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PubMed1985

[Radioimmunological system for the quantitative determination of progesterone in human blood serum].

A methodical approach to development of a radioimmune system for quantitative assay of progesterone in the blood serum of man is described. It implies investigation of every component in the system and conformity of these components by the physico-chemical properties and concentrations. An antiserum to the progesterone conjugate with bovine serum albumin was prepared. The "fitting density" of the heptaene was 35 mol/mol of the protein. The linkage with the carrier protein in it was performed by the functional group at C(ii) of the steroid molecule. The antiserum had a high titre (28000), high specificity and affinity to progesterone (Ka (3.5 +/- 0.4) X 10(9) M-1). The antiserum characteristics did not depend on the hydrogen ion concentration in the solution at pH 3.5-10.0 and the ionic strength of the buffer solutions (0.05 to 1.0 M). This allowed performing the radioimmunological assay at pH 3.5. Under such conditions complete inhibition of the endogenic binding proteins of the blood serum was possible. 125I preparations of progesterone made in the USSR were used in the study. The progesterones were prepared by adding the molecule of histamine containing the 125I atom to the molecule of progesterone modified at C(3). The quality of the 125I preparations of progesterone was estimated by their immune reactivity. The standard solutions of progesterone were prepared with the donor blood serum. It was shown that quantitative determination of progesterone in the blood serum at clinically significant ranges of the hormone concentration was possible at the final antiserum dilution of 1:28000, the label activity of 20000-30000 imp/min per a sample and the content of the blood serum in the analyzed sample equal to at least 10 per cent. A simple and reliable procedure for the assay of progesterone in the blood serum of man was developed.

Animals↗
PubMedDate not supplied

[Combined effects of estradiol, progesterone and tamoxifen on the synthesis of specific proteins in the oviduct of the chicken].

Tamoxifen, an antiestrogen with non estrogenic effect on growth, differentiation and egg white protein synthesis in the chick oviduct, has been administered together with progesterone. In estrogen differentiated, hormone withdrawn animals, biochemical and ultrastructural data indicated that the combined treatment with progesterone plus tamoxifen increased conalbumin (+ 100%) and ovalbumin (+ 30%) more than progesterone alone, and displayed estrogen-like growth promoting properties (+ 50% in DNA content/oviduct). Moreover, administered with progesterone and estradiol, tamoxifen lost part of its antiestrogenic activity. No obvious change of progesterone clearance from the blood or progesterone metabolism was recorded. The presence in amounts similar to those resulting in optimal activity when injected alone (greater than or equal to 1mg/kg) was necessary to obtain progesterone plus tamoxifen effets. The simultaneous presence of progesterone and tamoxifen was required in order to observe their combined effects and it was observed that progesterone did not have to commit cells on which tamoxifen could subsequently act. In the immature "undifferentiated" oviduct, contrary to tamoxifen and progesterone alone, their combination induced cytodifferentiation of tubular gland cells synthesizing conalbumin and ovalbumin, an event never observed before in the absence of estrogen. These results support the suggestion that the hormonal milieu interferes with antiestrogen action.

Animals↗
PubMed1993

Influence of the conjugation site on the specificity of monoclonal antibodies to progesterone and on the performance of direct enzyme immunoassay.

Monoclonal antibodies against progesterone conjugated to carrier protein through substituent on the A-ring (C3 position ) or the C-ring (C11 position) of progesterone were used in the enzyme immunoassay. Antibody specificities were determined by testing the ability of 11 represensitive steroids to displace labelled progesterone in a competitive enzyme immuno-assay (EIA) and a radioimmunoassay (RIA). Immunization with progesterone conjugated to BSA through substituent on the A-ring (C3) resulted in the formation of monoclonal antibody (mAb) which fairly specific for progesterone. While immunization with progesterone at the 11-position (C11) resulted in the mAbs which were very specific for progesterone. The best EIA system was developed by using mAb against progesterone-11 alpha -hemisuccinyl BSA and tracer of progesterone-3(O-carboxymethyl oxmine)-horseradish peroxidase. Our approach is to confirm the overall orientation of the steroid in the binding site. Immunobiochemical analysis of mAb suggested that the D-ring is substantially more buried in the binding pocket than the A-ring. Our assay was designed to use the reagent inactivating cholesterol binding globulin in serum so that the extraction of the hormone into organic solvent was unnecessary. Therefore, our assay system can detect directly and rapidly the progesterone level in serum within one hour.

Animals↗
PubMed1993

Progesterone receptor expression in meningiomas.

