal↗

PubMed1986

Cholinergic mediation of feminine sexual receptivity: demonstration of progesterone independence using a progestin receptor antagonist.

Feminine sexual receptivity can be elicited by the intracerebral administration of muscarinic cholinergic substances in ovariectomized female rats treated with small amounts of estrogen. These experiments were designed to investigate whether this facilitative cholinergic mechanism is dependent upon progesterone. In the first experiment, estrogen-primed animals were injected with RU 38486, a progestin receptor antagonist, 1 h before progesterone or vehicle injection. Injection of RU and progesterone on Day 1 of testing produced animals which were sexually unreceptive. Furthermore, injection of the antagonist without concomitant progesterone produced animals which were unresponsive to progesterone given 24 h later. Thus, RU 38486 itself may be binding to progestin receptors in critical brain regions, preventing progesterone from binding to these same receptors. In Experiment 2, animals made sexually unreceptive by treatment with RU 38486 subsequently became receptive when injected intracerebrally with oxotremorine, a muscarinic agonist. Therefore, activation of progestin receptors does not appear necessary for the cholinergic mediation of sexual receptivity. As in Experiment 1, animals receiving progesterone on Day 1 showed a diminished response to a second progesterone injection 24 h later; an even greater decrease in this response was observed in animals that also received the antagonist on Day 1.

Animals↗

PubMed1986

Effect of the antiprogestin RU486 on progesterone production by cultured human granulosa cells: inhibition of the ovarian 3B-hydroxysteroid dehydrogenase.

Recent studies suggest that the antiprogestin RU486 may have a direct effect on human ovarian luteal function. To further examine this possibility, we have studied the effect of RU486 on ovarian steroidogenesis using human granulosa cells obtained from women undergoing in vitro fertilization. RU486 at concentrations of 0.1, 2, 5, 10 and 100 nM was incubated with 10(5) granulosa cells over 72 hours. Significant suppression of progesterone production occurred following treatment of cultured cells with 2, 5, 10, and 100 nM RU486 at 24 hours (p less than 0.05) and 48 hours (p less than 0.01). At 72 hours, significant decreases in progesterone production were observed with 10 nM (p less than 0.05) and 100 nM RU486 (p less than 0.01). The greatest effect of RU486 on progesterone production occurred at 24 hours of incubation (slope = -8.03) compared with 48 (slope = -4.71) or 72 (slope = -2.31) hours (p less than 0.01). Maximal suppression of progesterone production occurred using 10 nM RU486 with no further significant suppression observed with 100 nM RU486. Other steroids (R5020, DHA) failed to suppress progesterone production suggesting that the observed inhibitory effect on progesterone was specific to RU486. To better understand how RU486 decreases progesterone production in granulosa cell cultures, we measured human ovarian 3B-hydroxysteroid dehydrogenase (3BHSD) in the presence and absence of RU486 in vitro. A significant dose-dependent decrease in the activity of 3BHSD was observed at concentrations of RU486 that were equal to or greater than substrate concentration. Taken together, these findings suggest that RU486 may directly affect human ovarian progesterone production through a mechanism that involves a reduction in 3BHSD activity.

3-Hydroxysteroid Dehydrogenases↗

PubMed1988

Changes in progesterone metabolism in the chicken hypothalamus during induced egg laying stop and molting.

In the present study, we have established and validated a radioenzyme assay which permits us to quantify progesterone metabolism in the chicken brain. Progesterone metabolism was then studied in five brain areas obtained by microdissection from the telencephalon (part of the lobus paraolfactorius immediately rostral to the preoptic area), the preoptic area, and the hypothalamus. Three metabolites of progesterone were produced in large amounts in these brain regions and were quantified in this study: 5 beta-pregnane-3,20-dione (5 beta-DHP) as well as its metabolite 3 alpha-hydroxy-5 beta-pregnane-20-one (5 beta,3 alpha-ol) and 5 alpha-pregnane-3,20-dione (5 alpha-DHP). The unmetabolized progesterone was also recovered and quantified. The 5 beta-reduction of progesterone (production of 5 beta-DHP and 5 beta,3 alpha-ol) was very active but its 5 alpha-reduction (production of 5 alpha-DHP) was almost absent in the lobus paraolfactorius. An opposite pattern of metabolism was found in the preoptic area and the hypothalamus (higher 5 alpha- but lower 5 beta-reductase activity). The changes in progesterone metabolism in these brain areas were then studied in groups of hens submitted to induced egg laying stop and molting. A significant decrease in progesterone 5 alpha-reduction was found in the median hypothalamus of hens during the period of molt. Simultaneously, the experimental procedures induced significant decreases in the production of 5 beta-DHP by the lobus paraolfactorius, anterior, and medial hypothalamus but induced a significant increase in the production of this metabolite in the preoptic area. These changes are likely to be involved in the control of reproductive functions including sexual behavior and secretion of luteinizing hormone-releasing hormone, and a number of possible causal mechanisms are presented. These should now be tested experimentally especially in view of the very limited information which is now available on the biological effects of the metabolites of progesterone.