The possibility that the female sex steroid progesterone plays a role in meningioma proliferation has been suggested by a number of investigators; and it had been shown that many meningiomas have high affinity progesterone binding sites. The aim of this study was to examine the expression of progesterone receptor mRNA and correlate it with the nuclear localization of progesterone receptor by immunocytochemistry in a large number of meningiomas. Thirty-three meningiomas were examined for the presence of measurable amounts of progesterone receptor mRNA by Northern blot analysis and 11 of these were analyzed for receptor protein by immunohistochemistry. These were compared with normal arachnoid and an arachnoid cell line. Sixty-four % of the meningiomas expressed progesterone receptor mRNA. This occurred in a similar pattern to what has previously been shown for T-47 D cells, a breast carcinoma cell line with 11.4-, 6.1-, 5.2-, 4.5-, 3.2-, and 2.5-kilobase mRNA species. This suggests that progesterone receptor expression is not tumor specific. There was a marked predominance of women among those patients whose tumors expressed progesterone receptor; 81% were female and 19% were male. The immunohistochemistry data correlate well with the Northern blot analysis. The staining was clearly nuclear, suggesting that the receptor is in a location to be activated. These data suggest that progesterone receptor mRNA and protein is expressed in meningiomas and support the concept that progesterone may play an important role in meningioma growth.

Adult↗
PubMed1996

Progesterone regulates gamma-aminobutyric acid B (GABAB) receptors in the neocortex of female rats.

After discovering that binding to GABAB receptors in rat neocortex varied as a function of the estrous cycle of the rat, we asked whether either or both of the major ovarian steroids could affect binding to GABAB receptors in the same way, namely, by regulating the apparent density (Bmax) of GABAB receptors. We report here that in ovariectomized rats, subcutaneous injection of progesterone alone, without the necessity of estrogen priming, increased the Bmax of baclofen binding to GABAB receptors in the neocortex. Radioimmunoassay of plasma progesterone before and after progesterone injections revealed that plasma progesterone levels similar to those reached during the progesterone surge in proestrus were associated with increased baclofen binding. The effect of progesterone upon baclofen binding was evident 4 h but not 1 h following progesterone treatment. There was some specificity with respect to the cortical receptors affected by progesterone in that under our conditions, progesterone did not increase agonist binding to 5-HT1A or GABAA receptors. We interpret our results to indicate that progesterone variation during the estrous cycle could be responsible for a component of the regulation of GABAB receptors that occurs in neocortex during the estrous cycle of the rat.

Adrenalectomy↗
PubMed1997

Effect of blood sampling and administration of ACTH on cortisol and progesterone levels in ovariectomized zebu cows (Bos indicus).

Four zebu cows were bilaterally ovariectomized through lateral laparotomy. Three months after ovariectomy, blood samples were collected by jugular venipuncture daily for 5 consecutive days prior to a single injection of ACTH to establish baseline concentrations of cortisol and progesterone. Baseline concentrations of cortisol and progesterone were 31 +/- 5 nmol/L and 0.3 +/- 0.01 nmol/L, respectively. On the day of ACTH treatment the cows were allowed to rest for 2 h to reduce the stress of cannulation before the sampling period started. Blood samples were collected every 30 min from 2 h before until 2 h after the injection of 6 micrograms ACTH and hourly between 2-6 h after ACTH injection. A significant increase was observed in cortisol secretion from 90 min before until 120 min after ACTH injection. No significant increase was observed in progesterone secretion before ACTH injection. After ACTH injection progesterone was significantly elevated for 120 min. Four weeks after the ACTH treatment the cows were cannulated again and blood samples were collected following the same bleeding schedule used during the ACTH experiment. Instead of ACTH a saline injection was given via the catheter. A significant increase in cortisol concentration was recorded 90 min before saline injection. This increase was not accompanied by an elevation in progesterone concentration. No significant changes were observed in cortisol and progesterone levels after saline injection. When cortisol was added to a plasma pool having a progesterone concentration of 0.3 nmol/L and a cortisol concentration of 25.4 nmol/L and assayed for progesterone in 2 different assays no increase in progesterone concentration was observed. We conclude that the adrenal glands can be an extra-ovarian source of progesterone during stress in Zebu cows.

Adrenocorticotropic Hormone↗
PubMed1990

Progesterone receptor antagonists and prostaglandins in human fertility regulation: a clinical review.

Progesterone receptor antagonists have been developed by substitutions at the 11-beta and 17 side-chain positions of the progestagen norethisterone. The most studied progesterone receptor antagonists are mifepristone (Mifegyne; Roussel-UCLAF; RU486) and ZK98734 and ZK98299 (Schering AG). These compounds bind avidly to the progesterone receptor and glucocorticoid receptor but have essentially no binding to the mineralocortocoid, oestrogen or androgen receptors. Mifepristone also binds avidly to albumin, resulting in a half-life of approximately 24 h after oral administration. Progesterone receptor antagonists can induce menstruation by a direct action upon the endometrium. They have also been shown to exert weak progesterone agonist actions in certain circumstances and to modulate pituitary hormone secretion by antagonizing the feedback actions of progesterone. Moreover, they release prostaglandin F2 alpha and E2 from human endometrium or early pregnancy decidua and reduce the metabolism of these eicosanoids. Clinically, progesterone receptor antagonists have been used in trials of menstrual regulation, abortion and induction of labour, and during treatment of breast or ovarian cancer, some forms of hypertension and meningioma. Progesterone receptor antagonists have been administered to approximately 70,000 women in 18 countries as medical abortifacients. They have been proven, especially when combined with prostaglandin analogues, to be as effective as surgical methods of termination of pregnancy. Progesterone receptor antagonists have focussed international attention on menstrual regulation, abortion and the rights of women to regulate their fertility.

Abortifacient Agents, Steroidal↗
PubMed1986

The effect of exogenous progestins on endogenous progesterone secretion in pregnant mares.

Twenty-four pregnant, light horse mares were used in a study to determine if an exogenous progestin or progesterone would alter serum concentrations of progesterone. On day 40 of gestation, mares were randomly assigned to one of three administration groups: 1) 250 mg of progesterone in oil every other day, 2) 22 mg of Altrenogest (Regumate, American Hoechst, Somerville, NJ 08876) orally every day, or 3) 10 ml of neobee oil (control) orally every day. The treatment period was from day 40 to 105. Pregnancy status was monitored on days 40, 60, 80, 100 and 105 and a single jugular blood sample was obtained daily from days 40 to 46, 69 to 75 and 99 to 105. Serum concentrations of progesterone were determined by radioimmunoassay. Concentrations of progesterone were similar (P>0.05) among groups at each sampling period. Overall concentrations of progesterone increased (P<0.001) from days 40 to 46. Injection of 250 mg of progesterone in oil failed (P>0.05) to maintain concentrations of progesterone in serum above baseline for 24 hr. Thus higher doses and/or more frequent injections would be needed in order to increase concentrations of progesterone above those seen in untreated controls. In summary, Altrenogest was found to be a nonstressful, convenient method of administering progestins to pregnant mares without altering their endogenous secretion of progesterone.

Journal Article↗
PubMed1990

Progesterone determinations and clinical examinations of reproductive organs in purebred and crossbred female Zebu cattle.

Five experiments were undertaken to investigate variation in progesterone concentrations as related to the collection and handling of samples of milk and blood, to determine reference values for progesterone and to evaluate clinical findings in relation to progesterone data from pure- and crossbred Zebu cattle reared in the Peruvian tropics. Whole-milk progesterone concentrations obtained from 12 Holstein x Nellore pregnant cows at hourly intervals over a 24-h period were highest immediately after milking; this peak was followed by a sharp drop over the next 3 h. Milk-fat content from 28 Brown Swiss x Nellore pregnant cows increased from 2.4% before milking to 6.7% afterwards (P < 0.05), whereas progesterone concentrations in whole milk increased from 18.4 to 59.5 nmol/1 (P < 0.05). Progesterone concentrations in fat-free milk were stable, with the exception of the fore-milk sample, which was lower than subsequent samples collected during milking (P < 0.05). Blood samples collected from 23 purebred, pregnant Nellore cows, were divided into four aliquots, and plasma and serum were harvested periodically over the next 120 h of storage at +4 degrees C, or in the shade at ambient air temperatures. The results indicate that blood samples can be stored unseparated at both temperatures studied for up to 3 h without severe loss of progesterone. Milk samples collected at different intervals during the luteal phase of the estrous cycle and during early and mid-pregnancy from crossbred cows showed no significant differences in progesterone concentrations between Days 9 to 13 of the cycle and Days 9 to 13 of gestation. Progesterone levels during advanced gestation were higher (P < 0.05). Out of 2,607 clinical examinations, the level of agreement between palpatory findings and progesterone determinations was 95.6 and 81.9% in diagnosing non-luteal and luteal structures, respectively. Significant differences in the level of agreement between palpators were found (P < 0.01). It is concluded that milk samples, preferably composite milk or strippings, must be consistently collected at the same stage of milking, and that centrifugation of blood samples should be done as soon as possible and not later than 3 h after collection.