3-Hydroxysteroid Dehydrogenases↗

PubMed1986

Tamoxifen and progesterone effects in target tissues during the perinatal period.

The biological effects of tamoxifen (TAM), progesterone (P), or a combination of TAM + P were investigated in the uterus and vagina of newborn guinea pigs after short (2 days) and long (12 days) treatments. In both tissues, tamoxifen provoked a significant trophic effect which is indicated by the increase in weight, protein and DNA content. In the uterus, progesterone also provoked an increase in weight, protein and DNA content, but much less than that provoked by tamoxifen. In contrast, in the vagina progesterone had no effect on the weight, protein and DNA content, but progesterone did not block the agonistic effect provoked by tamoxifen. The situation was different when progesterone receptor was concerned. Tamoxifen in both tissues (particularly in the vagina) stimulated the progesterone receptor very significantly. Progesterone blocked the number of specific binding sites of progesterone and the stimulatory effect provoked by tamoxifen.

Animals↗

PubMed1986

Characterization of progesterone binding to nuclear receptors in rat placenta.

Exchange assays have been validated to study several forms of the progesterone receptor found to occur in nuclei of rat placenta after extraction with high salt. One form was solubilized by the extraction procedure (KCl extractable Rpn) and another form remained attached to nuclear structures (KCl resistant Rpn). Specific binding of progesterone was optimized in both forms using buffered media containing 0.01 M Tris, 30%-glycerol (v/v), 0.2 mM leupeptin, and 1 mM dithiothreitol (TDGL), pH 7.8, at 0-4 degrees C for 18-24 h. At 0-4 degrees C the nuclear receptors were stable and degradation was negligible even after 44 h of in vitro incubation. The binding reaction between progesterone and receptor demonstrated mass action principles of ligand exchange throughout this interval. Saturation analysis indicated the presence of a single binding moiety of high affinity (app Kd = 2.9-3.2 nM) for both forms of the receptor. However, the nuclear progesterone receptor was thermolabile and after a 10 min exposure to 30 degrees C no longer complexed ligand. At an intermediate incubation temperature of 22 degrees C the binding reaction was stable for about 30 min. The KCl resistant binding sites were markedly more thermolabile. Addition of 10 mM Na molybdate protected all forms of the nuclear progesterone receptor from thermal denaturation and extended the life of the complex 3-4-fold. The dissociation rate constant of progesterone-nuclear receptor complex in each preparation was 6-8 X 10(5) s-1 resulting in a half-life of about 3 h. The KCl resistant and extractable binding sites were sensitive to blockade by 1 mM N-ethylmaleimide which was reversed by co-incubation with a 2-fold molar excess of dithiothreitol. This suggested that reduced sulfhydryl groups located on or near the surface of the ligand binding domain of the receptor were necessary to bind hormone. These studies showed that the interactions between ligand and the KCl resistant and extractable receptor sites found in rat placenta were of high affinity, saturable, and heat sensitive. Thus, these binding moieties exhibited physicochemical behavior very similar to each other and to the placental receptor which has previously been partially purified from the cytosol. The conclusion is made that all of the nuclear receptor binding sites for progesterone are structurally identical. Thus, the distinctive physicochemical properties responsible for KCl resistant and extractable forms of the nuclear progesterone receptor must reside in other domains of the receptor molecule.

Animals↗

PubMed1988

Progesterone receptors regulate gallbladder motility.

The increased incidence of gallstones in multiparous women may be related to hormonal effects on the gallbladder and its contractility. The occurrence of estrogen and progesterone receptors were studied in the gallbladders of three groups of female guinea pigs (normals, oophorectomized, and oophorectomized treated with estrogen + progesterone for 14 days). Gallbladder contractile response in vivo to cholecystokinin (CCK) was related to the presence of these receptors. The gallbladders from normal females showed low progesterone and estrogen binding activity (4.9 +/- 2.0 and 2.4 +/- 0.8 fmoles/mg cytosol protein). Oophorectomized females had no detectable progesterone or estrogen receptors, but after treating oophorectomized females for 14 days with estrogen + progesterone, gallbladder concentrations of progesterone receptors increased significantly to 14.7 +/- 5.9 fmoles/mg and estrogen binding activity was minimally detectable at 1.4 +/- 0.8 fmoles/mg. The gallbladder contractile response to CCK was inversely related to the concentration of progesterone receptors in the gallbladder wall. These data suggest that the gallbladder contains progesterone receptors which are susceptible to circulating hormonal conditions and which have a regulatory effect on gallbladder contractility.

Animals↗