Journal Article↗
PubMed2001

Induction of the acrosome reaction in dog sperm cells is dependent on epididymal maturation: the generation of a functional progesterone receptor is involved.

In the current study we investigated the progesterone receptor exposure on the sperm from the testis and different parts of the epididymis, the relation to the sperm maturation stage, the functionality of the progesterone receptor and the capacity of sperm to undergo acrosome reaction. Exposed progesterone receptors on spermatozoa were detected using Progesterone-BSA conjugate labeled with fluorescein isothiocyanate (P-BSA-FITC) or a monoclonal antibody against progesterone receptor, C-262. Either progesterone or calcium ionophore was used to induce acrosome reaction. A high percentage (69 +/- 8%; mean +/- SD) of spermatozoa from the cauda epididymis showed P-BSA-FITC labeling at the onset of incubation, whereas only 0.1 +/- 1 and 4 +/- 2%, of spermatozoa from the testes, caput, and corpus epididymis, respectively, were labeled. There was no significant increase in P-BSA-FITC binding during the course of a 6 hr incubation. Treatment with either 10 microM progesterone or 5 microM calcium ionophore induced acrosome reaction in cauda epididymal sperm but not in testicular sperm, caput or corpus epipidymal sperm. It is concluded that the matured sperm of the dog from cauda epididymis and freshly ejaculated sperm demonstrate a functional membrane-bound progesterone receptor while less matured spermatozoa from the testicle, caput, and corpus epididymis fail to demonstrate such a receptor. Acrosome reaction of dog sperm can be induced using either progesterone or calcium ionophore; however, the maturation stages of spermatozoa influence this occurrence.

Acrosome Reaction↗
PubMed1995

Modulation of cardiac M2 muscarinic receptor binding by progesterone-related steroids.

We have previously shown that progesterone, but not estradiol or testosterone, can compete with [3H]N-methyl scopolamine (NMS) for the cardiac M2 muscarinic binding site. Experiments have been carried out to investigate the inhibitory effects of a large variety of progesterone-like steroids on the M2 muscarinic receptor. These studies were performed with the aid of a new binding assay which uses intact tissue in the form of ventricular micropunches. Our data show that synthetic, clinically-used progestins such as Provera, norgesterel and cyproterone are largely ineffective at the M2 binding site whereas the naturally occurring progesterone derivatives 17 alpha hydroxy progesterone and Reichstein's Substance S are highly active. (Ki values 5 x 10(-6)M and 1 x 10(-6)M, respectively). Minor structural modifications such as acetylation of the 17 alpha hydroxy group abolishes activity. Steroids known to exert cell membrane effects, such as alfaxalone and pregnenolone sulfate, had no influence on [3H] NMS binding. The progesterone antagonist RU-486 did not block the inhibitory effect of progesterone. Moreover, this putative receptor may be located in the cardiomyocyte membrane since 17 alpha hydroxy progesterone induces rapid dissociation of [3H] NMS from its binding site (30% reduction in 5 min). We attempted to further localize the inhibitory locus to the M2 receptor itself by means of the irreversible antagonist propylbenzilylcholine mustard (PrBCM). Ninety percent of the M2 receptor could be blocked by PrBCM (10(-6) M), an effect reversible by the specific muscarinic antagonist scopolamine methyl bromide but not by progesterone. These results suggested that progesterone does not interact directly with the [3H] NMS labelled M2 binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Allosteric Site↗
PubMed1984

High affinity progesterone binding sites of human uterine microsomal membranes.

Microsomal membranes sedimented at 40 000 g were prepared from human myometrium samples. The progesterone binding properties of microsomal suspensions were determined by incubating microsomes and [3H]progesterone at 4 degrees C. Dextran-coated charcoal was used for the separation of bound and free steroids. Membrane-associated progesterone binding sites of high affinity were identified in microsomes prepared from pregnant and nonpregnant uteri. The binding was saturable (Kd approximately 4 X 10(-9) M, concentration of binding sites 400-900 fmol/mg microsomal protein) and specific for natural progesterone. Of 21 steroids tested only 21-hydroxy-4-pregnene-3,20-dione, 17 alpha-hydroxyprogesterone and testosterone showed moderate competition against progesterone with relative affinities between 7.0-20.0% (R.A. of progesterone 100%). 5 alpha-Dihydroprogesterone and 5 alpha-dihydrotestosterone showed weak cross reaction (relative affinities 2.5 and 2.0%, respectively). Corticosteroids, estrogens and the 5 synthetic progestins tested showed only weak competition with relative affinities lower than 1.0%. These microsomal progesterone binding sites of high affinity and limited capacity resemble steroid hormone receptors but they are different from the soluble cytosolic progesterone receptor of human uterus in terms of steroid specificity. The physiological function of this microsomal progesterone receptor is unknown.

Binding, Competitive↗
PubMed1979

On the regulation and turnover of progesterone metabolites in rat uterus.

Recently the in vitro progesterone metabolism was shown to be inhibited in uterine tissue by association of the hormone with binding components. However, a dissociation of progesterone would impair the protection of the steroid hormone caused by complex formation. In order to study this effect, the influence of time was investigated on the metabolism of progesterone. Progesterone metabolites were analysed quantitatively from the recovered material of uteri and nutrient media by thin layer chromatography (TLC) at various time invervals. After finishing the incubation with the labelled steroid, the amount of progesterone metabolites produced increased continuously in the tissue during the following hour when the uteri were kept in nutrient medium. This indicated that the dissociation of progesterone from a hormone protein complex led to the subsequent metabolism of the unbound hormone. However, the metabolism was reduced markedly by an increase of the protein content in uterine tissue and with it by an increase of progesterone binding proteins in uterine cytosol as determined by charcoal adsorption technique. Additionally, the amount of progesterone metabolites was found to be much higher in uterine tissue than that released into nutrient medium during the time interval studied. Therefore, uterine tissue concentrates progesterone metabolites, and a rapid turnover of these substances does not occur.

Animals↗
PubMed1982

Characteristics of the nuclear translocation of progesterone receptor in fetal guinea pig uterus "in vivo", "in vitro" and in organ culture.

The translocation of progesterone receptor from the cytosol into the nucleus was studied under "in vivo" and "in vitro" conditions in the uteri of guinea pig fetuses exposed to progesterone or a synthetic progestin, R5020. Progesterone treatment of estrogen-primed fetuses leads to a rapid (before 1h) transfer of cytosol progesterone receptor into the nucleus which is, however, short-lived (less than 3h). A rapid decrease in the retention of the estrogen receptor in the nucleus also occurs. In the "in vitro" incubations of whole fetal uteri, translocation of progesterone receptor is temperature-dependent and specific for progesterone and R5020; estradiol and cortisol have no effect. Putative progesterone receptors can also be induced in explants of fetal guinea pig uteri in organ culture which translocate from the cytosol into the nucleus under the same "in vitro" conditions as in whole uteri. Fetal uterine progesterone receptor, either stimulated "in vivo" by estrogen-priming or induced in organ culture, translocates from the cytosol into the nucleus and this process seems to be accompanied by a decrease in retention of the estrogen receptor in the nucleus which appears to be the mechanism by which progesterone antagonises estrogen action in fetal guinea pig uterus.

Animals↗
PubMed1992

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 anti-hormonal 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.

Animals↗
PubMed2004

Porcine spermatozoa contain more than one membrane progesterone receptor.

Progesterone has been shown to be a physiologically relevant inducer of the sperm acrosome reaction. A novel protein intrinsic to microsomal membranes, membrane progesterone receptor (mPR, now termed progesterone membrane receptor component 1, PGMRC1) that binds progesterone with high affinity has been cloned from porcine liver previously, and corresponding antibodies mitigate the progesterone induced acrosome reaction. In this study we aimed at the localization of mPR in porcine spermatozoa. Immunostaining suggested the exclusive occurrence of mPR in a hardly accessible place, possibly the inner acrosomal membrane, with digitonin dramatically increasing the number of positively stained cells. Consistent with the structure prediction for mPR, its short N-terminus (NT) but not the large C-terminal part becomes accessible from outside after digitonin treatment as evidenced by the staining pattern of antibodies directed against different regions of the protein. However, digitonin treatment solubilizes a progesterone binding activity of approximately 140 kDa molecular weight, that is different from mPR, which remains in the cell membrane as demonstrated by Western blotting. Ligand binding studies confirm the dissimilarity of mPR and the digitonin-soluble progesterone binding protein. Chemical modification studies also indicate that the digitonin-soluble progesterone binding protein has a binding site that differs from that of mPR. It is concluded that more than one progesterone receptor is present in porcine spermatozoa.

Acrosome↗
PubMed2003

The significance of the 20-carbonyl group of progesterone in steroid receptor binding: a molecular dynamics and structure-based ligand design study.

Polar functional groups in the A- and D-ring (positions 3 and 17beta or 20) are common to all natural and synthetic steroid hormones. It was assumed that these pharmacophoric groups are involved in strong hydrogen bonding interactions with the respective steroid receptors. High resolution X-ray structures of the estrogen and androgen receptors have confirmed these assumptions. Also site-directed mutagenesis studies of the human progesterone receptor (hPR) suggest an important role for Cys891 in the recognition of the progesterone 20-carbonyl group. Surprisingly, the crystal structure of the hPR ligand binding domain (LBD) in complex with progesterone suggests that the carbonyl oxygen in position 20 (O20) is not involved in hydrogen bond contacts. To investigate these surprising and contradicting results further, we performed a molecular dynamics simulation of the hPR-progesterone complex in an aqueous environment. The simulation revealed hPR-Cys891 as the sole but weak hydrogen bonding partner of progesterone in the D-ring. In contrast to the site-directed mutagenesis data a major role of hPR-Cys891 in progesterone recognition could not be confirmed. Isolated hydrogen bond acceptors, such as the prosterone O20 group, in a relatively lipophilic environment of the receptor led to a decrease in affinity of the ligand. Based on this consideration and the structure of the PR, we designed compounds lacking such an acceptor function. If the X-ray structure and the calculations were right, these compounds should bind with comparable or higher affinity versus that of progesterone. E-17-Halomethylene steroids were synthesized and pharmacologically characterized in vitro and in vivo. Although the compounds are unable to form hydrogen bonds with the hPR in the D-ring region, they bind with superior affinity and exert stronger in vivo progestational effects than progesterone itself. Our investigations have confirmed the results of the X-ray structure and disproved the old pharmacophore model for progestogenic activity, comprising two essential polar functional groups on both ends of the steroid core. The 20-carbonyl group of progesterone is likely to play a role beyond PR-binding, e.g. in the context of other functions via the androgen and mineralocorticoid receptors and as a site of metabolic inactivation.

Animals↗
PubMed2004

Regulation of hepatic progesterone and estrogen receptors in the female turtle, Chrysemys picta: relationship to vitellogenesis.

Previous studies using the fresh water turtle Chrysemys picta have demonstrated the differential expression of the two progesterone receptor isoforms (PRA and PRB) in the liver during the turtle seasonal cycle, correlating with ER mRNA levels and hepatic vitellogenesis. During the inter-vitellogenic periods the ratio of PRB:PRA favors PRA, suggesting that the PRB:PRA ratio may be important in the regulation of vitellogenesis. Based on these and other studies we hypothesize that progesterone may have differential effects on the estrogen (E)-induced vitellogenin response (inhibitory or stimulatory), depending on the PRB:PRA ratio. In this study, we determined the expression pattern and the hormonal regulation of progesterone receptor (PR) isoforms in the liver, during the estrogen-induced vitellogenin response in female C. picta. Northern blot analysis was used to evaluate the changes in vitellogenin mRNA, estrogen receptor (ER) mRNA, and PR mRNA expression; Western blot to determine changes in PR isoform expression and a homologous ELISA for measurement of plasma vitellogenin. The expression of PR isoforms in the liver of female turtles at the mRNA and protein levels was differentially regulated by estradiol and progesterone. Estradiol treatment enhanced the transcription of PR mRNA isoforms and possibly translation and/or increased stability of PRB protein in the female turtle liver. In contrast, this hormone decreased PRA protein levels. Progesterone alone down-regulated progesterone receptor isoform A (PRA) and progesterone receptor isoform B (PRB) proteins equally, but did not affect PR mRNA levels. Estradiol markedly increased hepatic ER mRNA, vtg mRNA, and vtg in plasma, but this effect was not impacted significantly by progesterone. The results presented here indicate that in female turtles hepatic PRB:PRA ratios are markedly changed by estradiol treatment due primarily to a decrease in PRA. The change in the PRB:PRA ratio after hormonal treatment confirms that progesterone and estrogen exposure will be a determinant in the regulation of vitellogenesis, and, in turn, that the regulation of vitellogenesis will be determined by the ratio of PR isoforms and the physiological levels of steroid hormones.

Animals↗